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https://f1000research.com/articles/11-84/v1	24 Jan 22	{ "type": "Research Article", "title": "Assessment of the knowledge, attitudes and practices of Lebanese shoppers towards food labeling: The first steps in the Nutri-score roadmap", "authors": [ "Maha Hoteit", "Nour Yazbeck", "Ayoub Al-Jawaldeh", "Cecile Obeid", "Heba Abdul Fattah", "Marwa Ghader", "Hala Mohsen", "Nour Yazbeck", "Ayoub Al-Jawaldeh", "Cecile Obeid", "Heba Abdul Fattah", "Marwa Ghader", "Hala Mohsen" ], "abstract": "Background: Food labeling is a fundamental educational tool for advocating for public awareness. It emphasizes knowledge of the nutrient content of food and thus directs the choice towards the healthiest food products. This cross-sectional survey aimed to assess the knowledge, attitudes, and practices (KAP) regarding nutrition label use in Lebanon through a valid questionnaire. Methods: Overall, 768 participants (mean age: 30.8 ±12, males: 60.2%) were recruited randomly between February and May 2020. We used word of mouth and social media to recruit our sample population. Results: Social media was the most accessed tool to attain nutrition information by responders (39.8%). More than half the participants expressed positive attitudes to check information related to sugars (66.4%), vitamins (64.9%), total fats (61.7%), proteins (59.1%), and calories (58.7%) on the food label. Expiry date, price, and brand name were the top three considerations while reading food labels. About half (46.5%) reported to “always” look at the food label. Responders reported reading labels related primarily to sugars (44.3%), calories (38.8%), and total fats (36.8%). The optimal total KAP score was 46; our findings revealed a mean KAP score of 14.46 ±7 (31.4%). When categorizing the KAP scores, 15% had high scores, and 85% scored low. Spearman’s coefficients showed positive correlations between knowledge-attitude, knowledge-practice, and attitude-practice scores, with p<0.001. The regression analysis revealed that gender, age, BMI, residency area, educational level, university degree, health and diet statuses, and activity level were significant predictors of the KAP score. Being on a diet had the highest odds (OR=3.107, CI=1.904-5.072, p<0.001). Conclusion: The low awareness of food labels leads Lebanese people to choose unhealthy food options. A planned educational program is recommended to ease the interpretation of these labels.", "keywords": [ "Food labels", "knowledge", "attitude", "practices", "Lebanon" ], "content": "Introduction\n\nNon-communicable diseases (NCDs) account for over 35 million disease cases per year and two-thirds of the world's deaths.1 Nutrition-related non-communicable diseases (N-NCDs), mainly diabetes, cancer, hypertension, and cardiovascular diseases, are highly prevalent in most countries in the Eastern Mediterranean Region (EMR).2 In Lebanon, it is estimated that NCDs accounted for 91% of all deaths in 2016.3 Thus, as a preventive action plan, the Lebanese Ministry of Public Health (MOPH) developed a national NCD prevention and control plan (NCD-PCP), that is yet to be implemented.4 Dietary guidelines and labeling legislations are considered an effective tool for creating a healthy food environment to reduce the global burden of NCDs.5 Moreover, the nutrition facts provided on food labels could drive favorable consumers’ behaviors.6 In the United States of America (US), 98% of FDA-regulated packaged foods have nutrition facts panels (NFPs).7 As for Europe, 84% of products have nutrition labels.8 In Lebanon, nutrition labeling is regulated by mandatory standards: NL 206, NL 719 which address the labeling requirements for foods.9 Food labeling is required for most prepared foods such as breads, cereals, canned and frozen foods, snacks, desserts, drinks, among others. Ingredients list, nutrition facts, food allergen declarations and date marking must be available as well.9 The use of food label information is influenced by multiple elements, including comprehension difficulties, promotions, price, educational level, attention, and memorizing the information to apply it to a consequent food-related decision.10 Thus, a combination of these factors may propel the consumer to prefer one product over another.10 However, the presence of detailed nutrition information on the food package does not necessarily guarantee a healthy choice.6 For example, it was found that most consumers (78%) could notice nutrient differences between food products; however, fewer (20%) were able to calculate the contribution of food nutrients to the daily intake.10 Additionally, a recent cross-sectional study revealed that the Health Star Rating, a front-of-pack labelling system that rates the overall nutrition profile of packaged food and assigns it a rating from 0.5 to 5 stars, resulted in a greater willingness to pay for healthier products.6 The cognitive processing model considers decision-making as a high-level cognitive process defining how people think, perceive, remember and learn.10 In other words, it represents the acquisition and storing of knowledge related to any topic leading to a corresponding behavior.10 As for nutrition labels, this model showed that food purchasing behaviors depend heavily on having prior knowledge via three interlinked pathways.10 Prior knowledge could enable consumers to disregard marketing attributes.10 Besides, it facilitates the comprehension and memory of nutrition information.10 As a result, the stored information supports the choice of healthy food options.10 The model concluded that consumers who are more knowledgeable about food nutrients are more likely to develop positive attitudes and use label information productively and correctly.10 A previous study reported that two-third of the consumers with a particular interest in healthy eating actually payed attention to food labels when shopping.11 This result suggests that across most countries, the effectiveness of food labelling varies with culture, nutritional knowledge and demographic characteristics of the population.11 Thus, it is a high priority to point out the factors affecting Lebanese consumers’ interpretation of food labels, to formulate new regulations or update existing ones. The aim of this study was to assess consumers’ knowledge, attitudes and practices (KAP) regarding the information on food labels, and to investigate the correlates of low levels of KAP among Lebanese shoppers.\n\n\nMethods\n\nThis was a cross-sectional study conducted between February and May 2020. The estimated minimum sample size was 384 (as per the Epi-info statistical software developed by the Center for Disease Control and Prevention Version 7.2,), and we eventually recruited 768 participants from the different Lebanese governorates. Lebanese participants aged 15 to 64 years old were eligible to participate in the study. Non-Lebanese individuals and those who did not fit our age recommendations were disregarded while collecting data. Due to the national lockdown imposed due to the COVID-19 pandemic, a self-constructed and validated questionnaire was filled online. We used word of mouth and social media to recruit our population. We gave a brief explanation of our valuable research objectives for each responder. The collected data were solely used for scientific and research purposes.\n\nA 41-item questionnaire was adapted after conducting an extensive review of the literature and based on instruments used in previous studies.12–14 It was comprised of four parts: the first collected information on demographic, socio-economic and health-related data; the second part (11 questions) focused on the knowledge related to nutrition labels; the third (five questions) addressed the attitudes, and the fourth part (12 questions) investigated the responder’s practices. The questionnaire was drafted in English, subsequently translated into Arabic, the native language of the participants. It was filled online, and it took the participant around 20 minutes to fill it. The completion of the questionnaire was voluntary and anonymous. Firstly, our formulated questionnaire was piloted with 200 respondents to check its acceptability. Its internal consistency and validity were assessed using Cronbach’s alpha (α = 0.7); observed alpha values were high for all the questionnaire sections. As the pilot study justified its validity and reproducibility, the questionnaire was then employed for further data collection. The findings from the pilot study were not considered in the final analysis.\n\nIn regard to knowledge, a score of 1 point was granted for responders who answered correctly. Otherwise, those who reported a wrong answer, or failed to give any response (I don’t know response) were given no mark. For each respondent, an overall knowledge score was calculated, by adding the scores from all responses. The respondent could earn a maximum score of 14. The mean knowledge score for our sampled population was then calculated. Regarding the attitudes, and in a similar concept, one point was allocated for each appropriate response; assumed to be a positive attitude. However, a score of zero was issued for undesirable responses which were considered as negative attitudes. For each respondent, an overall attitude score was calculated, by adding the scores from all responses, with a maximum score of 16. The mean attitude score for the overall population was then derived. Likewise, the practice score was computed by adding the respondent's number of appropriate responses over a score of 16. The relevant practice warranted one score for the respondent, whereas an improper practice left the respondent with no score granted. Mean practice score for each respondent was calculated by dividing the total practice score by 16. The resulting scores for the KAP were summed up to generate individualized KAP scores. The respondents could get a maximum KAP score of 46. The KAP score, therefore, was categorized into two levels. A low KAP (<23) and a high KAP (≥23). It is worth noting that the evaluation of the appropriateness of the responders’ KAPs was literature-based, with no subjective judgment.\n\nData were analyzed using the Statistical Package for Social Sciences (SPSS) software version 25. Demographic, socio-economic, and health-related conditions and the responses related to the knowledge, attitudes, and practices were analyzed descriptively. Data were represented as means ± standard deviation (SD) for continuous variables, and as frequencies (N) and percentages (%) for the categorical ones. The Shapiro-Wilk test indicated a non-normal distribution for the KAP scores, and thus, we used the Mann-Whitney U and Kruskal-Wallis tests to assess differences in the mean KAP scores. The Mann–Whitney U test is suitable for the independent variables with two groups (such as gender), while the Kruskal–Wallis test was used for independent variables including more than two groups (residency, for instance). We also referred to the Spearman’s rank correlation coefficient (rho) to examine the associations between KAP scores. A binary logistic regression was conducted to determine the predictors of KAP scores. A confidence interval of 95% was applied, and the level of significance was determined at 5%.\n\nThis study received approval from the ethical committee of the Lebanese University (protocol code CUER # 22-2020). The study’s design and analyses were conducted according to the guidelines of the Declaration of Helsinki. Adult respondents and minors’ families provided a written informed consent before filling the questionnaire, and their confidentiality was protected. We received written informed consent from the adult respondents and minors’ family for the publication of this data.\n\n\nResults\n\nA total of 768 individuals were included in our analysis. The mean age of the overall sample was 30.8 ± 12 years. Adults (25-64 years old) represented 57.8% of the total population, while the remaining were youth (15-24 years old). Male participants constituted the dominant proportion (60.2%). More than half (59.4%) were single or divorced, while 40.6% were married. Moreover, the majority (61.3%) had no children. 49.5% of respondents were working, 35% had no job, 14.3% were housewives, and 1.2% were retired. As for the monthly wage, about half the participants (49.6%) had no salary at all, and another salient proportion (31.9%) were paid less than 1000$ a month. However, 17.3% had a salary of 1000$-3000$, and just 1.2% were earning more than 3000$ per month. Regarding their educational levels, 53.9% were university graduates or still studying at university, among which 76% had Bachelor’s degrees. Besides, 13.7% were studying at general secondary schools, 11.6% were studying at technical secondary schools, 11.2% were studying at elementary/intermediate schools, whereas 9.6% were not attending school at all. Among the final sample, around half the population (49.8%) were living in Beirut and the Mount Lebanon area. The remaining were living in South Lebanon (19%), and North Lebanon (31.2%) areas. Moreover, 45.5% of people in this study had normal BMI values, and 30.5% were overweight. Obesity and overweight status were more prevalent among the male participants (p<0.001). These findings are presented in Table 1.\n\nAs regards their health status, an appreciable proportion (42.7%) had medical conditions. Specifically, 22.9% had nutrition-related chronic diseases (diabetes, hypertension, cardiovascular and kidney disorders); 22.5% had gastrointestinal disorders (including gastroesophageal reflux disease (GERD), chronic constipation, and intolerances); 13.4% had thyroid disorders, and 3.2% were anemic. The remaining (38%) reported other health conditions (neurological, depression, polycystic ovarian syndrome, among others; Table 2). About one quarter of our sampled population were physically active (attending gym), active males (31.7%) were significantly higher than active females (20.8%), p=0.001. Additionally, 34.2% admitted that they were restricted to specific diets. It was shown that the most prevalent diets were weight loss (55.2%), low carbohydrate (15.2%), and weight gain (8.4%). Others were following intermittent fasting (6.6%), therapeutic (4.9%), low-fat (4.5%), vegan/vegetarian (4.5%), and gluten-free (0.4%) diets. More than half of the female dieters (61.4%) were following a weight loss diet, which was higher than that of males by 18.6%, p<0.001 (Table 2).\n\nNutrition information may be accessed from varied sources, and therefore, we asked our participants to report their frequently used sources to obtain nutrition-related information. Our findings showed that social media platforms were used more frequently (39.8%) (Figure 1). Almost 36.4% of Lebanese shoppers relied on Internet and magazines as data sources, based on our survey (Figure 1). On the other hand, a salient proportion of our sampled population (31%) accessed accurate nutrition facts from specialists, such as physicians and dietitians (Figure 1). Family (27.7%), friends (21.8%), and TV/radio channels (18.2%) were also substantial information sources. Otherwise, the minority reported referring to gym coaches (4.6%) or taking nutrition courses (3.8%) (Figure 1).\n\nParticipants’ responses to knowledge questions are described in Table 3. For knowledge scoring, participants were asked to calculate the number of calories contained in 325 grams of condensed milk (calories per 100 grams were given). They also had to choose the order in which food ingredients appeared on the front of the package. Their familiarity with the “sugar-free” claims indication was also assessed. Additionally, we assessed their awareness of common food additives’ function (xylitol, sorbitol, and aspartame), and whether these sweeteners could have a laxative effect or not. Furthermore, they had to decide if monosodium glutamate (MSG) can be consumed by hypertensive patients and if hydrogenated oils were healthy or unhealthy food ingredients. Moreover, we examined their familiarity with six nutrition symbols: “vegetarian”; “non-vegetarian”; “vegan”; “gluten-free”; “trans-fat-free”; and “genetically modified organisms (GMO)” symbols. The final assessed knowledge area focused on E-numbers and their corresponding functions when added to food.\n\nAccordingly, the understanding of nutrition labels was rated by grading an overall knowledge score for our study population, with a maximum possible score of 14. The mean ± SD knowledge score was 2.46 ± 1.93 (17.6%) for the overall population, 2.68 ± 2.07 for females, and 2.13 ± 2.68 for males, p=0.001 (Table 4).\n\n(a) Total KAP score is the sum of the knowledge, the attitude and the practice score.\n\n(b) Overall mean (%) is the product of the overall mean by hundred divided by the upper limit of each score, e.g., knowledge overall mean (%) = (2.46*100)/14= 17.6%.\n\nParticipants’ responses to attitude questions are described in Table 3. We observed how our participants do perceive the beneficial use of the nutrition labels and their mandating. Also, we inquired about participants’ interests in looking over information related to calorie’s, macronutrients, and micronutrients on the nutrition facts panel. Study findings showed that the attitude of Lebanese shoppers towards nutrition facts in food product were mostly to check information related to sugars (66.4%), vitamins (64.9%), total fats (61.7%), proteins (59.1%), calories (58.7%), cholesterol (47.3%), fibers (46.6%), carbohydrates (44.7%) minerals (44.3%) among which most importantly sodium (39.2%). In contrast, they gave less consideration for information related to saturated fats (16.8%), trans-fat (12.5%), monounsaturated fatty acids (MUFA) (5.6%), and polyunsaturated fatty acids (PUFA) (4.8%).\n\nTherefore, by responding to these questions, participants can optimally get an attitude score of 16. The mean ± SD attitude score was 6.24 ± 2.78 (39%) for the overall population, 6.39 ± 2.83 for females, and 6.01 ± 2.68 for males, p=0.072 (Table 4).\n\nIn practice, the top three information searched for when looking at the nutrition facts panel were: expiry date (75.2%), price (60.6%), and brand name (50.8%) (Table 5). Whereas the least sought for information was: nutrition and health claims (24.8%), nutrition content (24.3%), food weight (13.6%), and presence of preparation and cooking instructions (9.4%) (Table 5). Practice scores were based on 16 questions, including the frequency of checking nutrition labels, the act of comparing the nutrient content of food products, and looking at calories, macronutrients, and micronutrients on the food label. As for the frequency of checking the food label, about half of our sample population (46.5%) reported to “always” read the food label, whereas 43.8% of the respondents reported reading them occasionally and only 9.7% admitted not to read it at all (Figure 2, Table 3). Besides this, when hesitating between two food products, the majority (64.7%) reported that they don’t refer to the NFP to base their choices (Table 3).\n\nIn contrast to their attitudes towards the most searched information when looking at the NFP, in practice, Lebanese shoppers looked for the following nutrient information: sugars (44.3%), followed by calories (38.8%), total fats (36.8%), proteins (33.5%), and vitamins (32.9%). Others read labels related to saturated fats (11.5%), carbohydrates (26.6%), cholesterol (26.4%), fibers (25%), minerals (21.5%), sodium (20.7%), and trans-fats (7.8%). A minority were concerned to check information about monounsaturated fatty acids (MUFA) (3%), and polyunsaturated fatty acids (PUFA) (2.9%) (Table 3).\n\nRespectively, by responding to these questions, participants could get a maximum practice score of 16. The mean ± SD practice score was 5.76 ± 3.78 (36%) for the overall population, 6.35 ± 3.982 for females, and 4.89 ± 3.28 for males, p<0.001 (Table 4).\n\nThe obtained knowledge, attitudes, and practices scores were summed up to determine the total KAP score. Subsequently, the respondent could get a maximum total KAP score of 46. Our findings showed a total KAP score with a mean ± SD of 14.46 ± 7 (31.4%) for the overall population, 15 ± 7 for females, and 13 ± 6 for males, p<0.001 (Table 4).\n\nThe Spearman’s coefficients (rho) are presented in Table 6. There was a positive correlation between knowledge and attitude scores (rho = 0.356, p<0.001). Similarly, the knowledge and practice scores were positively correlated (rho=0.38, p<0.001). Correlation findings revealed a strong positive association between attitudes and practices with a Spearman’s coefficient rho= 0.562, p<0.001. These findings demonstrate that knowledge, attitude, and practice scores increased simultaneously, and these scores were significantly correlated.\n\nThe KAP score was significantly lower for men (13 ± 6) than women (15 ± 7), p<0.001. Adults had higher scores when compared to youth (15 ± 7 vs. 14 ± 7, p=0.419). Although underweight, overweight, and obese participants had similar scores (14 ± 7), those with normal BMI had a higher mean score (15 ± 7), p<0.001. In addition, the KAP score was significantly lower for North Lebanon residents (13 ± 6, p<0.001). Further, medical sector workers had higher mean scores, as opposed to non-medical sector workers (18 ± 7 versus 14 ± 6, p=0.012). Participants earning the highest income (>3000$/month) had the highest mean score (19 ± 8), p=0.008. Non-married (15 ± 7) and childless (15 ± 7) respondents had better mean KAP scores, as opposed to their counterparts, p=0.068, and p=0.074, respectively (Table 7).\n\nUniversity students had a higher mean score of 16 ± 7 when compared to lower educational level students, p<0.001. Ph.D. degree holders (22 ± 7) had higher mean KAP scores than those holding Masters’ (18 ± 9) and Bachelors’ degrees (15 ± 7), p<0.001. Those who were restricted to specific diets (17 ± 8) and physically active participants (17 ± 7) had higher mean scores than their counterparts (p<0.001). Healthy respondents had a higher mean KAP score than diseased participants (15 ± 7 versus 14 ± 7, p=0.184; Table 7).\n\nThe KAP score was categorized into two levels: a low KAP (<23) and a high KAP (≥23). The descriptive analysis revealed that 15% of our overall study population had high KAP scores, and 85% scored low for KAP (Figure 3). Based on these findings, we determined the risk factors contributing to having either a low or a high score by running a binary logistic regression test.\n\nTable 8 shows the binary regression analysis findings, with no adjustment. Males (versus females, OR=0.291, CI=0.159-0.321) were less likely to have a high KAP score by 71%, p<0.001. Adult participants were 1.2 times more likely to have a high KAP score, as opposed to young participants, (OR=1.21, CI=0.727-2.013, p=0.539). In addition, being overweight (OR=1.343, CI=0.349-5.171, versus underweight) and obese (OR=1.322, CI=0.32-5.453, versus underweight) increased the possibility of having a high KAP score by 1.3 times (p=0.668 and p=0.7, respectively). Regarding the residency area, North Lebanon residents had the lowest total KAP scores. In particular, North area residents (versus Beirut residents, OR=0.359, CI=0.161-0.804) were less likely to have high scores by 64.1%, p=0.013.\n\nIn terms of the educational level, university students (versus lower education level students) were two times more likely to score acceptably (OR=2.04, CI=1.126-3.696), p=0.019. Although studying at university appeared to be a significant predictor, a higher university degree contributed more significantly to the KAP score prediction. Postgraduates had had a two-fold greater probability to have a high KAP score, as opposed to undergraduates (OR=2.068, CI=1.148-3.725), p=0.016.\n\nInterestingly, our findings show that those who were restricted to specific diets (versus non-dieters, OR=3.107, CI=1.904-5.072) and those who were physically active (versus non-actives, OR=2.245, OR=1.364-3.696) were 3 and 2.2 times more likely to score acceptably, respectively (p<0.001). For the final predictor, i.e., the health condition, it was revealed that diseased participants (versus healthy) were less likely to obtain high score by 21% (OR=0.790, CI=0.480-1.302), p=0.355.\n\n\nDiscussion\n\nThis study assessed the KAPs related to the reading of nutrition labels among Lebanese people, using a validated questionnaire. Overall, the mean KAP score was low (14.46 ± 7, 31.4%) which might indicate a lack of public awareness of nutrition label use. In this study, the mean knowledge score was 2.46/14 points (17.6 over 100 points). Social media were the most used platforms (39.8%) to access nutrition information. However, a lower proportion (31%) accessed accurate and reliable nutrition facts from physicians, dietitians, and other specialists. A previous study aiming to assess the relationship between knowledge and the use of nutrition information on food packages reported that Croatian participants had an average nutrition knowledge of 70% (70 over 100 points).15 According to Koen et al., 2018, the knowledge scores of African participants was estimated around 44%.16 As for Italy, a mean nutrition knowledge score of 46% was observed.17 In Arabic countries, a recent observational study by Arfaoui et al., 2021 showed that adult Saudi adult consumers had a total knowledge score of 5.8/13 points (45%), and about 77% of the Saudi participants had an average knowledge score (50th-75th percentiles using the percentile threshold method).18 Between December 2013 and February 2014, a cross-sectional study was conducted among 748 Lebanese shoppers.19 In a previous study, Lebanese shoppers had an average food label knowledge score of 63.1%.19 The massive gap between our obtained knowledge score (17.6%) and that observed in a previous year (63.1%), might be partly related to the study protocol, as we carried out an online survey, which might have affected the comprehension of the questions; however, in the previous study, shoppers were surveyed in supermarkets. Moreover, the latest economic crisis might have driven the interest of Lebanese shoppers into seeking price over quality. In particular, our respondents had low knowledge scores about the E-number additives (E200, E700 …). E-numbers are the chemical names of certain food additives, and they appeared to have a bad reputation among consumers.20 Products containing food additives are usually perceived as unhealthier products.20 A study was conducted to compare E-number labels with “clean” labels, and showed that consumers find “clean” label ingredient lists the safest, healthiest, and the easiest to read.20 Concerning the ability to interpret nutrition symbols on food labels, especially the vegetarian and vegan symbols, only 2.3% and 4.2% (respectively of our sample were able to do it. A study by Berich, H. (2015) done among Kent State University students found that vegetarian dieters were more familiar with such symbols.21 It should be noted that only 1.8% of our participants were on a vegetarian/vegan diet, explaining the unfamiliarity with their corresponding food symbols. The “GMO” symbols corresponding to food containing genetically modified organisms were also unfamiliar for the majority (92.8%) of our respondents. Foods that contain genetically modified organisms was introduced to the US market and appeared on supermarket shelves in 1994.22 Comparing Lebanon to other countries, a cross-cultural survey assessing the knowledge of consumers in the US, Japan, and Italy showed that US consumers were more familiar with GMOs (40.9%) compared with Italian (28.0%) and Japanese (33.3%) consumers.22 Similarly, only 15% of our participants were familiar with the “gluten-free” food symbol. This finding is unsurprising, as only a few of our study population reported following a gluten-free diet. In addition, another important explanation is that we exposed our participants to the “crossed grain gluten-free” symbol, and not to worded gluten-free claims on packages. In Poland, an eye-tracking study of gluten-free cookies showed that consumers were more uncertain about the crossed grain symbol, as compared to verbal statements and gluten-free claims.23 In contrast, about half of our sampled population agreed that “sugar-free”-labeled products are artificially sweetened. However, they had little idea about the artificial additives which act as sugar substitutes: 75.6% of the overall responders were not familiar with the role of xylitol, sorbitol, and aspartame sweeteners. In a different setting, a qualitative study exploring consumer knowledge and understanding of added sugars in the United Kingdom demonstrated that saccharin and aspartame sweeteners were correctly classified by the majority (60% and 80%, respectively) of respondents.24 Sweeteners have been used to increase food flavor and were adopted because they contain few to no calories compared to the high caloric content of sugars.25 Also, an important proportion of our respondents (40.4%) were aware of the laxative effect of these sweeteners. We can assume this is due to having had previous experiences with this side effect after consuming any sugar-free product. As to MSG, the majority (93.2%) of our sample population did not know whether MSG has blood pressure-elevation effects. A nutrition study on Chinese adults observed that MSG intake was associated with a significant increase in blood pressure levels, especially among patients chronically taking antihypertensive medications.26 A previous study aimed to assess the KAP towards the use of MSG in Pakistan explored that 98.3% of the respondents were using MSGs as food flavor enhancers while cooking stews, soups, pottages, sauce, and others (6.6 g/person/week).27 Contrarily to our findings, 42.5% of the Pakistani consumers had high knowledge levels about MSG.27 Regarding hydrogenated vegetable oils, 42% of Lebanese shoppers in our study agreed that they caused detrimental health side effects. In Saudi Arabia, a study was conducted to assess the trans-fat-related knowledge among Saudis; its results showed that around 35.1% of the participants were familiar with the term “hydrogenated oils”, and only 4% classified these ingredients as unhealthy.28 Even though Lebanese shoppers showed high knowledge regarding hydrogenated oils’ health impacts, practice results differed when a cross-sectional survey was conducted on 657 Lebanese adults who completed the US National Institute of Health diet history questionnaire.29 This study found that the mean trans-fatty acids consumption among Lebanese people was double the World Health Organization (WHO) recommendations of 1 percent of total daily energy.29 Partially hydrogenated oils have been found to contain extremely harmful fatty acids, and they cause inflammation and calcification of the arterial cells, increasing the risk of coronary heart disease (CHD).30 When we asked our participants to calculate the energy density of a sweetened condensed milk, the majority either gave a wrong answer or failed to do any calculation (74.4%). Similarly, several studies reported a low understanding of serving size labeling among consumers.31 A study done by Persoskie et al., 2017, showed that Americans could not determine the calorie content of a full ice-cream container.32 Additionally, 21% could not estimate the number of servings equal to 60 g of carbohydrates, 42% could not estimate the effect on the daily calorie intake of one serving, and 41% failed to calculate the percentage daily value (DV) of calories in a single serving.32 The mean attitude and practice scores for our population were 6.24 ± 2.778 (over 16) and 5.76 ± 3.787 (over 16), respectively. Our findings have shown that the knowledge, attitude, and practice scores were positively correlated. It is known that the better the knowledge, the better the attitudes will be, and thus, more appropriate practices will take place.33 Although knowledge itself does not necessarily guarantee a behavior change, it shapes the attitudes towards favorable practices.33 Considering this, our research findings highlight the importance of educating Lebanese shoppers on food labels to ease their interpretation, and this, in turn, may enhance attitudes and practices in the long term. A clear evidence-based front-of-pack label like the nutri-score could be an effective tool to help improve consumers’ diet quality and mitigate the risk of NCDs.34 The Nutri-Score is a front-of-pack label that provides user-friendly information on the nutritional quality of food products.34 It is based on the British Food Standards Agency nutrient profiling system (FSAm-NPS) score.34 The higher the FSAm-NPS score, the lower the nutritional quality of the food. Nutri-score is a rating system that uses five different colours to categorize food products into five groups.34 For example, category A (dark green) suggests higher nutritional quality; however, category E (dark orange) indicates lower nutritional quality.34 The nutri-score is the only front-of-pack nutrition label in Europe having some strong scientific evidence for its effectiveness.34 It has already been adopted by several European countries (Belgium, France, Germany, the Netherlands, Spain, Luxembourg and Switzerland).34 Regarding the observed attitudes, the majority of our participants (72.5%) supported the beneficial use of the NFP. Furthermore, our participants seemed to positively perceive the nutrition labels’ importance, in contrast with a previous cross-sectional study recruiting 748 supermarket Lebanese shoppers in 2014.19 Less than half (44.4%) of the recruited shoppers agreed that reading food labels is very important.19 Besides, our findings are consistent with previous investigations from a cross-sectional study recruiting Iranian students from five different academic majors (including Nutrition, Public Health, Health Services Administration, Paramedical, and Engineering).35 Among 332 students, 89.2% believed that food labels affect nutritional awareness, and 77.4% agreed with their beneficial use.35 Similarly, a US survey aiming to examine the influence of 1990 Nutrition Labeling and Education Act food labels on college students found that 95% of participants perceived the NFP to be useful.36 Moreover, consumers were more concerned about reading nutritional information when they planned to lose weight or follow specific dietary regimens.36 Further, it was mentioned that 81% of participants who read the nutritional panel on product labels were on a weight-loss diet.37 This supports our results, since 55.2% of our sample were dieters, which explains their positive attitudes towards nutrition label use. In addition, our study findings showed that female participants had higher mean attitude scores than males (6.39 ± 2.834 vs. 6.01 ± 2.680, p=0.072). This is because women experience more food-related conflict, and more dissatisfaction with their body weight and shape than men do.38 With regards to nutrition labels mandating, the majority (89.5%) discerned the necessity of such legal actions. In addition to transparency, nutrition facts on food products enable people to determine, choose, and meet their dietary needs.39 The FDA provides effective guidance to the food industry regarding labeling information, depending on the product type.39 In an attempt to assess consumers’ valuation of nutrition labels, data was collected from food shoppers to observe their willingness to pay a premium cost for a box of cookies with a nutritional label.40 Interestingly, that study found that the mean willingness to pay for a box of cookies with a nutritional label was about 11%higher than that of a cookie box without a nutritional label.40 Moreover, expiry date marking (75.2%), price (60.6%), and brand name (50.8%) were the prioritized considerations for of our sample, whereas a smaller proportion admitted considering food ingredients (47%), and nutritional content (24.3%). Similar to our findings, the majority (84.7%) of the shoppers in Tabriz, Iran have been found to look for the expiry and production dates on the food label.41 In addition, 51.6% were looking at food price, and only 8.7% of the participants read food labels to find information about the food additives and artificial colors.41 Further, the date of minimum durability (i.e. best-before or use-by date) was rated the most important piece of mandatory labelling information, with 81% of Irish consumers scoring it as very important.42 Our respondents ranking the food price as a priority consideration was a predictable finding: at the time of our data collection, Lebanon was in a financial crisis, and more than half the population lived below the poverty line. According to the latest data in July 2021, a family's budget for food was around five times the minimum monthly wage.43 In contrast, a survey examining the awareness of food labeling among consumers in groceries in Al-Ain, United Arab Emirates reported that consumer’s responses showed general tendencies for reading the food label (89.5%); however; they read basic information related to production and expiry dates.44 Another study in India showed that the taste, quality, convenience and ease of use were the main reasons for purchasing food among the study participants.45 The majority (81%) looked for the expiry date, and only one third purchased food based on its nutritional value.45 Moving to South Africa (Lesotho), an observational study reported that 40.5% of the participants were interested in reading the nutrition information on food labels, rather than facts related to price, taste, appearance, habit, convenience, or brand name.46 When asked about the frequency at which they read nutrition labels, about half of our responders (46.5%) reported to “always” read the food labels. These findings are considered reasonable when compared to other studies in Arabic countries and worldwide. A research survey showed that only 27.4% of Saudi female college students stated that they always read food labels when purchasing food products.47 In China, however, 59.2% of a survey respondents indicated to “sometimes” look at the label, and only 28.7%” always” read nutrition label.48 Besides, only 21.6% of university Malaysian students reported to “often” use the food label during food purchasing decisions.49 To specify, our responders claimed to read labels relating primarily to sugars (44.3%), followed by calories (38.9%), total fats (36.8%), proteins (33.5%), and vitamins (32.9%). Chinese people had similar reading practices, and were found to look at proteins (51.5%), vitamins (49.8%), and fats (29.4%).48 A preliminary review aimed at assessing the types of label formats that could influence the use of nutritional label among consumers showed that the majority of label users were interested in checking information related to calories, fat, sodium, and cholesterol, and in deciding whether to buy fresh fluid milk.50 Most importantly, the majority (64.7%) of our responders reported that they did not refer to the NFP to choose between two food products. These results are not unusual in Lebanon: only 22.9% of recruited Lebanese shoppers admitted to checking the food labels every time they bought a food product.19 On the other hand, 65% of Ghanaian consumers read food labels before purchase, and 75% based their food selection according to its nutrition content.51 Besides, more than half of Singhalese people recruited in a cross-sectional survey could select the healthiest option with the better food label, when hesitating between two snack options.52 Consistent findings were obtained from our univariate analysis and binary regression analysis regarding the mean KAP score. Our findings revealed that female (15 ± 7), adult (15 ± 7), healthy (15 ± 7), and overweight/obese participants (14 ± 7) were more likely to have a high KAP score. In addition, those with higher educational levels (university level), and those who had completed higher university studies (Master, Ph.D.) had higher KAP scores. Other interesting findings are that those who were restricted to certain diets and the physically active participants had better KAP scores than their counterparts. On the other hand, North Lebanon residents had a lower KAP score (13 ± 6), as opposed to other residency areas. Supporting our findings, a sex-specific analysis of nutrition label use in the US showed that females used nutrition labels more than males (40.7% versus 54.3%, p<0.001).53 Similarly, another study in Malaysia reported that females, adults, tertiary level-educated, and physically active participants had increased odds of nutrition label use.54 Going back to the cognitive processing model, it revealed that some complex tasks related to reading food labels demand high comprehension and interpretation skills.10 Thus, this model explains why a higher educational level was a significant predictor of better KAP in our study findings.10 In addition, knowing more about food labels and consulting them before food purchase can help dieters and those who care about their body shape to pick healthy food options.55 Upon this, having better knowledge about nutrition labels can protect from many NCDs, which explains why our healthy responders had higher KAP scores than diseased participants. This study has identified the KAP scores and the associated factors of KAP related to nutrition label use among the Lebanese public. The findings of this study might advocate for future educational programs clarifying the meaning of crucial nutrition claims and symbols. Although increasing consumers’ awareness is key in leading to better KAP, food manufacturers should also invest in simplifying their nutrition labels presentation to attract more consumers. As discussed before, concerning the nutri-score’s promising application, epidemiological findings among European cohorts found that people who consume more food with higher FSAm-NPS scores (lower nutritional quality) had a higher risk of developing cancers, and a 6% increase in the risk of overall mortality.56 In France, seven out of 10 people check the Nutri-Score, and 84% say they are very likely to pick food products with higher scores.57 Besides, a cross-sectional study recruiting 501,594 adults from ten European countries mentioned that a FSAm-NPS score was calculated for each participant based on the nutritional quality of the food they consume.58 Individuals with a higher score revealed an increased risk of all-cause mortality as well as the incidence of circulatory and gastrointestinal diseases.58\n\nOur study includes some limitations. Firstly, the cross-sectional design of the survey itself limited the ability to reach causal inference. In addition, the online distribution of the questionnaire in the second period of the study (after COVID-19 pandemic emergence) may pose information and selection biases. Thus, the self-reported data may overestimate the understanding, the positive attitudes, and the appropriate practices regarding nutrition label use. On the other hand, this study has critical strengths. It is the first study in Lebanon adopting a valid and reliable questionnaire to assess the knowledge, attitudes, and practices of Lebanese consumers regarding nutrition label use. Besides, responders were recruited from different areas, had different educational levels, and were of various ages so that the study’s findings could be generalized to the whole population.\n\n\nConclusion\n\nThe low awareness of nutrition labels leads Lebanese people to choose unhealthy food options. This study showed an association between the participants’ attitudes, practices and self-reported knowledge. Because nutrition and chronic diseases are interrelated, a planned educational program is recommended to help Lebanese people pick healthy options mindfully. It is necessary to establish educational campaigns about the association between reading nutrition labels and health outcomes. The Food and Drug Administration (FDA) has implemented a “Read the Label” campaign to support children, families, and community leaders in analyzing nutrition labels and to use them effectively. In conclusion, advocating for a nutrition rating system like Nutri-Score in Lebanon is fundamental to mitigate obesity and chronic disease burden. However, one should take into account that nutrition labeling is only one approach to a public health nutrition policy, and it should be complementary with other public health measures and, in particular, nutrition education and communication.\n\n\nData availability\n\nOSF: Assessment of the Knowledge, attitudes and practices (KAP) of Lebanese shoppers towards food labeling: The first steps in the Nutri-score roadmap, https://osf.io/3nh559\n\nThis project contains the following underlying data:\n\n• Data-Excel-Nutrition Label.xlsx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWorld Health Organization: Non communicable Diseases.2021. October 16, 2021. Reference Source\n\nWorld Health Organization, Regional Office of East Mediterranean: Regional data on non-communicable diseases risk factors.2016. 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Publisher Full Text\n\nBarreiro-Hurle J, Gracia A, De-Magistris T: The effects of multiple health and nutrition labels on Consumer Food Choices. Journal of Agricultural Economics 2010; 61(2): 426–443. Publisher Full Text\n\nNurliyana G, Norazmir MN, Khairil an MI: Knowledge, attitude and practices of university students regarding the use of nutritional information and food labels. Asian Journal of Clinical Nutrition 2011; 3(3): 79–91. Publisher Full Text\n\nAzman N, Sahak SZ: Nutritional label and consumer buying decision: A preliminary review. Procedia - Social and Behavioral Sciences 2014; 130: 490–498. Publisher Full Text\n\nDarkwa S: Knowledge of Nutrition Facts on food labels and their impact on food choices on consumers in Koforidua, Ghana: A case study. South African Journal of Clinical Nutrition 2014; 27(1): 13–17. Publisher Full Text\n\nTalagala IA, Arambepola C: Use of food labels by adolescents to make healthier choices on snacks: A cross-sectional study from Sri Lanka. 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Reference Source\n\nDeschasaux M, Huybrechts I, Julia C, et al.: Association between nutritional profiles of foods underlying nutri-score front-of-pack labels and mortality: Epic cohort study in 10 European countries. BMJ 2020; 370. PubMed Abstract \| Publisher Full Text\n\nHoteit M: Assessment of the Knowledge, attitudes and practices (KAP) of Lebanese shoppers towards food labeling: The first steps in the Nutri-score roadmap.2022, January 4. Publisher Full Text" }	[ { "id": "127381", "date": "21 Mar 2022", "name": "Zahra Esfandiari", "expertise": [ "Reviewer Expertise Food Safety 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\nThe paper: Evaluation of Knowledge, Attitudes and Practices toward the knowledge, attitudes and practices of Lebanese shoppers towards food labeling. It is a good study and a worthy research topic. It is quite relevant to F1000Research topics. However, the article cannot be considered for indexing as it is. It needs a strong revision, as there are serious readability problems and methodological issues that the author(s) must manage effectively. I offer some questions and comments with the hope of helping s/he/them improve their work. I wish the research team the very best of luck as they continue work in this domain.\nOverall evaluation.\nIt is a worthy research topic and an interesting study but the paper needs a strong revision. I hope that the comments are fair and constructive. All Best!\nIntroduction: section needs revision. The author(s) must underline the initial assumptions of the paper and the originality of the paper – contribution to knowledge. I would suggest considering the following five (5) basic elements in the section of introduction as subheadings of Introduction:\na) Research aim: b) Initial assumptions of the paper c) Reasoning for the focus of the paper, d) Research objectives, e) Originality of the paper and contribution to knowledge.\nIn the last paragraph of the Introduction, the authors should clearly mention the weaker point of former works (identification of the gaps) and describe the novelties of the current investigation to justify the paper deserves to be indexed.\nMethod and material: This Section needs revision based on the following comments.\nThere is some parts in method and material such as the number of question and scores that needed more explanation; the number of phrases in table for knowledge is 11 but it is mentioned score 14. How is the calculation for knowledge, attitude and practices? It needs more clarification.\n\nAuthors must explain more about the process of validity and reliability of questionnaire.\nDiscussion.\n\nAuthor(s) must explain what is the difference of his study with others and if they are same, why they are similar? Comparison must be with papers after 2013. It needs to expand the importance of the findings. Most of the discussion was overstated and should be revised.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8099", "date": "05 May 2022", "name": "Maha Hoteit", "role": "Author Response", "response": "Reviewer 1 The paper: Evaluation of Knowledge, Attitudes and Practices toward the knowledge, attitudes and practices of Lebanese shoppers towards food labeling. It is a good study and a worthy research topic. It is quite relevant to F1000Research topics. However, the article cannot be considered for indexing as it is. It needs a strong revision, as there are serious readability problems and methodological issues that the author(s) must manage effectively. I offer some questions and comments with the hope of helping s/he/them improve their work. I wish the research team the very best of luck as they continue work in this domain. Overall evaluation. It is a worthy research topic and an interesting study but the paper needs a strong revision. I hope that the comments are fair and constructive. All Best! Introduction: section needs revision. The author(s) must underline the initial assumptions of the paper and the originality of the paper – contribution to knowledge. I would suggest considering the following five (5) basic elements in the section of introduction as subheadings of Introduction: a) Research aim: b) Initial assumptions of the paper c) Reasoning for the focus of the paper, d) Research objectives, e) Originality of the paper and contribution to knowledge. In the last paragraph of the Introduction, the authors should clearly mention the weaker point of former works (identification of the gaps) and describe the novelties of the current investigation to justify the paper deserves to be indexed. Reply: This is absolutely important and could be more highlighted in the manuscript. We add information about the relevance and the novelty of the current research study in the last paragraph of the Introduction. The study aims and the focus of the paper are clear too. “The usage and understanding of nutrition labels are inadequately registered before in Lebanon, with a current data shortage on this topic. Thus, it is a high priority to point out the factors affecting Lebanese consumers’ interpretation of food labels, to formulate new regulations or update existing ones. The aim of this study, the first of its kind in Lebanon, was to assess consumers’ knowledge, attitudes and practices (KAP) regarding the information on food labels, and to investigate the correlates of low levels of KAP among Lebanese shoppers. The study findings could serve as an initiative to motivate the national implementation of effective food labeling approaches like the Nutri-score front-of-pack label”. Method and material: This Section needs revision based on the following comments. There is some parts in method and material such as the number of question and scores that needed more explanation; the number of phrases in table for knowledge is 11 but it is mentioned score 14. How is the calculation for knowledge, attitude and practices? It needs more clarification. Reply: The questionnaire includes 14 knowledge-related questions. However, the knowledge scoring was not based on all of them. For example: The statements inquiring about the “vegetarian”; “non-vegetarian”; “vegan”; “gluten-free”; “Trans-fat”; “GMO” symbols are multiple parts of one question. The same is applied to the attitudes and practices parts of the questionnaire. This is why the total number of the questions and the total scores may differ. We chose the questions which seem reasonable to formulate individuals KAP scores. Authors must explain more about the process of validity and reliability of questionnaire. Reply: More details about the validity and reliability are added under the “Questionnaire” subsection of the Methods parts. “Its internal consistency and validity were assessed using Cronbach’s alpha in a preliminary analysis after conducting a pilot study. The observed alpha values were high for all the questionnaire sections (0.909 for knowledge questions, 0.841 for the attitude questions, and 0.836 for the practice questions)” Discussion. Author(s) must explain what is the difference of his study with others and if they are same, why they are similar? Comparison must be with papers after 2013. It needs to expand the importance of the findings. Most of the discussion was overstated and should be revised. Reply: The discussion section is updated and supported by recent studies by highlighting similarities and differences with other study findings. Reviewer 2: The topic is of interest. The study is complete and many important variables are studied to assert a conclusion. Therefore the paper is suitable for indexing: With reference to the study design and the questionnaire: Please provide an explanation about why the inclusion criteria for age was set to 15-64 years. Reply: The youth and adult Lebanese consumers were the target population of the current study. Thus, the age range of 15 to 64 years old was one of the eligibility criteria for our participants. Please demonstrate that the overall number of participants is representative of the population size of participants between 15-64 years. Please demonstrate that the sample size is representative to justify the discussion and conclusion based on the obtained results. Reply: According to the Lebanese Ministry of Public Health (MOPH), the total population aged 15-64 years across all Lebanese governorates in 2020 was 3,079,431. Based on the Epi-Info statistical software, with an acceptable margin of error of 5% and a confidence level of 95%, the minimum representative sample size was 384. Consequently, we recruited 768 participants as a representative sample size in the current study. It would be better to include rural and urban residence for participants. This is a major factor that would affect the knowledge and attitude of participants, rather than knowing in which governate they are coming from. Reply: This is definitely valuable and consistent with the study aims. However, we presented our data after reviewing the literature and in referral to other studies in Lebanon which were often based on the governorates of the participants. You mentioned that you used a self-constructed and validated questionnaire that was filled online. And that you used word of mouth and social media to recruit your population. Based on the above, please justify how for the questionnaire that was filled online, how the adult respondents and minors’ families were able to provide a written informed consent before filling the questionnaire. Reply: On the first page of the online form of the questionnaire (google form), the participants had to choose if they were consented to participate in the study to continue filling the remaining questions after being informed about study aims, risks, benefits, and confidentiality issues. Please specify in Table 1 that the Numbers in this question are calculated based on the rate of “Sayrafa” during the same day of filling this questionnaire. Reply: To record their monthly income, participants were asked to determine the exact choice (<1000$; 1000$-2000$; 2000$-3000$;>3000$) according to the rate of “Sayrafa” at the date of investigation. For example, if 1$= 3000 L.L. (Lebanese Lira), a participant with a monthly income of 3,000,000 L.L. had to choose the option “1000$-2000$”." } ] }, { "id": "127701", "date": "29 Mar 2022", "name": "Mireille Serhan", "expertise": [ "Reviewer Expertise Food safety", "Dairy processing", "Nutrition and Food Science research", "Consumer studies." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic is of interest. The study is complete and many important variables are studied to assert a conclusion. Therefore the paper is suitable for indexing:\nWith reference to the study design and the questionnaire:\nPlease provide an explanation about why the inclusion criteria for age was set to 15-64 years.\n\nPlease demonstrate that the overall number of participants is representative of the population size of participants between 15-64 years. Please demonstrate that the sample size is representative to justify the discussion and conclusion based on the obtained results.\n\nIt would be better to include rural and urban residence for participants. This is a major factor that would affect the knowledge and attitude of participants, rather than knowing in which governate they are coming from.\n\nYou mentioned that you used a self-constructed and validated questionnaire that was filled online. And that you used word of mouth and social media to recruit your population. Based on the above, please justify how for the questionnaire that was filled online, how the adult respondents and minors’ families were able to provide a written informed consent before filling the questionnaire.\n\nPlease specify in Table 1 that the Numbers in this question are calculated based on the rate of “Sayrafa” during the same day of filling this questionnaire.\n\nIs the work 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": [] } ]	1	https://f1000research.com/articles/11-84
https://f1000research.com/articles/11-50/v1	17 Jan 22	{ "type": "Case Report", "title": "Case Report: ASI intervention on a child with autism in Saudi Arabia", "authors": [ "Shahad Alkhalifah", "Susan Allen", "Hesham Aldhalaan", "Susan Allen", "Hesham Aldhalaan" ], "abstract": "Background: Ayres Sensory Integration (ASI) is widely employed by occupational therapists working with clients who experience challenges in sensory integration, including those with autism spectrum disorder (ASD). However, there is a dearth of research examining the feasibility of ASI outside of Western nations. This study documented the barriers associated with ASI in Saudi Arabia and assessed whether the intervention could improve process and participation skills. Methods: A pre-test/post-test case study design was used. The participant was a 4-year-old girl with ASD from Saudi Arabia. Data were gathered on sensory processing, motor skills, and participation in activities of daily living. The study used semi-structured interviews and assessments (Sensory Integration and Praxis Tests, the Sensory Processing Measure-Preschool, and the Peabody Developmental Motor Scale-2) to develop goals, identify outcome measures, and plan an ASI intervention. Results: Despite the limited availability of resources (e.g., toys, treatment spaces) and Arabic measures, improvements were observed on motor and sensory tasks and in occupational performance. Conclusion: ASI that adheres to the ASI fidelity tool can be of value for Saudi Arabian children with ASD. Additionally, the study provides a stepping-stone to further research for occupational therapists in Saudi Arabia working with children with ASD.", "keywords": [ "Autism", "sensory integration", "occupational therapy", "Saudi Arabia" ], "content": "Introduction\n\nSensory integration (SI) is a neurobiological process for finding, assimilating, organizing, and employing sensory information, which helps individuals to interact with the world (Parham et al., 2011). SI involves sensory perception and sensory reactivity. Sensory perception identifies the quality of sensory input to provide meaning; for example, recognizing an object’s shape or size without using vision. Sensory reactivity is the ability to organize and regulate one’s responses to sensory information (Schaaf & Mailloux, 2015). SI challenges are common among people with autism spectrum disorder (ASD) (Tomchek & Koenig, 2016). According to the American Psychiatric Association’s DSM-5 guidelines (2013), hyperreactivity and hyporeactivity to sensory input are features of ASD. Such issues can cause poor concentration and sensory over-reaction (Jorquera-Cabrera et al., 2017). Al-Heizan et al. (2015) reported that 84.8% of children in their ASD sample in Saudi Arabia (SA) had definite sensory processing challenges. However, such challenges can be difficult to identify and may be overlooked, especially by occupational therapists (OT) not trained in SI (Al-Heizan et al., 2015).\n\nThe existing literature shows that services for children with ASD are underdeveloped in SA (Alkhalifah & Aldhalaan, 2018). The Saudi government has recently encouraged healthcare professionals to train in Western countries to acquire evidence-based, up-to-date information concerning ASD interventions (Alnemary et al., 2016). Alshehri et al. (2019) found that Saudi OT practitioners are frustrated by limited resources; the lack of Arabic assessment tools, materials, and insufficient clinical knowledge about intervention protocols act as barriers to evidence-based practice (Alkhalifah, 2019).\n\nThe Ayres Sensory Integration® (ASI) intervention utilizes “individually tailored sensory–motor activities contextualized in play at the just-right challenge to promote adaptive responses and foster functional skills as a foundation for participation in occupations” (Schaaf et al., 2018, p.1). International research suggests that ASI is an evidence-based way to improve communication, social interaction, cognitive, academic/pre-academic, adaptive/self-help, behavioural, and motor skills in children aged 4-12 years with ASD (Schoen et al., 2019).\n\nSA has a unique culture, and there has been little exploration of whether existing SI interventions and measures, primarily developed in Western contexts, are suitable for its population (Al-Heizan et al., 2015). The differences between Arab and Western countries could affect interpretations of SI (Alkhakifah, 2019). For instance, Arabs “tend to interact with a direct body orientation, stand close together, touch frequently, and demonstrate unique use of paralinguistics” (Al-Khateeb et al., 2014, p.240). On the low energy/weak and movement sensitivity items of the Short Sensory Profile, more Australian children with ASD scored in the typical range than Saudi children with ASD (Al-Heizan et al., 2015). Parenting culture in SA is protective, possibly impacting children’s opportunities for motor, proprioceptive, and/or vestibular development (Al-Heizan et al., 2015). Alnemary et al.’s review (2017) indicated that, in the context of ASD, there has been little research on either the services available or treatment outcomes in Arab countries. With 167,000 Saudis estimated to have ASD (Alnemary et al., 2017), there is a clear need for OT services (Alshehri et al., 2019) to examine the effectiveness of treatment options, such as manual ASI. The rationale for the current study is to contribute towards filling these gaps in the research in the context of SA.\n\nBefore assessing the efficacy of OT-ASI in SA, it is necessary to examine such an intervention’s feasibility when applied to individuals and to address implementation obstacles (Portney & Watkins, 2000). While previous studies based on children with impaired SI suggest that OT-ASI can improve SI and occupational performance, such studies did not focus on Arab countries (Schaaf & Nightlinger, 2007). Moreover, many such studies failed to use replicable protocols (Schaaf et al., 2018). The current study hypothesized that participation challenges of children with ASD were linked to SI impairments. Therefore, this study had the following objectives: (1) identify barriers associated with providing OT-ASI to a child with ASD in SA and (2) establish the efficacy of this intervention in the SA context. In line with these objectives, this research set out to answer the following question: to what extent is OT-ASI appropriate and effective in the Saudi context?\n\n\nCase report\n\nThe inclusion criteria were: a) diagnosis of ASD by a multidisciplinary team using standardized tools, such as the The Autism Diagnostic Observation Schedule (ADOS) (Lord et al., 2000); b) living in Riyadh; c) parents providing voluntary consent; d) attending a mainstream preschool; e) experiencing challenges with activities of daily living (ADLs); f) aged 4-12 years. Individuals with other medical conditions were excluded. Recruitment was through parent invitation at Riyadh’s Centre in October 2018 at Autism Research (CFAR). The study was presented to CFAR at King Faisal Hospital and ethical approval granted for it to proceed.\n\nOne Saudi girl, referred to as L, aged 4 years and 7 months, met the criteria. L was diagnosed with ASD at 2 years and 3 months. She attended school full-time, where she participated in small groups of students with special educational needs. Her teachers had not undertaken ASD-specific training, and she was not receiving ASD-specific interventions. However, she had participated in a 2-month home-based speech intervention, completing 3-6 sessions weekly. While this appeared to improve her communication skills, L’s interactions with her peers and teachers were limited, and she exhibited frequent tantrums during transitions or changes to routine. Despite her poor communication skills, her cognitive development was typical. There was no relevant family medical or social history.\n\nThe study was guided by data-driven decision making (DDDM), a systematic process that aids OTs in clinical reasoning while addressing client needs (Schaaf & Mailloux, 2015). The study involved eight steps.\n\nThe first step involved a semi-structured interview with L’s mother, using occupational profiles, to identify how L interacts with her environment (AOTA, 2017) and her participation strengths and challenges (Schaaf & Mailloux, 2015). These profiles were translated into Arabic so the mother could understand them. L was reported to not initiate actions independently and often seem clumsy. She was described as a fussy child. She was unable to put on socks and shoes. She struggled to play with her peers and bumped into others, impacting social relationships. She also had difficulty writing. L’s teacher reported that L had a limited attention span and struggled to accept rules and use crayons.\n\nIn step II assessments were completed to identify factors affecting participation. To assess impairment related to SI and praxis, we used the Sensory Integration and Praxis Tests (SIPTs) and the Arabic Sensory Processing Measure-Preschool (SPM-P; Centre for Autism Research). The SIPTs comprise 17 individual sub-tests for children aged 4-8 years, measuring neurological ability to integrate the sensory inputs required for coordination, motor planning, visual-spatial actions, and perception (Schaaf & Lane, 2015). The SIPTs discriminate between normal and dysfunctional children in the United States at a statistically significant level (Schaaf & Mailloux, 2015). Of the 17 test items, 13 show reliability scores of.70 or more, which indicates heigh reliability. Moreover, the test has high inter-rater reliability due to the detailed scoring and because therapists using the SIPTs undertake training (Schaaf & Mailloux, 2015). Each SIPT results in a standard score that reflects the child’s performance compared to age-matched norms; the average score for a group of a given age is 0 (Schaaf & Mailloux, 2015). Scores below -1 standard deviation are considered evidence of dysfunction.\n\nThe Arabic SPM-P consists of home and classroom forms, which are suitable for children aged 2-5 years (Alkhalifah et al., 2020). The home form has 75 items and the classroom form has 62. The home form has excellent internal consistency when used with ASD (α = .93) and typically developing (α = .95) children (Alkhalifah et al., 2020). The Arabic classroom form has not yet been assessed, though the English version has excellent internal consistency when used with clinical (α = .93) and typical (α = .94) samples (Jorquera-Cabrera et al., 2017). Both forms use Likert scales to measure sensory behaviour frequency in sensory processing, praxis ability, and social participation (Alkhalifah et al., 2020; Jorquera-Cabrera et al., 2017). Both were used in this study because assessing children with ASD in different environments gives a comprehensive understanding of SI function (Jorquera-Cabrera et al., 2017).\n\nL attempted 13 of the 17 SIPTs, completing 12 (see Figure 1). Scores indicated difficulty with tactile and kinesthetic processing, especially manual form perception, finger identification, and graphesthesia. L struggled in motor planning as measured by postural praxis, oral praxis, sequencing praxis, design copy, and bilateral motor coordination. Similarly, she scored low for space visualization. L was unable to participate in the localization of tactile stimuli, kinesthesis, and constructional praxis tests, and unable to fully participate in motor accuracy and postrotary nystagmus tests.\n\nThe findings from the Arabic SPM-P home and classroom forms are illustrated in Table 1. The environmental difference score demonstrated consistency across environments. Both measures indicated that L struggled with body awareness, balance and motion, social participation, hearing, and touch. The SPM-P revealed problems in planning and idea items and suggested her tactile, body awareness, and auditory systems were hyper-responsive.\n\nAdditionally, we used the Peabody Developmental Motor Scale-2 (PDMS-2), a task-observation test designed to evaluate fine and gross motor skills in children aged 0–5 years (Folio & Fewell, 2000). We selected the PDMS-2 because L’s challenges were associated with impaired fine and gross motor skills, which are common in ASD (Provost et al., 2007). Using a motor skills measure that assesses visual-motor integrations was important as visuomotor connections seem to be aberrant in ASD (Sharer et al., 2015).\n\nL performed very poorly on tests of both fine and gross motor skills on the PDMS-2, and her total motor quotient score was very poor (Table 3). Additionally, on clinical observation, L was unable to maintain a flexion position for more than 10 seconds, while prone extension was achieved for 3 seconds. She showed difficulty with sequential finger touching and ramped arm movements. She also avoided touch in the absence of vision.\n\nIn step III, hypotheses were generated to link the assessment findings to L’s performance on ADLs (Schaaf & Mailloux, 2015). Overall, the findings suggested L’s participation challenges were due to impairments related to SI and praxis. Specifically, it was hypothesized that L had somatodyspraxia (Schaaf & Mailloux, 2015).\n\nIn step IV, goals were established using the Goal Attainment Scale (GAS; King’s College London) (Ruble et al., 2012). The GAS is both systematic and sensitive to changes in functioning associated with ASI. It entails describing the individual’s present level of functioning for a given goal and then scaling it for the anticipated level of function over the intervention period. GAS helps assess outcomes that are challenging to measure with traditional instruments (Mailloux et al., 2007), and is a valid and reliable tool (Steenbeek et al., 2007) for individuals with ASD (Ruble et al., 2012). In a recent systematic review of ASI, Schaaf et al. (2018) concluded that “GAS is a strong, sensitive, and meaningful outcome” (p. 7). GAS uses a five-point scale, with values ranging from -2 to 2, scaled with equally spaced probability intervals (Kiresuk et al., 2014). A score of 0 reflects the anticipated level of function, while -1 and -2 suggest a level of attainment less/much less than anticipated, respectively, while +1 and +2 suggest better/much better than anticipated, respectively. Scores for each goal are used to calculate T-scores, representing the extent to which functioning improved to the degree anticipated.\n\nThe first author worked with L’s mother to identify specific and measurable goals reflecting L’s functional challenges (Schaaf & Mailloux, 2015). L’s mother knew about her difficulties in school and could contribute to setting both school- and home-based goals. As recommended by Ruble et al. (2012), goals were quality checked by the second author.\n\nL’s goals were:\n\n1. Dressing: Improve participation in dressing, to independently put on shoes after verbal prompts with fewer than two physical prompts.\n\n2. Fine motor skills: Improve participation in learning activities, to draw or reproduce a circle with two verbal prompts.\n\n3. Play: Improve participation in social play, engaging with a peer in age-appropriate activities for 10 minutes with two adult redirections.\n\n4. Safety: Improve participation in playtime, navigating the playground without bumping into objects or people.\n\nThese goals were based on the premise that improving sensory processing would increase participation in everyday activities (Schaaf & Mailloux, 2015).\n\nIn step V, the authors determined the proximal and distal outcomes to use to track progress.\n\nDistal Outcome Measure: Changes in L’s goals were measured by GAS, enabling the evaluation of progress toward specific, measurable, and time-dependent goals\n\nProximal Outcome Measures: The Arabic SPM-P and the PDMS were used as secondary outcome measures. The former was used to measure sensory reactivity, praxis, and social participation, the latter to measure fine and gross motor skills.\n\nIn step VI, the intervention was planned. We agreed that L would participate in 1-hour ASI sessions twice weekly for 10 weeks, meeting her mother in person and her teacher over the phone every week for 30 minutes. Each session would provide sensory-rich experiences to elicit changes in the aberrant SI hypothesized to underlie L’s participation challenges. For therapy room sessions, interventions would consist of a beginning, middle, and end (Schaaf & Davies, 2010). Schaaf and Davies (2010) emphasized that ASI entails attention to meaningful activity, requiring adaptive responses and active participation from the child. Accordingly, L’s sessions were designed to provide a challenge level that was “just right” for her sensory systems (Schaaf & Lane, 2015).\n\nIn step VII, the intervention was conducted. The first author, a licensed OT with certification in ASI and six years of experience working with children with ASD, delivered the intervention activities tailored to L’s needs. L had numerous opportunities to play with tactile-rich apparatus, to improve her proprioceptive and tactile perception, and praxis. Moreover, through pulling, pushing, and hanging activities, she was encouraged to stretch and engage her muscles. She participated in various active sensory–motor activities. She used a scooter board to experience proprioceptive and vestibular sensations and increase body awareness. She also engaged in jumping into a ball pit, climbing, rolling, and crawling. There were opportunities to change the apparatus and the rhythm, duration, frequency, and/or intensity of sensory experiences, based on L’s responses. In line with ASI principles, the first author and child cultivated an active, trusting relationship, with the former monitoring activity demands to ensure a just-right level of challenge (Schaaf & Mailloux, 2015).\n\nThe use of the ASI Fidelity Measure also ensured the intervention was in line with ASI principles (Parham et al., 2011). This measure defines the structure of the intervention and process elements (Parham et al., 2011, 2011; Schaaf & Mailloux, 2015). A score of 80 is a tentative cut-off point for adherence to ASI (Parham et al., 2011). The measure has high interrater reliability for both total fidelity scores (.98) and individual items (.94–.99). The validity of the measure is strong as raters can distinguish ASI sessions from other interventions with 92% accuracy (Parham et al., 2011).\n\nAll 20 sessions were videotaped, and the second author, trained in the application of the measure and with 30 years of experience working with children with ASD, assessed four randomly selected tapes. Additionally, the second author provided weekly consultations to the first author and was available throughout to discuss intervention challenges. Only one session produced an unacceptable fidelity score, after which the authors collaborated to ensure acceptable fidelity in all other sessions. The mean fidelity score across the four sessions assessed was 85 (SD = 9).\n\nIn step VIII, outcomes were measured and progress monitored, which included the mother’s ratings for each goal and consultation with L’s teacher to calculate a T-score using Kiresuk et al.’s (2014) formula. Outcomes for the dressing goal (rating = 1) exceeded expectations. Outcomes for the other goals (ratings = 0) were consistent with expectations. L’s post-intervention T-score was 62, suggesting she performed better than expected.\n\nWhen interviewed six weeks post-intervention, L’s mother described her as happier, more sociable, better able to play safely, and less impulsive. She said:\n\n“L has started to play more. Yesterday, she created a restaurant menu and when she was upset, she drew a sad face! She can sit and play for more than 20 minutes. In addition, she hugs me without using too much force. L does not react to unfamiliar tasks with crying anymore! I wish many centres could offer the ASI… L cannot wait for your sessions!”\n\nWhen interviewed six weeks post-intervention, L’s teacher said that her “attention in the classroom was so much better and she is starting to participate in making choices during activities.” Both reported that L’s fine motor and social skills had improved and she showed fewer difficulties when playing.\n\nThe secondary outcomes showed that L improved in various domains. The results for the Arabic SPM-P home and classroom show L improved in sensory reactivity, praxis, and social participation in both home and classroom settings (Table 1). Similarly, the PDMS-2 data indicated L’s overall motor skills improved (Table 2). In a conversation six months post-intervention, L’s mother reported that, despite receiving no additional sessions, L’s behaviour at home and school continued to improve.\n\n\nDiscussion\n\nGiven the lack of research describing ASI in SA, this case study examined the feasibility and effectiveness of this intervention. The study suggests ASI is feasible within SA and can lead to improvements in individualized functional goals in ADL and performance on sensory and motor tasks.\n\nWhile OT-ASI in SA appears feasible, its delivery was not straightforward. Since OT-ASI has only recently been introduced to SA, few parents were aware of its potential, making it challenging to find willing participants. This was partially addressed by translating materials that explained what ASI is and why it is valuable, which was a labour-intensive process.\n\nTeachers in SA rarely participate in interventions and have limited knowledge of ASD and sensory issues (Haimour & Obaidat, 2013). Therefore, the first author spent time explaining to L’s teacher how to complete the Arabic SPM-P and was available to answer questions. Additionally, the first author was available to provide L’s mother and teacher with guidance to understand the intervention. However, therapists in SA working with individuals with ASD and their families often lack time to model interventions or address families’ complex medical and behavioural needs (Alnemary et al., 2016).\n\nA few Arabic language resources are available, such as a translation of the SPM-P (Alkhalifah et al., 2020). It is necessary to translate more resources into Arabic to increase understanding of ASD and SI among teachers in SA, to inform their practice and help them identify pupils who might benefit from OT interventions.\n\nAccording to Serbin et al. (2014), a child’s family is the main influence on their health and well-being. However, the parents of disabled children are especially susceptible to stress (Dempsey et al., 2009), potentially limiting their contributions to GAS. Indeed, L’s mother struggled to imagine goals that would reflect better regulation and participation in family life. Moreover, Saudi mothers often place great trust in healthcare providers and may feel it inappropriate to engage in the therapeutic process (Alkhalifah & Aldhalaan, 2018); this appeared true of L’s mother, who was hesitant to participate in GAS. Nonetheless, this study suggests that GAS is useful for quantifying individual outcomes (Mailloux et al., 2007). GAS also enables researchers to measure changes in tailored, functional, and parent-generated goals. Therefore, it is a valuable supplement to other assessments when measuring outcomes associated with individual interventions (Schaaf & Mailloux, 2015).\n\nThe use of proximal and distal goals was helpful in demonstrating links between the underlying challenges in SI and daily occupation. The SIPTs had not previously been applied in an Arab nation, so this was the first exploration of their suitability in SA. However, some SIPTs can be difficult to administer to non-English-speaking children (Bodison & Mailloux, 2006). Roley et al. (2015) highlighted that SI tests can be challenging for children with ASD, with only 63% of their sample finishing most tests. Therefore, proxy measures (e.g., observations) are recommended (Schaaf & Mailloux, 2015). Other limitations of the SIPTs are that they rely on 40-year-old normative data and their software is incompatible with modern operating systems (Mailloux et al., 2018). The SIPTs are also only suitable as pre-post-test measures for periods in excess of eight months (Mailloux et al., 2018). In the current study, L could not complete all of the SIPTs; therefore, additional measures were utilized, with GAS completed post-intervention.\n\nFuture related studies should utilize the Evaluation of Ayres Sensory Integration (EASI) tool, to be released in 2020. Suitably qualified therapists will have open access to the EASI, which will incorporate normative data collected internationally. The EASI will meet the demand for the assessment of ASI constructs with psychometrically validated and internationally applicable measurement tools (Mailloux et al., 2018).\n\nTreatment integrity was confirmed using the Ayres Sensory Integration® Fidelity Measure (Parham et al., 2011). This presented some challenges. For instance, it was necessary to videotape the sessions. While L’s mother agreed to this, many Saudi families may not, due to stigma and religious reasons (Alotaibi & Almalk, 2016). L’s mother expressed concerns and wanted to ensure no one beyond the first author and the training group would see the video; she personally refused to be videoed, or audio recorded, for religious reasons. Furthermore, accessing suitable training was difficult. Some Saudi professionals do not share alternative treatments with families because of the limited hands-on support available for ASD interventions (Alshehri et al., 2019). This shortage of qualified trainers could reduce the quality of services in SA for ASD (Alnemary et al., 2016). The first author undertook training abroad for over one year. To complete this study, the therapist required confidence and experience in navigating the local protocol and ethical consent processes (Schaaf & Nightlinger, 2007).\n\nNeuroscientific evidence suggests OT-ASI interventions elicit increased neurological adaptations if they simultaneously target multiple sensory systems (Lane & Schaaf, 2010). However, acquiring apparatus suitable for stimulating L’s various sensory systems was challenging; few items were available in SA, so they had to be ordered internationally. The ASI fidelity tool requires a suitable room, points of suspension, and equipment; negotiating additional space and financing for this was time-consuming.\n\nPost-intervention, L performed better on sensory and motor tasks and, in the context of ADLs, she demonstrated improvements in fine, gross, and overall motor skills. Previously, her performance was characterized as “very poor”, subsequently it was “average” or “below average”. These findings are in line with the review of Schaaf et al. (2018).\n\nJasmin et al. (2009) found that sensorimotor deficits in those with ASD underlie their day-to-day functioning. Congruent with this, L’s improvements in sensory processing and motor skills were reflected in enhanced performance in ADLs. For all tasks identified by GAS, she exhibited increased ability. She improved, as much as anticipated, her capacities to draw, play appropriately, and dress herself, positive changes that were reflected in the comments of L’s mother and teacher.\n\nAs the study focused on OT–ASI with one child, the results are limited with respect to generalizability. While case histories only provide level V evidence, they are useful for exploring transitions to different cultures (Schaaf & Nightlinger, 2007). Another limitation is that the evaluation was completed by the therapist delivering the treatment (not independent assessors).\n\n\nConclusion\n\nThis study hypothesized that participation challenges observed in a child with ASD were linked to SI impairments. In support of this, improvements in her participation were seen after an intervention targeting SI. Therefore, this study provides initial evidence that Saudi children with ASD could benefit from ASI treatments and offers insights into the factors affecting delivery.\n\nThis study highlights the lack of normative data on SI in SA and a need for further Arabic assessment tools. Moreover, there is little awareness of how individuals with ASD face challenges in SI, and relevant resources are insufficient. The government could address these issues through ASD and ASI workshops, establishing scholarships for training, and committing financial resources to assessment tools and treatment spaces.\n\nCreating videos was particularly valuable as the child’s responses could be reviewed, helping the therapist to reflect. Similarly, the intervention was facilitated by DDDM (Schaaf & Blanche, 2012). Post-graduate training in ASI and GAS supports service delivery and the evaluation of outcomes, mentored training is recommended for newly qualified OTs.\n\nHowever, the study is limited by only including one participant and by the lack of blinding, issues that future studies should address. Nevertheless, this study provides a stepping-stone to further research in this area for OTs in SA working with children with ASD whose functioning is impacted by aberrant SI.\n\n\n\n• There is a need for more data on SI in SA and Arabic assessment tools.\n\n• Government-funded workshops, training scholarships, assessment tools and treatment spaces would help address challenges in SA.\n\n• Post-graduate and mentored training in ASI and GAS is recommended for newly qualified OTs.\n\nThe study was presented to the Centre for Autism Research (CFAR) at King Faisal Hospital and ethical approval was granted for it to proceed. L’s mother gave written informed consent for L to be involved in the study and L’s information to be published in this manuscript. L’s mother also gave permission for L to be recorded during her sessions. All the recorded sessions are stored in CFAR’s system, in a separate, secure (locked) folder to ensure data protection. Further, in accordance with research data management policy, all recorded sessions have been secured in password protected files only accessible to the researchers. The videos will be retained for 10 years, before being securely disposed.\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\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nCompeting interests\n\nNo competing interests were disclosed.", "appendix": "Acknowledgments\n\nWe thank Eric Fombonne, M.D. and Roseann C. Schaaf, PhD, OTR/L, FAOTA for reviewing the manuscript and providing suggestions. We thank L for her willingness to play with us and L’s mother for her input on this project. 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Autism Dev. Disord. 2012; 42(9): 1974–1983. PubMed Abstract \| Publisher Full Text\n\nSerbin LA, Hubert M, Hastings PD, et al.: The influence of parenting on early childhood health and health care utilization. J. Pediatr. Psychol. 2014; 39(10): 1161–1174. PubMed Abstract \| Publisher Full Text\n\nSchaaf RC, Davies PL: Evolution of the sensory integration frame of reference. Am. J. Occup. Ther. 2010; 64(3): 363–367. PubMed Abstract \| Publisher Full Text\n\nSchaaf RC, Dumont RL, Arbesman M, et al.: Efficacy of occupational therapy using Ayres Sensory Integration®: A systematic review. Am. J. Occup. Ther. 2018; 72(1): 7201190010p1–7201190010p10. PubMed Abstract \| Publisher Full Text\n\nSchaaf RC, Lane AE: Toward a best-practice protocol for assessment of sensory features in ASD. J. Autism Dev. Disord. 2015; 45(5): 1380–1395. PubMed Abstract \| Publisher Full Text\n\nSchaaf RC, Mailloux Z: Clinician’s guide for implementing Ayres Sensory Integration: Promoting participation for children with autism. American Occupational Therapy Association; 2015. Publisher Full Text\n\nSchaaf RC, Nightlinger KM: Occupational therapy using a sensory integrative approach: A case study of effectiveness. Am. J. Occup. Ther. 2007; 61(2): 239–246. PubMed Abstract \| Publisher Full Text\n\nSharer E, Crocetti D, Muschelli J, et al.: Neural correlates of visuomotor learning in autism. J. Child Neurol. 2015; 30(14): 1877–1886. PubMed Abstract \| Publisher Full Text\n\nSchoen SA, Lane SJ, Mailloux Z, et al.: A systematic review of ayres sensory integration intervention for children with autism. Autism Res. 2019; 12(1): 6–19.\n\nSteenbeek D, Ketelaar M, Galama K, et al.: Goal attainment scaling in paediatric rehabilitation: A critical review of the literature. Dev. Med. Child Neurol. 2007; 49(7): 550–556. PubMed Abstract \| Publisher Full Text\n\nTomchek SD, Koenig KP: Occupational therapy practice guidelines for individuals with autism spectrum disorder. AOTA Press; 2016. Publisher Full Text" }	[ { "id": "120181", "date": "07 Feb 2022", "name": "Sayyed Ali Samadi", "expertise": [ "Reviewer Expertise Intellectual and developmental disabilities" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is important from the perspective of the cultural aspects of autism spectrum disorders (ASD). It is important because of the shortage of available knowledge on the applicability of the Western data, scales, and ASD protocols on non-Western cultures.\nThe manuscript presented some justification on the importance of considering sensory issues for individuals with ASD and reconsidered the sensory issue in the context of Saudi Arabia. The main aim, which is attainable through meeting two objectives, is cited at the end of the introduction part under a sub-heading. This study has aims and objectives which are clearly indicated and although this is a case report, it needs a part allocated to the methodology - this is important. The primary goal of the case study is to give readers an opportunity to see how the guidelines apply in the context of an actual situation (i.e. SI as an applicable approach for the Saudi individuals with ASD). The study methodology is the specific approach or technique that is used to identify, select, process, and analyze information about a topic and allows the reader to critically evaluate a study's overall validity and reliability. It is very helpful to understand why a case report is selected to answer such important research questions. Which design of the single case study was adopted? What is the logic behind the selection? All this information shapes a paragraph that is very helpful to understanding the study.\nThe other issue is mentioning ADOS, but not ADOS-2 (Lord et al., 20121, Pruette, 20132) as an evaluation for diagnosis without presenting any information about the applicability of this sophisticated scale on the Saudi Arabia children with ASD population. The irony is that the study tackles the issue of applicability of intervention approaches on non-Western cultures. I think since the diagnosis has already been done by a multidisciplinary team of experts, this scale might be excluded in this part. If no data is available, it deserves to be stressed (probably in the limitations part).\nIt is very important to understand how parents were approached, how did they receive information about the study? How many were approached and how many finally accepted? How many children were also excluded because of the exclusion and inclusion criteria?\nAutism implies to a very heterogeneous group of individuals and since this is a single case report, presenting some information about the level of ASD (level 1, 2, or 3 based on DSM5 ASD classification) or even the level of severity of the core symptoms of ASD will be very helpful.\nThis is very interesting to see the level of application of different scales to attain goals to understand the fidelity, but I think what is missing in the findings part is the visual presentation of the attained objectives in the duration of the intervention. This is included in most of the single-subject designs. This is generally in the form of graphs showing the baseline and also impacts of the intervention in different stages of the rehabilitation process. Single-subject research, opposite to the sample recruitment approach, relies heavily on visual inspection. This means plotting individual participants’ data recorded at the end of each session, looking carefully at those data, and making judgments about whether and to what extent the independent variable (in this study SI) affected the dependent variable (which is L’s performance in each domain). The visual inspection also shows the pace of changes over time and the expected time or sessions which is needed to see the changes.\nI do not agree with the justification that is presented in the limitation part regarding the generalization of this study. The researchers intentionally adopted a single case design, therefore, they should consider all the safeguards to make sure that the finding of the design is generalizable. But if this is called a preliminary or pilot study and the authors intend to perform a study with a group of Saudi children with ASD using the ASI approach I agree that this is the limitation of the study.\nI also think that other issues need to be considered as the imitation of the study. Most of these issues have already been mentioned by the authors in the different parts of the manuscript, such as lack of trained teachers, applicable diagnostic scales, and similar issues that might impact the results.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "8116", "date": "04 May 2022", "name": "Shahad alkhalifah", "role": "Author Response", "response": "Dear Dr. Sayyed, We are pleased to submit a revision of our manuscript entitled: “Case Report: ASI intervention on a child with autism in Saudi Arabia” for publication in F1000Research. We have revised it to take into account your valuable comments. Thank you for these appreciative comments; we now addressed all your comments and feedback. In the results section, we unfolded the results, adding tables and charts to offer a visual presentation of the attained goals. Moreover, we inserted a summary of the OT observations of L’s progress across the pre-assessment, intervention, and post-assessment phases. We are looking forward to receiving an answer from you at your earliest convenience. Sincerely, Shahad Alkhalifah" } ] }, { "id": "124289", "date": "28 Feb 2022", "name": "Gustavo A. Reinoso", "expertise": [ "Reviewer Expertise Children and youth", "disability", "sensory processing", "sensory integration", "handwriting", "measurement and statistics", "Rasch analysis", "assessment tool development." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease see my comments below:\nCase reports are very important to build evidence based practice. I would suggest some changes to the structure and how data were presented. The authors may find case report standards (e.g. https://www.care-statement.org/) useful in structuring the report. The manuscript would also benefit from establishing a more fluid connection between a) clinical findings, b) outcome measures, and c) GAS goals. Similarly, a more comprehensive description of the intervention (what was done) and how fidelity was achieved may also enhance your case. I am also adding a few recommendations below. Thank you for your work and contribution.\n\nDiscuss the limitations of using the SIPT. This is a 33 year old test with unknown parameters in other countries such as SA.\n\nSpell check \"heigh reliability.\"\n\nValidity and reliability are NOT properties of a test (they are parameters that refer to a specific sample. Thus, the parameters listed do not directly apply to your study). \"Moreover, the test has high inter-rater reliability due to the detailed scoring and because therapists using the SIPTs undertake training (Schaaf & Mailloux, 2015). Each SIPT results in a standard score that reflects the child’s performance compared to age-matched norms; the average score for a group of a given age is 0 (Schaaf & Mailloux, 2015). Scores below -1 standard deviation are considered evidence of dysfunction.\"\n\nUse primary references when discussing assessment tools (e.g. SPM and (Jorquera-Cabrera et al., 2017).\n\nSpelling \"kinesthesis\" vs Kinesthesia\n\nSIPT Scoring: Provide a qualitative summary of her overall performance during testing (e.g. organization of behavior, difficulties following directions, ability to imitate/copy, understanding instructions, etc). This will be more useful than scores derived by comparing your case with an older American reference sample.\n\nDescribe why unstructured observations were not completed if the child presented with difficulties with imitation/copying, following directions, etc. or provide a summary of how they were administered, scored, etc. \"Additionally, on clinical observation, L was unable to maintain a flexion position for more than 10 seconds, while prone extension was achieved for 3 seconds. She showed difficulty with sequential finger touching and ramped arm movements. She also avoided touch in the absence of vision\"\n\nStep VI - Consider rephrasing: \"... in the aberrant SI hypothesized'\n\nReconsider using a questionnaire as a post test measure.\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? No", "responses": [ { "c_id": "8117", "date": "04 May 2022", "name": "Shahad alkhalifah", "role": "Author Response", "response": "Dear Dr. Gustavo, We are pleased to submit a revision of our manuscript entitled: “Case Report: ASI intervention on a child with autism in Saudi Arabia” for publication in F1000Research. We have revised it to take into account your valuable comments. Thank you for these appreciative comments; we now addressed all your comments and feedback. We are looking forward to receiving an answer from you at your earliest convenience. Sincerely, Shahad Alkhalifah" } ] } ]	1	https://f1000research.com/articles/11-50
https://f1000research.com/articles/11-492/v1	04 May 22	{ "type": "Research Article", "title": "Kikuchi-Fujimoto disease in a tertiary care teaching hospital in Coastal South India: A 8-year retrospective study.", "authors": [ "Basavaprabhu Achappa", "Nipuni Chamathka Herath", "Bodhi Sebastian", "Nikhil Victor Dsouza", "PAVAN MANIBETTU RAGHURAM", "Ramesh Holla", "Nithyananda Chowta", "Jyoti Ramanath Kini", "Basavaprabhu Achappa", "Nipuni Chamathka Herath", "Bodhi Sebastian", "Nikhil Victor Dsouza", "Ramesh Holla", "Nithyananda Chowta", "Jyoti Ramanath Kini" ], "abstract": "Background: Kikuchi-Fujimoto disease (KFD) is a rare, benign condition of unknown etiology, presenting as cervical lymphadenitis. Lymphadenopathy is usually tender and maybe associated with systemic symptoms. Despite the extensive literature on this disease, it continues to be misdiagnosed owing to its misleading clinical presentation. Methods:\nA retrospective hospital-based descriptive cross-sectional study was conducted in tertiary care hospitals from 2011 to 2019. All patients with confirmed KFD diagnosis were included and after ethics committee approval the clinical details and histopathological data was retrieved from the medical records department and analyzed. Results: A total of 67 cases were included. The mean age was 26.9±11.3 years with a female: male ratio of 1.9:1. There were 50 patients with tender cervical lymphadenopathy which was the most common clinical presentation. The mean length and width of palpable lymph nodes were 2.3±1.0 cm and 2.2±0.7 cm respectively. Histology revealed proliferative stage in majority of patients (n=40, 59.7%). Lymphadenopathy resolved in 83.6% within 2 months. There were 42 patients who had complete recovery with symptomatic treatment within a period of 9 months. Conclusions: KFD is prevalent in young, female patients of Asian descent and often presents as tender cervical lymphadenopathy. Early diagnosis with excisional lymph node biopsy is crucial to avoid unnecessary investigations and treatment. Treatment is symptomatic unless complicated, where steroid therapy is considered. KFD has an excellent prognosis with almost no risk of fatality.", "keywords": [ "Kikuchi-Fujimoto disease", "Histiocytic necrotizing lymphadenitis", "Lymphadenopathy", "Fever", "Kikuchi disease", "Rare diseases." ], "content": "Introduction\n\nKikuchi-Fujimoto disease (KFD), also known as “histiocytic necrotizing lymphadenitis” is a rare condition of unknown etiology. This benign condition presents as cervical lymphadenopathy, usually tender and is often associated with systemic symptoms like fever.1 Identified first by two Japanese pathologists, independent of each other in the year of 1972, KFD was noted to have a higher incidence in Asian patients2 and continues to be misdiagnosed till date.1 Therefore, the awareness of this condition amongst clinicians and pathologists alike would be fruitful as it would aid in the early detection and prevention of life-threatening sequelae such as malignancies.3\n\nKFD is seen more frequently in young adults, with a mean age between 20–30 years,4 but it can occur in any age group.1 Even though a female predominance is reported in many previous cases, some studies done in Asia show a male to female ratio of 1:1.1 The rare involvement of the heart, liver and lungs increases the fatality of an otherwise self-limiting disease.2\n\nThe most common clinical feature is cervical lymphadenopathy, with or without systemic features such as fever, fatigue, headache and night sweats.5 The lymphadenopathy is usually under 4 cms,6 and in most cases can be tender or painful.2 Hepatosplenomegaly has been reported in a few cases.1\n\nBoth the etiology and pathogenesis of this disease is still unknown.1 Clinically and histologically, the disease has overlapping features with tuberculosis, lymphoma or systemic lupus erythematosus (SLE)4 resulting in dilemmas in diagnosis and management. KFD can be seen in patients with previous history of SLE, it can coexist with SLE or complicate into SLE.7 This association with SLE was more common in Asian than European patients.6 Meanwhile, a significant number of patients were found to have underlying viral infections.6\n\nThe gold standard investigation for diagnosis is by histopathological examination of an excisional biopsy obtained from the affected lymph node.7 Histologically, altered lymph node architecture by nodules of necrosis in the cortex, paracortical expansion, apoptotic cells with accumulation of crescentic histiocytes, and the absence of granulocytes especially neutrophils are typical fine-needle aspiration cytology features seen in KFD.6,8 Due to challenges faced in diagnosing KFD, three evolving histological patterns have been proposed by pathologists based on key morphologic features. They are proliferative, necrotizing and xanthomatous based on the dominant histological pattern.7 Most patients have normal laboratory findings.1 However, a few cases with mild anemia, elevated Erythrocyte Sedimentation Rate (ESR) and C-reactive protein (CRP) along with leucopenia and elevated Lactate Dehydrogenase (LDH) have been reported rarely.3\n\nEven though, this disease almost always runs a benign course and resolves in several weeks to months in most patients,6,9 it is seen to increase the risk of SLE and lymphoma.7 Treatment if necessary is symptomatic with analgesics and antipyretics.1 KFD has a very low recurrence rate and only a few fatalities have been reported.1 The use of corticosteroid therapy is still under debate for the management of recurrent cases.1\n\nDespite the extensive studies done on KFD, it continues to be misdiagnosed till date. The objective of this study was to promote awareness among clinicians and pathologists alike. Therefore, this study discusses the clinico-epidemiological presentations of the disease, the histomorphology commonly found in these cases and the importance of follow-up of these patients to ensure complete recovery and avoid possible sequelae and complications.\n\n\nMethods\n\nThe study was a retrospective hospital based descriptive cross-sectional study carried out among all patients diagnosed with KFD in Mangalore. The diagnosis was confirmed following histopathological examination of the excision biopsy of the lymph nodes of suspected patients in the Pathology Department in Kasturba Medical College (KMC), Mangalore. The inclusion criteria consisted of all the patients diagnosed with KFD in KMC teaching hospitals from 2011–2019, while exclusion criteria were patients not diagnosed with KFD and cases diagnosed before 2011 and after 2019.\n\nThe Institutional Ethics Committee (IEC) of Kasturba Medical College, Mangalore (Manipal Academy of Higher Education) approval (Reference No. IEC KMC MLR 03-17/41) was obtained. The permission to access the medical records of KFD patients and a waiver of consent was obtained from the Institutional ethics committee.\n\nThe records of KFD patients during the period of April 2011 to April 2019 were reviewed and information on demographics, clinical profile and histopathological data of KFD patients seeking medical care was recorded in a data extraction sheet. The proforma included the relevant clinico-epidemiological information about the patient inclusive of clinical presentation, associated co-morbidities, examination findings, investigations done, pathological morphology as well as any complications or sequelae on follow-up over a period of 9 months.\n\nHistopathological examination of the excision biopsy of all suspected cases of KFD was carried on formalin-fixed paraffin embedded sections stained with Hematoxylin and Eosin, Ziehl–Neelsen stain, Periodic acid-Schiff and Gomori Methenamine silver stains in the Pathology Department. The results obtained were categorized into 3 phases as proliferative phase, xanthomatous phase and necrotizing phase. The classic presentation of the proliferative stage was histiocytes, dendritic cells, lymphocytes and nuclear fragments. In the xanthomatous phase the predominant feature was foamy histiocytes within the lesions. Necrotizing phase typically showed extensive necrosis with karyorrhectic nuclear debris and coagulative necrosis and complete loss of lymph node architecture. The diagnosis of KFD was confirmed based on these morphological findings.\n\nData collected was analyzed using IBM Corp. Released 2017. IBM SPSS Statistics (RRID:SCR_016479) for Windows, Version 25.0. Armonk, NY: IBM Corp to analyze the clinico-epidemiological data, clinical presentation, local examination findings, histopathological presentation as well as outcomes on follow-up. This data was expressed as proportions, mean, standard deviation, median and Inter Quartile Range (IQR).\n\n\nResults\n\nThe baseline characteristics of the patients are shown below in Table 1. The age of our study population ranged from 4 to 77 years. The mean age was 26.9±11.3 years and a median age of 27 years (IQR: 18–32 years). As shown below KFD commonly affects younger adults between the ages of 21 to 40 years (n=44, 65.7%) and was seen more in female patients (n=44, 65.7%). When the occupation of the study population was analyzed, most of the patients were found to be homemakers (n=27, 40.3%).\n\nTable 2 demonstrates the distribution of chief complaints at presentation in KFD patients. A vast majority of the patients presented with tender swelling over the side of the neck (n=50, 74.6%). Following local examination, excision biopsies were performed on patients with positive findings. These biopsies revealed, the length and width of the cervical lymph nodes ranged from 0.5 to 5 cm and 1 to 5 cm respectively. The excised cervical lymph nodes had a mean length of 2.3±1.0 cm and mean width of 2.2±0.7 cm.\n\n* Multiple responses.\n\nPatients also presented with complaints of fever (n=35, 52.2%) and hepatosplenomegaly (n=10, 14.9%) due to the systemic involvement of the disease. Hepatosplenomegaly was assessed by abdominal examination in all the patients. Computerized tomography (CT) scan of the abdomen was used for the patients with positive findings on clinical examination. Axillary lymph nodes were assessed by clinical examination and were found to be enlarged in 6 patients.\n\nThe histopathological profile of all patients was analyzed after classifying the findings of excision biopsies into the three morphological stages (Table 3). Most of the patients presented during the proliferative stage (n=40, 59.7%). The duration of lymphadenopathy in KFD patients ranged from a few days to 6 months (Table 4). In most patients, lymphadenopathy resolved in less than 2 months (n=56, 83.6%).\n\nThe patients were followed-up over a period of 9 months to observe the outcomes of KFD. A vast majority of the patients recovered with symptomatic treatment (n=42, 62.7%). Here, the lymphadenopathy was assessed using ultrasound examination of neck once the symptoms improved following treatment. Meanwhile steroid use was successful in treatment of KFD patients with systemic symptoms (n=17, 25.4%), and one such patient presented with signs of neurological involvement which also resolved following treatment with steroids. A few patients were found to have a self-limiting course of KFD (n=6, 12.2%). Recurrence of KFD was observed after treatment in 2 patients in this study. Both these patients developed tuberculosis over the next 2 to 3 months. Antinuclear antibody (ANA) levels were elevated in 3 patients, one of these patients was later diagnosed with SLE (positive for dsDNA). One elderly male patient who was diagnosed with necrotizing lymphadenitis of axillary nodes developed generalized lymphadenopathy, a repeat fine needle aspiration cytology (FNAC) and excision biopsy 6 months later confirmed a diagnosis of Non-Hodgkin Lymphoma.\n\n\nDiscussion\n\nKFD is a rare, self-limiting condition that is usually prevalent in Asian countries, clinically presenting as tender cervical lymphadenopathy with or without systemic symptoms. The etiology of the disease is yet to be uncovered, although certain studies have suggested an underlying viral infection or autoimmune disease to trigger the onset of KFD.2 A retrospective hospital-based descriptive cross-sectional study was conducted on 67 patients diagnosed with KFD, to determine the sociodemographic profile, clinical presentation, and the outcome of the disease.\n\nThe mean age of KFD prevalence was found to be 27.1 years in this study while in a study done in Sub-Saharan Africa, an average of 21 years was recorded.10 The youngest patient in our study was a 4-year-old girl and the eldest, a 77-year-old man. Of the study population, 16 were children (ranging from 4–18 years old) with confirmed cases of KFD. A study done on pediatric cases of KFD by Guleria S et al. reported a mean age of 10.8 years.11 A study based on the characteristics of KFD based on age showed the mean age of children and adults to be 13.2 ± 4.8 and 32.7 ± 8.8 years respectively, which was similar to the range observed in our study.12\n\nOn analysis of the gender prevalence, a female predominance of 1.91:1 was observed which was similar to a study done in South India which had a significant female majority of 2.4:1.9 A ratio of 1:1 was observed in the results obtained from a study conducted by Pepe F et al.5 in Italy. A reversal of the gender ratio was observed in two studies carried out in Korea and North India with a significant male majority of 2.8:113 and 2:111 respectively.\n\nYounger females were noted to be more predisposed to KFD in our study with ages ranging from 4 to 38 years with all considerably younger than their male counterparts. A case series of 9 patients was conducted by Abeysekara RA et al., where all the cases were female patients in the age group of 12–30 years.14 Adhikari RC et al. carried out a similar study where 5 out of the 6 cases were females and the age range were 13–32 years.15\n\nThe most common clinical presentation of these patients was tender swelling on the side of the neck (74.6%). Similar results were obtained from a study done in Michigan with 60–90% of the cases having posterior cervical lymphadenopathy.1 Systemic symptoms such as fever (52.2% of the cases) and hepatosplenomegaly (14.9% of the cases) was found abundantly in this study. Fever was noted to be associated frequently with tender cervical lymphadenopathy (n=30, 44.8%). This coexistence of symptoms was replicated in the Michigan study in 35–77% of the patients.1 Tender cervical lymphadenopathy was the most common symptom in studies done in Italy5 (60–98%), Saudi Arabia2 (56–98%) and Michigan1 (60–90%). The involvement of axillary lymph nodes was encountered in 9% of the cases in this study, whereas 13% were reported in a study conducted by Supari D et al.9\n\nOne 22-year-old male patient in our study presented with symptoms of cervical lymphadenopathy for 15 days duration associated with systemic symptoms of fever, neck stiffness and meningitis. He was treated with steroids and tapered over a period of one month. Follow-up over 2 years revealed that lymphadenopathy subsided in 3 months and the patient recovered fully with no recurrence reported. In correlation to the above case, a study done in Japan reported 5 cases of recurrent KFD associated aseptic meningitis which resolved within several months with 3 of the cases requiring steroids.16 Thus, the use of steroids in patients with recurrence of KFD with neurological involvement is considered beneficial following extensive investigation to ensure its safety.16,17\n\nThe histopathology findings of the excisional lymph node biopsy remains a gold standard for confirming KFD. In this study, the findings were grouped into 3 morphological phases as done in previous studies5,7,8 and a vast majority of the confirmed cases presented in proliferative phase of KFD (n=40, 59.7%). Two of the patients whose lymph nodes showed proliferative phase with histiocytic aggregates later developed tuberculosis. Thus, histology of early tuberculosis can mimic KFD resulting in misdiagnosis. Similarly, two patients with predominance of necrosis went on to be diagnosed with SLE and Non-Hodgkin’s Lymphoma. Follow-up is thus crucial in KFD patients. Immunohistochemistry studies are ancillary techniques to support or exclude a diagnosis of lymphoma in suspicious cases.\n\nA study conducted in Bangalore revealed that the duration of lymphadenopathy lasted from 1 week to 3 months,9 however our study results suggested a longer duration of symptoms and it varied from 2 weeks to 6 months. The follow-up outcomes our patients over a period of 9 months revealed that all the cases completely recovered with no complications.\n\nRegarding the treatment aspect, majority of our patients recovered after symptomatic management (62.7%), which was the choice of initial treatment in previous studies as well.1,11,16 One fourth of the patients in this study were treated successfully with steroids (25.4%) and a similar result was obtained in two more studies where 2 out of the 6 patients required steroid therapy11 and the utilization of methylprednisolone exhibited a drastic response within 24 hours.6\n\nStudies conducted in Florida had self-limiting KFD which typically lasted 1 to 4 months6 and in England their symptoms resolved spontaneously within 6 months.7 On the contrary, even though KFD is known to be a self-limiting condition, our study showed that only a small fraction of patients recovered completely without any treatment (9%).\n\nRecurrence is always a matter of concern for the treating physician. Studies done have reported a recurrence rate of 3 – 4 %1,2,18 in adults which was similar to our study recurrence rate of 3%. In comparison to children, we observed no recurrence which was a paradoxical finding when compared to a study by Han HJ et al.16 which had a recurrence of 27% after a one year follow-up. Recurrence can present as late as 8 years after initial presentation according to Deaver D et al.6 Therefore, long-term follow-up is essential to determine the rate of recurrence of KFD.\n\n\nConclusion\n\nKFD is a rare, idiopathic condition which presents as a diagnostic challenge to both pathologists and clinicians due to its misleading presentation. Demographically, the disease is prevalent in young, Asian, female patients and often presents as cervical lymphadenopathy. Early diagnosis with excisional lymph node biopsy is crucial to avoid unnecessary investigations and treatment for this self-limiting condition. Treatment is only symptomatic unless complicated, where steroid therapy is considered. KFD has an excellent prognosis with almost no risk of fatality. Long term follow-up of patients is vital to look for recurrence, complications of this condition and a few of them can evolve into SLE, lymphoma and TB.\n\n\nData availability\n\nFigshare: Underlying data for ‘Kikuchi-Fujimoto disease in a tertiary care teaching hospital in Coastal South India: A 8-year retrospective study. https://figshare.com/s/b11d971a3f430af2f3d4. CC BY 4.0 license.\n\nThis project contains the following underlying data:\n\n- Data sheet in excel format\n\n\nAuthor contributions\n\nBasavaprabhu Achappa - Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Resources; Supervision; Writing - original draft, review & editing.\n\nNipuni Chamathka Herath - Data curation; Formal analysis; Resources; Writing - original draft, review & editing.\n\nBodhi Sebastian - Data curation; Formal analysis; Writing - original draft.\n\nNikhil Victor Dsouza - Data curation; Formal analysis; Resources; Supervision; Writing - original draft, review & editing.\n\nPavan Manibettu Raghuram - Conceptualization; Formal analysis; Methodology; Project administration; Supervision; Writing - original draft, review & editing.\n\nRamesh Holla - Formal analysis; Methodology; Project administration; Resources; Supervision; Writing - original draft, review & editing.\n\nNithyananda Chowta - Formal analysis; Project administration; Resources; Supervision; Writing - original draft, review & editing.\n\nJyoti Ramanath Kini - Data curation; Formal analysis; Resources; Supervision; Writing - original draft, review & editing.", "appendix": "References\n\nPerry AM, Choi SM: Kikuchi Fujimoto disease: A Review. Arch. Pathol. Lab. Med. 2018; 142(11): 1342–1346.\n\nJamal AB: Kikuchi fujimoto disease. Clin. Med. Insights Arthritis Musculoskelet. Disord. 2012; 5: 63–66. PubMed Abstract \| Publisher Full Text\n\nDalugama C, Gawarammana IB: Fever with lymphadenopathy - Kikuchi Fujimoto disease, a great masquerader: a case report. J. Med. Case Rep. 2017; 11(1): 349. PubMed Abstract \| Publisher Full Text\n\nTariq H, Gaduputi V, Rafiq A, et al.: The enigmatic kikuchi-fujimoto disease: a case report and review. Case Rep. Hematol. 2014; 2014: 1–4. Publisher Full Text\n\nPepe F, Disma S, Teodoro C, et al.: Kikuchi-Fujimoto disease: a clinicopathologic update. Pathologica. 2016; 108(3): 120–129. PubMed Abstract\n\nDeaver D, Horna P, Cualing H, et al.: Pathogenesis, diagnosis, and management of Kikuchi-Fujimoto disease. Cancer Control. 2014; 21(4): 313–321. PubMed Abstract \| Publisher Full Text\n\nSalamat S, Chan J, Jolly K, et al.: Kikuchi-Fujimoto Disease and Prognostic Implications. Head Neck Pathol. 2020; 14(1): 272–275. PubMed Abstract \| Publisher Full Text\n\nDas DK, Mallik MK, Hawaraa A, et al.: Kikuchi-Fujimoto disease in fine-needle aspiration smears: a clinico-cytologic study of 76 cases of KFD and 684 cases of reactive hyperplasia of the lymph node. Diagn. Cytopathol. 2013; 41(4): 288–295. PubMed Abstract \| Publisher Full Text\n\nSupari D, Ananthamurthy A: Kikuchi-fujimoto disease: a study of 24 cases. Indian J. Otolaryngol. Head Neck Surg. 2014; 66(1): 69–73. PubMed Abstract \| Publisher Full Text\n\nLame CA, Loum B, Fall AK, et al.: Kikuchi-Fujimoto disease, a rare cause of lymphadenopathy in Africa. Description of the first case in Senegal and review of the literature. Eur. Ann. Otorhinolaryngol. Head Neck Dis. 2017; 134(5): 347–349. PubMed Abstract \| Publisher Full Text\n\nGuleria S, Gupta A, Pilania RK, et al.: Kikuchi-Fujimoto Disease: An Under Recognized Cause of Fever with Lymphadenopathy. Indian J. Pediatr. 2020; 87(1): 85. PubMed Abstract \| Publisher Full Text\n\nKim HY, Jo HY, Kim SH: Clinical and Laboratory Characteristics of Kikuchi-Fujimoto Disease According to Age. Front. Pediatr. 2021; 9: 745506. PubMed Abstract \| Publisher Full Text\n\nHan HJ, Lim GY, Yeo DM, et al.: Kikuchi's disease in children: clinical manifestations and imaging features. J. Korean Med. Sci. 2009; 24(6): 1105–1109. PubMed Abstract \| Publisher Full Text\n\nAbeysekara RA, Kularatne SA, Waduge R, et al.: Kikuchi-Fujimoto's disease: a case series from Sri Lanka. Ceylon Med. J. 2013; 58(1): 31–33. PubMed Abstract \| Publisher Full Text\n\nAdhikari RC, Sayami G, Lee MC, et al.: Kikuchi-Fujimoto disease in Nepal: a study of 6 cases. Arch. Pathol. Lab. Med. 2003; 127(10): 1345–1348. PubMed Abstract \| Publisher Full Text\n\nKomagamine T, Nagashima T, Kojima M, et al.: Recurrent aseptic meningitis in association with Kikuchi-Fujimoto disease: case report and literature review. BMC Neurol. 2012; 12: 112. PubMed Abstract \| Publisher Full Text\n\nSekiguchi S, Yamamoto Y, Hatakeyama S, et al.: Recurrent Aseptic Meningitis Associated with Kikuchi's Disease (Histiocytic Necrotizing Lymphadenitis): A Case Report and Literature Review. Intern. Med. 2021; 60(11): 1779–1784. PubMed Abstract \| Publisher Full Text\n\nBosch X, Guilabert A, Miquel R, et al.: Enigmatic Kikuchi-Fujimoto disease: a comprehensive review. Am. J. Clin. Pathol. 2004; 122(1): 141–152. PubMed Abstract \| Publisher Full Text" }	[ { "id": "139409", "date": "13 Jun 2022", "name": "Thejaswi K. Poonacha", "expertise": [ "Reviewer Expertise Hematology/ Oncology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have done an excellent retrospective observational study on Kikuchi-Fujimoto disease (KFD). While the sample size is relatively small, and no conclusions of real statistical significance can be concluded, it is still a landmark article considering the 8 year study, and the similarities noted in other studies. The authors' conclusion cannot be overstated with regard to the need for follow up in these patients. Nevertheless, this study deserves full merit.\nPlease consider the following points:\nThe authors particularly focus on the clinical signs and symptoms, histopathology and the demographics. For the sake of completeness, they should include pathophysiology of the disease. 1\n\nThe authors could elaborate on the viral etiology of disease for academic significance. 2\n\nAs a follow-up project, I urge the authors to consider doing a meta-analysis study for this disease. There has been a meta analysis done on the causative agents.\n\nIs the work 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": "139408", "date": "23 Jun 2022", "name": "Chaitanya Tellapragada", "expertise": [ "Reviewer Expertise Microbiology", "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\nThe article by Achappa B et al. is well written and provides important information regarding the baseline demographic and clinical features of patients with Kikuchi-Fujimoto disease (KFD) in South India.\n\nI have few minor suggestions for the authors to add in their discussion:\nHow many of these 67 patients were subjected to microbiological investigations for infectious aetiology (mainly viral infections) at the time of their initial presentation.\n\nHistopathological profiles of the study subjects are described in table 3 and duration of lymphadenopathy in table 4. It would be interesting if the authors could further discuss (based on their experience or previous studies) whether patients with any one or more of the three morphological phases (by histopathology or based on duration of lymphadenopathy) would benefit more from the steroid therapy.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "139410", "date": "28 Jun 2022", "name": "Farhan Fazal", "expertise": [ "Reviewer Expertise Infectious disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a retrospective study of a very important misdiagnosed entity. The authors have elaborated on the clinical as well as histological features of the disease. Since the study was retrospective it is not clear how the follow-up was done?. The authors have commented on the duration of lymph nodes that the patients had, but did not mention if the patients came for regular follow-up or was it done whenever the patient came for follow-up. Also what type of steroids and their dosage are used have not been mentioned. Overall the paper is well written.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-492
https://f1000research.com/articles/11-491/v1	04 May 22	{ "type": "Research Article", "title": "Burnout and anxiety levels in human medicine teachers, COVID-19 context", "authors": [ "Jorge Guillermo Morales Ramos", "María Susana Picón Pérez", "Freddy Albaro Manayay LLaguento", "Enaidy Reynosa Navarro", "Jorge Guillermo Morales Ramos", "María Susana Picón Pérez", "Freddy Albaro Manayay LLaguento" ], "abstract": "Introduction: In the COVID-19 context, university teachers have had to face the most complex educational demands, psychosocial risks, and the anxiety of responding to limitations in terms of connectivity and fulfillment of academic objectives. To identify the levels of Burnout and anxiety in the COVID-19 context and determine how these levels are manifested in the participating teachers. Methods: This was an analytical non-experimental, cross-sectional study. The population was 150 teachers of the Human Medicine Program of the University of San Martín de Porres, Chiclayo, Peru, and the sample was 66 teachers. The survey consisted of three sections: 1. Informed consent, 2. Maslach's Burnout Inventory, 3. Beck's Anxiety Inventory. Data processing was performed using the SPSS V.27 statistical software and all citations and bibliographical references were processed using Mendeley Desktop 1.19.8. Results: In the variable burnout syndrome, 25% of the participants were in the high level downwards; they present anxiety in 30.30% of the total. It was found that 50% of teachers presented mild to moderate anxiety. Conclusions: the largest number of teachers surveyed present anxiety due to burnout syndrome in the COVID-19 context. Finally, it is found that there is a correlation between anxiety and the sociodemographic variables sex, age, and marital status.", "keywords": [ "Anxiety", "Burnout Syndrome", "Distance education", "E-learning", "COVID-19" ], "content": "1. Introduction\n\nBurnout syndrome (SBO) is defined as a psychological syndrome resulting from certain interrelated experiences, characterized first by emotional exhaustion, as a response to stress; second, by depersonalization, which usually presents as an adverse change in how the person feels about work and other people (cynicism); and third, by low personal fulfillment, which is when people begin to feel negative about themselves, about their ability, their competence, their motivation to work.1–4 The World Health Organization (WHO) officially recognized this syndrome as a disease associated with work and as an occupational risk factor due to its ability to affect quality of life, mental health and even put lives at risk of the individual who suffer from it; however, this resolution of its incorporation will enter into force in 2023.5 SBO is also known as emotional exhaustion syndrome or burnout syndrome (SQT) and is considered a psychological response of an interpersonal and emotional nature, exclusive to professionals who provide services with high social or help demand. In short, it is one of the most damaging types of stress that arises as a result of chronic work stress, which is influenced by individual, social and organizational variables.6 There are factors associated with Burnout for teachers, including sociodemographic variables (sex, age, marital status, level taught and type of center), personality (external locus of control, higher level of self-awareness, self-efficiency and self-control, behavior pattern, low-level self-esteem), and work and organizational factors, such as work overload.7\n\nAnxiety is defined as a response system at a cognitive, physiological, behavioral, and affective level, which occurs when anticipating circumstances that are usually judged as very aversive when perceived as unexpected events, which are out of control and that can enhance threats to the interests in people's lives.8 Anxiety manifests itself differently in subjects due to biological and learned factors. Some individuals may react with anxiety when faced with threatening situations, while others, in similar situations, will respond by attaching less importance to the facts.9,10 Most anxiety theory and research now recognize five subtypes of anxiety disorders: these are panic attacks, generalized anxiety disorder, social phobia, obsessive–compulsive disorder (OCD), and post-traumatic stress disorder (PTSD).8 According to WHO, anxiety is one of the factors that contributes to global disability, placing it in sixth position (3.6%), being more common in women than in men (4.6% and 2.6%); in 2015, according to his calculations, almost 264 million people suffered from some type of anxiety disorder. Anxiety is a common mental disorder that involves behaviors or manifestations that include physical, mental, or emotional agitation in the face of specific events or daily life that involve uncertainty and instability.11,12 Rates can vary, from 2.9% in the Western Pacific region to 5.8% in the Americas region.13\n\nIn the coronavirus disease 2019 (COVID-19) context, the population's mental health has been compromised, especially in those groups present on the front lines. Among these groups, we can find the professionals who work as teachers in the Human Medicine Programs (PMH), who, in the conditions of the pandemic, have had to face the demands, the psychosocial risks, the anxiety caused by responding to the limitations in terms of connectivity and fulfillment of academic objectives, all of this in a context marked by academic and technological barriers, conditions of spaces in the home, interruption of work by relatives, among other problematic manifestations.14–16\n\nThere is still insufficient research to analyze the impact caused by COVID-19 on people's mental health. A study carried out in China, whose objective was to evaluate the psychological impact of COVID-19 on its inhabitants to understand better their levels of psychological impact, anxiety, depression, and stress during the initial stage of the pandemic—based on 1,210 respondents from 194 cities—found that 53.3% of the participants rated the psychological impact of COVID-19 as moderate or severe, 16.5% reported moderate-to-severe depressive symptoms, 28.8% reported anxiety symptoms moderate to severe and 8.1% reported moderate-to-severe stress levels.17\n\nAlso, a systematic review, which included 13 studies, concluded that mental health and mental functions of health professionals were compromised, mainly in those on the front line. The results of this study were as follows: medium-high levels of anxiety (26.5–44.6%), depression (8.1–25%), and worry and insomnia (23.6–38%).18 Another study in university teachers, whose purpose was to identify the prevalence and factors associated with SBO, found worrying levels of Burnout; the most frequent associated factors were: accumulation of extra-teaching activities, work overload, high labor ties, low remuneration, and devaluation of the work performed.19\n\nA cross-sectional investigation that included 459 teachers (61% women); found a 20.8% prevalence of SBO, 10.6% for the rate of depression, and, regarding anxiety, the rate was 30.9%. Likewise, the comorbidity among the participants was 19.7%. The teachers with SBO presented increased depression and anxiety, significantly associating emotional exhaustion with anxiety and depression more than with depersonalization or personal fulfillment.20 Another study in Spanish university teachers (621 university teachers), whose objective was to evaluate the psychosocial risks of university teachers and identify areas for improvement for a healthy organization; found that these teachers presented an unfavorable situation for their health in five psychosocial dimensions: low self-esteem, high job insecurity, high psychological demands, high double presence and low social support.21 Likewise, an investigation focused on determining Burnout in teaching workers of the Faculty of Nursing of the Cooperative University of Colombia (sample of 30 teachers, population, 56); found that 66.6% presented low emotional exhaustion; 26.6%, average emotional exhaustion; 96%, low level of depersonalization; and 80%, higher level of personal fulfillment.22\n\n1.1.1 Burnout and anxiety: COVID-19 context\n\nPressley's research (2021), focused on obtaining information regarding the impact of COVID-19 and its association with anxiety in teachers, found that the closest significant predictors to Burnout according to the COVID-19 Anxiety Scale (CAS). These were: anxiety-related teacher burnout, anxiety when communicating with parents, current anxiety about teaching, and administrative support. The research points out that, to limit teacher burnout, it is necessary to monitor them during the pandemic and provide them with permanent educational, technological or emotional support. In addition, educational institutions must provide clear communication and protocols to help educators feel safe amid this complex pandemic.23\n\nAnother study in Canadian teachers where the authors investigate whether teachers' attitudes toward change, teaching effectiveness, and attitudes toward technology are correlated with resilience and Burnout during the early stages of a pandemic; also whether perceptions of principal support, teaching effectiveness, attitudes towards ICTs, teacher resilience and Burnout have changed during the early stages of a pandemic; found that attitudes towards change and administrative support were positively correlated with teachers' Burnout and resilience at the start of the pandemic, and that, during the three months following the start of the pandemic, teachers showed an increasing increase in tiredness and cynicism, but at the same time greater efficiency for classroom management and a greater sense of achievement; attitudes, on the contrary, became more negative. On the other hand, the implications of the scarcity of resources were evidenced, which resulted in stress and exhaustion over time. Research suggests that teachers have been burned out by these disruptive changes.24\n\nA recent study in Moldova to evaluate the Burnout of teaching staff during COVID-19 revealed significant relationships between Burnout and some sociodemographic variables such as age, gender, and years of professional experience, showing that female teachers presented higher exhaustion less personal fulfillment than teachers. The temporary closure of educational institutions and the transition to virtual teaching brought some restrictions that produced sudden changes in the teaching-educational environment and, consequently, negative consequences and an impact on teachers' health. The study warns about a high level of Burnout due to these changes in teaching methods. The fundamental concerns of the teachers were located in aspects such as the quality of the lesson, which depends on the teacher being competent to achieve quality electronic learning in the student, beyond the consequences that this means for their comprehensive health.25\n\nFor their part, Pressley and Ha (2021) explored the new approaches and requirements of virtual teaching and their impact on the effectiveness and participation of teachers in the USA, based on the variables participation and instruction, taking into account the Scale of Teacher Effectiveness (TSE). The results showed that the mean efficacy scores on the instruction and participation variables were lower than the TSE scores of instruction and participation compared to previous studies. The study also indicates that the teachers of the virtual modality presented lower scores than those who taught following a blended or face-to-face model. The regional educational administration should have provided more support and instructional guidance.26 Using this same scale, another study evaluated changes in Burnout and its relationships with changes in ERT during COVID-19, applying a regression model of latent change in German teachers, namely the implementation of the learning materials during the pandemic it became a relevant process where the evaluation of attitudes and self-efficacy towards virtual learning was taken into account and whether the two variables were related to changes in Burnout and self-efficacy. This study showed that the components of Burnout, depersonalization, and lack of personal fulfillment increased significantly from pre to post COVID-19, while emotional exhaustion did not show significance. Regarding changes in Burnout, the study itself showed a negative correlation with changes in the TSE and established that the lack of resources had an important implication.27\n\nFinally, an investigation in the USA, through an online survey involving 47 volunteer teachers from 18 countries whose objective was to analyze the problems of teacher burnout in general during the pandemic; found that the average level of pre-pandemic Burnout reached 3.8 points (out of a maximum of 10) and during the pandemic, it was calculated at 5.5 (out of a maximum of 10). In addition, the highest level of pre-pandemic Burnout was found in the group with 11 to 20 years of experience, while the level of pandemic burnout was recorded in the group with 21 to 30 years of experience. Among the most relevant characteristics of Burnout found were the psychological ones (stress, discouragement, depression, exhaustion, guilt, frustration, panic, and tiredness), those that were related to health (weakness, headache, insomnia, spine, and eyes), educational (lack of resources, increased workload, face-to-face contact, multitasking, cheating and low motivation of students), technological (spending too much time on the computer and the quality of the internet), management (lack of student participation, insufficient reward system, and excessive administrative control), health (excessive administrative control and lack of students), lifestyle, family and financial problems.28\n\nThe research results consulted so far show worrying rates of Burnout associated with anxiety in teachers globally. This reality is not unrelated to the situation that the professors of the Human Medicine Program of the University of San Martín de Porres, Chiclayo, Peru (PMH-USMP-Chiclayo, Peru; by its acronym in Spanish), have been facing; who, since the beginning of COVID-19, have been facing multidimensional difficulties. In the personal aspect, they faced insecurity to face the new educational forms that included using technological tools hitherto unknown or little-used; according to the results obtained, this situation generated high anxiety levels in the participants. In the labor aspect, the main limitations were work overload, the lack of adaptation to new schedules, the lack of economic resources, teaching materials, modern technological equipment, and connectivity problems. In the academic aspect, the lack of training in the management of educational technology, lack of experience in virtual teaching, deficiencies in the development of evaluation processes in the student, deficiencies in the feedback of content with the student as a result of the technological barriers between teacher-student-parents, deficiencies in the management of new educational methodologies for virtual teaching.\n\nAll these manifestations were decisive to problematize: what are the levels of Burnout and anxiety in the COVID-19 context in teachers of the Human Medicine Program of the USMP, Chiclayo, Peru? The objective of this research was to identify the levels of Burnout and anxiety in the COVID-19 context; also determine how these levels are manifested in the teachers participating in the study. As a hypothesis, it is argued that the Burnout Syndrome increases the anxiety of the teaching staff of the PMH-USMP-Chiclayo, Peru.\n\n\n2. Methods\n\nThe study uses an analytical, non-experimental, and cross-sectional design to respond to a positivist paradigm. A diagnostic evaluation was carried out based on online survey studies, using Google Forms, to measure the effect of COVID-19 on Burnout and anxiety in teachers from the PMH-USMP-Chiclayo, Peru; made up of two academic departments: 1. Basic Sciences and 2. Clinical Sciences; analyzing the variables in their natural context, without manipulating them. SBO was defined as study criteria variables and sociodemographic variables as predictor variables. The SBO variable was classified into three dimensions: emotional exhaustion (nine questions), depersonalization (five questions), and personal accomplishment (eight questions), for a total of 22 items with a Likert-type response scale with seven levels (never, a few times a year or less, once a month or less, a few times a month or less, once a week, a few times a week, and every day). While the anxiety variable was made up of 21 items with a Likert-type response scale with four levels: normal (0–7), mild,8–15 moderate,16–25 and severe (26–63); taking into account that there will be anxiety when the levels are mild, moderate or severe. The Chi-squared test was applied to correlate the variables Burnout Syndrome and anxiety; These data are shown in Table 6, which allowed us to establish the null (H0) and alternative (H1) hypotheses.\n\n• H1: The Burnout Syndrome increases the anxiety of the teaching staff of the PMH-USMP-Chiclayo, Peru, in the COVID-19 context.\n\n• H0: The Burnout Syndrome decreases the anxiety of the teaching staff of the PMH-USMP-Chiclayo, Peru, in the COVID-19 context.\n\nThe population was composed of 150 teachers. The sample was non-probabilistic for convenience, consisting of 66 teachers from the PMH-USMP-Chiclayo, Peru. To obtain the sample size, the formula was used to estimate proportions of a known population, considering a Cronbach's alpha of 5% and an allowed error of 9%; obtaining a sample value of 66.4. Inclusion criteria: practicing teachers (full-time dedication, part-time dedication) who have taught uninterruptedly from 2018 to the present; they are registered in the National Superintendence of Higher University Education; and belong to the teaching staff of the PMH-USMP-Chiclayo, Peru. Exclusion criteria: teachers who do not meet the above criteria or whose teaching work has been intermittent from 2018. The distribution of teachers according to sociodemographic variables of age, sex, marital status behaved as follows: age range, 26–32 years (1.52%), 33–40 years (22.73%), 41–50 years (30.30%), and 61–74 years (15.15%). Gender: 76% men and 24% women. Marital status, divorced (1.52%), widowed (4.55%), married (80.30%), and single (13.64%).\n\nData collection took place from March 3, 2021 to April 5, 2021. The data collection technique was the survey, through two questionnaires. The first was the Maslach Burnout Inventory (MBI), which evaluates the manifestation of not being able to give more of oneself, both physically and mentally (exhaustion), the presence or absence of a negative attitude of devaluation and loss of interest in work (depersonalization), and the existence of doubts about one's ability to do academic work (lack of personal accomplishment).4,29 The second was the Beck Anxiety Inventory, which effectively measures the degree of anxiety in children and adults.30,31\n\nFirst, the data was processed, eliminating all the subjects with missing values in all the variables and with scores out of range; second, the total score of the variables SBO (in its three dimensions) and anxiety was obtained, with which the level of the scales for each variable was obtained (adhering to the recommendations of the authors of the instruments); and, finally, the descriptive and inferential data analysis was carried out, through the statistical software SPSS V.27. To limit possible bias in data processing, parallel processing of the same data was used, but with other specialists from the statistics area of the institution that supports this study. In this sense, no significant differences were found.\n\nThe research complies with the ethical principles proposed in the Declaration of Helsinki. The principles of autonomy and informed consent are considered fundamental elements within the scientific research process.32 All teachers freely participated in the research. They were informed promptly through a meeting via Zoom, notified through institutional email, and explained the investigation's purpose. In the end, all signed an informed consent letter where the limits of their participation were established. The Ethics Committee approved the research of the Universidad De San Martín de Porres, Chiclayo, Peru. Date: February 22, 2021, Official Letter No. 139-2021 - CIEI-FMH-USMP.33\n\n\n3. Results\n\nTable 1 shows that in the burnout syndrome variable, 50% of teachers from the PMH-USMP-Chiclayo, Peru, are in the middle level downwards, the same thing happening with the third quartile. However, we have that the last quartile is in high levels downwards. In the anxiety variable, 50% of teachers from the PMH-USMP-Chiclayo, Peru, were normal, indicating that they do not have anxiety. In addition, the third quartile of teachers was at the mild level, which also indicates that they do not have anxiety. However, the last quartile of teachers was at levels less than or equal to 4 (severe downwards) of anxiety.\n\nIn Table 2, 57.58% of the teachers are in the medium level of SBO due to emotional exhaustion, and 13.64% are at the high level. Regarding the depersonalization dimension, 46.97% of teachers are at the medium level of SBO and 13.64% at the high level. Regarding the personal achievement dimension, 80.30% of teachers are at the medium level, while 4.55% are at the high level.\n\nIn Table 3, gender indicator, it can be seen that the largest number of teachers who belong to the male sex, who present Burnout syndrome, have a medium level in emotional exhaustion,28 low level,22 and medium21 in emotional exhaustion. Depersonalization and medium42 in personal fulfillment; in the same way, in the case of females, the medium level predominates10: in emotional exhaustion,10 in depersonalization, and11 in personal fulfillment. In the Age indicator, the largest number of teachers by age, who present burn syndrome, have a medium level: in emotional exhaustion in the age ranges: 33-40,9 41-50,13 and 51-6010; in depersonalization in the age ranges: 33-40,7 41-5012 and 51-609; and, in personal fulfillment in the age ranges: 33-40,13 41-50,17 51-6013 and 61-74.9 Finally, in the Marital Status indicator, it is seen that the largest number of teachers, by marital status, who present burnout syndrome, are married and have a medium level: in emotional exhaustion,34 in depersonalization,25 and personal fulfillment.44\n\n* M=Male; F=female; S=single; Ma=married; W=widowed; D=divorced.\n\nIn Table 4, anxiety was observed at mild (15.50%), moderate (12.12%), and severe (3.03%) levels, which indicates that teachers presented anxiety in 30.30% of the total. It can also be seen that 69.70% of teachers did not present anxiety.\n\nIn Table 5, the largest number of teachers belong to the male gender (75.75%). Anxiety is observed in male teachers: 5 (7.58%) are at the mild level; 8 (12.12%), at a moderate level; and 2 (3.03), at the severe level. This indicates that, of the 16 female teachers, only 5 (7.58%) present anxiety at the mild level. In addition, the largest number of teachers belong to the age ranges between 33 to 50 years, with the following distribution: 7.58% in the light level; in the moderate, 9.09%; and 1.52% in severe. For teachers in the range of 51 to 60 years: 7.58% are included in the light level; at the moderate level, 1.52%; and at the severe level, 1.52%. Concerning teachers in the range of 61 to 74 years, 1.52% are at the moderate level. On the other hand, it must be added that teachers whose age range is between 25 and 32 years do not present anxiety (Table 3).\n\n* M=Male; F=female; S=single; Ma=married; W=widower; D=divorced.\n\nRegarding marital status, the following is noted: 83.30% of teachers belong to married marital status, of which 13.64% of them are in the mild level, 10.60 in the moderate level, and 3.03% in the severe level. In addition, mild anxiety is observed in 1.52% of teachers with single marital status. In the widowed, marital status, only 1.52% are moderate (Table 3).\n\n\n4. Discussion\n\nBased on the results found, the research hypothesis is confirmed given that the most significant number of teachers surveyed from the PMH-USMP-Chiclayo, Peru, present anxiety due to burnout syndrome (medium and high levels) in the COVID-19 context. Also, the null hypothesis is rejected.\n\nBelow the median is 50% of participating teachers. A similar situation can be seen in the third quartile. However, the last quartile is at a high level downwards (Table 1). Various studies found high prevalence rates of burnout: 67.5%,34 42.1%,35 20.8%,20 and 40.0%.7 It is necessary to emphasize that the most recurrent causes such as becoming too internalized in the problems of the students, the disproportionate preparation of academic and administrative documentation, work overload, low salary compensation, poor working conditions, and poor recognition of the effort of the teacher,35 must be taken into account to prevent Burnout from affecting not only teachers but also their teaching performance. This study rescues other characteristics: psychological (stress, discouragement, depression, exhaustion, guilt, frustration, panic and tiredness), health (weakness, headache, insomnia, spine and eyes), educational (lack of resources, increased workload, face-to-face contact, multitasking, cheating, and low student motivation), technological (spending too much time on the computer and internet quality), and managerial (lack of student engagement, insufficient reward system and excess administrative control).28 It is also inferred that the rate of teacher burnout is increasing because current educational policies are not aligned with teachers' expectations, in the same way, teacher resilience is depleted due to decreased self-efficacy, the quality of professional relationships and an increase in the pace of technological integration.\n\nHigh values of 57.58% and average 13.64% were obtained for the emotional exhaustion dimension. In agreement, a consulting study found levels of Burnout of 66.66%.22 These high levels of Burnout are associated with a high commitment at work, work stress, youth and inexperience, academic work, research activities, teaching experience, and teachers' employment status.36,37 These results allow us to agree that online classes—already in the COVID-19 context— contributed to the social isolation of the teacher caused them emotional exhaustion because they had to develop and comply with new teaching activities to which they were not accustomed, meaning the search for greater motivations and efficient, but stressful, time management.38 In the depersonalization dimension, the teachers presented high values of 13.64%) and average values of 46.97%, showing signs of indifference and cynicism towards the students. These results are corroborated by current research that found burnout levels of 35%, 37.7% and 80% respectively.34,35,39 These values help explain why the depersonalization dimension increased significantly in the COVID-19 context compared to the pre-COVID-19 stage.\n\nThe personal accomplishment dimension was located at the medium level in a higher proportion concerning the other two dimensions (80.3%) and, at the high level in a lower proportion (4.55%), which are contrasted with the results presented by Araoz & Ramos (2020): 32.8% at the medium level and 27.6% at the high level,35 and those achieved in a study carried out on medical personnel in which they obtained values of 87.1% high level.40 These results show a weak work organization, a weak work structure, as well as a low capacity to face teaching work in the COVID-19 context41; however, it is necessary to understand that the changes from learning in the traditional classroom to the virtual one; meant an enormous challenge for teachers, not having the technological infrastructure, not having had enough time to achieve the appropriate skills for remote teaching or emergency distance education, could have a balance with overwhelming results and frustration.42 A recent study argued that COVID-19 demanded huge sacrifices from teachers, who in record time went from traditional face-to-face education to a new educational modality that directly impacted their self-efficacy.26 It is confirmed that teacher self-efficacy influences a high level of confidence in their skills and abilities for the development of teacher education.\n\nWhen analyzing the sociodemographic variables according to age, the data were distributed at the average level in all age ranges in terms of emotional exhaustion, depersonalization, and personal accomplishment. It should be noted that in the research by Araoz & Ramos (2020), it was explained that younger teachers have to deal with instability and work overload35; also, Aveiro-Róbalo et al. (2021) argue that at a younger age there is a greater perception in terms of repercussions that are associated with anxiety that translate into exhaustion, fear, worries, and perception of abuse.43 Another study consulted found that the level of Burnout marked a reduction with increasing age in men and women. The bimodal association in women between 20 and 35 and older than 55 showed the highest level of Burnout.44 Regarding the marital status of teachers, there is greater personal fulfillment at the average level in married teachers than single, divorced, and widowed; This is because married people would have acquired strategies to deal with problems and thus feel better about themselves.36,45\n\nRegarding the anxiety variable, 30.3% of the participants presented anxiety, which corroborates the data found for anxiety in educators of 30.9%,20 and 42.7% in the medical staff.44 Likewise, 50% of teachers presented anxiety of a mild-to-moderate level, which coincides with the data obtained in a recent study carried out on university teachers at the Ibero-American level in the pandemic scenario, where it was observed that the level of anxiety in the majority of them was medium/low, which was associated with a negative institutional perception of teachers.14 Another investigation analyzed the impact of the COVID-19 outbreak on the mental health of health sector professionals, detecting medium-high levels of anxiety of 26.5–44.6%.18 Other factors also contribute to anxiety, such as lifestyle in urban areas,46 and the confinement that is presented as another significant factor.47 The long period of quarantine that the Peruvian population went through may have increased the possibility of acquiring mental and psychological problems,43,48 like anxiety (16%).49 Other research indicated that anxiety symptoms increased by 28% in the general population.50\n\nRegarding age, the teachers who showed higher anxiety levels were those in the range of 33–50 years, with 18.19%, of which 9.09% presented moderate level anxiety. This result is consistent with two investigations indicating that the age group that presented the highest level of anxiety in the pandemic was those under 30 years of age.43,51 A factor that would contribute to psychosocial risk is the years of professional experience since younger teachers with little work experience have not obtained the sufficiency to handle the job,52 especially in the age group of 21–30 years of experience.28 The study of Ozamiz-Etxebarria et al. (2020) maintains that symptoms were low. The younger age group and those with chronic diseases reported higher symptoms than the rest of the population.53 It should be taken into consideration that the results of this research are based on a population where the majority of teachers from the PMH-USMP-Chiclayo, Peru, are doctors and that, due to the characteristics of the pandemic, those who were in the front line of care were those included in the range between 25–50 years, who performed care work.\n\nRegarding marital status, married teachers exhibit a moderate-to-severe level; however, various studies warn that there is no relationship between anxiety and marital status.54 However, COVID-19 forced governments to implement distance education through the virtual modality, which could generate feelings of dissatisfaction and loneliness in the participants, generating depression due to the increase in family demands vs. labor demands; the latter with the aggravating circumstance of not having enough technological tools to carry out educational work.55\n\nFinally, the research hypothesis is confirmed given that the largest number of participating teachers present anxiety due to burnout syndrome (medium and high levels) in the COVID-19 context. Therefore, according to the anxiety scale, there is a significant direct relationship between burnout syndrome and its closest predictors.\n\n\n5. Conclusions\n\nFrom the analysis and interpretation of the data related to Burnout in teachers of the PMH-USMP-Chiclayo, Peru, it is concluded that the most recurrent causes that caused Burnout and therefore affect the performance of the participating teachers are based on the level of involvement of these with the problems of the students, the elaboration of the academic and administrative documentation in a disproportionate way, the work overload, the low salary compensations, the bad working conditions, as well as the deficient recognition of the effort. In addition, other psychological causes (stress, discouragement, depression, exhaustion, guilt, frustration, panic, and fatigue), health (weakness, headache, insomnia, spine, and eyes), education (lack of resources, increased workload, face-to-face contact, multitasking, cheating, and low student motivation), technological (spending too much time on the computer and internet quality), and managerial (lack of engagement of students, insufficient reward system and excessive administrative control).\n\nBeyond work stress, inexperience in disruptive change, mandatory compliance with academic work, research work, social isolation, and the aforementioned emotional exhaustion, teachers have shown commitment to their role, assuming the fulfillment of their new teaching activities to which they were not accustomed. This meant looking for new motivations through personal self-management, which meant an enormous challenge for them as they did not have the technological infrastructure and had enough time to achieve the appropriate skills for virtual teaching.\n\nParticipating teachers showed anxiety at medium and high levels due to confinement and negative institutional perception. These anxiety levels were associated with the negative institutional perception of the teachers, who did not have the technological equipment and the necessary training to face the new educational challenge from the beginning. Likewise, other factors prevailed with the alterations in their lifestyles and compulsory confinement, which increased the possibility of being exposed to psychological problems that meant a biopsychosocial risk that influenced teaching, especially in younger teachers with little work experience.\n\nThe results of this research were obtained during the COVID-19 context in Chiclayo, Peru. These results could vary as time progresses and this pandemic situation is overcome. On the other hand, as teacher burnout is increasing because current educational policies are not aligned with their expectations, there is a depletion of their coping reserves, self-efficacy, and professional and family relationships that must be addressed in successive studies.\n\n\nData availability\n\nZenodo: Burnout and anxiety levels in Human Medicine teachers, COVID-19 context https://doi.org/10.5281/zenodo.632654856\n\nThe project contains the following underlying data:\n\n➢ Statistical Results.xlsx\n\n➢ Processed data.pdf\n\nExtended dataZenodo: Burnout and anxiety levels in Human Medicine teachers, COVID-19 context https://doi.org/10.5281/zenodo.632654856\n\n➢ Burnout Syndrome Questionnaire\n\n➢ Anxiety Instrument\n\n➢ Validation by Expert Judgment\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nContribution of the authors (taking as reference CRediT – Contributor Roles Taxonomy) https://casrai.org/credit/\n\nJorge Guillermo Morales Ramos: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Supervision; Validation; Writing – original draft; Writing – review & editing.\n\nMaría Susana Picón Pérez: Conceptualization; Formal analysis; Funding acquisition; Investigation; Writing – review & editing.\n\nFreddy Albaro Manayay LLaguento: Data curation; Formal analysis; Investigation; Validation; Writing – review & editing.\n\nEnaidy Reynosa Navarro: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Supervision; Writing – original draft; Writing – review & editing.", "appendix": "Acknowledgment\n\nThe research team is especially grateful to the Universidad De San Martín de Porres, Chiclayo, Peru for its commitment to scientific research and the permanent support to the research team during the development of the study until its completion.\n\n\nReferences\n\nOlivares-Faúndez V: Christina Maslach, comprendiendo el burnout. 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Publisher Full Text" }	[ { "id": "149090", "date": "19 Dec 2022", "name": "Juan Luis Rodríguez Vega", "expertise": [ "Reviewer Expertise Education and Comprehensive Health of the teacher" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research collects the knowledge of how university teachers have assumed specific educational challenges that, at the same time, have caused high levels of Burnout and anxiety during the COVID-19 pandemic context. To achieve this purpose, they presented a non-experimental study with teachers from the medical school of a private university in the north of Peru. The study finds that the participants showed a high level of 25% cases of burnout and 50% presented mild or moderate anxiety.\nTheoretically, the study is based on current research through which it is possible to precisely define the variables, dimensions, and indicators that intervene in the same COVID-19 context; in addition to the methodological precision that the study presents, it may be replicable in other mental health contexts.\nThe results of this research are relevant to the international scientific community, especially in the area of public health and intervention.\nAmong the strengths of this study, the causes that cause Burnout in teachers and that affect their general professional performance are identified; among them: the excessive level of involvement of the teacher with the students, the disproportion of work generated by the academic part and the administrative functions that lead to work overload in the teacher. In addition, the need for more technological equipment and teacher training to adequately handle the complex educational scenario that COVID-19 has meant. These findings are fundamental so that, currently and looking to the future, educational institutions promote comprehensive policies that allow the complete professional development of teachers, the fulfillment of their essential functions, and that avoid the depletion of coping, self-efficacy, and teacher professional relations.\n\nIs the work 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": "136730", "date": "12 Jan 2023", "name": "Víctor Eduardo Horna Calderón", "expertise": [ "Reviewer Expertise Psicología de la educación (Psychology of Education)", "Educación (Education)", "Burnout" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reveals how university teachers have faced complex educational demands that have caused high levels of Burnout and anxiety in the context of COVID-19; therefore, the current researchers set out to determine how these levels are manifested in the teachers under study. To do this, they proposed a non-experimental cross-sectional analytical study with teachers from the Human Medicine Program at the University of San Martín de Porres, Chiclayo, Peru; finding that, regarding Burnout, 25% of the participants were at a high level downwards; they present anxiety in 30.30% of the total. Likewise, 50% of teachers presented mild to moderate anxiety.\nFrom a theoretical point of view, the manuscript defines and bases the study variables in the context of COVID-19 based on reliable academic information extracted from databases such as Scopus, Web of Science, and Springer, among others, citing authors with results and recognized trajectory in the area. Methodologically, the study design and population are accurately described. Likewise, it describes how the processing and analysis of the research data were carried out, as well as the ethical considerations that governed said research.\nThe researchers concisely describe the results, discuss them based on theories related to the subject, and identify the scientific background and results found during the process, which allows reaching conclusions that support the results, contributions, and the main gaps and limitations found, which allows opening new horizons of study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/11-491
https://f1000research.com/articles/11-490/v1	03 May 22	{ "type": "Research Article", "title": "Shifting the water paradigm from social good to economic good and the state’s role in fulfilling the right to water", "authors": [ "Arinto Nurcahyono", "Fabian Fadhly Jambak", "Abdul Rohman" ], "abstract": "Background: The problem currently faced is the availability of water to meet human needs, which is decreasing in number. On the one hand, there is a view that water is a commodity (economic good) while on the other hand says that water is a social good. These different approaches influence state policies in water management on how the state regulates and manages water in fulfilling the right to water for their citizens. The right to water implies that everyone should have access to water without discrimination. Methods: This study uses a normative approach, which reviewing water as social economic issues and adjusting by the legal rules relating to water and human rights. Results: Changing the paradigm of water from social goods to economic goods, this change is also based on water scarcity faced not only by one country but has become a global issue. Economic issues are the main topic in the global conversation, by reducing water in a social perspective, which should be the responsibility of the state as Article 33 paragraph (3) of the Constitution of the Republic of Indonesia 1945. This legal basis has logical consequences for the state to keep water as a social commodity oriented to the public interest. Conclusions: The pattern of economic approaches through privatization in the management of water resources has resulted in co-opting water as a collective source of life. Water is forced in commercial motivation rather than as a constitutive element that regulates water for the \"sustainability of collective life\" and \"to the maximum extent possible for the prosperity of society.\" The fulfillment of the right to water by the state certainly cannot be directly fulfilled, the important thing is that there are progressive efforts to fulfill it, with community involvement in the management of water resources, especially local communities.", "keywords": [ "rights to water", "economic good", "social good", "the role of the state" ], "content": "Introduction\n\nWater in the history of human life has a central position and is a guarantee for the continuity of human life on earth. According to Sayyid Qutub, water is the basis of life. The elements in it can only be formed according to the Islamic decree.\n\nWhat is the fundamental role of humans in the formation of water? Its role is simply to consume it. It is only He who has sent it down from Islamic Thought. It is possible to argue that its existence is a mandate and a gift from the Creator to be used and that its preservation should also be preserved for the sake of human survival. Therefore, the management, control, and ownership of water sources should also be established.\n\nInitially, water was a common property (res commune) passed down from generation to generation. According to Vandhana Shiva, there is a foundation of water democracy which includes mentioning that water is essential for humans, it also said that water is public property because water is not a human invention, cannot be owned as private property and sold as a commodity.1\n\nWater is a basic human need whose existence is guaranteed by the constitution, namely Article 33 of the 1945 Constitution paragraph 3, which reads, “Earth and water and the natural resources contained therein are controlled by the state and used for the greatest prosperity of the people.” This constitution clearly shows a social contract between the government and its citizens.2\n\nArticle 33 of the 1945 Constitution is a provision that forms the basis for the control and management of natural resources by the state to be used for the greatest prosperity of the people. Mohammad Hatta was the person who influenced the substance of the provisions of Article 33 of the 1945 Constitution. Article 33 of the 1945 Constitution seems to have become its legal terminology.3\n\nMohammad Hatta formulated that being controlled by the state in Article 33 of the 1945 Constitution does not mean the state itself becomes an entrepreneur or an ordernemer. It is more accurate to say that the state’s power lies in making regulations for the smooth running of the economy, regulations that prohibit the exploitation of the weak by people with capital.4\n\nWater is mentioned in the provisions of Article 33 paragraph (3) of the 1945 Constitution, although it does not explicitly mention the water right. Other articles on human rights in the 1945 Constitution also do not mention water rights. However, in the Constitutional Court’s Decision on Judicial Review of Law Number 7 of 2004 concerning Water Resources, the water right has been stated to derail the right to life guaranteed by the 1945 Constitution. Like the right to life, which is one of the human rights, the leading state is the government is obliged to respect, protect, and fulfill it. This obligation was constitutionally affirmed in 1945.5\n\nThe Constitutional Court, in its decisions, emphasizes the role of the state that must be maximized in fulfilling the rights of citizens to water. The water right is recognized as a distinct human right in both the constitution and international law. As a human right, the state must fulfill these rights. For this reason, in its decision, the Constitutional Court also imposes restrictions on the management of water resources with an emphasis on the protection of citizens’ human rights.6\n\nHowever, the role of the private sector is not closed in water exploitation. The first limitation in the decision of the Constitutional Court stipulates that the exploitation of water must not interfere, negate or override the people’s right to water. The last aspect that becomes a sign regarding the involvement of the private sector in water exploitation is that with a licensing mechanism, the private sector can be allowed to cultivate water. However, granting this permit is the last thing to be done once it has been ascertained that all citizens’ water needs are fulfilled. This means that while emphasizing the role of the state in fulfilling the water right, the private sector is still possible with strict terms and conditions.7\n\nAfter the decision of the Constitutional Court, through the initiative of the DPR, the Draft Law on Water Resources was drafted. On September 17, 2019, through the plenary session of the DPR, the bill became the Water Resources Law. Law Number 17 of 2019 concerning Water Resources revokes and does not apply Law Number 11 of 1974 concerning Irrigation (State Gazette of 1974 Number 65), Supplement to State Gazette Number 3046). Although Law Number 11 of 1974 concerning Irrigation was re-enacted after Law Number 7 of 2004 concerning Water Resources was annulled by the Constitutional Court.8\n\nThe formulation of water as a human right is explicitly stated in Article 6 of Law no. 17 of 2019 concerning Water Resources, which states that “The state guarantees the people’s right to water to meet the minimum daily basic needs for a healthy and clean life in sufficient quantity, good quality, safe, sustainable and affordable”.\n\nIn this century, the world issue of water resources is almost always faced with the strong current of liberalization of natural resources. No doubt water is then considered a new commodity. Even Donald Water said that water is oil in the XXI century; whoever controls water will rule the world.9\n\nAccess to water becomes a problem when experiencing a paradigm shift. Water is no longer considered a social good but an economic good. The current discourse on water resources today also concerns how to give value to these resources and then to whom the management will be entrusted. Resource liberalization is an almost inevitable contestation. The value of a natural resource is only measured economically. This capitalization process is a symbolic conquest of natural resources.10\n\nThe International Conference on Water and Environment in Dublin, Ireland in January 1992, issued four principles known as The Dublin Principles, one of which reads: “Water has an economic value in all of its competing uses and should be recognized as an economic good.”11\n\nThe strong current of capitalism that places water as an economic good raises question and is the focus of this article, how does the paradigm shift in water from social good to an economic good affect the state’s role in managing water resources for the fulfillment of the right to water?\n\n\nMethods\n\nThis study uses a normative approach, which reviewing water as social economic issues and adjusting by the legal rules relating to laws and regulations concerning Water Resources, Law No. 17 of 2019 concerning Water Resources, Decision of the Constitutional Court Number 85/PUU-XI/2013. Besides that, the laws, and regulations about Human Rights, especially the Right to Water, both international and national instruments, namely the International Covenant on Economic, Social and Cultural Rights along with General Comments, as well as Law no. 39 of 1998 concerning Human Rights. The type of data used is secondary data in the form of primary and secondary legal materials with literature study data collection techniques, then the data is analyzed qualitatively. The legal rules and regulations data collected from Ministry of Law and Human Rights and Constitutional Court of Republic of Indonesia, United Nation Human Rights Office of The High Commissioner (OHCHR), and Komisi Hak Asasi Manusia Republik Indonesia (KOMNAS HAM RI). Qualitative data obtained through literature is used to analyze water phenomena as a public interest that turn into an economic paradigm by specifically looking at the problem in Indonesia.\n\nList of the primary and secondary legal materials:\n\n1. Primary:\n\na. Law No. 17 of 2019 on Water Resources.\n\nb. Law No. 39 of 1998 on Human Rights.\n\nc. Decision of the Constitutional Court Number 85/PUU-XI/2013.\n\n2. Secondary\n\nthe International Covenant on Economic, Social and Cultural Rights along with General Comments.\n\n\nResults and discussion\n\nIn the conventional paradigm, water, apart from being a basic human need, is also a social good that is not owned by anyone but in the form of global commons, which is managed collectively, not to be sold or traded for profit. This traditional view has changed and been abandoned because water is not just a ‘social good’ but has become an economic commodity. This conventional paradigm contradicts the modern water management paradigm based on the intrinsic economic value of water, based on the assumption of limited and scarcity of water and the need for investment or the provision of clean water, as the fulfillment of the rights of every citizen.12\n\nNo water, no civilization is a phrase that may be appropriate to describe the importance of water in life. Without water, life will be extinct, and thus civilization will also be destroyed. Therefore, everyone feels they have a right to this one resource. It was this sentiment that contributed to the complexity of water resource management in the first place. Certain groups which perceive water as a commodity are increasingly viewing this situation as a water resources business opportunity. There is a clear shift in the perspective of certain groups on the value of water, where water is no longer considered a social good but an economic good. No doubt, water is then considered as a new commodity whose management is approached with economic principles. This is the beginning of the commodification, commercialization, and privatization of water resources being taken for granted.13\n\nThe availability of clean and widely accessible water is vital for all living things on earth. Hence, water is a social good. According to Peter H. Gleick, the definition of social good is:\n\nOne widely used definition is that social goods have significant “spillover” benefits or costs. Literacy is a social good{XE “social good”}, for example, because it benefits not just literate individuals but also makes possible a higher level of civilization for all members of society.14\n\nFor Gleick, water as a social good has benefits for many people, and social goods open up a better life.\n\nWater as a public good is the marginal cost of producing a social good is zero, according to economic literature. In addition, social goods have the nature in use and the need for city parks. Another trait is lacking excludability so that none of the members of society do need it. The market price of these goods and services can be equal to (or close to) zero. Therefore, goods as social goods needed by the state’s people must strive for it. It does not mean that water has no value, but that is inappropriate to use market forces to describe the sacrifices that consumers of clean water have to pay.15\n\nThe shift in water as an economic good found its momentum in January 1992 when the International Conference on Water and Environment took place in Dublin, Ireland. The conference produced four principles - which became known as the Dublin Principles -, one of which was\n\n“water has an economic value in all its competing uses and should be recognized as an economic good {XE “economic good”}. Within this principle, it is vital to recognize first the basic right of all human beings to have access to clean water and sanitation at an affordable price. Past failure to recognize the economic value of water has led to wasteful and environmentally damaging uses of the resources. Managing water as an economic good is an important way of achieving efficient and equitable use, and of encouraging conservation and protection of water resources”.16\n\nThe Dublin Principal argument at a glance shows there is a concern for the protection of increasingly limited water resources. Water is an economic good that departs from wasteful use efforts are needed to use it efficiently and fairly and encourage conservation and protection of water resources.\n\nHowever, if we look further, there are global powers that play a role. The World Bank has a huge role in the water resources sector. And not the only role, but also power. This role and power are obtained through the policies and conditions that accompany the loans, which unfortunately greatly encourages the privatization of water in developing countries such as Indonesia.\n\nThis new perspective on watermarks is the start of a paradigm shift about who owns water, how to understand its function, and how to use it. Especially after many international organizations gave their support for a new perspective on water, as Budds and McGranahan (2003) said,\n\n“… In the wake of Dublin, many international organizations realigned their position in the water sector, and the World Bank came to play a central role in developing and promoting new approaches consistent with its interpretation of the Dublin Principles, in particular the treatment of water as an economic good. … Bilateral development agencies also started to promote private sector participation in their recipient countries, including DFID and USAID.”17\n\nEven the World Bank is actively encouraging the governments of developing countries to minimize their role in public services and shift them to the private sector. The sequence of World Bank policies concerning the water resources sector demonstrates this effort to broaden the task of the private sector.18\n\nFor privatization groups, they argue that if a product is available for free, the market function that should allocate resources efficiently will not be achieved, it means there is no guarantee that the water availability is proportional to the level of consumption carried out. People tend to overuse water. The way that the government can control this is by limiting its use through regulations, tax, or by imposing it as a private good, namely goods that are excludable and rival.19\n\nThe changes in the perspective of water as an economic good then brought about a tremendous transformation in approach through the economic law of supply and demand. The consequence is that price becomes the key to control demand, which in turn makes the reallocation of water use to uses with a higher water value.\n\nIndonesia has imposing water as an economic good since the early 1980s, the government’s efforts to encourage and develop community participation in water resource management, especially in the sub-irrigation sector and clean water supply. Subsequently, in the 1990s, efforts to attract interest and cooperate with the private sector in the irrigation sector appeared to expand. The attempt to withdraw the private sector from managing water resources is inseparable from the policies of the new order government at that time were controlled by foreign capital forces.20\n\nThe intense privatization of water resources has become complicated, pitting the public sector against a group of capital owners. It is the same as confronting resources that control human life versus the subject of ownership for trading. It seems justified when Law (UU) number 7 of 2004 on water resources has been promulgated. The enactment of this law on February 19, 2004, was followed by the issuance of several regional regulations (Perda) related to water privatization. The privatization of water in Indonesia highly contributes to the clean water crisis because Law no. 7 of 2004 provides opportunities for the privatization of the drinking water sector and the control of water sources (groundwater, surface water, and some river bodies) business entities and individuals. As a result, the water rights of every individual are threatened with the agenda of privatization and commercialization of water in Indonesia.21\n\nThe cancellation of the Water Resources Law was carried out because it did not guarantee restrictions on water management by the private sector, hence, it violated Article 33 of the 1945 Constitution concerning Indonesian natural resources that must be controlled by the state for the benefit of Indonesian citizens. So that there is no legal vacuum, the Irrigation Law is enacted again. On September 17, 2019, the House of Representatives had ratified the Draft Water Resources Law (RUU SDA) into Law No. 17/2019 concerning Natural Resources.22\n\nIt is awaited that the making of the new Water Resources Law will not repeat the old law so it can create water management that can maximize the people’s right to water and protect the state against that right. Then in the new Water Resources Law, it can be seen how far it can eliminate the opportunity for clean water to be privatized. Therefore, the core issue is whether water should be treated as a public good, the fulfillment of which must be carried out by the state, or water as an economic good which is understood as a commodity whose fulfillment would be better if carried out by the private sector. In this case, the government’s role in supporting and implementing the new Water Resources Law is necessary, so the fulfillment of water needs during the dry season is met without incurring huge costs.23\n\nThe paradigm shift framework for water policy can be described in Figure 1 by referring to Thomas Kuhn’s Paradigm Theory.\n\nFollowing the lines of Thomas Kuhn’s Paradigm Shift Theory, first, water was initially positioned as a Res Commune as a shared property that was passed down from generation to generation. This phase can be categorized as the Pre-Paradigmatic Stage or Pre-Science, namely the phase where science does not yet have a foundation of methods, theories, and concepts; in other words, it does not yet have a paradigm. In this context, the juridical regulation of water has not yet emerged and still places water as common property. The state has no role and is still purely managed by the community simply or traditionally.\n\nThe second phase in Kuhn’s terminology is called Normal Science; the theory began to emerge, which then dominated other disciplines. This is the result of a paradigm that has been agreed upon by the scientific community. In the context of the Indonesian legal system, Legal Positivism is thought to place positive law as the main thing. Regarding water regulation in Indonesia, for the first time, regulations related to water management were the birth of the Algemeen Water Regulation (AWR) 1936. The origin of the AWR 1936 departed from the interests of the Dutch colonial government through the politics of cultuur stelsel (forced cultivation). The AWR 1936 does not provide a solid basis for efforts to develop the use/utilization of water and or water sources to improve people’s living standards. Still, in the normal phase of science after 29 years of independence, Law No. 11 of 1974 concerning Irrigation was born. The contents of the 1974 Irrigation Law, which amounts to 17 articles, are based on the principle and basis of regulations, namely viewing water as a social function aimed at the greatest prosperity of the people. The regulatory substances include, among others, state control over water, the concept of the right to water, and management of water resources.\n\nThe third phase is termed the critical phase, a contradictory situation. In terms of water resources management, there is a paradigm of water as an economic good which is contradictory to water as a social good. The enactment of Law No. 7 of 2004 concerning Water Resources was inseparable from the situation where global power through world financial institutions was very influential. The impact of the 1998 economic crisis increased Indonesia’s dependence on international financial institutions. The Dublin Principle emerged in 1992 when water emerged as an economic good. The World Bank has a huge role in the water resources sector. And not the only role, but also power. This role and power are obtained through the policies and conditions that accompany the loans, which unfortunately greatly encourages the privatization of water in developing countries such as Indonesia. Law No. 7 of 2004 concerning Water Resources has the character of water as an economic good was finally revoked by the Constitutional Court through Decision No. 85/PUU-XI/2013.\n\nThe last phase is the need for new ideas to emerge to get out of the crisis. The establishment of Law no. 17 of 2019 reaffirms the relationship between the state, the people, and water, emphasizing the necessity of state intervention in water regulation. Consideration of the state’s obligation to respect, protect and fulfill the human right of access to water. However, Law no. 17 of 2019 has a weak point epistemologically, which remains characterized by purely positivistic strengths. Water in its initial position has a religious value as something sacred for human life. For that, we need the concept of incorporating spiritual values into water resource management and safeguarding water resource management by local or traditional communities.\n\nIt is necessary to build a legal framework between God, the State, and Society. The relationship between God, the State, and Society was inspired by Werner Menski’s idea, known as the Triangular Concept of Legal Pluralism Theory.24 For Werner, the legal product is a triangular synergy between Religion, State, and Society. In terms of water resources management, see Figure 2.\n\nPlacing water as God’s property, the state and society only must manage it. It can be said that water is again placed as a sacred item for human life. The state should fulfill the right to water which places water as a social good and must also have religious values. Religious values provide awareness of the state’s responsibility in fulfilling the water rights.26\n\nThe role of the private sector in water resources management should be limited. This restriction avoids exploitation which threatens water resources. This restriction also provides opportunities for the private sector to participate without leaving religious values, the absence of greed, or in the other hand, water has economic and religious value, and the last is the protection of water resources in indigenous or local communities so that potential water sources are maintained and utilized for the sustainability of the ecosystem. Indigenous peoples have a long history of local wisdom, and the state must protect and maintain their existence from attacks by capital owners who are only profit oriented.\n\nThe state is an organization held by a nation and inhabits a particular area based on the law for the common good. A country certainly has the ideals of shared prosperity based on justice and humanity value. A democratic state fights for the realization of the public interest or res republica. Indonesia, as a country in the form of a republic, must establish itself to be oriented to the public interest.\n\nThe water right is a human right that does not come from the state; it is the specific ecological context of human existence that gives rise to the water rights. As a result, the inclusion of the state in water management as a form of control rights contained in Article 33 paragraph (3) of the Republic of Indonesia’s 1945 Constitution is a form of protection of these human rights, which cannot be eliminated by anyone, because the water right is a natural right. As a result, water as a human need is a right that the State must fulfill in order to recognize the right to life itself.27\n\nThe water right is a dimension of rights that stems from the “right to an adequate standard of living” and the “right to health.” In the Universal Declaration of Human Rights (DUHAM, this right is affirmed in Article 25 of the DUHAM as follows:\n\nEveryone is entitled to a decent standard of living for the health and well-being of himself and his family, including the right to food, clothing, boards, and health services, necessary social services, and the right to security when unemployed, sick, disabled, abandoned by his partner, old age, or other circumstances that result in a deterioration in the standard of living that occurs beyond his power.28\n\nFurthermore, the explanation of the water right contained in General Comment No. 15 on the Right to Water, the word “included” in Article 11 paragraph (1) of the Covenant on Economic, Social and Cultural Rights, the list of rights in an adequate standard of living is not a limited list.\n\nArticle 11 paragraph 1 of the EKOSOB Rights Covenant establishes several rights derived from, and are necessary for, the realization of the right to a decent standard of living “including food, clothing and decent housing.” The use of the word “included” indicates that the list of rights is not intended to be a limited list. The water right falls into the category of necessary guarantees to ensure a decent standard of living, especially since water is one of the necessary conditions for survival. Furthermore, the Committee has previously recognized that water is a human right included in Article 11 paragraph (1).29\n\nGeneral Comment No. 15 also explains that the water right is an inseparable right concerning the right to achieve the highest standards of health (and the right to adequate housing and food) and being a bridge for other rights.\n\nThe water right is inclusive. The inclusive nature makes this right to be owned individually and jointly by everyone. One person’s claim to this right does not make another person lose his claim to this right. The universality of this right is demonstrated by the recognition made by the world community. Many rights do not necessitate any special treatment to be held. Every human being can automatically have this right.30\n\nThe implication of acknowledging the water right is to give the State the task to organize such a mechanism so that public access to water can be made available. This mechanism must be regulated so that it does not provide an opportunity for the State to transfer its responsibilities to other parties. Consequently, it does not mean that everyone has to get water for free without any limits on the amount of use, which will create the possibility for those who are vital to get more water resources, but rather the recognition of the water right provides an opportunity for the State to make arrangements for restrictions on water resources. Certain restrictions on certain people or groups of people to ensure that the right to water for each person must be fulfilled.31\n\nNormatively, the formulation of the recognition of the water right is also contained in Law no. 17 of 2019 concerning Water Resources in Article 6, which means that in the regulation phase, the government has an instrument for recognizing water rights. Then what about the fulfillment aspect or implementation aspect?\n\nAt the implementation stage, it is still far from expectations, as can be seen from budget politics in the clean water sector. Adequate development budget to encourage access to safe drinking water in 2030, so far, the government’s allocations for the last five years for the clean water sector have been around Rp. 3.5-6.5 trillion with an average per year of Rp. 4.5 trillion. If this amount can be maintained every year until 2030 there will be around Rp. 45 trillion. It is still very far from the development needs until 2024, which is Rp. 147 trillion, even funding needs in 2030 amounting to Rp. 238 trillion.32\n\nUntil 2018, access to safe drinking water in Indonesia has reached 87.75%. However, only 6.8% of the population has enjoyed secure access. There is still a gap of 80.95% of the population in 2018 whose access still needs to be improved from decent access to safe access. Overall, as many as 93.2% of the population have not had secured access. If this figure translated into funding needs, the estimated amount up to 2030 could be Rp. 238 trillion if the inflation rate is considered. It is expected that 30% of funding needed can be allocated by the regional government.33\n\nThe challenge of meeting the amount of funding is not easy. If the APBN is still unable to provide the necessary funds for the sector development budget, it is almost impossible for local governments to allocate APBD (as expected by the central government). Field data collected by the Water and Environmental Health Working Group indicate that the average APBD size for the water and sanitation sector is only around 2% per year. Fulfilling the need for the development budget for the drinking water sector is still an extensive homework for sector managers.34\n\nHomework for the government to fulfill the right to water for its citizens does not mean leaving it to the private sector or shifting responsibility by doing privatization. In principle, the state assigns the task to the state to implement such a mechanism so that public access to water can be provided.\n\nThere is a tug of war between the strengthening of the state’s role in the management of water resources on the one hand and the other hand the power of capital owners who place water as an economic good, so an alternative needed to emphasize community participation in the management of water resources {XE “Sumber Daya Air”}.\n\nIn the context of justice, strengthening the aspect of justice that places cultural factors as an important factor is also formulated in this goal, namely the protection and empowerment of communities, including indigenous people to conserve water and water resources. Indigenous peoples are people with knowledge, habits, and culture passed down from generation to generation by utilizing water sources in their territory to meet their daily needs. They manage these resources together and live in harmony with the nature around them.\n\nIn conditions of limited water, arrangements are needed so that all community members have access to water in a mutually agreed manner and place. In Law Number 17 of 2019 concerning Water Resources, Article 9 paragraph (2) provides space for local wisdom in the management of Water Resources, which states that Control of Water Resources is carried out by the Central Government and, or Regional Governments while still recognizing the Ulayat Rights of local Indigenous Peoples and rights similar to that, as long as they do not conflict with national interests and the provisions of laws and regulations. The need for community participation in maintaining water resources is also emphasized in Law Number 37 of 2014 concerning Soil and Water Conservation. In Article 46 paragraph (1), it is stated that the Community has the same opportunity to participate in the implementation of Soil and Water Conservation carried out by the Government and, or Regional Government per their authority, and paragraph (2) states that the implementation of community participation is carried out with due observance of the local wisdom.\n\nLocal wisdom can be used as social capital that deserves attention in managing water resources in an area. Local wisdom has two critical roles: fulfilling the right to water for life and maintaining the relationship between humans and water resources and the surrounding environment.\n\n\nConclusion\n\nWater has experienced a paradigm shift from social goods to economic goods, starting with the global situation regarding water scarcity. The pattern of economic approach through privatization in the management of water resources has resulted in the co-optation of water as a source of collective life. Water is imposed in a commercially motivated rather than as a constitutive element that regulates water for “sustainability of collective life” and “to the greatest extent possible for people’s prosperity.” The water right is a human right for citizens. The existence of the state’s role aims for the prosperity of the people themselves. The fulfillment of the right to water by the state certainly cannot be directly fulfilled, what is important is that there is a progressive effort to fulfill it. The state’s limited role necessitates community involvement in water resource management, particularly the local communities with a long history of environmental-based water management. The role of the State should respect, protect, and recognize the human rights of others to a good and healthy environment.\n\n\nData availability\n\nThe legal rules and regulations data are publicly available and accessible at:\n\n1. https://peraturan.go.id/peraturan/view.html?id=ae26fe65e5dc3f2acfb42714eaf4272a\n\n2. https://www.komnasham.go.id/files/1475231474-uu-nomor-39-tahun-1999-tentang-$H9FVDS.pdf, https://www.mkri.id/index.php?page=download.Putusan&id=2131\n\n3. https://www.ohchr.org/en/instruments-mechanisms/instruments/international-covenant-economic-social-and-cultural-rights, https://tbinternet.ohchr.org/_layouts/15/treatybodyexternal/Download.aspx?symbolno=E%2fC.12%2f2002%2f11&Lang=en.", "appendix": "References\n\nShiva V: Water Wars: Privatisasi, Profit, dan Polusi. Uzair A, trans. Jogjakarta: INSIST Press; 2002; p. 41.\n\nSanim B: Sumber Daya Air dan Kesejahteraan Publik: Suatu Tinjauan Teoritis dan Kajian Praktis. Bogor: PT Penerbit IPB Press; 2011; 4.\n\nArizona Y: Perkembangan Konstitusionalitas Penguasaan Negara atas Sumber Daya Alam dalam Putusan Mahkamah Konstitusi. Jurnal Konstitusi. Juni 2011; Volume 8(nomor 3): p.260.\n\nHatta M: Penjabaran Pasal 33 Undang-Undang Dasar 1945. Mutiara Jakarta; 1977; 28.\n\nKasim H, Anindyajati T: Perspektif Konstitusional Kedudukan Negara dan Swasta dalam Pengelolaan Sumber Daya Air Menurut UUD 1945. Jurnal Konstitusi. June 2016; 13(2): 455. Publisher Full Text\n\nIbid. p. 458.\n\nKasim H, Anindyajati T: Perspektif Konstitusional, Op. Cit. p. 458.\n\nUU 17 tahun 2019 tentang Sumber Daya Air. accessed January 2021. Reference Source\n\nWorster D: Rivers of Empire. New York: Oxford University Press;\n\nToly NJ: Globalization and the Capitalization of Nature: A Political Ecology of Bodiversity in Mesoamerica. Bulletin of Science. 24(1 February): 47–54. Publisher Full Text\n\nInternational Conference on Water and the Environment (ICWE): The Dublin Principles. 1992. accessed January 25, 2016. Reference Source\n\nSanim B: Pengelolaan Sumberdaya Air Dalam Menopang Negara Mandiri Dan Berdaulat, Makalah Pembicara Pada KIPNAS X Di Jakarta Atas Kerjasama Lembaga Ilmu Pengetahuan Indonesia (LIPI) Dan Kementerian Pendidikan Dan Kebudayaan Nasional Pada Tanggal 8-10 November 2011.\n\nRusmadi TI: Menyisir Keadilan Air Di Tengah Liberalisasi Alam. AICIS 12 Tahun 2012, 5-8 Nopember 2012, Surabaya. accessed June 1, 2015. Reference Source\n\nGleick PH, Wolff G, Chalecki EL, et al.: The New Economy Of Water: The Risks And Benefits Of Globalization And Privatization Of Fresh Water. California: Pacific Institute For Studies In Development, Environment, And Security; 2002; 5.\n\nSantika R: Air Sebagai Barang Publik: Studi Empiris Pengaruh Faktor Sosial Ekonomi Terhadap Pemekaian Air Bersih di Kota Bandung, Majalah Ekonomi, Tahun XIX, No. 2.Agustus 2009; p. 140.\n\nThe Dublin Statement On Water And Sustainable Development. Accessed May 15, 2020. Reference Source\n\nBudds J, Mcgranahan G: are the Debates On Water Privatization Missing The Point? Experiences From Africa, Asia and Latin America. Environment Urbanization. October 2003; 15(2): 87–113. Publisher Full Text\n\nHadad N, Water I’s: Op. Cit. p. 60.\n\nKelompok Kerja Air Minum Dan Penyehatan Lingkungan (AMPL): Kerjasama Pemerintah - Swasta Dalam Pelayanan Air Minum Di Indonesia: Buku Putih, Jakarta, The Water Dialogues Indonesia.2009.\n\nIbid.\n\nBatubara M: “Menggugat Penjajahan Sumber Daya Air dengan Modus Privatisasi,” dalam. accessed January 18, 2020. Reference Source\n\nPrivatisasi dan Hak Atas Air. accessed January 18, 2020. Reference Source\n\nIbid.\n\nMenski W: Fuzzy Law And The Boundaries Of Secularism. Potchefstroom Electronic Law Journal. Jan. 2010. Faculty of Law, Private Bag X6001, North-West University, PER vol.13 n.3 Potchefstroom.\n\nThe author’s illustration refers to Werner Menski’s Theory, known as the yesTriangular Concept of Legal Pluralism Theory.\n\nThis statement comes from the development process of Menski’s theory which is used to relate to water shown in figure no. 1.\n\nHarjanti W: Hak Atas Air Dalam Konstitusi Negara dan Pengelolaannya di Indonesia (Right of Water in Indonesian Constitution and its Managements), Risalah Hukum: Jurnal Hukum UP. Fakultas Hukum Universitas Mulawarman. 2009; 5(2).\n\nUniversal Declaration of Human Rights, Article 25.\n\nGeneral Comment No 15. (3).\n\nSetianto BD: Sesat Pikir Hak Atas Air. accessed August 20, 2020. Reference Source\n\nSetianto BD: Ketika GTZ Melakukan Penyesatan Terhadap Hak Atas Air; Menyesatkan -Penyesat- Hak Atas Air. accessed 29 November 29, 2020. Reference Source\n\nBappenas P: Baseline dan Target RPJMN 2020-2024 Bidang Air Minum, Bahan Paparan Direktorat Perkotaan, Perumahan, dan Permukiman, 17 Februari 2020. dikutip dari Eko Wiji Purwanto, Pembangunan Akses Air Bersih Pasca Krisis Covid-19. The Indonesian Journal of Development Planning. Juni 2020; IV(2).\n\nPurwanto EW: Pembangunan Akses Air Bersih Pasca Krisis Covid-19. The Indonesian Journal of Development Planning. Juni 2020; IV(2).\n\nIbid." }	[ { "id": "139959", "date": "27 Jun 2022", "name": "Moch Faisal Karim", "expertise": [ "Reviewer Expertise Political science", "social science", "political economy" ], "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 is interesting however, at this moment, it is rather descriptive and does not provide any meaningful insights into the role of the state in the fulfilment of water rights.\nI suggest the authors to provide questions and arguments as well as what debate the authors want to engage in.\n\nI would also suggest the authors to provide more literature review in order to situate the research within broader debate regarding water and human rights. Doing this will allow readers to understand the novelty of the research.\nThe Analysis can also be enhanced to provide more interesting insights. The discussion of the shifting of the water paradigm indeed provides us with the context of the commercialisation of water. However, the discussion of how such shifting happens in the context of Indonesia should be discussed more. For instance, how the law no 7 of 2004 can be interpreted within this shift?\n\nThe analysis can also be enhanced by providing a deeper analysis of the dynamic roles of the state in fulfilling water rights. At the moment, it is primarily stating prepositions without providing any meaningful evidence or analysis. For instance, the manuscript stated that \"There is a tug of war between the strengthening of the state’s role in the management of water resources on the one hand and the other hand power of capital owners.\" But there is no discussion about it further.\nConclusion should also be enhanced by providing what the implications of the study to broader debate in the literature.\nThere are many abbreviations that are not fully explained (APBB, APBD).\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "145690", "date": "30 Aug 2022", "name": "Priscila Neves-Silva", "expertise": [ "Reviewer Expertise Water", "sanitation", "Hygiene and public health", "WASH Public Polices", "Human Rights to water and sanitation", "Evaluation of Public Polices" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Shifting the water paradigm from social good to economic good and the state’s role in fulfilling the right to water \" aimed to analyze how the shift in water paradigm, from water as social good to economic good, can influence state's role in what concerns the right to water.\n\nThe author present the question and the problem in the introduction but there is many references missing, such as the Report about water privatization made by the former UN Special Rapporteur at the Human Rights to Water and Sanitation published in 2020.\nAlso, the authors did not present the method of analyses they used to analyze the data they have collected. Which means that the way it is presented it does not allow replication.\nThe authors also need to explain better the elements in normative content of the Human Rights to Water and Sanitation (HRWS). The implication of having water as a human rights is not only relayed to the availability but also to other important elements as physical accessibility, affordability, quality and acceptability. All of them is important.\nAlso the HRWS do not oblige the State to be the only service provider. However, the State need to guarantee that all aspects of the HRWS will be considered. Therefore, the author need to rewrite this sentence and organize better the information. Also, The State not only need to fulfill, but also to protect and respect the HRWS.\nConsidering all that, I recommend that the authors implement the article with more references and also modify some of the information presented in the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-490
https://f1000research.com/articles/9-1029/v1	24 Aug 20	{ "type": "Research Article", "title": "Translation, validity and reliability of the Danish version of the Adolescent Insomnia Questionnaire", "authors": [ "Alessandro Andreucci", "Christian Lund Straszek", "Michael Skovdal Rathleff", "Clara Guldhammer", "Rocio de la Vega", "Tonya M. Palermo", "Christian Lund Straszek", "Michael Skovdal Rathleff", "Clara Guldhammer", "Rocio de la Vega", "Tonya M. Palermo" ], "abstract": "Background: The Adolescent Insomnia Questionnaire (AIQ), English version, is the only validated screening measure developed specifically to identify insomnia symptoms in adolescents. To date, no specific screening tool for adolescent insomnia is present in Danish language. The aim of this study was to translate and validate the AIQ in a sample of Danish adolescents. Methods: The AIQ underwent a process of forward-backward translation and pilot testing. Subsequently, data were collected at baseline and two-week follow-up from adolescents aged 11-19, who completed both the AIQ and an available adult measure of insomnia (the Athens Insomnia Scale, AIS). The internal consistency, test-retest reliability and convergent validity were assessed. Exploratory factor analysis was conducted to identify the latent factors underlying the questionnaire. Results: At baseline 185 adolescents (18% males and 82% females, mean age 16.0 years) and 102 (55.1%) at two-week follow-up completed the questionnaires. The AIQ showed excellent internal consistency for the total score (Cronbach's a: 0.88) and good convergent validity with the AIS total score (Pearson’s correlation value= 0.86, P<0.001). The test-retest reliability at two weeks was very satisfactory (ICC coefficient = 0.89; 95% CI 0.84, 0.92). Results from the exploratory factor analysis identified a three-model solution corresponding to the same three-model solution identified within the original development sample. Conclusions: The Danish version of the AIQ demonstrated satisfactory psychometric properties in terms of internal consistency, test-retest reliability and validity, which supports its use as a screening tool for the identification of insomnia symptoms in adolescents, including Danish-speaking adolescents.", "keywords": [ "Insomnia", "Adolescent", "Screening", "Sleep", "Translation", "Validation" ], "content": "Abbreviations\n\nAIQ, Adolescent Insomnia Questionnaire; AIS, Athens Insomnia Scale.\n\n\nIntroduction\n\nInsomnia is a common sleep disorder during adolescence that affects between 10 and 40% of adolescents, depending on the diagnostic criteria used1–3. Insomnia is associated with broad negative impact on emotional, social, cognitive and academic functioning1,3–5, and poor quality of life. There are also potential health problems associated with insomnia, such as anxiety, depression, obesity, substance abuse and chronic musculoskeletal pain3,6,7. In addition, insomnia symptoms in adolescents may lead to use of sleep medications and other substances (e.g. alcohol, illicit drugs), which could result in additional risk for other disorders3. Insomnia can persist into adulthood1 with increased potential burden over the long-term8. The estimated average six-month cost (including direct and indirect costs) of insomnia in adults in the U.S.A. (2003 figures) was about $1,253 greater compared to individuals without insomnia9. Insomnia is, therefore, an important public health issue3.\n\nThe prevalence of insomnia during adolescence has increased during the last several decades7,10. It is hypothesized that this is due to increased availability of electronic devices in the bedroom and consumption of caffeinated beverages3,7,11,12. These changes in lifestyle affect the biological homeostasis, together with the hormonal changes occurring in a crucial developmental period such as puberty, impacting sleep patterns and resulting in changes in the sleep architecture13. Additional changes related to the social domain, such as academic pressure and early start time for school, can contribute to insomnia symptoms3,11,13. Altogether, these changes result in adolescents getting less sleep than needed. The identification of insomnia symptoms early on in life is extremely important7. This would allow for early referral for intervention to address the symptoms and reduce potential health problems associated with insomnia. However, sleep disorders such as insomnia often go unrecognized in pediatric primary care3,14.\n\nPediatricians need a developmentally appropriate measure for the assessment of insomnia in adolescents that has adequate psychometric properties and high clinical utility (i.e., is brief, simple to use). In order to fill this gap, the Adolescent Insomnia Questionnaire (AIQ) has been recently developed to identify insomnia symptoms in adolescents and guide treatment decisions. This questionnaire was tested in a case-mix of 314 English-speaking American adolescents (11–18 years old) recruited from a sleep clinic, pain clinic, headache clinic and the community. In this heterogeneous sample of adolescents, the questionnaire showed good internal reliability, convergent and discriminant validity, and high criterion validity. However, it is not known if such a questionnaire will prove to be effective in other languages and cultural contexts such as Denmark.\n\nTo date, there is no valid insomnia questionnaire for adolescents available in Danish language. Due to the lack of validated tools for the assessment of adolescent insomnia, other measures originally developed for the assessment of insomnia in adult populations are used in Denmark, such as the Athens Insomnia Scale (AIS). However, the AIS has not undergone proper psychometric testing in a Danish adolescent population, and includes fewer items that do not capture the whole range of problems with sleep maintenance and sleep onset and also the specific impairments in the adolescents’ life (e.g. problems at school or with friends), which can be assessed with the AIQ. Testing the properties of the AIQ in an additional sample of adolescents in a different country, language and culture would provide additional support for the AIQ as an appropriate screening measure to assess insomnia in adolescents. Therefore, the aim of this study was to translate the Adolescent Insomnia Questionnaire into Danish and validate it in a sample of Danish adolescents attending a primary care clinic and from the community.\n\n\nMethods\n\nThis is a prospective study with data collected at two time-points: baseline and two-week follow-up. Due to the non-interventional nature of the study and the regulation of the Scientific Ethics Committee for Region North Jutland, the study was exempt from ethical approval. Written informed consent was obtained at baseline and self-completed if participants were 15 years or older, otherwise it was completed by one parent/guardian if participants were younger than 15, and the minors provided their assent.\n\nData were collected from adolescents aged 11–19 years old recruited from one general practice clinic in the city of Aalborg (n = 10, recruited in February 2020), Denmark and through social media advertisement (i.e. Facebook, n = 175, recruited in March/April 2020). Adolescents who attended primary care for any type of symptom/condition were provided with the questionnaire (together with the informed consent) to be completed in the waiting room of the general practice. Likewise, adolescents who responded to the Facebook advertisement were provided with the questionnaire (together with the informed consent) to be completed through a web application (RedCap)15 accessible by a link in the advertisement. The Facebook post was tailored in order to be advertised to the population of interest (adolescents aged 11–19 and parents of adolescents) in the country of Denmark. After two weeks, an e-mail was sent to all the adolescents containing a link to a re-administration of the questionnaire to be completed at home in order to assess test-retest reliability. In both the baseline and follow-up questionnaire it was clearly stated that the questionnaire was intended to be completed by the adolescents. If participants did not complete the follow-up questionnaire, they were contacted by phone, SMS, or e-mail reminders. If four unsuccessful contact attempts were made or 10 days had passed without obtaining a response, no further contact was made.\n\nDemographic information. The participants’ age and sex were collected.\n\nAdolescent Insomnia Questionnaire (AIQ). The AIQ (Table 1) is a 13-item self-report screening measure of insomnia symptoms developed specifically for adolescents; it contains three subscales (sleep onset, sleep dissatisfaction and impairments, sleep maintenance)1. The AIQ was validated in a sample of adolescents aged 11–18 years old with and without chronic pain conditions who were recruited from the community and clinical settings. The AIQ showed acceptable convergent (range .47–.88, p <.01) and discriminant (r = .06, p = .334) validity and strong reliability for both the total score (α = .91) and the subscale scores (α = .79 - .89)1. A confirmatory factor analysis (CFA) revealed three factors consistent with the three subscales. Total scores range from 0 to 52, with higher scores indicating more severe insomnia symptoms. A provisional cut-off score of 15 for identifying insomnia was suggested following receiver-operator curve analysis of the development sample1. The AIQ can be completed in approximately five minutes and it is relatively easy to score (there are only four reversed items)1. The original English version of the AIQ was obtained through contact with the authors who developed the tool at the Seattle Children's Research Institute.\n\nAthens Insomnia Scale (AIS). The Athens Insomnia Scale (AIS) is an eight-item self-administered tool developed to assess insomnia severity in adults16, which has also been used in adolescent populations in a few limited validation studies17,18. The AIS includes a variety of insomnia symptoms (difficulty with sleep initiation, awakenings during the night, early morning awakening, total sleep time, overall quality of sleep, sense of well-being during the day, physical and mental functioning during the day and sleepiness during the day) occurring on a frequency of at least three times per week during the last month16. Each item can be rated with a number from 0 (no problem at all) to 3 (very serious problem). The resulting total score ranges from 0 (no sleep-related problems) to 24 (most severe degree of insomnia). The AIS showed good internal consistency (α = .89) and good test-retest reliability for both the total score (Pearson's correlation coefficient = .89) and for each item (Pearson's correlation coefficients = .70 - .86). The AIS also showed good external validity when compared to the Sleep Problems Scale (Pearson's correlation coefficient = .90)16. The cut-off score for defining insomnia based on the AIS is 619. A Danish translated version of the AIS has been previously used in a study conducted in a Danish general practice where participants >12 years old were assessed for insomnia symptoms20.\n\nThe Adolescent Insomnia Questionnaire was translated to Danish by a panel composed by A.A., C.L.S., M.S.R., and M.A. Following translation, a preliminary pilot test was conducted and then the measure was validated in a larger sample of adolescents. Guidelines for the translation and validation of a questionnaire were followed21.\n\nTranslation of the questionnaire to Danish. A process of forward-backward translation of each item included in the AIQ from English to Danish was applied. This was done to ensure that the wording of the items in Danish was conceptually equivalent to the wording of the items in English. Two forward translations were initially produced by two native Danish researchers (C.L.S. and M.S.R.). The two translations were then discussed within the research team until a final decision on the translation of each item was reached. This version was then translated back to English (backward translation) and compared to the original version in English to assess potential differences between the two versions. The backward translation process was carried out by a bilingual researcher native in English. During the process of translation, items of the AIQ were properly worded for the age of adolescents in order to ensure comprehensibility and face validity.\n\nPreliminary pilot testing of the questionnaire. After the initial translation stage described above, the AIQ was tested with volunteer participants (n = 11, age range 10 - 19) recruited through advertisement of the study on Facebook. Children and adolescents who were interested in the study (or their parents) contacted the primary investigator of this study (A.A.), and a date was arranged for testing the tool and carrying out cognitive interviews with a research assistant (C.G). Study procedures were carried out at the Center for General Practice at Aalborg University, and participants were given a cinema ticket as a reward for participating in the cognitive interviews. The initial translated version of the AIQ was delivered to participants who were instructed to complete it by themselves. After completion of the questionnaire, cognitive interviews were carried out to assess the comprehensibility of the items. The aim was to improve the face and content validity of the tool at this stage. The feedback received through cognitive interviews indicated that no change to the Danish translated version of the AIQ was needed (Table 2). The translated version was subsequently validated through the process described below.\n\nValidation of the questionnaire. Several parameters were assessed during the validation of the questionnaire. Cronbach's α coefficient was calculated to assess the internal consistency of the AIQ total score and of the AIQ subscales. Test-retest reliability was assessed by comparing the responses to each item of the AIQ between baseline and two-week follow-up in order to evaluate the short-term stability of the questionnaire in adolescents22,23. Convergent validity was assessed by comparing the scores obtained with the AIQ with those obtained with the AIS. Exploratory factor analysis was conducted in order to identify the latent factors underlying the questionnaire, and compare them to the original English version of the measure24.\n\nDescriptive analysis of the study sample was performed. Results are shown as means and standard deviations (SD) or as counts (%) depending on the type of variable (continuous or categorical). T-tests or Pearson χ2-tests were used to compare groups on continuous and categorical variables, respectively. Cronbach's α for the AIQ total score and for the AIQ subscales (sleep onset subscale, sleep maintenance subscale, sleep dissatisfaction and impairments subscale) was calculated in order to assess internal consistency reliability. Test-retest stability was evaluated by assessing the relationship between the AIQ total score and subscale scores between baseline and follow-up by means of the intraclass correlation (ICC) coefficient, using a two-way mixed-effects model. If the ICC values are <0.5, this is indicative of poor reliability, while values between 0.5 and 0.75 show moderate reliability. ICC values between 0.75 and 0.9 show good reliability, and values > 0.90 excellent reliability22. Limits of agreements were also used to express the agreement between the two measurements (i.e. baseline vs. follow-up). The limits of agreement represent the mean difference between the two measurements ±1.96 times the standard deviation of the differences25. Convergent validity was assessed by calculating Pearson correlations between the AIS total score and the AIQ total score (and scores for subscales). Exploratory factor analysis (EFA) using principal factor extraction and oblique rotation was conducted to identify the factor loadings. Results of EFA were compared to those from the original development sample. Analyses were conducted with STATA version 15.0.\n\n\nResults\n\nParticipant characteristics are shown in Table 3. The sample included 185 participants at baseline, 33 (18%) males and 151 (82%) females26. One participant did not report their sex. The mean age of participants was 16.0 years (± 1.4, range 11–19 years). One-hundred and two participants (55.1%) completed the questionnaires at two-week follow-up. The average AIQ score at baseline was 30.14 (± 8.90). Females had a statistically significant (31.1 ± 8.1, p = 0.003) higher mean score than males (26.0 ± 10.8). Higher AIQ scores were significantly associated with older age (linear regression coefficient = 1.48; 95% CI 0.58, 2.39; P = 0.001). Ninety-two percent of the sample (N = 169) had AIQ values above the suggested cut-off of 15 for defining insomnia symptoms. The average AIS total score was 11.4 (± 4.8), and 83% of the sample (N = 169) had AIS values higher than the cutoff of 6 for defining insomnia symptoms. Thus, this sample represented those adolescents with high levels of insomnia symptoms.\n\nSD, standard deviation; AIQ, Adolescent Insomnia Questionnaire; AIS, Athens Insomnia Scale.\n\nThe internal consistency of the AIQ was calculated for both the AIQ total score and the AIQ subscales (Table 4). The internal consistency for the AIQ total score was excellent (Cronbach's α: 0.88). The internal consistency for both the AIQ sleep onset subscale (items 1, 4, 7, 9) and AIQ sleep dissatisfaction and impairments subscale (items 3, 5, 10, 11, 12, 13) was excellent as well (Cronbach's α: 0.84 and 0.87, respectively). The internal consistency for the AIQ sleep maintenance subscale (items 2, 6, 8) was slightly lower (Cronbach's α: 0.73).\n\nAIQ, Adolescent Insomnia Questionnaire; AIS, Athens Insomnia Scale.\n\n* = statistically significant.\n\nThe convergent validity of the AIQ was evaluated using correlations with the total score of the AIS (Table 4). A large, positive significant correlation between the AIQ total score and the AIS total score was found (Pearson’s correlation value= 0.86, P<0.001). The positive correlation between the AIQ total score and the AIS is also illustrated in Supplementary Figure 1 (see Extended data26). Subscale scores of the AIS were also significantly related to the AIS with the largest correlation between the sleep dissatisfaction and impairment subscale (Pearson’s correlation value = 0.83, P<0.001) and the AIS and smaller correlations for the sleep onset subscale (Pearson’s correlation value = 0.59, P<0.001) and the sleep maintenance subscale (Pearson’s correlation value = 0.58, P<0.001).\n\nResults of the test-retest reliability analysis at two-weeks are shown in Table 5. The ICC coefficient for the AIQ total score was 0.89 (95% CI 0.84, 0.92), while it was 0.86 (95% CI 0.79, 0.90) for the sleep onset subscale, 0.86 (95% CI 0.80, 0.90) for the sleep impairment subscale and 0.80 (95% CI 0.71, 0.86) for the sleep maintenance subscale. These values demonstrate strong test-retest reliability.\n\nICC, intraclass correlation coefficient; 95% CI, 95% confidence intervals.\n\nThe limits of agreement calculation showed limits of agreement from – 7.371 to 9.553 for the AIQ total score, and a mean difference of 1.091 (95% CI: 0.247, 1.935). This shows a good agreement between the two measurements (i.e. baseline vs. follow-up), as there was a mean difference of 1 point in the AIQ score, which can be considered small on a scale ranging from 0 to 52.\n\nResults from EFA showed a three-factor solution that supported the model identified in the original development sample and accounted for 65.45% of the variance among items. Values of the rotated individual items ranged from 0.60 to 0.84 (Table 6) and were similar to those identified within the original development sample. Factor cross-loadings of the AIQ items are provided in Supplementary Table 1 (see Extended data26).\n\n* = Item is reverse scored.\n\n\nDiscussion\n\nThe objective of this study was to translate and validate a self-report screening measure of insomnia symptoms for adolescents, the AIQ, to Danish, providing additional support for the AIQ and allowing its use in a broader population. The translated AIQ showed good content validity, good to excellent internal consistency, and very satisfactory test-retest reliability. The translated questionnaire demonstrated good convergent validity as evidenced by positive strong correlations between the AIS total score and the AIQ total score. Exploratory factor analysis resulted in a three-model solution corresponding to the same three-model solution identified within the original development sample1, providing additional support for construct validity. Altogether the findings demonstrated that the AIQ has excellent reliability and validity and high clinical utility in a Danish translation of the measure.\n\nAccording to the Cohen criteria for establishing the quality of pediatric questionnaires27 the AIQ would be considered a “well-established” measure; this means it has been assessed by at least two studies by two different research teams, which have both evaluated the psychometric properties with information on validity and reliability and have provided details that allow critical evaluation and replication. Therefore, the AIQ has the potential to be used for the assessment of insomnia symptoms in both research and clinical settings. This might be relevant especially for the detection of adolescents’ insomnia symptoms in the primary care setting, where they are usually under-diagnosed2,14. However, further studies of the AIQ may help to provide evidence of validity in other settings with different populations. The original version of the AIQ (in English) was tested in a mixed population of adolescents from a pediatric sleep clinic, a pediatric pain clinic, a pediatric headache clinic and healthy adolescents recruited from the community. Further data across the full range of demographics (e.g. a sample with equal proportions of males and females, younger adolescents) and patients with conditions that are associated with insomnia (e.g. chronic pain, depression)28 are needed before a cut-off for the Danish version of the AIQ can be established. In addition, other studies where the score obtained with the AIQ is compared to other sleep tools such as sleep diaries, which might also be filled electronically29, might provide more insights on the stability of the AIQ on a longer time-period.\n\nThe results of the study should be interpreted in light of several limitations. First, there are limitations in terms of external validity30. The majority of participants in this sample were female (82%), most of them (N = 175) were recruited through a Facebook advertisement, and the sample had severe insomnia symptoms, as shown by the high AIQ and AIS total score values, which were both well above the respective proposed cut-offs for clinically significant insomnia symptoms. This might be due to a self-selection bias of participants responding to an ad related to sleep research in social media. Indeed, in our sample most adolescents were recruited through a Facebook post, which might have attracted adolescents with severe symptoms of insomnia. While this highlights the relevance and need for further identifying and treating insomnia in adolescents, it does limit the psychometric evaluation. Therefore, further research with other pediatric samples with a broader range of insomnia symptoms (e.g. general population, medical or psychiatric samples) is needed to assess the stability of the findings. Second, in order to reduce participant burden very limited demographic information were collected. Because race and ethnicity were not collected it was not possible to assess whether the Danish translation of the AIQ performs well in ethnic minority populations. We also do not have information on adolescent’s concurrent health or mental health conditions and therefore are limited in understanding whether responses differ based on underlying health conditions. Future studies should include populations with specific conditions (e.g. chronic pain, depression) which might be at higher risk of concurrent severe insomnia symptoms6.\n\nIn summary, the AIQ is a tool that can potentially fill the need for a validated, brief, screening measure for insomnia symptoms in adolescents29,31. It is available now translated in Danish and has shown good psychometric properties that allow for the use in Danish research and clinical settings. The AIQ can be used for the identification of adolescents with insomnia symptoms who can subsequently be referred for sleep intervention such as cognitive-behavioural therapy for insomnia for improvement of the condition early on6,32. In addition, the findings confirm the three‐factor structure identified in the development sample.\n\n\nConclusions\n\nThe AIQ was translated to Danish and demonstrated satisfactory psychometric properties in terms of internal consistency, test-retest reliability and validity, which supports its use as a screening tool for the identification of insomnia symptoms in adolescents. Future studies for the exploration of its validity in populations with specific conditions and potentially different levels of insomnia symptoms are needed.\n\n\nData availability\n\nHarvard Dataverse: Danish Translation of the Adolescent Insomnia. https://doi.org/10.7910/DVN/VQFT9W26.\n\nThis project contains the following underlying data:\n\n- Final dataset.tab\n\nHarvard Dataverse: Danish Translation of the Adolescent Insomnia. https://doi.org/10.7910/DVN/VQFT9W26.\n\nThis project contains the following extended data:\n\n- Supplementary Figure 1.docx (Correlation between the AIQ total score and the AIS total score)\n\n- Supplementary Table 1.docx (Factor cross-loadings of the AIQ items)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors would like to thank Mrs. Mai Andreasen for her aid during the process of forward-backward translation of the questionnaire. Mrs. Mai Andreasen has given permission for her name to be included in this publication.\n\n\nReferences\n\nBromberg MH, de la Vega R, Law EF, et al.: Development and Validation of the Adolescent Insomnia Questionnaire. J Pediatr Psychol. 2019; 45(1): 61–71. PubMed Abstract \| Publisher Full Text\n\nHonaker SM, Meltzer LJ: Sleep in pediatric primary care: A review of the literature. Sleep Med Rev. 2016; 25: 31–39. PubMed Abstract \| Publisher Full Text\n\nde Zambotti M, Goldstone A, Colrain IM, et al.: Insomnia disorder in adolescence: Diagnosis, impact, and treatment. Sleep Med Rev. 2018; 39: 12–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Dyk TR, Becker SP, Byars KC: Rates of mental health symptoms and associations with self-reported sleep quality and sleep hygiene in adolescents presenting for insomnia treatment. J Clin Sleep Med. 2019; 15(10): 1433–1442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOwens J: Insomnia in Children and Adolescents. J Clin Sleep Med. 2005; 01(4): e454–e458. Publisher Full Text\n\nBadawy SM, Law EF, Palermo TM: The interrelationship between sleep and chronic pain in adolescents. Curr Opin Physiol. 2019; 11: 25–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOwens J, Au R, Carskadon M, et al.: Insufficient sleep in adolescents and young adults: An update on causes and consequences. Pediatrics. 2014; 134(3): e921–e932. PubMed Abstract \| Publisher Full Text\n\nMorin CM, Bélanger L, LeBlanc M, et al.: The natural history of insomnia: a population-based 3-year longitudinal study. Arch Intern Med. 2009; 169(5): 447–53. PubMed Abstract \| Publisher Full Text\n\nOzminkowski RJ, Wang S, Walsh JK: The direct and indirect costs of untreated insomnia in adults in the United States. Sleep. 2007; 30(3): 263–273. PubMed Abstract \| Publisher Full Text\n\nMatricciani L, Olds T, Petkov J: In search of lost sleep: Secular trends in the sleep time of school-aged children and adolescents. Sleep Med Rev. 2012; 16(3): 203–211. PubMed Abstract \| Publisher Full Text\n\nCarskadon MA: Sleep in Adolescents: The Perfect Storm. Pediatr Clin North Am. 2011; 58(3): 637–647. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHale L, Guan S: Screen time and sleep among school-aged children and adolescents: a systematic literature review. Sleep Med Rev. 2015; 21: 50–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarskadon MA, Tarokh L: Developmental changes in sleep biology and potential effects on adolescent behavior and caffeine use. Nutr Rev. 2014; 72 Suppl 1(Suppl 1): 60–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeltzer LJ, Johnson C, Crosette J, et al.: Prevalence of Diagnosed Sleep Disorders in Pediatric Primary Care Practices. Pediatrics. 2010; 125(6): e1410–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\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 Inf. 2009; 42(2): 377–381. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoldatos CR, Dikeos DG, Paparrigopoulos TJ: Athens Insomnia Scale: validation of an instrument based on ICD-10 criteria. J Psychosom Res. 2000; 48(6): 555–60. PubMed Abstract \| Publisher Full Text\n\nSiomos KE, Avagianou PA, Floros GD, et al.: Psychosocial correlates of insomnia in an adolescent population. Child Psychiatry Hum Dev. 2010; 41(3): 262–273. PubMed Abstract \| Publisher Full Text\n\nYen CF, King BH, Chang YP: Factor structure of the Athens Insomnia Scale and its associations with demographic characteristics and depression in adolescents. J Sleep Res. 2010; 19(1 Pt 1): 12–18. PubMed Abstract \| Publisher Full Text\n\nSoldatos CR, Dikeos DG, Paparrigopoulos TJ: The diagnostic validity of the Athens Insomnia Scale. J Psychosom Res. 2003; 55(3): 263–267. PubMed Abstract \| Publisher Full Text\n\nSørensen L, Jensen MSA, Rathleff MS, et al.: Comorbid insomnia, psychological symptoms and widespread pain among patients suffering from musculoskeletal pain in general practice: A cross-sectional study. BMJ Open. 2019; 9(6): e031971. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsang S, Royse CF, Terkawi AS: Guidelines for developing, translating, and validating a questionnaire in perioperative and pain medicine. Saudi J Anaesth. 2017; 11(Suppl 1): S80–S89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoo TK, Li MY: A Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research. J Chiropr Med. 2016; 15(2): 155–163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTerwee CB, Bot SDM, 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\nOsborne JW, Costello AB, Kellow JT: Best Practices in Exploratory Factor Analysis. 2011.\n\nBland MJ, Altman DG: Statistical Methods For Assessing Agreement Between Two Methods Of Clinical Measurement. Lancet. 1986; 1(8476): 307–310. PubMed Abstract \| Publisher Full Text\n\nAndreucci A: \"Supplementary Table 1.docx\", Danish Translation of the Adolescent Insomnia Questionnaire. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/VQFT9W/MGVSB7\n\nCohen LL, La Greca AM, Blount RL, et al.: Introduction to special issue: Evidence-based assessment in pediatric psychology. J Pediatr Psychol. 2008; 33(9): 911–915. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinan PH, Smith MT: The Comorbidity of Insomnia, Chronic Pain, and Depression: Dopamine as a Putative Mechanism. Sleep Med Rev. 2013; 17(3): 173–183. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpruyt K, Gozal D: Pediatric sleep questionnaires as diagnostic or epidemiological tools: A review of currently available instruments. Sleep Med Rev. 2011; 15(1): 19–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelgado-Rodríguez M, Llorca J: Bias. J Epidemiol Community Health. 2004; 58(8): 635–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde la Vega R, Miró J: The assessment of sleep in pediatric chronic pain sufferers. Sleep Med Rev. 2013; 17(3): 185–192. PubMed Abstract \| Publisher Full Text\n\nPalermo TM, Beals-Erickson S, Bromberg M, et al.: A single arm pilot trial of brief cognitive behavioral therapy for insomnia in adolescents with physical and psychiatric comorbidities. J Clin Sleep Med. 2017; 13(3): 401–410. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "88082", "date": "06 Jul 2021", "name": "Jefferson Santos", "expertise": [ "Reviewer Expertise Chronobiology", "Adolescent Sleep" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 article achieved the aim to provide a good insomnia measurement to the adolescent population. The text is objective and the procedures are adequate to questionnaire validity and reliability.\n\nTherefore, I would like to point two questions in order to improve the quality of the manuscript.\n\nMajor point:\nOnly exploratory factor analysis was performed. The factor loadings are important to comprehend the contribution of each item into the three-factor model extracted by EFA, however, the CFA is also important to measure the factor latent structure model of the AIQ. I suggest that CFA should be included in the analysis as well as the graphic demonstration of the CFA. Lastly, it would be recommended to report the KMO and Bartlett tests to show the suitability of the sample for factor analysis in EFA and the model adjustment indexes in CFA (RMSEA, CFI, GFI, and TLI). These values ensure robustness to the models.\n\nMinor points:\nIn table 6, the rotated factor loading of item 1 is missing. I suggest including the lack of a social jetlag measurement as a limitation of the study. It would be relevant information to better sample description.\n\nIs the work 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": "6966", "date": "03 Aug 2021", "name": "Alessandro Andreucci", "role": "Author Response", "response": "Dear Editorial Team, F1000 Research, We were delighted that the Reviewer of our submission \" Translation, validity and reliability of the Danish version of the Adolescent Insomnia Questionnaire \" (ID: 25832) provided us with their very helpful feedback on our paper. This has given us the opportunity to apply changes that has led to an improvement of the paper. Following the recommendations of the reviewer we have now modified the manuscript. We have set out below a response to the Reviewer’s specific comments, outlining point by point how we addressed the comments and changed the manuscript. We hope that F1000 Research and the Reviewer find the changes acceptable, and we look forward to publication in due course. None of the data has been published elsewhere. There are no conflicts of interest. Reviewer 1 The research article achieved the aim to provide a good insomnia measurement to the adolescent population. The text is objective and the procedures are adequate to questionnaire validity and reliability. Therefore, I would like to point two questions in order to improve the quality of the manuscript. Response: We thank the reviewer for the valuable feedback provided. We have addressed the comments and changed the manuscript, which we feel is now improved. Individual responses for each specific comment are outlined below. Major point: Only exploratory factor analysis was performed. The factor loadings are important to comprehend the contribution of each item into the three-factor model extracted by EFA, however, the CFA is also important to measure the factor latent structure model of the AIQ. I suggest that CFA should be included in the analysis as well as the graphic demonstration of the CFA. Lastly, it would be recommended to report the KMO and Bartlett tests to show the suitability of the sample for factor analysis in EFA and the model adjustment indexes in CFA (RMSEA, CFI, GFI, and TLI). These values ensure robustness to the models. Response: We thank the reviewer for the comment and have now performed the Kaiser-Meyer-Olkin (KMO) Measure of Sampling Adequacy test and the Bartlett test of sphericity. Results show a value of 0.877 for the KMO test and a p-value <0.001 for the Bartlett test of sphericity, indicating that the sample is adequate for the Exploratory Factor Analysis. Regarding the Confirmatory Factor Analysis (CFA), the main goal of our study was to present a preliminary validation of the Danish version of the Adolescent Insomnia Questionnaire (AIQ) and explore how many factors the Danish version of the AIQ has. However, our aim was not to confirm that its factor structure is the same in a different language, as it can be assessed with the CFA. In addition, the AIQ is still in an early phase of instrument development. Therefore, a CFA will be conducted when further validity evidence will be obtained on the internal structure of the scale from similar populations and contexts. We have now modified the text in the methods, result and discussion sections, to strengthen these points. The text now reads: Methods section, subsection “Statistical analysis”: “Exploratory factor analysis (EFA) using principal factor extraction and oblique rotation was conducted to identify the factor loadings. Results of EFA were compared to those from the original development sample. The Kaiser-Meyer-Olkin (KMO) Measure of Sampling Adequacy test and Bartlett’s Test of Sphericity were performed to assess the suitability of the data for factor analysis.” Results section, subsection “Exploratory factor analysis”: “Results from EFA showed a three-factor solution that supported the model identified in the original development sample and accounted for 65.45% of the variance among items. Values of the rotated individual items ranged from 0.60 to 0.84 (Table 6) and were similar to those identified within the original development sample. Factor cross-loadings of the AIQ items are provided in Supplementary Table 1. The value for the KMO test was 0.877 and the p-value for the Bartlett test of sphericity was <0.001, indicating that the sample is adequate for the Exploratory Factor Analysis.” Discussion section: “Second, a CFA was not performed as our aim was not to confirm that the AIQ factor structure is the same in a different language, but to explore what the factor structure would be in such language, without making assumptions, given that the languages and culture are different. In addition, the AIQ is still in an early phase of instrument development. Therefore, future plans will be to conduct a CFA once there is further validity evidence on the internal structure of the scale from similar populations and contexts.” Minor points: In table 6, the rotated factor loading of item 1 is missing. I suggest including the lack of a social jetlag measurement as a limitation of the study. It would be relevant information to better sample description. Response: We thank the reviewer for the comment. The rotated factor loading of item 1 (0.78) has now been provided in Table 6. In addition, we have added the lack of a social jetlag measurement as a limitation of the study. We have now modified the text discussion section, which now reads: “Finally, adolescence is a period where changes in bedtime occur due to both biological and social factors 7, which, together with the effect of the inborn chronotype, might result in a social jetlag and consequential reduced sleep duration 32. Therefore, future studies should also try to measure the social jetlag that the adolescent might be experiencing.”" } ] } ]	1	https://f1000research.com/articles/9-1029
https://f1000research.com/articles/11-485/v1	03 May 22	{ "type": "Brief Report", "title": "How did the US and UK markets react to the COVID-19 vaccines’ announcements? A preliminary assessment", "authors": [ "Manar Abushosheh", "Shabbir Bohara", "Davide Contu", "Elgilani Elshareif", "Ikhlaas Gurrib", "Manar Abushosheh", "Shabbir Bohara", "Elgilani Elshareif", "Ikhlaas Gurrib" ], "abstract": "Background: The COVID-19 pandemic has caused major public health and economic disruption. At the same time, a pandemic allows researchers to assess market efficiency; namely, whether, to what extent, and how swiftly stock markets incorporated information related to COVID-19. Soon after the outbreak of the pandemic, research on this front was conducted, with a particular focus on the United States of America (US) market. However, new major events linked to the pandemic have unfolded: a number of vaccines were announced and authorized. The research available in relation to market efficiency relative to vaccine availability is scant. The aim of this study was to assess market efficiency hypotheses with regards to the US and United Kingdom (UK) markets, investigating the impact of the promising news of the vaccines’ successful trials and, subsequently, their authorization. Methods: This work considered data from the S&P500 for the US market and the FTSE100 for the UK market. The time interval considered ranged from the date positive results of vaccines’ trials were first announced, 18 November 2020, until up to two months after the vaccines’ authorization that happened later in December 2020. For both markets, we analyzed the daily returns, cumulative returns, standard deviation and average returns. Results: In both the US and the UK, there was a positive effect of the vaccines’ announcements in terms of increase in the daily returns. However, the standard deviation was not found to increase substantially, notwithstanding the increase in the COVID-19 cases worldwide and the potential lockdown in several countries, as well as the fear from new coronavirus strains that the new vaccines might not be protect against.\nConclusions: Whilst both markets displayed an increase in the average return following the vaccines’ announcements, the UK market seemed to reflect vaccines’ announcements faster than the US market.", "keywords": [ "Market efficiency", "COVID-19", "COVID-19 vaccines" ], "content": "Introduction\n\nThe coronavirus disease 2019 (COVID-19) pandemic has had devastating, manifold impacts across the world which rapidly triggered a quest for a vaccine. In April 2020, it was estimated that 115 candidate vaccines were in research and development (Le et al. 2020). Promising news for an upcoming vaccine, at first, came from the United States of America (US). On 18 November 2021, Pfizer and BioNTech reported the final results of their vaccine trials, stating an effectiveness rate of 95%. Subsequently, the US Food and Drug Authority (FDA) issued the first emergency authorization to use the Pfizer–BioNTech Vaccine on 11 December 2020. Shortly after, on 18 December 2020, an additional vaccine, in this instance developed by Moderna, received emergency authorization by the FDA. Later that month, on 31 December 2020, WHO authorized the use of the Pfizer vaccine. The United Kingdom (UK) also saw encouraging news of vaccine development. The University of Oxford, in collaboration with AstraZeneca, announced on 23 November 2020 the completion of the phase 3 trial with a 70.4% effectiveness rate; this vaccine was later approved for use in the UK on 30 December 2020 (Gallagher and Triggle 2020) and then subsequently by WHO on 15 February 2021.\n\nThese vaccines present remarkable differences in terms of cost and storage requirements. Governments around the world have secured different prices for the vaccines, but it appears the Oxford-AstraZeneca vaccine is the cheapest, followed by Pfizer and Moderna (Dyer 2021). With regards to the storage requirements, the Pfizer vaccine requires an extremely cold environment of −70°C. In contrast, the Moderna vaccine needs to be stored at −20°C. Finally, the Oxford-AstraZeneca vaccine requires the storage temperature to stay between 3–8°C, making its handling process easier as opposed to the other vaccines. However, all of these vaccines have been approved by the relevant health authorities indicating that they are considered helpful in the fight against COVID-19.\n\nThe announcement and authorization of COVID-19 vaccines has obvious health and economic implications. It is an example of positive economic shock that allows to assess the market efficiency hypothesis: do asset prices reflect all available information? The market efficiency hypothesis is classified in weak, semi-strong or strong, depending on how rapidly markets integrate new information. A weak market would reflect all data of past prices, a semi-strong market would reflect all publicly available information, while a strong market would reflect all public and private information (Fama 1970). During the COVID-19 pandemic, research was conducted to test the US market efficiency, with a focus on the market’s reaction following the outbreak of the pandemic. This research has shown that the US market did not react as swiftly as was expected, as it maintained positive returns and relatively low standard deviation until 21 February 2020. The news of the virus outbreak appear to have been reflected only from 24 February 2020 onwards (Vasileiou 2020).\n\nWe expect markets to be positively affected by the news of available vaccines, displaying higher average stock returns and higher standard deviation. Notably, the standard deviation is expected to be higher when new information is reported, regardless of whether it is considered to be positive or negative. We focused on the US and the UK stock market from the period of their successful vaccine trials announcements, until February 2021.\n\n\nMethods\n\nThe Research Committee at Canadian University Dubai exempted this study from the requirement of ethical approval as it does not involve the use of human, animal or plant data.\n\nWe analyzed1 the daily returns, cumulative returns, standard deviation and average returns across time intervals of interest related to the vaccines’ announcements. Time ranges are specified in the next section. The returns at a given time ‘t’ (rt) are computed as a function of the current (Pricet) and previous day’s prices (Pricet−1), as follows:\n\nwhereas the cumulative returns (Cr) from period 1 to period T is given as follows:\n\nFinally, the standard deviation (SDr) is derived as shown below:\n\nWith regards to the US, we referred to the S&P500 Index during the period between 18 November 2020 to 19 February 2021. We retrieved the information relative to market prices using Yahoo Finance (Yahoo Finance 2021). The dates considered were motivated as follows: the first interval starts from 18 November 2020, when Pfizer announced their encouraging results about the vaccine. The second interval started on 11 December 2020, when the FDA authorized the Pfizer vaccine to be used in the US. The third interval started on 31 December 2020, when WHO authorized the use of the Pfizer vaccine.\n\nFurthermore, for the US we considered the Dow Jones Industrial Average (DJIA), again accessed through Yahoo Finance, and looked at index movements around important events happening at the time of vaccine announcements. We further detailed our analysis by breaking our research period into 3 November 2020 to 9 November 2020, being the period of US Presidential elections; 10 November 2020 to 11 December 2020, corresponding to the first announcement by a pharmaceutical company to enter phase 3 of COVID-19 vaccine trials; finally, 12 December 2020 to 31 January 2021, corresponding to the announcement of the US FDA approval for emergency use of a COVID-19 vaccine, and post-announcement vaccine rollout.\n\nWhen considering the UK, we inspected the FTSE 100 Index between 18 November to 19 February 2021. We retrieved the information relative to market prices from Yahoo Finance. The dates considered were motivated as follows. In a similar fashion as for the case of the US, the first interval started from 18 November, when Pfizer announced their encouraging results about their vaccine trials. The second interval starts on 24 November, when the Oxford-AstraZeneca news of successful vaccine trials was released.\n\n\nResults and discussion\n\nResults are presented in Table 1. Whilst the average of daily returns for the entire time period considered averaged 0.2%, it can be noticed that the cumulative returns definitely improved from 2.81% at the beginning of the period (18 November), up to 9.5% at the end of the period considered. Hence, it appears as the market took a few days to reflect the vaccine announcement dates.\n\nIt is important to notice that during the time period considered in this work, markets could have also been affected by the US election (Financial Times 2020). The presidential election took place on 3 November 2020, and on 14 December 2020, the winners were declared across the country. In Figure 1, we also present data relative to the DJIA, indicating key events around the US elections.\n\nBy assessing specific time intervals, the following should be noticed:\n\n1) 3 November 2020 to 9 November 2020: The 2020 US Presidential elections were concluded with a change in evident administration. This was altogether seen as a positive development (The New York Times 20202). However, there was growing uncertainty of the validity of the election results, which provided a lot of counter-arguments stating that the markets were volatile during the period (Chicago News 20213).\n\n2) 10 November 2020 to 11 December 2020: The Pfizer-BioNTech COVID-19 vaccine was announced to have completed phase 3 trials with a 95% efficacy in COVID-19 disease prevention. A surge of investment in the US stock markets could have been expected, particularly in the stocks of major pharmaceutical companies. However, only an increase of 3.05% and a standard deviation of 0.82% were observed, indicating that the market participants, though optimistic about the upcoming vaccine introduction, were pessimistic about the conclusion of the US Presidential power transfer.\n\n3) 12 December 2020 to 31 January 2021: the Pfizer vaccine obtained emergency use authorization and began rolling out for the general public. However, there were several events that might have contributed, at least partially, in the negative returns for the period, which are as follows:\n\ni) On 6 January 2021: riots occurred at the US Capitol which resulted in the deaths of four civilians and one law enforcement personnel;\n\nii) On 20 January 2021: the US President Joe Biden was inaugurated and sworn in as the 46th President of the United States. He pledged a USD 1.9 trillion package to fight the COVID-19 pandemic and to revive the US economy. Furthermore, there was growing uncertainty around the impeachment trial of former US President Trump for encouraging the US Capital riots.\n\nResults are presented in Table 2. It can be noticed that the cumulative returns improved significantly from negative 0.80% at the beginning of the period, compared to 3.74% at the end of the period considered. The average value of the daily returns, taking into consideration the entire period, is 0.03%. It appears that the market reacted swiftly to the news of successful trials, as average returns increased from −0.3% to 0.2%, and the standard deviation increased from 0.44 to 0.87.\n\n\nConclusions\n\nBoth the US and the UK appear to have reacted to the encouraging news of the vaccines’ availability. This can be seen from the increase of the average daily returns. It can be noted that the UK market seems to have reflected the announcements faster as opposed to the the US market: the FTSE100 took one day to reflect the vaccine’s announcements, while the S&P500 took several days. It is important to highlight that when vaccines were announced, COVID-19 case numbers were still increasing, with new lockdowns being put in place: this might have counter-balanced, to some degree, the impact of the vaccines’ announcements.\n\n\nData availability\n\nZenodo: ‘How did the US and UK markets react to the COVID-19 vaccines' announcements? A preliminary assessment’ https://doi.org/10.5281/zenodo.5733141 (Contu 2021).\n\nThe project contains the following underlying data:\n\n• Data 2021. xlsx (The file contains data from S&P 500, DJIA, FTSE100 employed in this research)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nContu D: 2021. Publisher Full Text\n\nDyer O: Covid-19: Countries are learning what others paid for vaccine. BMJ. 2021; 372(281). Publisher Full Text\n\nFama EF: Efficient capital markets: A review of theory and empirical work. J. Financ. 1970; 25(2): 383–417. Publisher Full Text\n\nFinancial Times: The US election and your investment portfolio.2020.\n\nGallagher J, Triggle N: Covid-19: Oxford-AstraZeneca vaccine approved for use in UK.2020. Reference Source\n\nLe TT, Andreadakis Z, Kumar A, et al.: The COVID-19 vaccine development landscape. Nat. Rev. Drug Discov. 2020; 19: 305–306. Publisher Full Text\n\nVasileiou E: Efficient Markets Hypothesis in the time of COVID-19. Rev. Econ. Anal. 2020; 13(1): 45–63. Publisher Full Text\n\n\nFootnotes\n\n1 Data was analyzed in Microsoft Office Excel online, which is available free of charge.\n\n2 https://www.nytimes.com/2020/11/13/business/stock-market-record.html\n\n3 https://news.uchicago.edu/story/lasting-impact-trumps-attempts-challenge-2020-election-results" }	[ { "id": "139666", "date": "18 Jul 2022", "name": "Chong Choy Yoke", "expertise": [ "Reviewer Expertise Economics", "Finance", "Statistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study has good intention of studying the impact of vaccine news on stock prices in US and UK. However, the time frame for the study is kind of short and the conclusion is made solely based on some descriptive statistics which might not be very convincing. Why study the stock price of USA and UK? Why not other countries? It should be mentioned in the introduction.\nIs the study design appropriate and is the work technically sound? Are the statistical analysis and its interpretation appropriate? Are the conclusions drawn adequately supported by the results?\nThe design is partly OK. Since the study is meant to look at the impact of the vaccine news on the stock price, hence I believe the period before the vaccine news announcement should be included in the study too.\n\nThe time period is quite short as it is only around 3 months time in the study. As a result, this study only able to compare the descriptive statistics which I think is a bit not so convincing. If the period is long enough, perhaps you may consider studying the volatility of stock price.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-485
https://f1000research.com/articles/10-1221/v1	01 Dec 21	{ "type": "Research Article", "title": "Priorities for research in child and adolescent anxiety and depression: a priority setting partnership with youth and professionals", "authors": [ "Brynhildur Axelsdóttir", "Lise Mette Eidet", "Ragnhild Thoner", "Sølvi Biedilæ", "Ingrid Borren", "Mari Elvsåshagen", "Kristine Horseng Ludvigsen", "Astrid Dahlgren", "Lise Mette Eidet", "Ragnhild Thoner", "Sølvi Biedilæ", "Ingrid Borren", "Mari Elvsåshagen", "Kristine Horseng Ludvigsen", "Astrid Dahlgren" ], "abstract": "Background: A starting point for evaluating the effectiveness of treatments should be to identify evidence gaps. Furthermore, such evaluations should consider the perspectives of patients, clinicians and carers to ensure relevance and potentially influence future research initiatives. Methods: Our approach, inspired by the James Lind Alliance methods, involved three steps. First, we performed a document analysis by identifying interventions and outcomes in two recently published overviews of systematic reviews, which summarised the effects of interventions for anxiety and depression in children and adolescents. Second, we surveyed children and adolescents with personal experiences of depression or anxiety as well as clinicians, and asked them to suggest treatments and outcomes associated with uncertainty. Finally, we facilitated a consensus process where clinicians and youth mental health patient representatives were invited to prioritise research uncertainties in separate consensus processes. Results: The survey included 674 respondents who reported a total of 1267 uncertainties. Independent coding by four investigators revealed 134 suggestions for treatments of anxiety, 90 suggestions for treatments of depression, 84 for outcomes of interventions for anxiety and 71 suggestions for outcomes of interventions for depression. Two separate priority setting workshops with eight clinicians and ten youth resulted in four independent top ten priority lists. Conclusion: Top ten lists of treatments and outcome domains of anxiety and depression in children and adolescents was identified by youth and clinicians. The results may influence the research agenda, and ultimately benefit patients.", "keywords": [ "Anxiety", "Depression", "Children", "Adolescents", "Research priorities", "Consensus." ], "content": "Introduction\n\nAnxiety and depression are common mental disorders in adolescence. Anxiety is characterised by restlessness or nervousness, poor concentration, and irritability. Depression is characterised by persistent low mood, loss of interest and enjoyment.1\n\nThe prevalence of anxiety and depression increases during adolescence, and the comorbidity between these diagnoses is high among young people.2 Almost 10% of adolescents will meet the criteria of an anxiety disorder.3 The one-year prevalence rate of adolescent depression is estimated to be 5.6%.4 In Norway, the prevalence of diagnosed depression in girls 15-17 years has increased from 1.5% to 2.3% from 2010-2013.5\n\nBoth anxiety and depression in adolescence are associated with functional impairment and can affect academic achievement, which may have a lifelong effect on employment.6,7 According to the WHO’s Global Burden of Disease, the leading cause of years lost due to disability (YLDs) for both genders 10-24 years is unipolar depressive disorders.8 The serious consequences of anxiety and depression in adolescence highlights the need for efficient interventions, and the importance of including perspectives of their own experiences.\n\nCurrently, recommended treatments for anxiety and depression are psychological therapy, pharmacotherapy, or a combination of both.9–11 There are also many other treatments used for both anxiety and depression, some based on well-founded scientific research while others can be regarded as treatment uncertainties. Such uncertainties are either consequences of a lack of research, or that the research is not adequately performed and therefore the evidence is weak.12 A starting point for new research on treatment uncertainties should be to identify evidence gaps, in order to shape future research priorities.13,14\n\nResearch uncertainties can be identified through user involvement. The purpose of user involvement in research is to ensure that research becomes as relevant to the population in question as possible. When initiating research on treatment effects, it has not always been common practice to obtain the perspectives of patients, clinicians or carers.15,16 Thus, important research questions remain unanswered, and research funding may not be used where most needed.17 A recent systematic review, based on 83 studies involving 15,722 participants, demonstrated how uncommon it is to involve children and their caregivers in setting research priorities in the field of childhood chronic disease.16\n\nA recent publication by Chevance et al.,18 published in 2020, described a similar process with adult participants in an international survey, identifying outcomes for depression that matter to patients, informal caregivers, and health-care professionals. Another process of developing an Overall Paediatric Health Standard Set [OPH-SS] of outcome measures which matters to young people and their families, internationally, was also published in 2020.19 The current study complements both papers, as this paper looks at both children and adolescents, as well as desired research priorities in terms of treatments, as well as outcomes.\n\nJames Lind Alliance (JLA), a non-profit making initiative, is funded by the National Institute of Health Research (NIHR) and the Medical Research Council in the UK. The purpose of the alliance is to facilitate cooperation between clinicians and patients to identify and prioritise essential research uncertainties. Their method is transparent, accountable, and systematic, and has inspired the study described in this paper.20 We recently produced two overviews of systematic reviews on the effects of interventions for anxiety and depression in children and young people, respectively.10,11 This left us with a momentum for inviting users and those providing mental health services to identify and prioritise research uncertainties associated with these conditions.\n\nThe objective of this study was to identify research priorities judged as additional priorities, beyond those previously identified, which may guide the research agenda moving forward.\n\n\nMethods\n\nREK, Regional Committees for Medical and Health Research Ethics, Norway was contacted for approval of the project. They concluded that the project did not require their approval as there was no registered personal data. All information was collected through Nettskjema (a web-based survey system), ascertaining a high level of data security and safety.\n\nAll respondents were given information about the purpose of the study and how the results would be managed and presented and were informed that by responding to the survey, they consented to participation in the study. The questionnaire was anonymous and once submitted, the information could not be traced back to the respondent.\n\nIn the current study, we included both qualitative and quantitative methods in three stages:\n\n1. Document analysis: identification of interventions and outcome measures used for treating children and adolescents with anxiety and depression in two previously published overviews of systematic reviews.10,11\n\n2. Mapping study (surveys): we encouraged identification by clinicians and patient representatives (children and adolescents who have, or have had, anxiety or depression) of additional priorities outside of those previously identified.\n\n3. Consensus process: prioritisation of research uncertainties by clinicians and patient representatives.\n\nOur approach was inspired by a method developed by the JLA.20 The method involves patients and clinicians in suggesting research priorities. Their input is given equal weight. The method is designed to raise awareness of important evidence gaps, with the potential of influencing new research initiatives.15\n\nThe stages of the prioritisation process are outlined in Figure 1.\n\nIn two recently published overviews of systematic reviews, we have summarised the effects of interventions for anxiety and depression in children and adolescents.10,11 Although these publications are in Norwegian, the methodology of the review process have been published in registered protocols and is available in English through the PROSPERO database; CRD42020159883 (depression) and CRD42020159884 (anxiety). To provide context to this paper, we briefly describe the inclusion criteria and search strategy of the reviews here. Both overviews adhered to the PRISMA guidelines21 and to the following inclusion criteria:\n\nPublications: Systematic reviews published 2012 and later, fulfilling the DARE-criteria.\n\nLanguage: English, Norwegian, Danish, or Swedish.\n\nParticipants: Children and adolescents under the age of 18, with or without an identified risk of developing mental health problems or those who have already developed these problems.\n\nIntervention: Any intervention aimed at preventing or reducing mental health problems or welfare interventions, including psychological therapy, pharmaceutical interventions, psychosocial interventions etc.\n\nComparison: Other relevant interventions, treatment as usual (TAU), no treatment or wait list.\n\nOutcomes: All outcomes of mental health problems and child welfare evaluated in children and adolescents, including other health outcomes, quality of life, function, use of health care, attitudes and adverse effects of interventions.\n\nThe search for reviews that were included in these two overviews was largely based on the IN SUM database and was performed in April 2018, with an updated search in December 2018. IN SUM is a recently developed database of systematic reviews of the effects of interventions relevant to children and young people’s mental health and welfare. The database indexes systematic reviews from the following databases: Cochrane Database of Systematic Reviews, Campbell Library, PsycINFO, Medline, Embase, Web of Science, Database of Abstracts of Reviews of Effects (DARE) and Evidence-Based Mental Health. IN SUM is continuously updated monthly with the latest systematic reviews. In addition to IN SUM, we hand searched the websites of the Norwegian Institute of Public Health, the Swedish Agency for Health Technology Assessment and Assessment of Social Services, the Danish Health Authority for Systematic Reviews and the National Institute for Health and Care Excellence for evidence-based guidelines, UK. For complete search strategies see Extended data.28\n\nThe first author (BA) extracted all interventions and outcomes reported in these two overviews in a simple document analysis and second author (AD) double-checked the extraction.\n\nWe created three surveys, each including four questions asking the respondents to report what treatments and outcomes ought to be topics for research, in their opinion. For each question, the recipients were presented with a list of the treatments and the outcomes already addressed in existing research (see Table 1, Table 2), based on the two overviews of reviews.10,11 The three surveys were distributed to clinicians and users as an electronic questionnaire via Nettskjema.\n\n* The quality of the evidence is graded as low or very low.\n\n** Treatments and outcomes in 2018, more treatments and outcomes are described in the 2021 update.\n\n* The quality of the evidence is graded as low or very low.\n\n** Treatments and outcomes in 2018, more treatments and outcomes are described in the 2020 update.\n\nThe survey questions had an open answer option (see Extended data28). Respondents can be unfamiliar with research, and we therefore considered it more appropriate to let respondents formulate their need for research in their own words.20 The purpose of the surveys was to collect suggestions for research uncertainties, consequently, the sample did not need to be representative.20 Instead, we used convenience sampling to recruit the participants. Anyone living in Norway with experience and understanding of living with anxiety or depression was eligible to participate in the identification of uncertainties. This included children and adolescents with anxiety and/or depression, carers, family members and friends. Also, healthcare, and social care professionals who had worked with children and adolescents living with the conditions were eligible. We strived to ensure that professionals working in different levels in health and welfare services were represented, as well as users. No demographic data were collected as it is not a part of later analysis in priority setting partnerships. The JLA guidebook recommends that the purpose of collecting demographic information from respondents is for this purpose only.20\n\nThe first survey was sent on 22nd February 2019, to our institution's contacts working with children and young people's mental health in the municipalities (Eastern and Southern Norway), including employees in child welfare institutions/orphanages, special education teachers working in schools, child welfare services, child welfare guards, family protection offices, refugee and immigration departments.\n\nThe second survey was distributed on 19th March 2019, to professionals working in the specialist mental health service for children and adolescents. These were also contacted through our networks. In addition, we recruited respondents in collaboration with the Norwegian Association for Children and Young People’s Mental Health (NBUP) and from our institution’s newsletter.\n\nThe third survey was distributed on 25th April 2019, to children and adolescents having personal experiences with depression and/or anxiety, as well as to their carers, in collaboration with the Norwegian organisation for youth mental health, Mental Helse Ungdom (MHU). We also sought to recruit respondents through social media platforms of our institution, e.g., Facebook and Instagram. We posted a link of the survey on the platforms 2nd August 2019, with an invite to eligible participants to complete the survey.\n\nThe interventions and outcomes suggested by the respondents were coded independently by at least two investigators (IB, SB, LME and BA). This part of the process is both interpretative and subjective. Duplicates and similar submissions were combined to a common suggestion. Combining submissions can greatly reduce the volume of data in the process of finalising a top ten list.20 Based on this analysis we created four “master-lists” including all suggestions for:\n\n1) interventions for anxiety\n\n2) interventions for depression\n\n3) outcomes of interventions for anxiety\n\n4) outcomes of interventions for depression\n\nPreparations for the consensus process\n\nThe next step was to prepare for the consensus process, where selected professionals and users were asked to prioritise the suggested research uncertainties. There is no gold standard for conducting a consensus process. However, group composition can have an impact and may lead to different judgements.22\n\nA multi-disciplinary team of professionals were recruited through our networks through convenience sampling. We received help recruiting clinicians from a local child and adolescent psychiatric outpatient clinic. Our contact person there, reached out via e-mail on 21st August 2019, to clinicians with a request to participate in the consensus process. The criteria were clinicians who work, or have worked, with children and adolescents with anxiety or depression. A variety of professionals from different backgrounds and working at different levels of health and welfare services (such as psychologists, psychiatrists, physiotherapists, nurses, educators, and health nurses) came forward. Seven clinicians from the specialist mental health services and four from the municipal health services accepted the invitation to participate. For recruitment of user representatives, we contacted the Assistant General Secretary of MHU. She reached out via e-mail on 15th September 2019, to their members of staff and youth with experience of the conditions, and twelve participants accepted the invitation.\n\nOnce recruited, we received contact information of 10 participants proposed by the assistant general secretary of the organisation on October 10th,2019. We emailed the four lists with the suggested interventions and outcomes for anxiety and depression, respectively to the participants. They were individually asked to put the suggestions in ranked order, by selecting only 10 options that were assigned 1 point each. For the three most important options we asked them to assign these 2 points. This resulted in the first drafts of prioritised lists of interventions, and outcomes of interventions, for anxiety and depression.\n\nThe results from this pre-prioritisation were summarised by two members of the research team (AD and BA), and four lists were created with the highest-ranking suggestions. According to the JLA method, uncertainties must be checked and verified as true uncertainties before prioritisation can begin.20 The two overviews of systematic reviews documented which treatments and outcomes that lacked or had weak scientific evidence.10,11 The participants of the workshops were made aware of this before conducting the interim prioritisation, also enabling them to prioritise among those.\n\nThe workshops\n\nFor practical reasons, it was not possible to host a shared workshop for professionals and users. Instead, separate workshops were held.\n\nWhen conducting consensus processes, the criteria for establishing priorities should be applied using a systematic and transparent process.22 Furthermore, group discussions should follow some basic rules that the participants have chosen jointly. Participants should listen to each other and show respect for each other’s ideas.20\n\nAccording to the JLA, it is recommended to base the consensus process on the Nominal Group Technique, which we applied for both workshops.20 This approach is characterised as a structured method for group brainstorming, encouraging discussion and facilitating agreement on the relative importance of issues in question. The process should be led by someone who is not part of the project group, and according to JLA, who has no research background. The person will, therefore, have a more neutral role in the process. It is essential that the entire process has openness and justice as guiding principles.20 For this study, we invited an experienced expert in consensus processes to facilitate and host the workshops (RT), the rest of the team played the part of silent observers and handled all practical needs (LME, SB, AD, and BA).\n\nThe first workshop was held at our organisation’s location in Oslo, Norway on 26th September 2019, from 9:00 am to 3:00 pm. Three members of the project group attended the workshop in addition to the consensus host (LME, RT, AD, and BA). Eight out of 11 clinicians were able to participate in the workshop: psychologists, special educators, clinical social workers, and a physician. Three clinicians were unable to attend for various reasons such as sickness etc.\n\nFor the second workshop, we recruited youth from MHU. The workshop took place in their location on 11th November 2019, from 9:00 am to 3:00 pm and was administrated in the same way as the workshop with the clinicians. Ten out of 12 invited youth were able to participate in the priority setting, and three members of the project group facilitated the workshop (RT, SB and LME). Two participants were unable to attend.\n\nAfter formal introductions and light refreshments, the participants received an introduction for one hour, to the principles of research, systematic reviews, and evidence-based practice. They were also informed about the purpose and agenda of the day. Thereafter, the participants were divided into small groups based on their professional background, age and in the workshop with the youth, earlier experience with anxiety and/or depression. For each topic, the participants were then mixed in different groups with at least three participants in each group. This part of the workshops lasted for four hours with a half an hour lunch break.\n\nThe groups were assigned the task of selecting 10 options and prioritising these for each topic. The groups worked independently but were facilitated by the host when necessary. Other members of the project group were silent observers, taking notes. At the workshop with the professionals, the host used images of children and adolescents with depression and anxiety during this process, as a reminder of the perspectives of the target group involved.\n\nThe final hour of the workshops included individual prioritising. All four lists were entered into a voting app by one of the members of the project group and each participant was asked to anonymously rank the final top ten priorities per list. This resulted in four top ten lists of priorities ranked in order by their perceived importance [see Underlying data28].\n\n\nResults\n\nThe results of the document analysis were collated and made into 4 lists. In the surveys, the respondents were presented with these lists (see Table 1 and Table 2). Note that for several of these treatments and outcomes, the quality of the evidence is graded as low or very low (marked with * in the tables). Therefore, these could still be suggested as research uncertainties.\n\nOverall, 674 respondents submitted a total of 1267 research suggestions in the three surveys. After content analysis, 379 unique suggestions (134 treatments for anxiety, 90 treatments for depression, 84 outcomes for anxiety and 71 outcomes for depression), were sent for ranking via e-mail to the clinicians and youth participating in the workshops.\n\nIn response, the clinicians ranked and shortened the list to 70 suggestions. The youth ranked and shortened it to 51 suggestions. For full detail of the results of the process see Figure 2.\n\nEight clinicians participated in the first workshop: psychologists, special educators, clinical social workers, and a physician. Two of the clinicians worked in the mental health services in the municipalities, and the six others worked in the specialist mental health service for children and adolescents.\n\nThe 10 youth participants from MHU participated in the second workshop. See detailed results of the process in Figure 2 and the final results of the workshops priority setting in Tables 3, 4, 5 and 6.\n\n\nDiscussion\n\nThis study has demonstrated essential research priorities in terms of treatments that should be evaluated and outcomes that should be measured according to youth and clinicians. The top ten lists reflect both similarities and differences in what is considered important by the clinicians and the youth.\n\nClinicians ranked family and parent-based interventions as their top priority for both lists of treatments (anxiety and depression). Youth also ranked family and parent-based interventions as their top priority for treatments of anxiety. Functioning in daily life, and in the family are amongst the top ten treatment priorities by both groups. Other common priorities important to both clinicians and youth are increased cooperation between mental health services and schools, and multi-disciplinary cooperation.\n\nTop priority for depression treatment among the adolescents, were easy access to treatment. The clinicians also emphasize increased cooperation between mental health services and schools, as well as group treatment and school-based interventions. Thus, the clinicians seem to focus on strengthening the environment around the youth to a greater extent than the adolescents do. School-based therapies, school functioning and access to a school psychologist are also similar priorities. The youth seem, however, to display a greater need for interventions for forming relationships, resilience groups, and life coping strategies, which is not mentioned at all in the clinicians’ list.\n\nA unique priority suggested by the youth is therapy for transgender people, specifically regarding anxiety. This may demonstrate a difference between generations regarding the focus on gender identity and the need to cope with such issues.\n\nOn the lists of outcomes of interventions for both conditions, functioning in daily life, in the family, and at work were ranked very high by both the clinicians and the youth, as well as friends and social activities. Other important common suggestions are long-term follow-up of interventions, treatment satisfaction and user involvement. However, it is worth noting that the outcomes most important for the adolescents, for both anxiety and depression, were highly subjective/internal outcomes like resilience, faith in oneself, life skills, identity, daily life functioning and trust in other people. In contrast, the clinicians ranked friends and social activities on top of both lists, while this suggestion was not found on the adolescent’s lists. Thus, the clinicians seem to view the context the youth is in as more important than the youths do themselves, who to a greater extent emphasize personal coping skills, like faith in oneself and resilience. This difference may possibly tell us that contextual factors (friends, school or dropping out of school) are regarded less important for individuals struggling with mental health challenges, and that inner personal growth and mastery are key factors for these young people. The clinicians may, on the other hand, have been thinking more in terms of outcomes known to be preventive factors (like friendship and social structures).23\n\nClinicians rated adverse events as important for both conditions. The lack of research of unwanted effects of treatments for depression in children and adolescents has recently been demonstrated in a mapping of systematic reviews.24 Both the clinician’s views and Eidet’s article24 point to the need for more research, and thus address adverse events in these treatment groups as an important evidence gap.\n\nThis study builds on rigorous qualitative and quantitative methods, including two extensive systematic reviews on the effects of treatments for anxiety and depression. To our knowledge this is also the first mapping study in Norway exploring research uncertainties related to treatments and associated outcomes for anxiety and depression.\n\nThe current study is in line with evidence-based practice as it is defined as ‘The conscientious, explicit and judicious use of current best evidence in making decisions about the care of the individual patient’.25 Evidence-based practice highlights the consideration of the patient’s opinions in choice of treatment (alongside clinical opinions and research-based methods), and the current project contributes along these lines also, by letting patients voice their concerns regarding research gaps. We have integrated the best research evidence and involved clinical expertise both in the surveys and the workshop with clinicians. Furthermore, we have included the personal and unique values of the patients. All of these should be a part in any decision-making process concerning research and treatments for children and adolescents.\n\nThere has been increasing attention to patient-reported outcomes during recent years. Outcomes should be relevant and important to both patients, caregivers, health care professionals and other stakeholders making decisions about health care.26,27 For discovering what outcomes are important to patients and health care professionals, consensus processes, as demonstrated in this study, are vital. This study is especially important because we succeeded in including the views of young people, considering how rare patient and family engagement are in research priority setting.16\n\nThe importance of user involvement is demonstrated in feedback from participants in both workshops:\n\n“It feels very meaningful to be able to contribute to this project on behalf of all the patients I have been in contact with”.\n\n“Children and adolescents should always be involved in decision-making, not just clinicians”.\n\nThe limitation of consensus processes should be acknowledged. The current priorities are based on individual’s or groups’ point of views and their subjective opinions. We might, in our consensus process with a different pool of people in a different situation, reach a different result.20 However, involving people together in a quality discussion to reach genuine consensus is of great value, as it represents an important contribution to the debate on research priorities. Bringing people together in a workshop enables them to exchange knowledge and information and make decisions in their meetings with the health services, based on a wider set of experiences.\n\nInitially we intended to host only one priority setting workshop with both clinicians and the youth as recommended by JLA, however we were unable to find an appropriate date suitable for both groups. Although hosting a shared workshop would have had several benefits, we also found it useful to keep the groups separated. We were able to avoid challenges, such as ensuring the choice of participants being balanced, avoiding domination by one person, and reaching consensus when there may have been disagreement. The two separate processes allowed us to compare the results of professionals and the youth. It also provided a safe zone for professionals and the youth, where especially the latter could speak more freely and perhaps avoid feeling ‘led’ to conclusions by clinicians whom they perhaps could see as authority figures with more experience than themselves. However, keeping the groups separate meant that we also missed the opportunity of cross-fertilization of ideas and nuancing of perspectives, that mixing professionals and users may have contributed to.\n\n\nConclusion\n\nWe have demonstrated the possibility to develop an agreed four top ten lists of research priorities for anxiety and depression in children and adolescents, with contribution from youth experiencing anxiety or depression as well as clinicians. The perspectives from their individual lists, have the possibility to influence the research agenda according to the needs and opinions of both clinicians and the patients themselves.\n\n\nData availability\n\nHarvard Dataverse: Priorities for research in child and adolescent anxiety and depression: a priority setting partnership with youth and professionals https://doi.org/10.7910/DVN/UQPYVT.28\n\nThis project contains the following underlying data:\n\n• Coding_priorities from participants_Clinicians_final_25.09.2019.tab\n\n• Coding_Priorities_Adolescents_Final_07.11.2019.tab\n\nHarvard Dataverse: Priorities for research in child and adolescent anxiety and depression: a priority setting partnership with youth and professionals https://doi.org/10.7910/DVN/UQPYVT.28\n\nThis project contains the following extended data:\n\n• Tables 3-6 (in Norwegian, pdf.)\n\n• Appendix I (Copy of survey no.1, no.2. and no.3.)\n\n• IN SUM Search strategies_2021.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to thank the following for helping recruiting participants to the workshop with clinicians: Signe Revold, Akershus University Hospital, Morten Grøvli, Akershus University Hospital and a member of the RBUPs board and Kaja Kierulf, centre manager of RBUP. We would like to thank Thisbe Verner-Carlsson and Aida Tesfai at the Norwegian Mental Health Youth (Mental Helse Ungdom) for distributing the survey no. 3 and recruiting participants to the second workshop with the youth. We are grateful to NBUP, the Norwegian Associations of Mental Health Services for Children and Adolescents for help of distributing the survey no. 2. We also would like to thank our colleagues at RBUP, Siri Saugestad Helland, Kristian Rognstad and John Kjøbli for assistance with the first survey, distributed to persons working with children and young people's mental health in the municipalities. Finally, and most importantly, we would like to thank all the participants of both workshops.\n\n\nReferences\n\nAmerican Psychiatric Association: Diagnostic and statistical manual of mental disorders: DSM-5 Arlington, VA: American Psychiatric Association; Fifth ed.2013. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nObbarius A, van Maasakkers L , Baer L, et al.: Standardization of health outcomes assessment for depression and anxiety: recommendations from the ICHOM Depression and Anxiety Working Group. Qual. Life Res. 2017; 26(12): 3211–3225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliamson PR, Altman DG, Bagley H, et al.: The COMET Handbook: version 1.0. Trials. 2017; 18(Suppl 3): 280. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAxelsdottir B: Priorities for research in child and adolescent anxiety and depression: a priority setting partnership with youth and professionals. Harvard Dataverse, V2. 2021a. Publisher Full Text" }	[ { "id": "124299", "date": "22 Feb 2022", "name": "Kristina Staley", "expertise": [ "Reviewer Expertise I have worked on over a dozen JLA PSPs as an Information Specialist and have worked in the field of patient and carer involvement in research for over 20 years" ], "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 reports on a priority setting exercise which has drawn on the JLA approach but has changed so far from it that I question whether to make the links is appropriate. For example I'd challenge the use of the term priority setting partnership in the title.\nThe approach in this paper differs from the JLA process in two main ways:\nCategorising the uncertainties collected via survey of young people and professionals. In a JLA process the Steering Group, a mix of professionals and affected patients/carers, are heavily involved in interpreting the responses to generate a list of uncertainties using phrasing and language that summarise the responses. The aim is always to stay faithful to the original responses. In this paper the researchers have drawn out interventions and outcomes as separate lists - not whole questions. I do not understand the rationale for this and would like a clearer explanation in the article. As they have identified, the language used and the priority given to different ways of understanding the issues makes it difficult to combine the youth and professionals' priority lists of interventions/outcomes. In the JLA process, this is done in the partnership of the Steering Group to reach a shared agreement of the list of topics to be prioritised, a shared understanding of what these mean so that people from all perspectives can understand and prioritise the shared list.\nFurthermore, I'd like the authors to comment on how the prioritised lists of interventions and outcomes might be used to shape future research.\n\nThe final workshop - it is essential that all parties come together and reach a shared agreement of the Top Ten. It would seem important to find a date for such a meeting that all could attend rather than have separate meetings. And for the group discussion to inform the prioritised list rather than individuals voting on an app.\n\nSo in general there seems to have been limited shared decision-making at each of the stages of this process which makes me question whether this was genuinely a partnership or actually different groups prioritising topics separately. This is what makes it very different to the JLA process.\nThe outputs are quite distinct from those of a JLA process - so I suggest the authors refer to the JLA perhaps once, and instead describe their own process and the rationale for how they have approached it, what they expect the impact to be, and their perceived value of their outputs.\n\nDifferent does not mean better or worse - this is a different process to the JLA and may have strengths or weaknesses as a result. Perhaps these could be explored in the article. The JLA is not a set of methods, but the principles and values that underpin partnership working are absolutely key to it and these are not described in the approach in this paper and I therefore recommend that the suggestions that this process is linked to the JLA approach are reduced.\n\nIs the work 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": "8191", "date": "04 May 2022", "name": "Brynhildur Axelsdottir", "role": "Author Response", "response": "1.This paper reports on a priority setting exercise which has drawn on the JLA approach but has changed so far from it that I question whether to make the links is appropriate. For example I'd challenge the use of the term priority setting partnership in the title. The approach in this paper differs from the JLA process in two main ways: Categorising the uncertainties collected via survey of young people and professionals. In a JLA process the Steering Group, a mix of professionals and affected patients/carers, are heavily involved in interpreting the responses to generate a list of uncertainties using phrasing and language that summarise the responses. The aim is always to stay faithful to the original responses. In this paper the researchers have drawn out interventions and outcomes as separate lists - not whole questions. I do not understand the rationale for this and would like a clearer explanation in the article. As they have identified, the language used and the priority given to different ways of understanding the issues makes it difficult to combine the youth and professionals' priority lists of interventions/outcomes. In the JLA process, this is done in the partnership of the Steering Group to reach a shared agreement of the list of topics to be prioritised, a shared understanding of what these mean so that people from all perspectives can understand and prioritise the shared list. Furthermore, I'd like the authors to comment on how the prioritised lists of interventions and outcomes might be used to shape future research. Response: We thank the reviewer for pointing this out and we acknowledge the differences of our study and the James Lind Alliance framework. We have therefore changed the title of the article. In addition, we have elaborated on these differences in methods and discussion. As to the comment on how the prioritised lists of interventions and outcomes might be used to shape future research, we strongly believe that researchers can be inspired to see what interventions lack evidence (based on evidence gaps identified by the overviews of systematic reviews) as well as what outcomes should be measured when designing new studies on these subjects, based on the participants’ priorities. To highlight the desired interventions and outcomes of users and clinicians may hopefully bring awareness to researchers regarding the needs of these groups – potentially enhancing shared decision-making in future studies. 2. The final workshop - it is essential that all parties come together and reach a shared agreement of the Top Ten. It would seem important to find a date for such a meeting that all could attend rather than have separate meetings. And for the group discussion to inform the prioritised list rather than individuals voting on an app. So in general there seems to have been limited shared decision-making at each of the stages of this process which makes me question whether this was genuinely a partnership or actually different groups prioritising topics separately. This is what makes it very different to the JLA process. Response: There is no gold standard to priority setting of research uncertainties. JLA has however developed an extensive experience and evidence base in this area which has inspired our efforts. We acknowledge that our approach differs from that of the JLA. We have revised our manuscript to make this clearer and have made it explicit which of the methodological choices recommended by JLA we have applied. We have also added a paragraph to the discussion about the potential limitations and strengths of the choices we made. As the reviewer points out, the JLA is not a set of methods but suggests some principles and values that should be considered. The experts and patients taking part in our study were not able to meet in the same day for the consensus workshop, and thus our process resulted in two separate sets of priority lists. Although the resulting lists were not created in a partnership of patients and providers, the results of these two consensus processes provides the opportunity to compare the differences in priorities by patients and providers. This may have brought additional – and potentially valuable – information and possibly cover more evidence gaps. Even though our process differs from that of JLA, we have used methods of high quality, including basing our process on high-quality systematic reviews, including both qualitative and quantitative feedback from experts and patients, and applying a recognized consensus-process methodology. We believe that the priorities-lists resulting from our study is an important contribution to this research and should be used to shape future research efforts. 3. The outputs are quite distinct from those of a JLA process - so I suggest the authors refer to the JLA perhaps once, and instead describe their own process and the rationale for how they have approached it, what they expect the impact to be, and their perceived value of their outputs. Different does not mean better or worse - this is a different process to the JLA and may have strengths or weaknesses as a result. Perhaps these could be explored in the article. The JLA is not a set of methods, but the principles and values that underpin partnership working are absolutely key to it and these are not described in the approach in this paper and I therefore recommend that the suggestions that this process is linked to the JLA approach are reduced. Response: We accept and agree that our process varies from the one of JLA and we have erased the sentence of JLA in the abstract and reframed sentences where we mention JLA in the method section, as well as reduced the numbers of references to the JLA guidance. We have elaborated on strengths and weaknesses of the current study in the discussion and added some clarifications in the introduction about the differences between our approach and the JLA method. We would like to thank the reviewers for their valuable comments." } ] }, { "id": "126468", "date": "23 Mar 2022", "name": "Judith Borghouts", "expertise": [ "Reviewer Expertise Academic researcher in Digital Mental Health and Human-Computer Interaction with 10 years of experience in quantitative and qualitative 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\nThis paper describes an approach, inspired by James Lind Alliance (JLA) methods, to identify research priorities in child and adolescent anxiety and depression treatments. Strengths of the paper are the detailed descriptions of the methods used. I also appreciate the authors making the data available.\nIt was however quite unclear what the paper is trying to contribute, as the problem, objective and results do not seem aligned. The paper starts by highlighting the problem of treatment uncertainties, and that some treatments lack scientific evidence. The introduction then states that the objective of the study was to identify research priorities, which seems different from treatment uncertainties. Finally, it presents results of what types of treatments clinicians and youth would like to see. If this all relates to the same thing, the paper should do a better job explaining how these are all connected.\nRelated to my point above, the key terms are not well-defined. The abstract mentions treatment uncertainties but it is unclear what this is. It becomes a little bit clearer through examples given in the introduction (“uncertainties are either consequences of a lack of research, or the research is not adequately performed”), but it is then not clear how you ‘prioritize’ uncertainties? Do the authors mean which type of treatments should be given priority in future research? Furthermore, Table 4 and 6 mention the term ‘outcomes’, which in the context of treatment usually means treatment outcomes, such as measurable health symptoms. A number of the outcomes in these tables do not seem to be outcomes in the traditional sense; for example, how is ‘friends and social activities’ an outcome? Is this somehow related to social connectedness? The paper is currently lacking a clear explanation of all of these terms, concepts and how they relate to one another.\nLastly, if the objective was to identify research priorities, it was not clear to me why non-researchers were asked to identify uncertainties. As the paper states, respondents can be unfamiliar with research and may not be equipped to prioritize research. It seems that the paper instead collected a stakeholder perspective of important considerations in adolescent treatment for anxiety and depression, which is still important, but is not reflected in the paper’s objective at all.\nI recommend the authors to clearly define the key concepts, clarify the problem, aim of the study, how the results address this problem and aim, and make this consistent throughout 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? 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? Partly", "responses": [ { "c_id": "8192", "date": "04 May 2022", "name": "Brynhildur Axelsdottir", "role": "Author Response", "response": "1.This paper describes an approach, inspired by James Lind Alliance (JLA) methods, to identify research priorities in child and adolescent anxiety and depression treatments. Strengths of the paper are the detailed descriptions of the methods used. I also appreciate the authors making the data available. It was however quite unclear what the paper is trying to contribute, as the problem, objective and results do not seem aligned. The paper starts by highlighting the problem of treatment uncertainties, and that some treatments lack scientific evidence. The introduction then states that the objective of the study was to identify research priorities, which seems different from treatment uncertainties. Finally, it presents results of what types of treatments clinicians and youth would like to see. If this all relates to the same thing, the paper should do a better job explaining how these are all connected. Related to my point above, the key terms are not well-defined. The abstract mentions treatment uncertainties but it is unclear what this is. It becomes a little bit clearer through examples given in the introduction (“uncertainties are either consequences of a lack of research, or the research is not adequately performed”), but it is then not clear how you ‘prioritize’ uncertainties? Do the authors mean which type of treatments should be given priority in future research? Furthermore, Table 4 and 6 mention the term ‘outcomes’, which in the context of treatment usually means treatment outcomes, such as measurable health symptoms. A number of the outcomes in these tables do not seem to be outcomes in the traditional sense; for example, how is ‘friends and social activities’ an outcome? Is this somehow related to social connectedness? The paper is currently lacking a clear explanation of all of these terms, concepts and how they relate to one another. Response: We see the reviewers point; however, we see this as the one influencing the other. What we hope with our process is that identified treatment uncertainties (treatments that lack scientific evidence) should become priorities in future research. Research priorities should be based on research uncertainties established by systematic reviews of the existing evidence. We have added a sentence to make this more explicit. By “treatment” we refer to any action or intervention used to change an aspect of a young person’s mental health, that being medicines or school-based interventions. Such treatments may also have an impact on other aspects of the young person’s life that may be important to consider in research. As we state in the paper, the outcomes found to be important to evaluate in research by researchers often differs from those of providers and patients. Thus, in many cases effects on outcomes important to patients and providers are unknown. This study tries to address this issue. Such outcomes may include a person’s ability to participate in social activities and so on. Our aim was to enable the participants to suggest and prioritise preferred treatments and outcomes and thus highlight the needs of users and clinicians in hope that these needs could be met in future studies. The lists of priorities are the outcome of this whole process, presenting the interventions and outcomes that the involved groups would like to see in future studies. We acknowledge that this link may have not been sufficiently elaborated on and have therefore inserted some sentences that may help clarify the link between these stages of the process in the introduction and the discussion. The objectives have also been rephrased and hopefully appear clearer. 2. Lastly, if the objective was to identify research priorities, it was not clear to me why non-researchers were asked to identify uncertainties. As the paper states, respondents can be unfamiliar with research and may not be equipped to prioritize research. It seems that the paper instead collected a stakeholder perspective of important considerations in adolescent treatment for anxiety and depression, which is still important, but is not reflected in the paper’s objective at all. Response: The idea here is to involve the perspectives of the patients involved and the professionals that treat them. They have unique insights in their needs, which may deviate from the priorities of a researcher. Further, user involvement is one of the main principles of the JLA guidebook, which have partly inspired us in conducting this study. The JLA initiative was established to bring both patients, carers and clinicians together in priority setting partnerships. This ensures shared decision-making processes, which is a cornerstone of evidence-based practice. 3.I recommend the authors to clearly define the key concepts, clarify the problem, aim of the study, how the results address this problem and aim, and make this consistent throughout the paper. Response: We have, based on the reviewers’ responses, rephrased the aim, and sought to make the objective clearer. We believe that our responses to other remarks from the reviewers may also make the paper more accessible. In the introduction, we have described some of the key concepts for clarification. We would like to thank the reviewers for their valuable comments." } ] } ]	1	https://f1000research.com/articles/10-1221
https://f1000research.com/articles/11-45/v1	13 Jan 22	{ "type": "Review", "title": "Tourette syndrome research highlights from 2020", "authors": [ "Andreas Hartmann", "Cyril Atkinson-Clément", "Christel Depienne", "Kevin Black", "Cyril Atkinson-Clément", "Christel Depienne", "Kevin Black" ], "abstract": "We present here research from 2020 relevant to Tourette syndrome (TS). The authors briefly summarize a few reports they consider most important or interesting.", "keywords": [ "Tics", "Tourette syndrome", "2020" ], "content": "Introduction\n\nThis article is meant to disseminate recent scientific progress on Tourette Syndrome (TS).\n\n\nMethods\n\nWe searched PubMed during 2020 using the search strategy (“Tic Disorders”[MeSH] OR Tourette NOT Tourette [AU]) AND 2020[PDAT] NOT 1950:2019[PDAT]. On 15 Feb 2021 this search returned 292 citations. Colleagues also recommended articles, and we attended selected medical conferences (in 2020, mostly online). We selected material for this review subjectively, guided by our judgment of possible future impact on the field.\n\n\nResults\n\nHealth-related quality of life in 52 adults with TS was explained largely (79% of variance) by four self-report questionnaires measuring severity of depression, anxiety, obsessive-compulsive symptoms, and attention deficit hyperactivity disorder (ADHD), plus the total tic score (TTS) from the Yale Global Tic Severity Scale (YGTSS) (Isaacs et al. 2021). Depressive symptoms were the strongest predictor by far, and TTS was the weakest. This result supports previous studies concluding that attending to non-tic psychiatric symptoms is crucial in providing optimal care for tic patients.\n\nThree German centers drew attention to a spate of patients presenting with unusual, tic-like manifestations that appeared to be driven by a prominent social media influencer (Müller-Vahl et al. 2020b).\n\nBecause of the outbreak of SARS-CoV-2, the medical landscape shifted significantly in 2020. One consequence was the widespread deployment of telemedicine services, which was discussed specifically for TS by Cen et al. (2020).\n\nAt the beginning of the pandemic (spring 2020) worries and concerns were raised on how this could impact people with TS, albeit on a speculative basis (Robertson et al. 2020). Studies published later in the year sought to investigate this. In an Italian cohort, perhaps unsurprisingly, lockdown worsened symptoms in 67% of patients with TS (n = 238), ranging from tics to hyperactivity, rage attacks, obsessions/compulsions and anxiety. Of note, however, about one fifth of patients reported symptom improvement, maybe linked to lessened social exposure (Conte et al. 2020b). Similar observations were made by two further groups (Graziola et al. 2020a; Mataix-Cols et al. 2020).\n\nAnother analysis, of the Swedish population registry, revealed significantly more substance abuse and consequences—including substance-related death—in people with TS (Virtanen et al. 2021). This result was not explained by other psychiatric illness nor by familial effects (assessed by comparison with their siblings without TS). This result adds substance-related death to suicide and accidental deaths previously found by this same group to be elevated in TS, and suggests clinicians should assess substance use in patients and arrange appropriate treatment. From the same group, it was shown that serious transport accidents occur more frequently in people with TS/connective tissue disease (CTD) but that this is largely explained by comorbid ADHD (Mataix-Cols et al. 2021). Finally, Fernández de la Cruz and Mataix-Cols review the emerging data on higher rates of general medical illness and mortality in TS based on their comprehensive work using the afore-mentioned Swedish population registry (Fernández and Mataix-Cols 2020).\n\nThe question as to whether the prevalence of TS might vary across the globe remains open. Previous studies suggested that TS might be rarer in Sub-Saharian Africa (also Japan) than in North America and Europe, where most epidemiological studies have been conducted so far. Rodin et al. challenge this assumption, rather proposing that adequate training and increased public awareness might result in higher recognition of TS in Uganda and elsewhere. This seems to be a sensible proposition, given that TS was considered ultra-rare just a few decades ago in North America and Europe (Rodin et al. 2021).\n\nIn 61 people with TS, being aware of signals for emerging tics (quality and intensity of premonitory urges) seems to facilitate self-initiated tic suppression, while ruminative tic-associated sensations did not, which lends support to the use of premonitory urges in behavior therapy of tics (Matsuda et al. 2020).\n\nSensory hypersensitivity is a frequent feature in patients with TS and should not be associated uniquely or primarily with autism spectrum disorder. In 34 adults with TS, Isaacs et al. confirm what had previously been described mostly in youth with TS, and they further show that sensory hypersensitivity is associated with obsessive-compulsive symptom severity (Isaacs et al. 2020). By the same group, a comprehensive review was carried out on this topic with an accent on pathophysiology of sensory processing dysfunction in TS and associated disorders (Isaacs and Riordan 2020).\n\nA revised version of the PUTS (Premonitory Urges for Tic Disorders Scale - PUTS-R) was proposed by Baumung et al., with slight rephrasing compared to the original, and divided into two subscales regarding urge severity and urge quality (Baumung et al. 2021). Also, the psychometric properties of the original scale were tested in a very large cohort of children (n = 658, subdivided into three age groups: 3 to 7 years, 8 to 10 years, 11 to 16 years) within the European Multicentre Tics in Children Studies (EMTICS) study. Contrary to previous findings, the PUTS also displayed good internal reliability in children under the age of 10. In children 11 years or older, sensory phenomena related to tics and mental phenomena observed in obsessive-compulsive disorder (OCD) could be distinguished. The authors conclude that questionnaires assessing premonitory urges might need to be age-specific (Openneer et al. 2020b).\n\nEmotional dysregulation is frequently observed in TS and thought to be related to the co-occurence of ADHD, eventually predisposing to explosive outbursts. However, it has so far been mostly assessed in parent-reported questionnaires. Using an observational measure, Hagstrøm et al. directly examined children with TS only, ADHD only, TS+ADHD, and controls. Emotional dysregulation was clearly dependent on the presence of ADHD and could not be observed in TS only (Hagstrøm et al. 2021).\n\nA well-written and comprehensive review on one of the foremost therapeutic challenges in TS, i.e., rage attacks: many questions remain open and much work needs to be done (Conte et al. 2020a). On this topic, Müller-Vahl et al. propose a revised version of their Rage Attack Questionnaire for adults, this becoming the RAQ-R. Testing this new tool in 127 patients with TS (compared to 645 controls), it was found that rage attacks correlated with and ADHD but, interestingly, could also be observed in “TS only” patients (Müller-Vahl et al. 2019).\n\nAggressive symptoms in youths with TS (n = 47, compared to 32 controls) appear to correlate with the severity of ADHD; overall, there was - somewhat surprisingly - no difference between the TS and the control group. Note, however, that agression and rage attacks may be correlated but are not identical entities (Benaroya-Milshtein et al. 2020).\n\nTwo up-to-date and complete review of sleep disorders in TS appeared, covering both adults and children (Jiménez-Jiménez et al. 2020; Hibberd et al. 2020). Importantly, self-injurious behavior in TS was comprehensively reviewed by Stafford and Cavanna (2020).\n\nOpenneer and colleagues studied a cognitive control task in children with TS, ADHD, neither or both (Openneer et al. 2019). Their results suggest that executive control impairment in TS could be explained by ADHD, not the tic disorder itself.\n\nKurvits and colleagues present a wonderfully thorough and thoughtful review of disinhibition as a unifying summary of tics and more complex symptoms in TS (Kurvits et al. 2020). They note strengths and weaknesses of this formulation and suggest future studies that may help resolve the debate.\n\nA large study (N = 720) compared compared autistic and compulsive phenomena in children with a clinical diagnosis of either TS or autism spectrum disorder (ASD) (Gulisano et al. 2020). The Children’s Yale-Brown Obsessive Compulsive Scale (CY-BOCS) for ASD measure was abnormal in patients with ASD or TS+ASD, but not in TS patients without ASD. Low and high scores successfully separated ASD from non-ASD, with or without TS, but scores between 1 and 14 on the CY-BOCS ASD did not adequately discriminate the two groups.\n\nGenetics\n\nDespite several studies published in 2020, the genetic factors contributing to TS remain largely unknown. These studies are divided into three main approaches: 1) whole exome sequencing (WES); 2) microarrays, which aim to identify rare coding variants or copy number variants with large effects; and 3) association studies that mainly focus on common variants. WES sequencing in a Chinese family with several affected members identified a missense variant in chloride voltage-gated channel 2 (CLCN2) (G161S), which was enriched in a TS cohort (Yuan et al. 2020). Loss-of-function variants in CLCN2, encoding chloride channel 2 (CLC-2), cause a leukoencephalopathy with ataxia, a recessive monogenic disorder (Depienne et al. 2013). The association of G161S with TS remain to be confirmed and its functional impact on the channel investigated. Another WES study conducted on 15 Chinese child-parent trios led to the identification of 25 coding de novo variants including two that likely disrupt gene function. The same study also identified rare compound heterozygous variants in cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) in one proband (Zhao et al. 2020b). CELSR3 encodes the Cadherin EGF LAG seven-pass G-type receptor 3 that may have an important role in cell/cell signaling during nervous system formation and is one of the few genes significantly associated with TS using WES (Wang et al. 2018) (Willsey et al. 2017). A review by Zhang and colleagues highlighted the possible role of variants in ASH1 Like Histone Lysine Methyltransferase (ASH1L) in the etiology of psychiatric disorders including TS, autism spectrum disorders and intellectual disability (Zhang et al. 2021). ASH1L encodes a histone-lysine N-methyltransferase specifically trimethylating Lysine 36 of histone H3 forming H3K36me3. De novo mutations in this gene mainly cause intellectual disability with autistic traits (Krumm et al. 2015). A single study making use of array comparative genomic hybridization identified a 260-kb duplication on chromosome Xq28 comprising two genes (Vesicle-associated membrane protein 7 - VAMP7 and Sprouty RTK Signaling Antagonist 3 -SPRY3) in a single female patient, inherited from her healthy father. The same duplication has been reported many times as likely benign in Decipher and do not lead to increase expression of the gene in blood, thus association with TS remains speculative. Several association studies focused on candidate genes or candidate regions have suggested possible association of rare or common variants in CNR1 (cannabinoid receptor 1), Lim Homeobox 6 (LHX6), Inner Mitochondrial Membrane Peptidase Subunit 2 (IMMP2L) and Arylacetamide Deacetylase (AADAC) with TS (Szejko et al. 2020) (Pagliaroli et al. 2020). These association studies were performed on case-control populations limited in size and need further replication. Furthermore, a study showed that deletions altering IMMP2L (encoding the mitochondrial inner membrane protease subunit 2) do not lead to a substantial mitochondrial dysfunction in fibroblasts of TS subjects, thus questioning the biological relevance of variants in this gene (Bjerregaard et al. 2020). Finally, a recent study showed that socioeconomic status and education have to be taken into account when studying genetic factors involved in TS as these constitute potential confounders limiting the power of current genetic studies studies (Wendt et al. 2021).\n\nEnvironmental risk factors\n\nA monumental and definitive review (for the time being) on the immunology (immune pathways, neuroinflammation, microbiome) of brain development in general and TS in particular was written by one of the foremost specialists in the field (Martino et al. 2020).\n\nA fascinating study was driven by the clinical observation that blinking tics, a common first symptom of tic disorder, are often mistaken for allergic conjunctivitis (AC) by families and primary care physicians. A group in southwest China studied 70 children with provisional tic disorder (PTD) and 70 tic-free controls and found that AC was more than 4 times more common in PTD (74%) than controls (17%) (Chen et al. 2020). They showed it could not all be symptom confusion, as quantitative measures of dry eyes and allergic responses to a skin prick test were also about four times more common in patients with PTD. These results suggest interesting ideas about immune abnormalities leading to tics. We suggest another possibility based on the common patient report that tics developed after a behavior repeated for another reason outlived its provoking stimulus and became chronic, e.g., a child coughed due to an upper airway infection, but then the cough persisted and became a tic. Perhaps sometimes tics develop when underlying host factors turn an externally triggered repeated behavior (like blinking or coughing) into a chronic symptom; previously, a rodent study showed that this two-hit scenario could cause a different movement disorder, dystonia (Schicatano et al. 1997).\n\nAnimal models\n\nRecanatesi et al. offer the interesting observation that sequences of self-initiated movements can be phenomenologically consistent, but their timing may differ substantially from one instance of a sequence to another (Recanatesi et al. 2020, in press). They used a rat model and cortical electrical recordings to inform a hidden Markov model. They showed that a model “produced by reciprocally coupling a high dimensional recurrent network and a low dimensional feedforward one” can produce certain “metastable attractors” with both predictable phenomenological patterns but highly variable timing. Since tics also often occur in stable sequences, a similar model may provide useful, testable hypotheses for how the brain generates such tic sequences.\n\nElectrophysiology\n\nCagle and colleagues reported an interesting study based on LFP recording of both the centromedian thalamic nucleus and the primary motor cortex in four TS patients following bilateral deep brain stimulation surgery (Cagle et al. 2020). They highlighted that beta power (12-30 Hz) was reduced in the primary motor cortex after both a tic and a voluntary action, while low-frequency power (3-10 Hz) was increased after a tic but not after a voluntary movement. They concluded to the identification of a tics’ specific signal within the centromedian thalamic nucleus which could be a target for developing closed-loop deep brain stimulation.\n\nA vast resting-state EEG study comparing young (7-15 years old) TS patients, chronic tic disorders patients and healthy controls revealed many interesting results (Naro et al. 2020). Among them, they highlighted a disconnection of the fronto-parietal network which could contribute to TS motor symptomatology, while a sensorimotor disconnection was revealed for both TS and chronic tic disorders patients as related to tic severity only. In addition, they identified the dynamic of tics in both groups of patients as follow: (1) for TS patients only, tics are preceded by a gamma (30-70 Hz) frequency activation and a beta2 (20-30 Hz) frequency deactivation in the posterior cingulate cortex and the supplementary motor area; (2) for both, tics onset are associated with alpha (8-13 Hz) and beta (13-30 Hz) deactivation within the sensorimotor areas; (3) for TS patients only, tics are followed by a gamma (30-70 Hz) and beta (13-30 Hz) frequency activation in the left dorsolateral prefrontal cortex, while for chronic tic disorders patients they are followed by a delta (2-4 Hz) and alpha (8-13 Hz) deactivation within the posterior cingulate cortex. Therefore, the dynamic of tics in TS and chronic tic disorders patients are differently disturbed, and the fronto-parietal network disconnection result reinforces the known pathophysiology of TS as related to an impairment of the limbic, paralimbic and cortico-striatal-thalamo-cortical pathways.\n\nSun and colleagues explored the suppressive effect of the motor system on the sensory system in TS patients (Sun et al. 2020). They used a sham-controlled repetitive Transcranial Magnetic Stimulation (rTMS) protocol (1Hz, 90% of the resting motor threshold) and recorded the somatosensory evoked potentials before and 15 minutes after rTMS. If somatosensory evoked potentials amplitudes were decreased for both TS patients and healthy controls, the decrease was stronger for TS patients. They interpreted this finding as a suppressive effect of the motor-sensory system on the sensory system instead of a sole influence of the motor system, and therefore as TS resulting more from a sensorimotor disorder than a sole motor dysfunction.\n\nRae and colleagues provide a high-quality study of action inhibition in TS comprising 23 adults with TS and 21 healthy controls using the same intentional inhibition task (Rae et al. 2020). Importantly, the authors chose a task that did not directly involve tics, so both groups could participate equally in inhibiting a movement. Several inhibitory regions were more active in TS, especially right inferior frontal gyrus, plus insula and basal ganglia. Even though participants did not move, the primary cortex was more active during the task in the TS group but less active in the control group. Finally, during a task in which participants decide on their own whether or not to move a finger, premonitory sensations correlated with functional connectivity of the pre-supplementary motor area (SMA) region to caudate, globus pallidus and thalamus.\n\nHippocampal volume was increased both in TS and in 41 children with PTD compared to tic-free control children (Kim et al. 2020). Since the PTD group was studied a mean of only 4 months after tic onset, this difference cannot be due to living with or adapting to tics for years, an advantage over all studies in TS itself. Excitingly, in the PTD group, a larger hippocampus at the initial visit predicted worse tic severity at one-year follow-up, comprising the first predictive biomarker identified for PTD (Kim et al. 2020). Surprisingly, striatal volume at baseline did not predict tic severity at follow-up.\n\nBhikram and colleagues reported a resting-state functional magnetic resonance imaging (fMRI) study in TS that included 39 TS patients and 20 controls, analyzed using seed-based functional connectivity (fcMRI) methods (Bhikram et al. 2020). The TS group showed greater connectivity between the temporal gyri, insula and putamen, and between orbital frontal cortex (OFC) and cingulate cortex. Tic severity correlated with increased connectivity of the putamen with sensorimotor cortex. By contrast, OCD severity correlated with decresae connectivity between SMA and thalamus and between caudate and precuneus. Finally, premonitory urge severity correlated with decreased connectivity between OFC and primary sensorimotor cortext, and inferior frontal gyrus correlated with putamen and insula. Perhaps surprisingly, even though only the first symptom domain reflects actual movement, all three symptom-related networks include sensorimotor regions.\n\nZapparoli and colleagues reported two interesting studies of the experience of agency, i.e., the appreciation that we intentionally acted, and our actions caused the observed consequences (Zapparoli et al. 2020a,b). These studies involved 25 adults with TS and 25 tic-free control participants, and used an implicit, indirect measure of agency called the intentional binding phenomenon, in which people judge the delay between an action (e.g., pressing a button) and its effect (e.g., the turning on of a light) to be shorter with intentional than with passive movement. The earlier report gives results from tic-free participants studied with fMRI and TMS to the pre-SMA. The second report shows that the TS group did not show significant intentional binding or correlation with activity in the network identified in the control group. The authors interpret the results as consistent with impaired action monitoring and an impaired sense of agency in TS, which may contribute to the perception of some people with TS that tics are fully involuntary.\n\nRage attacks in TS were examined using structural and functional MRI network methods in 55 patients with TS, 47% of whom had intermittent explosive outbursts (IEOs) (Atkinson-Clement et al. 2020c). The group with IEOs (TS+IEO) was compared to the remaining (TS−IEO) group, and showed increased fractional anisotropy in the right SMA and right hippocampus, and decreased mean diffusivity in the left OFC. Those three regions were used as seeds for resting state fcMRI. The TS+IEO group showed lower connectivity within a sensorimotor cortical-basal ganglia network, and altered connectivity among OFC, amygdala, and hippocampus. These results suggest that IEOs are associated in TS with disrupted white matter and associated functional connectivity in circuits related to action selection, emotion regulation, impulse control, and aggression.\n\nUsing diffusion tensor imaging and subcortical regions of interest in 15 children suffering from TS and 15 healthy controls, Xia and colleagues found decreased fractional anisotropy (FA, related to white matter myelin integrity, fiber compactness and parallelism) in the left globus pallidus and the left thalamus and an increased apparent diffusion coefficient (ADC, related to the molecular diffusion rate) increased in the right caudate nucleus and the thalamus bilaterally in TS patients (Xia et al. 2020). Moreover, the decreased FA within the left thalamus was related to the YGTSS score.\n\nAn MRI surface-based study on an important sample of 60 TS patients and 52 healthy controls was also published this year (Kong et al. 2020). They identified several changes regarding cortical thickness and cortical curvatures, essentially distributed within the frontal, parietal and temporal cortices, but they found no difference regarding local gyrification.\n\nFrequency-specific regional homogeneity (ReHo) was also assessed in children’s patients (Lou et al. 2020). This study revealed an increased ReHo in the left precentral gyrus and a decreased in the right operculum. They also identified ReHo changes in some specific frequency bands within the superior frontal gyrus, the superior parietal gyrus, the anterior cingulate gyrus, the putamen, the superior temporal gyrus and the operculum.\n\nUsing graph theory on resting-state fMRI, the study of Openneer et al. demonstrated that TS is related to dysfunction within the default mode (for local efficiency and clustering coefficient) and that tic severity is correlated with dysfunction within both the fronto-parietal and the default mode networks without relation with ADHD comorbidity (Openneer et al. 2020a). This suggests an immature topological brain organization specifically related to TS.\n\nA fascinating study on endocannabinoids was reported using cerebrospinal fluid (CSF) from 20 adults with TS and 19 controls (Müller-Vahl et al. 2020a). The authors measured anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoyl ethanolamide (PEA), and arachidonic acid (AA). The key results were that “CSF AEA (p = 0.0018), 2-AG (p = 0.0003), PEA (p = 0.02), and AA (p < 0.0001) were significantly increased in TS compared with controls,” and that “levels of 2-AG correlated with the severity of comorbid ADHD (p < 0.01).” The authors note these differences could relate to compensation for chronic tics, or may be causative.\n\n137 children with CTD were assessed at baseline, during a tic exacerbation, and 2 months later (Addabbo et al. 2020). Serum anti-dopamine-2 receptors (D2R) antibodies were measured. 8% had anti-D2R antibodies during the exacerbation, and 8% of those with 2-month data at 2 months after the exacerbation. The αD2R antibodies were significantly associated with exacerbations, with or without correction for patient characteristics including medication use. These antibodies may possibly worsen tics via antibody receptor blockade. Further research is needed to clarify the causal role. See also the commentary by (Conceição 2020).\n\nImpaired associative learning was shown in 46 children with TS compared to 46 matched control children who performed the Rutgers Acquired Equivalence Test (face and fish test) (Eördegh et al. 2020). This test includes an acquisition phase (associating two visual stimuli based on feedback of correct vs. incorrect), which depends on intact basal ganglia function, and a test phase (retrieval of previous association and generalization to predictable new stimuli), which depends on the hippocampus and medial temporal lobe. The TS group performed worse on the acquisition phase (number of trials and accuracy), regardless of comorbid ADHD, OCD, autism spectrum disorder or medication status. However, they performed normally on retrieval and generalization. Compare two prior studies showing that people with TS have abnormal probabilistic classification learning, which also involves the dorsal striatum (Kéri et al. 2002; Marsh et al. 2004).\n\nInhibitory control continues to be a matter of debate in TS. In this topic, a first study explored reactive inhibitory control in adult patients by using a stop-signal task (Atkinson-Clement et al. 2020b). Reactive inhibition was not impaired in all TS patients but only in medicated patients (essentially aripiprazole). In addition, impairment in this group was underpinned by brain structures and functional connectivity of the fronto-temporo-basal ganglia-cerebellar pathway.\n\nA second study from the same group focused on another form of motor impulsivity called “waiting impulsivity” defined as the difficulty to withhold a specific action (Atkinson-Clement et al. 2020a). The authors demonstrated that this form of impulsivity is present in TS patients and correlates with tics severity but is normalised by medication (mainly aripiprazole). In addition, waiting impulsivity in unmedicated TS patients was related to abnormal gray matter intensity in deep limbic structures, and with connectivity between cortical and cerebellar regions.\n\nA third and very interesting study compared automatic and volitional inhibition in 19 adult patients with primary tic disorder in comparison to 15 healthy controls (Rawji et al. 2020). They used a conditional stop-signal task associated with motor cortex TMS to assess reactive volitional inhibition, and a masked priming task to assess proactive automatic inhibition. This opposition is of particular interest since volitional inhibition could be increased to prevent tics to reach the threshold for expression, while automatic inhibition could prevent the initial excitation of the striatal tic focus. The authors found that volitional movement preparation, execution and inhibition are not impaired in patients. Conversely, automatic inhibition was found as impaired in patients which was also correlated to tic severity.\n\nOn the same theme of voluntary movements, Mainka et al. (2020) published a follow-up study of a previous one on mental chronometry (Ganos et al. 2015). If they found no difference between TS patients and healthy controls on the estimated time of their own voluntary movements and the conscious intention to make a voluntary movement, they identified a linear association between both these variables and the disease duration. The longer the disease duration, the lesser the performances were changed from the data of the first study. For the authors, the chronic tics persistence at adulthood could be associated with developmental impairment of internal premotor processing.\n\nTo go further on the assessment of perception-action impairment in TS, members of the same group published an interesting study (Kleimaker et al. 2020). Based on the theory of event coding (Hommel et al. 2001; Hommel 2009), a visual-motor event file task and EEG recording, they found that perception-action binding was increased in Tourette patients and partially correlated with tic frequency. Interestingly, electroencephalography (EEG) results revealed that this process was not solely related to motor and perceptual processes, but also to cognitive processes (i.e., involving the inferior parietal cortex). Based on these results, they conclude that the investigation of perception-action binding in TS is more relevant than the assessment of only motor or perception alone.\n\nThe association between real and perceived action in TS was also assessed by using a finger-tapping synchronization task (Graziola et al. 2020b). Interestingly, the authors observed an impaired temporal control in two opposite ways for TS and TS+ADHD patients. The first were “behind the beat”, the second were “ahead of the beat”. This confirmed that TS is related to an impairment of temporal motor control.\n\nThis year, two articles also assessed reward evaluation in TS. The first one revealed that adolescents with TS present a higher delay discounting, specifically for large rewards (Vicario et al. 2020). In other words, if they have the choice between a large immediate reward and a larger delayed reward, TS patients are less likely to wait for the larger option than healthy controls. This result is of importance and could contribute to the debate on impulsivity from a more cognitive standpoint. The second one used a reinforcement learning task with various reward probabilities (Schüller et al. 2020). The authors showed that TS patients had lower learning curves than healthy controls, but also that reaction time of the healthy was influenced by the reward amount which was not the case for TS patients. In addition, EEG recording revealed an attenuated P3a (positive fronto-central peaking) modulation was found in TS, which was interpreted as an impaired coding of attention allocation.\n\nPsychological interventions\n\nBehavior therapy (BT) is considered to be the first line treatment since publication of the 2019 American Academy of Neurology guidelines, based on controlled randomized trials. In a naturalistic setting (children and adolescents with chronic tics, n = 74) and over a 12 month follow-up period, it could be demonstrated that BT is and remains effective in 75% of patients analyzed, attesting not only to its efficacy but durability (Andrén et al. 2021).\n\nInternet-based BT programs are investigated by multiple groups to make BT available to a larger number of patients, rendering it thus independent on the availability of trained practitioners and financial considerations in countries where psychotherapy is not reimbursed by social security. Rachamin et al. offer preliminary data on internet-based guided self-help comprehensive behavioral intervention for tics (I-CBIT) in 25 youths (passive control group/waiting list, n = 16), and show this approach to be both effective and well received over a 6-month period. Larger trials including an active control group are necessary to confirm these first positive results (Rachamim et al. 2020).\n\nAnother way to increase access to BT for tic treatment is group training. The “Tackle your Tics” program is an intensive four-day course based on an enhanced version of ERP (exposure and response prevention). First results in 13 youths offer promising results regarding tic reduction and increased quality of life, with a two month follow-up period. Larger controlled trials with longer follow-up periods are awaited (Heijerman-Holtgrefe et al. 2020).\n\nStill another approach is to train parents as therapists. For that purpose, an instructional video guide (on DVD) based on habit reversal training was developed and applied (n = 33) and compared to in-person training (n = 11) in children (mean age 10 years). Home-based, parent-administered HRT was as efficacious for tic reduction as traditional in-person training. However, the drop out rate in the former group was close to 50%, so that the authors advocate regular phone contacts during the DVD treatment course, which squares with other hybrid formats such at BipTic (Singer et al. 2020).\n\nA very small (n = 3) case series described an interesting new BT technique based on attention training to suppress tics: to be followed (Schaich et al. 2020).\n\nSo far, BT is usually proposed for children above the age of ten. In this very interesting proof of concept study, Bennett et al. test a comprehensive behavior intervention for tics (CBIT) format (“CBIT-Jr.”) for children ages 5-8 (n = 16) and show positive response (tic reduction) and acceptance. Moreover, they monitor these improvements over a one-year period and speculate that early BT might alter the chronic course of tics: this is a very important subject and should be investigated in larger, longitudinal cohorts (Bennett et al. 2020).\n\nRemarkably, CBIT was shown as also normalizing inhibitory control in a specific task of perception-action bindings (Petruo et al. 2020).\n\nNeurosurgery\n\nAn Italian center reports their experience with anterior globus pallidus internus (GPi) vs. thalamic centromedian-parafascicular complex (Cm-Pf) deep brain stimulation (DBS) for TS (Servello et al. 2020). Forty-one TS patients had DBS in ventro-oralis/centromedian-parafascicular thalamus and 14 had DBS in anteromedial GPi. The authors followed them for 4 years. YGTSS and Y-BOCS improved in both groups (p < .001), but Y-BOCS improved more in the GPi group. Hardware removal was limited to the thalamic DBS group (13/41, vs. 0/14 in the GPi group).\n\nThe DBS registry (n = 66 bilateral GPi, n = 32 centromedian [Cm] thalamus) has provided additional important information (Johnson et al. 2020). Probabilistic tractography from estimated volumes of tissue activated (VTAs) was used to identify networks correlated with improvement in tics or OCD symptoms. Cleverly, these networks were in turn used as seed regions for “reverse” tractography to identify local “hot spots” and “cold spots.” For GPi targets, connectivity to limbic and associative networks, caudate, thalamus and cerebellum predicted clinical improvement scores. The anteromedial GPi showed higher connectivity to this network, and the extent to which estimated VTA overlapped with this anteromedial region correlated with tic improvement. For Cm targets, connectivity to sensorimotor and parietal-temporal-occipital networks, putamen and cerebellum correlated with tic improvement. The anterior/lateral part of the Cm region was more highly connected to this network. For both sites, connectivity to prefrontal, orbitofrontal and cingulate cortex correlated with OCD improvement. These results suggest that structural connections of focal stimulation sites to specific networks may lead to clinical benefit. Interestingly, the identified networks may differ not only by symptom but also based on the surgical target region.\n\nA German study explained the effect of centromedian thalamic nucleus deep brain stimulation by using probabilistic tractography in 7 TS patients (Andrade et al. 2020). They highlighted that the tic reduction following DBS was related to the degree of stimulation-dependent connectivity between the centromedian thalamic nucleus and the pre-supplementary motor area. Conversely, non-responders had more active fibers that project into non-motor cortical areas.\n\nOther treatments\n\nA fascinating report from the University of Nottingham described a potential novel treatment for tics that uses the peripheral nervous system to induce changes in primary sensorimotor cortex (Morera Maiquez et al. 2020). The radical new idea arose from observations associating movement inhibition with 8-14 Hz activity in motor cortex. The authors first showed that rhythmic 12 Hz peripheral stimulation of the median nerve evoked synchronous contralateral EEG activity over primary sensorimotor cortex, whereas arrhythmic stimulation at the same mean rate did not. As hypothesized, median nerve stimulation (MNS) at 12 Hz created small but statistically significant effects on initiation of voluntary movements. Importantly, this stimulation did not meaningfully impair concentration, suggesting that the effect did not operate through simple distraction. Next, they tested 10 Hz MNS in 19 TS patients, and demonstrated using blinded video ratings a significant reduction in tic number and severity during 1-minute stimulation epochs vs 1-minute no-stimulation epochs. Videos accompanying the publication showed dramatic benefit during MNS in some subjects.\n\n(Sukhodolsky et al. 2020) report intriguing results from a controlled, crossover design, pilot study of real-time fMRI neurofeedback. Tics improved more with real than sham feedback, the improvement was clinically meaningful (3.8-point decline in YGTSS total tic score), and the effect size was 0.59. Surprisingly, however, the two treatment conditions did not differ in the putative mechanism of benefit, namely control over SMA activity. An accompanying commentary is also useful (Coffey 2020).\n\nThe role of the microbiome in the etiology and pathogenesis of various CNS disorders has attracted widespread interest over the past decade, with fecal transplantation being hailed as a potential treatment. Zhao et al. (2020a) report in an exploratory trial in children with TS that this approach resulted in a significant tic decrease (>25% on the YGTSS-TTS) in four out of five subjects during the 8-week trial period. However, there was no placebo group and larger, randomized trials are warranted.\n\nPhysical exercise is advocated as positive for a plethora of somatic and mental disorders these days, and TS is no exception. Jackson et al. propose that aerobic exercise training (kick boxing) decreased tic frequency in young people with TS (n = 18, age 10-20 years), likely through enhancement of cognitive control (Jackson et al. 2020). Interestingly, tic frequency reduction was less in a Tai Chi group, in which cognitive control enhancement was not significantly altered compared to controls. Thus, the type of physical exercise is important, “aerobic” being the key word here (Jackson et al. 2020). In the same vein, but on an observational basis, Pringsheim et al. report that in 110 children with TS, less vigorous physical activity indeed correlated with tic severity. This negative correlation could also be found for light exposure and subjective sleep quality (Pringsheim et al. 2021).\n\nSpanish researchers conducted an open trial of a gluten-free diet in 34 TS patients (mostly children) without celiac disease (Rodrigo et al. 2018). After a year, in the 29 patients who did not withdraw due to dietary noncompliance, tics, OCD symptoms and quality of life were all improved substantially compared to baseline. Prospective data on a dietary intervention, as in this study, are greatly needed. However, this study design cannot exclude improvement due to expectation effects or regression to the mean, so a randomized, controlled trial is essential before we can justify adding dietary restrictions to treatment recommendations.\n\nA survey of 90 respondents from 13 countries showed that online support communities offer valuable informational and emotional support to those living with tic disorders/TS and their families, especially in the light of local face-to-face support that is often lacking. However, some disadvantages also became apparent, such as the suggestible nature of tics and being reminded of the challenging nature of tic disorders. Also, some conflict arising within online communities was noted (Perkins et al. 2020).\n\nComplementary and alternative medicine (CAM) blossoms in all of medicine, treatment of TS is no exception. A survey of 110 patients with TS showed that more than two thirds used one or more CAM therapies. The most popular were: stress management, herbal medicine, homeopathy and meditation. 93% reported a decrease in tic frequency and 46% considered CAM more efficient than medication (Patel et al. 2020). Patients reported they often did not mention CAM treatments to their treating physicians, placing the onus on clinicians to ask patients specifically about them. These results also support the crucial need for randomized, controlled trials of any intervention.\n\n\nConclusions\n\n2020 was a rich year in terms of publications and confirms the impression that TS draws increased attraction in the neuroscience community. Hopefully, this will eventually lead to larger collaborative efforts and, especially, longitudinal studies on TS and comorbidities, with a special emphasis on transition from childhood to adulthood.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nAddabbo F, Baglioni V, Schrag A, et al.: Anti-dopamine D2 receptor antibodies in chronic tic disorders. Dev. Med. Child Neurol. 2020; 62: 1205–1212. PubMed Abstract \| Publisher Full Text\n\nAndrade P, Heiden P, Hoevels M, et al.: Modulation of Fibers to Motor Cortex during Thalamic DBS in Tourette Patients Correlates with Tic Reduction. Brain Sci. May 2020; 10(5): 302. PubMed Abstract \| Publisher Full Text\n\nAndrén P, Wachtmeister V, Franzé J, et al.: Effectiveness of Behaviour Therapy for Children and Adolescents with Tourette Syndrome and Chronic Tic Disorder in a Naturalistic Setting. Child Psychiatry Hum. Dev. 2021; 52: 739–750. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "119803", "date": "08 Feb 2022", "name": "Lorena Fernández de la Cruz", "expertise": [ "Reviewer Expertise Tourette syndrome", "OCD", "epidemiology", "CBT" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a scoping review of the published literature on the topic of Tourette Syndrome (TS) during 2020. The search is not designed to be exhaustive (e.g., only one database is exploded) and the highlighted articles, presented in narrative form, have been selected based on the subjective judgement of the authors. This, however, works very well as a text-book summary for those in and outside the field of TS, turning it into a compulsory yearly read. I commend and congratulate the authors for doing this for yet another consecutive year.\nMy only minor criticism (or rather a wish) is that this 2020 review could have appeared earlier. I sincerely appreciate the effort and time behind this manuscript, but the more than 1 year lapse between the review content and the current date makes some (not all) sections appear outdated when I read them now (e.g., functional tics in relation to the Covid-19 pandemic, and some of the treatment parts). On the other hand, some time lag is to be expected, and it is good to see the quick development of the field.\nI have some minor suggestions for improvement of the text, mainly referred to typos, if the authors believe that they are helpful:\nIn the Methods section, the authors link the output of their PubMed search. When clicking the link, this search returns 293 citations. However, in the text, the authors write that this number is 292. Please amend.\n\nIn the Results>Epidemiology section, CTD is incorrectly defined as “connective tissue disease”. Please change this to “chronic tic disorder”.\n\nResults>Other section: I would appreciate if the authors could add a more informative summary of the systematic review by Conte et al. about rage attacks (it currently reads: “many questions remain open and much work needs to be done”).\n\nPlease note that the two references by Conte et al. 2020a and 2020b have been cited in the wrong order. Conte et al 2020b (rage attacks in the Reference list) is cited in the Covid section, while Conte et al 2020a (Covid-19 survey in the Reference list) is cited in the rage attacks paragraph.\n\nResults>Other: “Two up-to-date and complete review of…” Please change “review” to “reviews”.\n\nPathophysiology>Electrophysiology section: I believe the following sentence does not read well: “They concluded to the identification of a tics’ specific signal within the centromedian thalamic nucleus which could be a target for developing closed-loop deep brain stimulation.” If I understand the conclusion correctly, it should probably say: “The authors identified a tics’ specific signal within the centromedian thalamic nucleus could be a target for developing closed-loop deep brain stimulation.”, or similar wording.\n\nClinical and neuropsychological studies section: Please revise the following sentence: “However, they performed normally on retrieval and generalization. Compare two prior studies showing that people with TS have abnormal probabilistic classification learning, which also involves the dorsal striatum.”\n\nTreatment>Psychological interventions: In the paragraph summarizing the paper by Singer et al., BIP TIC is mentioned, but it is not explained what it is and no reference for the reader is given (the reference by Singer is cited at the end of the sentence but this paper does not seem to mention BIP TIC).\n\nOther treatments section: The name of the authors Sukhodolsky et al. should be outside the parenthesis.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "7790", "date": "09 Feb 2022", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank Dr. Fernández de la Cruz for her thoughtful and thorough review. We will address her helpful suggestions after receiving those of a second reviewer." }, { "c_id": "7797", "date": "11 Feb 2022", "name": "Andreas Hartmann", "role": "Author Response", "response": "We thank Dr. Fernández de la Cruz for her thoughtful comments and careful review of our manuscript. To begin with, we fully agree that submission this year occurred much too late, for which we would like to present our apologies. Frankly speaking, 2021 was a bit of an annus horribilis for us, with delays due to COVID-related, family and authorship matters. Usually, these reviews appear in the first half of the upcoming year, as will be the case again in 2022 for the 2021 review. As to the comments raised, we agree with all of them, and they were accordingly addressed." } ] }, { "id": "127168", "date": "13 Apr 2022", "name": "Laura Zapparoli", "expertise": [ "Reviewer Expertise Cognitive Neuroscience", "Motor Control", "Motor Cognition", "Neuroimaging", "Movement Disorders." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, Hartmann and colleagues provide a thoughtful summary of the most relevant articles published on Gilles de la Tourette Syndrome in 2020. The included papers are adequately chosen and well-presented, with a clear and readable style. In my opinion, it may represent a useful tool for both researchers and clinicians in the GTS and related fields.\nI have only some minor comments.\np.2: The authors mentioned the deployment of telemedicine services due to COVID-19, which was explicitly discussed for GTS by Cen and colleagues (2020). I would appreciate some details on the domain of telemedicine and GTS.\np.2: please, define the “ruminative tic-associated sensations”.\np.3: please, add some details about the two subscales of the Revised Premonitory Urges for Tic Disorders Scale (e.g., what does “urge quality” refer to?).\np. 3: The authors mentioned two “up-to-date and complete reviews of sleep disorders in GTS”. I would add some details/results of these reviews.\np. 5: The authors reported a seed-based functional connectivity study by Bhikram et al. (2020): which seeds were considered?\np. 5: The authors mentioned the study on the sense of agency in GTS by Zapparoli et al. 2020b: I would add that the more reduced was the individual sense of agency, the more severe were the motor symptoms correlations, measured with Yale Global Tic Severity Scale.\np. 7: I would add some details about the durability of GTS's behavioral therapy.\np. 8: The acronym “HRT” is not defined in the manuscript.\np. 8: The authors mentioned a large-scale DBS study using the pallidus internus and the thalamic centromedian-parafascicular complex as target. Can the authors specify the timing of the follow-ups of the YGTSS/YBOCS improvement?\n\nIn general, I would be consistent in reporting (or not) the statistical details of each study (e.g., p-values).\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "8155", "date": "26 Apr 2022", "name": "Andreas Hartmann", "role": "Author Response", "response": "We thank Dr. Zapparoli for her thoughtful comments and careful review of our manuscript. Concerning the comments raised, they have been addressed to the best of our abilities. Nonetheless, we would like to point out that the aim of this yearly review is not to present studies in detail, but rather point interested readers to papers which they would like to read themselves if their interest has been peaked. However, since this is a multi-author paper, some differences in style and result presentation are inevitable (which includes the presentation of p values, CI etc.), despite our efforts to harmonize the paper. Also, apart from original studies, summarizing reviews is a complicated affair since they already represent summaries, but we tried to highlight the salient points." } ] } ]	1	https://f1000research.com/articles/11-45
https://f1000research.com/articles/10-1171/v1	18 Nov 21	{ "type": "Systematic Review", "title": "Smokeless tobacco use and reproductive outcomes among women: a systematic review", "authors": [ "A.G. Radhika", "Sutapa B. Neogi", "Preetha GS", "Sumant Swain", "Jaswinder Kaur", "Jagdish Kaur", "Sutapa B. Neogi", "Preetha GS", "Sumant Swain", "Jaswinder Kaur", "Jagdish Kaur" ], "abstract": "Background: Both smoked and smokeless tobacco use have deleterious effects on most major organ systems including the reproductive system. We conducted a systematic review on smokeless tobacco (SLT) use and reproductive outcomes among women. Methods: We searched Pubmed, ProQuest, Cochrane, Wiley and Emerald databases for studies involving smokeless tobacco use in women with any or a combination of three conditions: infertility, menstrual disorders and pelvic inflammatory disease (PID). Eligibility criteria included English language publications from 1st January 1990 - 31st October 2020. CADIMA software used for filtering the studies and modified SIGN checklist for the quality assessment. The findings are reported as per the PRISMA guidelines. The AXIS and ROBIN E tool were used for assessment of risk of bias. Results: In total, three studies addressed our research question. Two studies addressed infertility (prospective cohort: n=501, cross sectional: n=192) of which, the cross-sectional study compared the mean cotinine levels between those with infertility, menstrual disorders and PID. This study also explored the association between SLT and PID. PID was the most common gynecological complaint. Women with PID had significantly higher urinary cotinine levels = 24.95±12.259) ng/ ml (p=0.0144). Mean urinary cotinine in women with menstrual complaints was 19.32±10.29 ng/ml. The other study used population-based sampling of 501 couples who attempted pregnancy (enrolled in the LIFE Study). Results showed that only 2% (n=28) of men and none of the women used smokeless tobacco. Compared with never users of tobacco, smoking by females was individually associated with longer time-to-pregnancy; smoking among males remained significant when modeling partners together. Conclusions: Available studies exploring associations between SLT and reproductive outcomes are inconclusive due to limitations in the study methodologies. More studies with robust study designs are required from low- and middle-income countries with high prevalence of SLT use.", "keywords": [ "Smokeless Tobacco", "infertility", "menstruation", "abnormal uterine bleeding", "pelvic infections", "reproductive health", "women" ], "content": "Introduction\n\nUse of both smoked and smokeless forms of tobacco is a major cause of preventable morbidity and mortality. It kills half of all its lifetime users (WHO, 2011) and more than 8 million people each year, out of which, 1.2 million die due to second hand smoke (WHO, 2019). Toxic and carcinogenic chemicals in tobacco along with other ingredients that are added to them are known to be causally associated with several non-communicable diseases (NCDs) including cancer, especially oral cancer which is the leading cancer among men and the third most common cancer among women in India (Bhisey, 2012).\n\nSmokeless tobacco (SLT) is “consumed without combustion at the time of use” (WHO, 2015). It is generally used orally (sucked, chewed, dipped or held in the mouth, used as dentifrice or toothpaste) or nasally resulting in nicotine absorption across the mucous membrane, along with other chemicals. Majority of SLT users, approximately 286 million people, live in low and middle-income countries in South-East Asia region. Three countries, namely India, Bangladesh, and Myanmar, host around 86% of the global users (NCI &CDC, 2014). As per Global Adult Tobacco survey 2016-17, women accounted for 2% among around 99.5 million adults current smokers. In contrast, 12.8% of women used SLT out of 199.4 million adults (GATS, 2017).\n\nSLT use is addictive; it leads to oral health problems and plays a contributory role in the development of cardiovascular disorders, fatal ischemic heart disease, stroke, peripheral vascular diseases, peptic ulcers, type 2 diabetes, chronic rhinitis, foetal morbidity and mortality (WHO, 2015; Inamdar et al., 2015; Suliankatchi and Sinha, 2016; Hossain et al., 2017). The leading health consequences related to SLT use in Southeast Asia include cancers of numerous sites along with poor reproductive outcomes (World Health Organization. News release 11th Sept 2013).\n\nTobacco use in India is majorly considered a male-dominant behavior. However, over the past decade, the use of SLT products by Indian women is substantial and increasing, with adverse consequences for oral (Niaz et al., 2017; Singh et al., 2020) and perinatal health (Inamdar et al., 2015; Suliankatchi and Sinha, 2016; Nair et al., 2015). Women who use SLT are at risk of oral (Singh et al., 2020) and pharyngeal cancers (Niaz et al., 2017; Datta et al., 2014; Sinha et al., 2016; Awan and Patil, 2016), esophageal cancer (Niaz et al., 2017; Datta et al., 2014; Sinha et al., 2016; Awan and Patil, 2016), cervical cancer, ischemic heart disease (IHD) (Sinha et al., 2018) and osteoporosis (Ayo-Yusuf and Olutola, 2014). Compounds in SLT products such as nicotine act as neuro-teratogens as they can cross the placental barrier (Liao et al., 2012) affecting the fetal development along with other pregnancy complications like pre-term delivery, low-birth weight (Inamdar et al., 2015; Suliankatchi and Sinha, 2016) increased stillbirth risk (Hossain et al., 2017) and risk of cancers in the developing fetus (Rogers et al., 2009). However, there is little evidence that explores the association between SLT use and reproductive health of women. Therefore, we planned to systematically conduct a review on smokeless tobacco (SLT) use and reproductive health among women.\n\n\nMethods\n\nThe protocol was registered on PROSPERO on 2nd October 2020 (CRD42020207176). This paper is reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Radhika, 2021).\n\nWe did an extensive literature search that described the association between SLT use and selected reproductive outcomes in women. For this, we selected three common reproductive outcomes, namely infertility, menstrual disorders and pelvic inflammatory disease. The search terms revolved around P (population): women, E (exposure): exposure to SLT, C (control): women who were not exposed to SLT, O (outcome): reproductive outcomes in women that included infertility, menstrual disorders, and pelvic inflammatory disorder (PID). The reference period was from 01 January 1990 to 31st October 2020.\n\nPublications including reviews, original trials and conference proceedings documenting any form of SLT use along with the three selected reproductive outcomes in English language within the timeframe were considered for the inclusion.\n\nStudies were identified by searching electronic databases (Pubmed, Wiley, Cochrane Library, Emerald and ProQuest) in addition to scanning reference lists of articles using the chosen keywords for all three reproductive outcomes separately.\n\nSearch strings used for infertility among women and SLT were: (Smokeless OR Chew OR Chewing OR Dipping OR Snuff OR toothpaste OR dentifrice OR paan masala OR betel quid OR betelquid OR areca nut OR arecanut) AND (Tobacco OR Tobaccos OR Gutka OR Gutkas OR Snuff OR Mint) AND (Female OR Women) AND (Infertility OR Subfertility OR Sub Fertility OR sterility OR Infertile OR sterile).\n\nSearch strings used for Menstrual disorders and SLT were: (Smokeless OR Chew OR Chewing OR Dipping OR Snuff OR toothpaste OR dentifrice OR paan masala OR betel quid OR betelquid OR areca nut OR arecanut) AND (Tobacco OR Tobaccos OR Gutka OR Gutkas OR Snuff OR Mint) AND (Female OR Women) AND (menstrual OR menstruation OR bleeding OR menorrhagia OR Hypermenorrhea OR Hypermenorrhoea OR Hypomenorrhea OR Hypomenorrhoea OR dysmenorrhea OR dysmenorrhea OR Abnormal uterine bleeding OR amenorrhea OR menometrorrhagia OR metrorrhagia OR dysfunctional uterine bleeding.\n\nFor PID and SLT use, search strings used were (Smokeless OR Chew OR Chewing OR Dipping OR Snuff OR toothpaste OR dentifrice OR paan masala OR betel quid OR areca nut) AND (Tobacco OR Tobaccos OR Gutka OR Gutkas OR Snuff OR Mint) AND (Female OR Women) AND (infection OR inflammation OR pelvic inflammatory disease OR PID OR genital infections OR genital tuberculosis OR tubo-ovarian mass OR Salpingitis OR salpingo-oophoritis OR endometritis OR adnexitis OR parametritis).\n\nWe included studies irrespective of sample size. After duplicate removal by CADIMA, every record was screened by two reviewers independently using the title. Those accepted by both were subjected to abstract review. In case of any disagreement, arbitration was done by a senior member from the team. For the selected abstracts, full articles were obtained, and quality check was performed by two reviewers independently using the modified SIGN checklist. Those selected at this stage were eligible for the review. The study selection is mentioned according to reproductive outcomes selected and reported according to PRISMA guidelines. The AXIS tool was used to assess risk of bias (Ma et al., 2020) for cross sectional study and the ROBIN E tool was used for prospective studies.\n\nFull text appraisal for study selection was done by two authors independently. Title & abstract filtering was done with CADIMA. Information was extracted from all the eligible studies on a predesigned format (Tables 2–4) including a range of study variables relating to the design, objectives, and outcomes. For each of the reproductive outcomes, studies included were summarized separately.\n\n\nResults\n\nSearch for infertility among women and SLT use resulted in 1093 results which were run through CADIMA for removal of the duplicate studies, which gave a final of 1062 results (9 from Pubmed; 44 from Wiley; 6 from Emerald; 11 from Cochrane and 992 from ProQuest). that for Menstrual disorders and SLT gave 1330 results. These results were run through CADIMA for removal of the duplicate studies, which gave a final of 1294 results (44 from Pubmed; 19 from Wiley; 25 from Cochrane and 1206 from ProQuest). For PID and SLT use, there were 3929 results. These results were run through CADIMA for removal of the duplicate studies, which gave a final of 3808 results (205 from pubmed; 27 from wiley; 46 from Cochrane and 3530 from proquest) (Table 1).\n\nThe search revealed a total of two studies (prospective cohort, n = 501 and cross sectional, n = 192) addressing our research question related to infertility (Figure 1, Table 2).\n\nIVF = in vitro fertilization.\n\nTTP = time-to-pregnancy; FOR = fecundability odds ratio; OR = odds ratio; CI = confidence interval.\n\nThe prospective cohort study used population-based sampling with 501 couples who attempted pregnancy in Michigan and Texas, 2005–2009 (enrolled in the LIFE Study). Results showed that only 2% (n = 28) of men were SLT users and no women used smokeless tobacco. Smokers showed higher cadmium levels than SLT, adjusted for cadmium attenuated the cigarette–time-to-pregnancy (TTP) association, especially among women. Shorter TTP was observed among SLT users in comparison to smokers (FOR [fecundability odds ratio]: 2.86, 95% confidence interval [CI]: 1.47, 5.57). Compared with never users of tobacco, smoking by females (FOR: 0.53, 95% CI: 0.33, 0.85) was individually associated with longer TTP; for males, smoking remained significant (FOR: 0.46, 95% CI: 0.27, 0.79) when modeling partners together (Sapra et al., 2016).\n\nA cross sectional study carried out in India to evaluate the urinary cotinine levels in three common categories of gynecological complaints, among 192 women of reproductive age, who were not pregnant, and sought treatment from a Government Medical college. Results showed that mean urinary cotinine level in women exposed to secondhand smoking (SHS) was 23.82±12.67 ng/ml. PID was the most common gynecological complaint. Mean urinary cotinine levels in infertile women were 22.42±12.72 ng/ml. The limitations of this study were that the sample size was not enough, other confounding variables were not considered and none of the participants admitted to smoking or use of SLT (Radhika et al., 2017).\n\nThe same study found that mean urinary cotinine levels in women with menstrual complaints was 19.32±10.29 ng/ml. Out of 1330 articles obtained on initial search, this was the only study selected in our review to study the association between SLT and menstrual problems (Figure 2, Table 3). For the question related to PID, another study (Simen-Kapeu et al., 2009) in addition to this study was selected (Figure 3, Table 4). Women with PID had significantly higher urinary cotinine levels = 24.95 (±12.259) ng/ml (p = 0.0144) (Radhika et al., 2017).\n\nPID = pelvic inflammatory disease. SD = standard deviation.\n\nHPV = human papillomavirus; OR = odds ratio; CI = confidence interval; LSIL = low-grade squamous intraepithelial lesion; HSIL = high-grade squamous intraepithelial lesion.\n\nAnother study (Simen-Kapeu et al., 2009) compared the association between tobacco use (smoking and chewing) and the risk of multiple human papillomavirus (HPV) infections and cervical squamous intraepithelial lesions (SILs) in two populations with different exposure. For this, baseline data from 2144 women from Cote d'Ivoire, West Africa and 415 women from Finland, Northern Europe regarding cervical screening, HPV positivity and tobacco use (smoking and chewing habits) was re-analyzed to determine the association between tobacco chewing in Cote d'Ivoire and tobacco smoking in Finland and the age stratified risk of multiple HPV infections and cervical SIL. Results show that in Côte d'Ivoire tobacco chewing (2.6%) was more common than tobacco smoking (1.4%). In 236 cases (eligible women with SIL), mean age of the women was 28.4±6.6 years with low-grade SIL in 165 and high-grade SIL in 71. Tobacco users (smokers and chewers) showed an increased risk of LSIL. Tobacco chewers were at 5 times higher risk for HSIL in both younger age group (<30 years) with OR: 5.5, 95% CI: 1.2-26 and older age group (≥30 years of age) with OR: 5.5, 95% CI: 2.1-14) in comparison to non-chewers. Age-adjusted OR of cervical HSIL was significantly higher among tobacco chewers. Increased risk of LSIL and HSIL (not significant) was found in HPV positive women ≥ 30 years of age who were actively exposed to tobacco through smoking or chewing was seen.\n\nHaving multiple HPV infections was common in HPV16 and/or HPV18 infected women (60.4% in Finland and 47.2% in Côte d'Ivoire). There was no increased risk of multiple HPV infections among tobacco consumers. It was found that women ≥ 30 years of age exposed to tobacco through smoking in Finland (OR: 2.2, 95% CI: 0.5-8.7) and chewing in Côte d'Ivoire (OR: 5.5, 95% CI: 2.1-14) had a moderately or highly increased risk of high-grade SIL, respectively. In the latter, the risk was statistically significant. Sampling bias was seen in the study as very few Ivorian women reported smoking evaluation, for this habit alone, and the regression analysis was restricted to Finnish women (Simen-Kapeu et al., 2009).\n\nThe risk of bias assessed using appraisal tool for cross sectional studies (AXIS) for one and ROBIN E for observational studies. Risk of bias for cross sectional study was high due to the small sample size but the those for the prospective studies was low.\n\n\nDiscussion\n\nThis systematically conducted rapid review to study association between SLT use and reproductive health of women yielded a total of three studies with findings from four different countries, namely India, USA, Finland and Cote d’Ivore and Finland. The differences in the study methodologies precluded us from combining the study findings.\n\nInfertility was measured in terms of TTP and Urinary cotinine levels and both were seen to be higher in SLT users. Women with PID had highest mean urinary cotinine levels among the three, followed by infertility and menstrual complaints respectively (Radhika et al., 2017). Another study showed longer TTP for cotinine levels more than 10ng/ml for SLT users (Sapra et al., 2016). Another study comparing association between tobacco smoking and chewing with risk of PID in two populations with different exposures showed that SLT users were at a five times higher risk of SIL in comparison to non-chewers irrespective of age (Simen-Kapeu et al., 2009). Results were however inconclusive regarding strong associations between SLT use and reproductive health in women.\n\nBiologic fertility can be assessed using TTP. A study based in a community setting showed a remarkably comprehensible association between female smoking and sub-fecundity during the most recent waiting TTP (Kassa and Kebede, 2018).\n\nCigarette smoking was also seen to be associated with an adverse effect on ovarian function and hence on fertility among women. Evidence suggests that there is an association between cigarette smoking and reduced fertility (risk of delayed conception), even at low doses (Hatch et al., 2012; Gormack et al., 2015). Since the active metabolite is similar, it is natural to expect some effects on the reproductive outcomes i.e. menstrual function, infertility, PID and pregnancy outcomes with SLT use. However, there is a dearth of good quality studies to ascertain such an association.\n\nNicotine levels are found to be highest in bidis, followed by chewed tobacco and cigarettes (Amith et al., 2018). Nevertheless, SLT products are more often abused than smoked tobacco products (Sharma et al., 2015) and serum nicotine levels remain in significant amounts for a longer time period (Mala et al., 2016). Like smoked form, SLT use causes alteration of the immune response to infections and has a damaging effect on majority of organ systems in the body including the reproductive system (Willis et al., 2012). This justifies the biological plausibility between SLT and reproductive outcomes.\n\nTo conclude, the number of studies about an association between SLT use and reproductive health of women were very few despite the high prevalence of use in the South East Asia region. Impact of SLT on reproductive health of women requires more research. In this rapid review, we followed the principles of systematic review that offers strength to the study. We have considered the major reproductive outcomes comprehensively. However, we restricted our search to English papers that poses a limitation. In addition, our reference period was from 1990, though it possibly would not have made a difference even if we went beyond that timeline. Though our results show that there might be an association between the SLT and poor reproductive outcomes, we recommend more studies on this topic with robust study designs for conclusive results.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReporting guidelines\n\nFigshare: PRISMA checklist for ‘Smokeless tobacco use and reproductive outcomes among women: a systematic review’. https://doi.org/10.6084/m9.figshare.16819102 (Radhika, 2021).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAmith HV, Agrawal D, Gupta A, et al.: Assessing the nicotine content of smoked and smokeless forms of Tobacco Available in Bhopal. Indian J. Dent. Res. May 2018; 29(3): 341–346. PubMed Abstract \| Publisher Full Text\n\nAwan KH, Patil S: Association of Smokeless Tobacco with Oral Cancer – Evidence From the South Asian Studies: A Systematic Review.2016; vol. 26: p. 6. PubMed Abstract\n\nAyo-Yusuf OA, Olutola BG: Epidemiological association between osteoporosis and combined smoking and use of snuff among South African women. Niger. J. Clin. Pract. Apr. 2014; 17(2): 174–177. PubMed Abstract \| Publisher Full Text\n\nBhisey RA: Chemistry and toxicology of smokeless tobacco. Indian J. Cancer. Dec. 2012; 49(4): 364–372. PubMed Abstract \| Publisher Full Text\n\nDatta S, Chaturvedi P, Mishra A, et al.: A review of Indian literature for association of smokeless tobacco with malignant and premalignant diseases of head and neck region. Indian J. Cancer. 2014; 51(3): 200–208. PubMed Abstract \| Publisher Full Text\n\nGATS2 (Global Adult Tobacco Survey): Fact Sheet, India, 2016-17.2017. Reference Source.\n\nGormack AA, Peek JC, Derraik JGB, et al.: Many women undergoing fertility treatment make poor lifestyle choices that may affect treatment outcome. Hum. Reprod. Jul. 2015; 30(7): 1617–1624. PubMed Abstract \| Publisher Full Text\n\nHatch EE, et al.: Caffeinated Beverage and Soda Consumption and Time to pregnancy. Epidemiol. Camb. Mass. May 2012; 23(3): 393–401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHossain MS, Kypri K, Rahman B, et al.: Smokeless tobacco consumption and stillbirth: Population-based case–control study in rural Bangladesh. Drug Alcohol Rev. 2017. Accessed: Jun. 29, 2021. Publisher Full Text\n\nInamdar AS, Croucher RE, Chokhandre MK, et al.: Maternal Smokeless Tobacco Use in Pregnancy and Adverse Health Outcomes in Newborns: A Systematic Review. Nicotine Tob. Res. Sep. 2015; 17(9): 1058–1066. PubMed Abstract \| Publisher Full Text\n\nKassa EM, Kebede E: Time-to-Pregnancy and Associated Factors among Couples with Natural Planned Conception in Addis Ababa, Ethiopia. Afr. J. Reprod. Health. 2018; 22(3). Art. no. 3. Publisher Full Text\n\nLiao C-Y, Chen Y-J, Lee J-F, et al.: Cigarettes and the developing brain: Picturing nicotine as a neuroteratogen using clinical and preclinical studies. Tzu Chi Med. J. Dec. 2012; 24(4): 157–161. Publisher Full Text\n\nMa LL, Wang YY, Yang ZH, et al.: Methodological quality (risk of bias) assessment tools for primary and secondary medical studies: what are they and which is better?. Mil. Med. Res. 2020 Dec; 7(1): 1–1. Publisher Full Text\n\nMala D, Nallapu V, Ambati M, et al.: Serum nicotine level among various tobacco users: A study. Journal of Indian Academy of Oral Medicine and Radiology. 2016 Apr 1; 28(2): 129. Publisher Full Text\n\nNair S, et al.: Use of Smokeless Tobacco by Indian Women Aged 18–40 Years during Pregnancy and Reproductive Years. PLoS ONE. Mar. 2015; 10(3): e0119814. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Cancer Institute and Centers for Disease Control and Prevention: Smokeless tobacco and public health: A global perspective. Bethesda: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Institutes of Health, National Cancer Institute; 2014.\n\nNiaz K, Maqbool F, Khan F, et al.: Smokeless tobacco (paan and gutkha) consumption, prevalence, and contribution to oral cancer. Epidemiol. Health. Mar. 2017; 39: e2017009. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRadhika AG: PRISMA_2020_checklist FILLED.pdf. figshare. Dataset. 2021. Publisher Full Text\n\nRadhika AG, Bhaskaran S, Kaur J, et al.: Assessment of urinary cotinine levels in women with gynecological complaints at a tertiary care hospital: A pilot study. Indian J. Public Health. 2017; 61(Suppl 1): S63–S65. PubMed Abstract \| Publisher Full Text\n\nRogers JM: Tobacco and pregnancy. Reprod. Toxicol. Elmsford N. Sep. 2009; 28(2): 152–160. PubMed Abstract \| Publisher Full Text\n\nSapra KJ, Barr DB, Maisog JM, et al.: Time-to-Pregnancy Associated With Couples’ Use of Tobacco Products. NICTOB. 2016; 18(11): 2154–2161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharma P, Murthy P, Shivhare P: Nicotine quantity and packaging disclosure in smoked and smokeless tobacco products in India. Indian J. Pharm. 2015; 47(4): 440–443. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimen-Kapeu A, et al.: Tobacco smoking and chewing as risk factors for multiple human papillomavirus infections and cervical squamous intraepithelial lesions in two countries (Côte d’Ivoire and Finland) with different tobacco exposure. Cancer Causes Control CCC. Mar. 2009; 20(2): 163–170. PubMed Abstract \| Publisher Full Text\n\nSinha DN, et al.: Global burden of all-cause and cause-specific mortality due to smokeless tobacco use: systematic review and meta-analysis. Tob. Control. Jan. 2018; 27(1): 35–42. Publisher Full Text\n\nSingh S, Jain P, Singh P, et al.: White paper on smokeless tobacco & women’s health in India. Indian J. Med. Res. 2020; 151(6): 513–521. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nSuliankatchi RA, Sinha DN: The Human Cost of Tobacco Chewing Among Pregnant Women in India: A Systematic Review and Meta-analysis. J. Obstet. Gynecol. India. Oct. 2016; 66(S1): 161–166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWillis D, Popovech M, Gany F, et al.: Toxicology of Smokeless Tobacco: Implications for Immune, Reproductive, and Cardiovascular Systems. J. Toxicol. Environ. Health B Crit. Rev. Jul. 2012; 15: 317–331. Publisher Full Text\n\nWorld Health Organization: Smokeless Tobacco: Essential Facts.Feb. 2015.\n\nWorld Health Organization: Tobacco Factsheet.Jul. 26, 2019.\n\nWorld Health Organization: WHO report on the Global tobacco epidemic, 2011: The MPOWER package. Geneva: 2011.\n\nWorld Health Organization: 90% of smokeless tobacco users live in South-East Asia. News release.11th September 2013. Accessed on 15th Oct 2021.Reference Source" }	[ { "id": "108658", "date": "05 Jan 2022", "name": "Ruchika Gupta", "expertise": [ "Reviewer Expertise Smokeless tobacco and health impact" ], "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 objective of the systematic review, as stated by the authors, is to review the association of SLT use and reproductive health among women. The methodology, search strings, eligibility criteria are all suitable to the objective. However, I have major concerns about the results.\n\n1. What were the exclusion criteria? If the E in PECO is an exposure to SLT, then studies reporting on smoking or secondhand smoke should be excluded from the review.\n\n2. Of the three studies that the authors claim to have 'addressed' their research question, the study from India addresses secondhand smoke. Neither of the participants admitted to smoking or SLT use. Hence, this study does not fulfill the eligibility criteria. In the report from Michigan and Texas, none of the female participants used SLT while only a small fraction of males consumed SLT in the form of snuff or chewing products. A close review of their results shows that impact of SLT on infertility is compared to smokers but not to never-users (could have been insignificant due to the low numbers of SLT users). Hence, this study also, in my opinion is not worth being included in the review.\n\nThat leaves only one study from Cote d'Ivoire, West Africa and Finland, Northern Europe that evaluated the risk of HPV and SIL in tobacco users, including SLT users.\n\n3. The discussion section also pertains to smoking rather than SLT which is the actual topic of the review.\n\n4. When talking of reproductive outcomes, why did the authors not include pregnancy outcomes like prematurity, stillbirth, low birth weight etc.?\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? No", "responses": [] }, { "id": "123938", "date": "11 Mar 2022", "name": "Sonali Jhanjee", "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\nThe authors use the term reproductive outcomes in the title and then use the terms “reproductive outcomes” and “reproductive health” interchangeably throughout the review. It may be beneficial to use standardized definitions in the review. Rationale also needs to be provided why these 3 particular outcomes namely infertility, menstrual disorders, and pelvic inflammatory disease have been the focus of the review rather than the more commonly reported reproductive outcomes low birth weight, stillbirth, spontaneous abortion, preterm birth and so on in studies related to smokeless tobacco.\n\nIn two of the studies, included in the review, the women participants were not consuming smokeless tobacco which is the focus of the review. So the authors need to clarify whether these studies fulfilled their inclusion and exclusion criteria.\nAll the above points have a bearing on the conclusions drawn from the review and hence need to be addressed.\nThe discussion also focuses more on smoking rather than smokeless tobacco.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-1171
https://f1000research.com/articles/10-1084/v1	25 Oct 21	{ "type": "Research Article", "title": "A pilot study assessing the uptake of core outcome sets in health technology assessments", "authors": [ "Peter Cox", "Paula R. Williamson", "Susanna Dodd", "Paula R. Williamson", "Susanna Dodd" ], "abstract": "Objective: Core outcome sets (COS) are an agreed standardised collection of outcomes created with representation from all key stakeholders (such as patients, clinicians, researchers), which should be reported as a minimum for all trials in that corresponding clinical area. There has been little research investigating the use of core outcomes in Health technology assessments (HTAs) and none in non-oncology HTAs. This study aimed to assess the similarity between COS and HTA outcomes. Methods: Ten COS published between 2015 and 2019 were selected, with patient participation taken as a proxy measure for a high quality COS. The INAHTA database was used as a source to identify relevant HTAs, which were accessed through the hyperlinks provided. Outcomes selected for these assessments were categorised as either a specific, partial or no match compared to the COS. An additional cohort of non-oncology HTAs published between 2019 and 2021 were identified from the NICE website and compared against a relevant COS. Results: Six hundred and fifty-one HTAs were matched to the ten COS areas, of which 119 were reviewed. Of a possible 1318 core outcome matches, there were 562 (43%) matches, 413 (31%) specific and 149 (11%) partial. NICE HTA matches against corresponding COS ranged from 44% to 100%, with a total of 78% (73/94) matches, 57 (61%) specific and 16 (17%) partial. Conclusion: Further work is required to promote the awareness and implementation of COS within HTAs. Improved uptake across NICE HTAs is encouraging, demonstrating acceptance of COS by HTA producers.", "keywords": [ "Core outcome set", "COS", "health technology assessment", "HTA", "outcome research" ], "content": "Introduction\n\nClinical trials are performed to evaluate the effects of treatment interventions, with the gold standard being randomised controlled trials.1 Fundamentally this includes a test and a control treatment, with random assignment of treatment groups and at least one outcome measure.2 Researchers decide on outcomes which best answer their research question, and consequently differences arise between trials in the same field as the chosen outcome measures are often of particular relevance to each study. The lack of uniformity amongst trial designs is abundant, demonstrated by a review of 8942 oncology trials which revealed that over 25,000 outcomes occurred only once or twice3; furthermore the top cited and most accessed Cochrane reviews in 2009 described problems due to inconsistences in the reported outcomes in included studies.4 Discrepancies are also observed when examining how outcomes are measured; for example, a survey of 10,000 trials investigating schizophrenia discovered 2194 different measurement scales were employed.5 This produces a substantial challenge in data analysis, limiting the ability to compare studies, synthesise the available evidence and perform meta-analysis, ultimately leading to avoidable research waste.6 There are also questions concerning whether trial outcomes are always relevant to patients or clinicians, meaning statistically significant results may have limited clinical bearing, hence not translating into improved clinical care.7\n\nIn January 2010 the Core Outcome Measures in Effectiveness Trials (COMET) Initiative was launched with the aim of bringing together people interested in the development and application of core outcome sets (COS). COS address the issue of inconsistency and outcome reporting bias, while reducing the difficulties facing systematic reviewers due to heterogeneity in outcome measures.8 COS are an agreed standardised collection of outcomes which should be reported as a minimum for all trials in the corresponding clinical area. The Core Outcome Set-STAndard for Development (COS-STAD) provides criteria against which to assess the quality of COS.9 Researchers are not restricted to the COS, but there is an expectation it will always be reported.8 The importance of COS has become increasingly acknowledged over time, with COMET endorsed by trial funders such as the National Institute for Health Research (NIHR), the Cochrane Collaboration and National Institute for Health and Care Excellence (NICE) . There is currently ongoing work assessing the representation of COS in US Food and Drug Administration (FDA) and European Medicines Agency (EMA) regulatory guidance.10\n\nHealth technology assessments (HTAs) are multidisciplinary processes which use explicit methods to determine the value of a health technology at different points in its lifecycle.11 They are produced to inform decision makers and promote an equitable, efficient and high-quality health system.11 The technologies are often interventions designed to prevent, diagnose or treat medical conditions. The International Network of Agencies for Health Technology Assessments (INAHTA) is a network of 51 different HTA agencies which support healthcare decision making. This network produces the international HTA database, which provides a free, single point of access to information about ongoing and published HTAs.\n\nRecent work has assessed the International Consortium for Health Outcomes Measurement (ICHOM) outcome sets against oncology HTAs, finding that HTAs tend to focus on generic measures which allow comparison across disease areas. However, it should be noted that these outcome sets are developed for routine care, unlike COS which are produced specifically for research so are more relevant for HTAs.12 Additionally, a review of technology appraisal oncology scopes found that in the majority of cases there was complete overlap with those outcomes in COS; in a small number of exceptions the COS included two additional outcomes to those specified in the scope.13 Other work has investigated a trial funder and member of INAHTA, the National Institute for Health Research Health Technology Assessment (NIHR HTA), examining their recommendation for applicants to search for a COS to include in their trial. By examining research applications and then surveying applicants it was found that 38% (36/95) searched for a COS through either the COMET database or another method, e.g. review of the literature; where a published COS existed it was included in 29% (7/24) of cases.14 There is reason to believe that uptake may have increased, as a limitation was that no COS existed for 68 of the 95 studies assessed at the time of submission.14 However, this study assessed the uptake of COS in trials rather than HTAs, and to our knowledge, ours is the first study to examine the uptake of COS for research in HTAs. There is potential for COS to further improve the quality of HTAs, providing more uniform and patient-centred evidence to inform clinical guidelines and policy makers, such as NICE.\n\nThe purpose of this study was to assess the similarity between COS and outcomes included in HTAs.\n\n\nMethods\n\nThe COMET database, a database of studies relevant to the development of COS, was searched to select individual COS for this review. As a surrogate measure to ensure quality, only COS published between 2015 and 2019 where patients were included were selected, maximising the number of COS-STAD standards met.9 The methodology of producing COS has improved with time, hence this 5-year period was chosen to include the most recent COS, which had a greater likelihood of including outcomes most relevant to all appropriate stakeholders. Additionally, COS developed for common diseases and interventions were selected to ensure sufficient HTAs were available for assessment. COS outcomes were extracted by one author (PC) from a pre-existing COMET database of outcomes from all COS for research published up to 2019.\n\nThe INAHTA database was searched in February 2021 for relevant HTAs, using the disease name as the search term with the English language filter applied. Where an intervention was specified by the COS this was included as a search term, e.g. obesity AND surgery. Quotation marks were used to refine searches that returned a large number of HTAs or when the results lacked specificity to the desired condition, e.g. searching type 1 diabetes produced HTAs relating to type 2 and gestational diabetes. There were no restrictions by year or country. MeSH terms were employed when there were variations in how search terms were spelt, for example postpartum haemorrhage versus postpartum hemorrhage.\n\nHTAs were accessed using the hyperlinks provided in the INAHTA database. Where the hyperlink did not direct to the HTA but to the publisher’s website, this was searched using the title of the HTA. If a hyperlink did not work, the abstract in the INAHTA record was screened for outcomes if available. HTAs which were irrelevant to the COS, inaccessible (e.g. due to a non-functional hyperlink or no hyperlink was present) or which summarised clinical evidence with no outcome measures stated were excluded. Outcome measures were then extracted from the HTA alongside study identifier, year and data source. Data extraction involved creating a table comprising the core outcomes as headings with each HTA listed below. For each HTA, outcomes were matched to the appropriate headings and then colour coded green, yellow or red to indicate specific, partial or no match respectively. For partial matches, bold and italic text indicated an outcome more general or specific than the COS respectively.\n\nOutcomes were categorised as either a specific match, a partial match or no match compared to the COS, following an approach used previously.15,16 We considered a match between an HTA outcome and a core outcome to exist if they were either specifically or partially related. We defined a specific match as one where both outcomes corresponded to each other exactly, while a partial match was defined as one where the outcomes correspond to each other non-specifically. Taking the example of swollen joint count as a core outcome, if the HTA included swollen joint count as an outcome it would be considered a specific match, whilst disease activity would be considered a partial match. Partial matches were further categorised according to whether the HTA outcome was either more general or more specific than the COS outcome. The outcome matching process was quality checked by a seconder reviewer (SD). The data extracted from each HTA included the HTA identifier, publisher, publication year and outcome measures. Outcomes were ascertained from stated outcomes of interest or from the PICO (population, intervention, comparator, outcomes) statement. Where outcomes were not explicitly stated, the HTA was reviewed to elicit them and on occasion outcomes had to be ascertained from a series of key research questions. Considering the research question “what is the expected beneficial effect of atezolizumab on mortality?” as an example, this was deemed a specific match for overall survival and a partial match for cancer-specific survival.\n\nWithin the UK, NICE HTAs are of particular importance. It was noted that no NICE HTAs had been registered on the INAHTA database beyond 2011, so a cohort of 10 recently published NICE HTAs were identified from their website. These HTAs were identified by searching the website sequentially back in time until 10 HTAs were identified for which there was a relevant COS for research with patient involvement published between 2015 and 2019, matching in terms of intervention and population scope. The outcomes included in these HTAs were compared to core outcomes included in the matching COS. Non-oncology conditions were selected given previous work has examined uptake across oncology HTAs.12,13\n\nThe results are presented through tables, graphs and using descriptive statistics. The analysis was performed using Excel 2019.\n\n\nResults\n\nTen COS were selected to assess against the INAHTA database. The search terms for each are displayed in Table 1. The search relating to the relapsing remitting multiple sclerosis and clinically isolated syndrome COS was performed as two individual searches to ensure all relevant HTAs were identified. The database search returned a cumulative total of 1057 HTAs, ranging from three for clinically isolated syndrome to 451 for breast cancer. A selection of 10% (45) of the breast cancer results were sampled, with the most recently published HTAs chosen as they were most likely to be accessible. This selection of HTAs comprised the most recently published HTAs until the 10% sample was reached, encompassing HTAs published between 2015 and 2020. This produced a possible 651 HTAs to be included in the report. Eight of the included COS applied to any intervention, with metabolic and bariatric surgery specified for obesity and treatment specified for postpartum haemorrhage. For each of the ten COS, the majority of the HTAs were not assessed as the hyperlinks listed in the INAHTA database were non-functional, detailed in Table 2. There were several HTAs irrelevant to the COS which were therefore excluded. Other reasons for exclusion included diagnostic accuracy study, HTA where no hyperlink was available, HTA not in English, HTA recommendations, HTA inaccessible on the source website, duplicates and where an updated version of the HTA had already been assessed.\n\n† The search relating to the relapsing remitting multiple sclerosis/clinically isolated syndrome COS was performed as two individual searches.\n\n‡ 10% (45) of the breast cancer HTAs were assessed and included in the report.\n\n† 10% (45) of the breast cancer HTAs were assessed and included in the report.\n\nOverall, 119 HTAs were assessed against core outcomes with a maximum of 1318 matches possible, shown in Table 3. In total, there were 562 (43%) core outcome matches, 413 (31%) specific and 149 (11%) partial, with the percentage values rounded to whole numbers. Rheumatoid arthritis had the greatest percentage of specific matches (52%), postpartum haemorrhage returned the greatest percentage of partial matches (50%) while epilepsy and psoriasis both had the greatest percentage of no matches (67%). Only rheumatoid arthritis, type 1 diabetes, acne and postpartum haemorrhage had more total core outcome matches compared to no matches. The number of HTAs assessed ranged from one for acne to 22 for rheumatoid arthritis. The COS for rheumatoid arthritis had the greatest median value of matches with 69% while epilepsy had the fewest with a median of 25%, detailed in Table 4.\n\nTo consider how the scope of the HTA compared to that of the COS, each HTA was assessed with regards to the investigated population and intervention. Table 5 shows the majority of the studies clustered around exact scope matches and the COS being broader, both in terms of the population and intervention.\n\nThe distribution of HTAs by year ranged from 1998 to 2020, with 69% of included HTAs published in 2012 and beyond. The most HTAs published in a single year was 13 in 2012. A mean of nine HTAs were published per year in the following years between 2013 and 2020. Figure 1 shows from 2009 onward there has been some fluctuation in the degree of match, though ultimately the trend is stationary, remaining close to the mean of 43%. This may reflect the fact that, although the clinical trials community are becoming more aware of COS, they are yet to be adopted by the HTA community.\n\nTo assess uptake of COS within recently published NICE HTA outcomes, 10 non-oncology HTAs published between 2019 and 2021 were identified from the NICE website and compared against a relevant COS, shown in Table 6. HTA matches ranged from 44% to 100%, with a total of 78% (73/94) outcome matches with NICE HTAs, including 57 (61%) specific matches and 16 (17%) partial matches.\n\n\nDiscussion\n\nThis review found that HTAs in the INAHTA database included 43% of core outcomes from COS, with specific matches accounting for 31% and partial matches for 11%.\n\nThis is a novel piece of work as there are few studies conducted assessing COS uptake, and none investigating uptake amongst non-oncology HTAs. Recent work has found that the uptake of COS in randomised controlled trials and systematic reviews varies greatly between different areas of health.27 Barriers to COS uptake are also noted, with lack of awareness, lack of validated measures or no consensus on measures, and lack of patient involvement being the most common reasons reported.27 Ideally with time, awareness will continue to grow, leading to the development of more high-quality COS which involve all key stakeholders including patients. The issue regarding a lack of validated measures, however, is a pertinent problem and one which goes hand in hand with the fundamentals of COS. Research to determine the most suitable outcomes to measure must be accompanied by work to determine how best to measure those outcomes. This is particularly important for life impact domains which tend to be patient reported. There are many well established methods to measure outcomes from clinical, resource use and adverse event domains as these are often linked to everyday work of professionals in healthcare, unlike life impact outcomes, the importance of which are becoming increasingly recognised as a result of COS.\n\nA potential reason for reduced COS uptake in this review may be the target populations selected when originally designing the COS. Eight of the ten COS related to any intervention for their given disease. The result is that studies investigating specific interventions, such bioabsorbable stents for coronary artery disease, are compared against measures designed for all coronary artery disease patients. A study investigating bioabsorbable stents may not have had the resources available to follow up their participants after five years to survey for outcomes such as stroke, depression or functional status, as this would have large time implications and ultimately may be of little relevance to their research question. Table 5 illustrates this point, with the distribution of HTAs around that of “COS is broader” for both population and intervention.\n\nAnother potential explanation for low representation of core outcomes could relate to the selection of COS published between 2015 and 2019. This was used as a proxy measure for quality COS, but the majority of the HTAs assessed (71/119) were published before 2015. While this could account for the lack of overlap prior to this year, Figure 1 demonstrates there has been no sustained increase in the percentage of outcomes included in HTAs.\n\nRecent work has compared ICHOM standard sets to oncology HTAs using a qualitative approach. They found that HTAs favour more generic outcome measures which allow comparison with other disease areas, rather than disease-specific outcomes recommended by the ICHOM standard sets.12 It has been shown that HTAs consider overall survival data to be most crucial when making decisions on the value of the technology.28 However, overall survival data are not always mature when submitted for HTA assessment and so intermediate outcomes, such as progression-free survival, may be suitable for HTA agencies. HTA agencies only accept validated intermediate outcomes and the level of validity varies between HTA agencies.12,28 However, it should be noted the study only focussed on five areas of oncology and that ICHOM standard sets are developed for routine care, whereas COS designed for research are therefore of more relevance for HTA agencies.\n\nDespite the accessibility difficulties, the INAHTA database remains a great resource for identifying HTAs. It can be easily navigated and continues to update details of new HTAs whenever they become available. In response to feedback on the difficulties accessing the HTAs, the managers of the database recognised the hyperlink issues, which arose as the records are self-administered and the producer of the report adds their own copy, noting that the correction of this issue will take time.\n\nA recent study surveying members of the INAHTA found that one of their biggest challenges is including stakeholders in HTAs, particularly where short deadlines offer little time for input.29 Implementing COS that have been developed with patient input could ensure HTAs included patient representation and reduce the time-consuming process of surveying patients themselves.\n\nThe finding that 78% of core outcomes were covered by NICE HTAs is very encouraging and notably greater than that for HTAs from the INAHTA database. This may be linked to the endorsement of COS by NICE or their rigorous approach of protocol design and stakeholder involvement.\n\nThe main limitation in this review was the large number of hyperlinks (474/651, 73%) in the INAHTA database which did not lead to a report, resulting in a large number of exclusions. This was an important finding in terms of the feasibility assessment in this pilot study. This allowed for feedback to the database providers, beginning the process of amending the accessibility of the reports so future research can make use of the resource. On occasion the hyperlink led to the source website from which the HTA could be searched for, but this did not always produce the desired HTA. Additionally, only one database was searched for HTAs to include in the review potentially omitting appropriate HTAs not registered on the INAHTA database. Another limitation is that many of the COS included in the review related to any intervention for their given disease. This may have exaggerated poor uptake of core outcomes in the context of very specific HTAs.\n\nAlthough the HTA outcome data extraction was conducted by a single researcher (PC), the opinion of a second researcher (SD) with extensive experience of outcome extraction and classification was consulted in all cases of doubt or ambiguity. In addition, all outcome matching assessments were double checked by this reviewer (SD).\n\n\nConclusion\n\nThis novel piece of research includes, to our knowledge, the first comparison of non-oncology HTAs and COS for research. This pilot study highlights that further work is needed to promote awareness and implementation of COS within HTAs, with less than half of core outcomes currently measured. Improved uptake across NICE HTAs demonstrates this is both practical and achievable with the additional benefit of increasing patient representation, a particular challenge for HTA agencies. Incorporation of COS will allow HTA findings to better reflect the opinions and preferences of all relevant stakeholders.\n\n\nData availability statement\n\nThe COMET database was utilised to identify suitable COS. Each of the 10 COS are accessible though the following hyperlinks: rheumatoid arthritis, relapsing remitting multiple sclerosis/clinically isolated syndrome, obesity, epilepsy, type 1 diabetes, acne, coronary artery disease, postpartum haemorrhage, psoriasis, breast cancer. HTAs can be accessed through the INAHTA database. The search terms are described in Table 1.\n\nThe 10 COS compared to the NICE HTAs can be accessed through the following hyperlinks: type 1 diabetes, type 2 diabetes, rheumatoid arthritis, age-related macular degeneration, relapsing remitting multiple sclerosis/clinically isolated syndrome, secondary progressive multiple sclerosis, Dravet syndrome, psoriasis, coronary artery disease, Crohn’s disease.\n\nThe corresponding NICE HTAs can be accessed through their website or through the following hyperlinks: type 1 diabetes, type 2 diabetes, rheumatoid arthritis, age-related macular degeneration, relapsing remitting multiple sclerosis/clinically isolated syndrome, secondary progressive multiple sclerosis, Dravet syndrome, psoriasis, coronary artery disease, Crohn’s disease.\n\nHarvard Dataverse: Data analysis for “A pilot study assessing the uptake of core outcome sets in health technology assessments” https://doi.org/10.7910/DVN/62NTAA.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nGartlehner G, Hansen RA, Nissman D, et al.: Criteria for distinguishing effectiveness from efficacy trials in systematic reviews.2006.\n\nMeinert CL: ClinicalTrials: design, conduct and analysis. USA:OUP;2012.\n\nHirsch BR, Califf RM, Cheng SK, et al.: Characteristics of oncology clinical trials: insights from a systematic analysis of ClinicalTrials. gov. JAMA Intern. Med. 2013; 173(11): 972–9. PubMed Abstract \| Publisher Full Text\n\nTovey D: The impact of cochrane reviews: The Cochrane Collaboration.2010. Publisher Full Text .\n\nMiyar J, Adams CE: Content and quality of 10 000 controlled trials in schizophrenia over 60 years. Schizophr. Bull. 2013; 39(1): 226–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan A-W, 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\nWebbe J, Sinha I, Gale C: Core outcome sets. Archives of Disease in Childhood-Education and Practice.2018; 103(3): 163–6.\n\nWilliamson PR, Altman DG, Blazeby JM, et al.: Developing core outcome sets for clinical trials: issues to consider. Trials. 2012; 13(1): 1–8. Publisher Full Text .\n\nKirkham JJ, Davis K, Altman DG, et al.: Core outcome Set-STAndards for development: the COS-STAD recommendations. PLoS Med. 2017; 14(11): e1002447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDodd S, Fish R, Gorst S, et al.: Representation of published core outcome sets for research in regulatory guidance: protocol. HRB Open Research. 2021; 4(45): 45. Publisher Full Text\n\nO'Rourke B, Oortwijn W, Schuller T: The new definition of health technology assessment: A milestone in international collaboration. Int. J. Technol. Assess. Health Care. 2020; 36(3): 187–90. PubMed Abstract \| Publisher Full Text\n\nKalf RR, Vreman RA, Delnoij DM, et al.: Bridging the gap: Can International Consortium of Health Outcomes Measurement standard sets align outcomes accepted for regulatory and health technology assessment decision-making of oncology medicines. Pharmacol. Res. Perspect. 2021; 9(2): e00742. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Institute of Health and Care Excellence: Health-related quality of life task and finish group report. Report 4: Core outcome sets 2020.Reference Source\n\nHughes KL, Kirkham JJ, Clarke M, et al.: Assessing the impact of a research funder’s recommendation to consider core outcome sets. PLoS One. 2019; 14(9): e0222418. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDodd S, Harman N, Taske N, et al.: Core outcome sets through the healthcare ecosystem: the case of type 2 diabetes mellitus. Trials. 2020; 21(1): 1–7. Publisher Full Text\n\nSaldanha IJ, Dodd S, Gorst SL, et al.: More than half of systematic reviews have relevant core outcome sets. J. Clin. Epidemiol. 2021; 136: 168–79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRadner H, Chatzidionysiou K, Nikiphorou E, et al.: 2017 EULAR recommendations for a core data set to support observational research and clinical care in rheumatoid arthritis. Ann. Rheum. Dis. 2018; 77(4): 476–9. PubMed Abstract \| Publisher Full Text\n\nMarrie RA, Miller A, Sormani MP, et al.: Recommendations for observational studies of comorbidity in multiple sclerosis. Neurology. 2016; 86(15): 1446–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoulman KD, Hopkins J, Brookes ST, et al.: A core outcome set for the benefits and adverse events of bariatric and metabolic surgery: the BARIACT project. PLoS Med. 2016; 13(11): e1002187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoble A, Marson A: Which outcomes should we measure in adult epilepsy trials? The views of people with epilepsy and informal carers. Epilepsy Behav. 2016; 59: 105–10. PubMed Abstract \| Publisher Full Text\n\nByrne M, O’Connell A, Egan AM, et al.: A core outcomes set for clinical trials of interventions for young adults with type 1 diabetes: an international, multi-perspective Delphi consensus study. Trials. 2017; 18(1): 1–8. Publisher Full Text\n\nLayton AM, Eady EA, Thiboutot DM, et al.: Identifying what to measure in acne clinical trials: first steps towards development of a core outcome set. J. Invest. Dermatol. 2017; 137(8): 1784–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcNamara RL, Spatz ES, Kelley TA, et al.: Standardized outcome measurement for patients with coronary artery disease: consensus from the International Consortium for Health Outcomes Measurement (ICHOM). J. Am. Heart Assoc. 2015; 4(5): e001767. Publisher Full Text\n\nMeher S, Cuthbert A, Kirkham JJ, et al.: Core outcome sets for prevention and treatment of postpartum haemorrhage: an international Delphi consensus study. BJOG Int. J. Obstet. Gynaecol. 2019; 126(1): 83–93. PubMed Abstract \| Publisher Full Text\n\nDuffin KC, Merola JF, Christensen R, et al.: Identifying a core domain set to assess psoriasis in clinical trials. JAMA Dermatol. 2018; 154(10): 1137–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOng WL, Schouwenburg MG, Van Bommel AC, et al.: A standard set of value-based patient-centered outcomes for breast cancer: the international consortium for health outcomes measurement (ICHOM) initiative. JAMA Oncol. 2017; 3(5): 677–85. PubMed Abstract \| Publisher Full Text\n\nHughes KL, Clarke M, Williamson PR: A systematic review finds core outcome set uptake varies widely across different areas of health. J. Clin. Epidemiol. 2020; 129: 114–23. Publisher Full Text\n\nKleijnen S, Lipska I, Alves TL, et al.: Relative effectiveness assessments of oncology medicines for pricing and reimbursement decisions in European countries. Ann. Oncol. 2016; 27(9): 1768–75. PubMed Abstract \| Publisher Full Text\n\nO'Rourke B, Werkö SS, Merlin T, et al.: The ‘top 10’ challenges for health technology assessment: INAHTA viewpoint. Int. J. Technol. Assess. Health Care. 2020; 36(1): 1–4. PubMed Abstract \| Publisher Full Text" }	[ { "id": "118406", "date": "18 Jan 2022", "name": "Catriona McDaid", "expertise": [ "Reviewer Expertise Applied health research", "systematic reviews", "randomised controlled trials", "health technology 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 peer review has been undertaken jointly with Andrew Mott, NIHR Pre-doctoral Research Fellow at the University of York. We each reviewed independently and then combined our reviews.\nThe article explores the extent of agreement/match between 10 individual COS published between 2015 and 2019 and HTA reports published on the INHATA database up to February 2021, supplemented by a comparison between COS and a cohort of UK NICE HTA reports. Overall, the study methods and results are comprehensively reported, tables are informative, and the data underlying the results are available. There is no reference to a publicly available protocol, though we have not identified any obvious sources of potential bias. Key limitations are addressed. There are a few areas where the reporting could be made clearer and some aspects of interpretation given further consideration.\nThe study is described as a pilot in the title and the conclusions. In the discussion there is also reference to “the feasibility assessment in this pilot study”. However, this is not apparent from the study aim/purpose or the methods so we suggest the term pilot is not used unless further information can be presented to show that it was indeed intended as a pilot – in which case the objective and methods would need amended to reflect this.\n\nWe were not totally convinced by the rationale for excluding oncology studies from the NICE cohort but not the INHATA cohort, when the stated purpose of including the former was to fill the gap identified in what was recorded on the INHATA database. It also looks as though a different process was used – as we interpret it, for the INHATA part of the study the COS were identified first then the reports and vice versa for the NICE cohort. We don’t think this will have introduced any important bias though possibly you could end up with the UK reports covering a different time period. You could consider adding some further descriptive information on the NICE cohort on range of publication year as you have for the INHATA cohort so that the similarity of the two datasets is clear.\n\nSelecting a subset of 10% of the breast cancer HTAs seems reasonable given the volume. Given that the most recent were selected it would be worth picking up in the discussion what effect this might have on the results as these are more likely to have been undertaken after the COS were published than any of the other conditions. This might be of particular interest as it appears to be amongst the lower matching (median 38%) conditions.\n\nThe methods state that the HTAs were taken from the INAHTA database. This database includes a variety of study designs and the term HTA can be defined in different ways. As reviewers we both interpreted differently whether or not trials were included so it would be useful to be explicit about what study designs were included under the term HTA. If all study designs were included some discussion around how the different designs may have affected the use of COS should be added.\n\nIf available, the date of searching the NICE website should be added as the content is regularly changing.\n\nThe methods state that COS were selected from the COMET website based on year of publication (2015-2019) and whether they addressed a common disease or intervention. Is there any further information on how the decision about being common was applied? A study selection flow diagram would also be a helpful addition to the text in the first paragraph of the results.\n\nFurther information in the methods on how outcomes were identified/established/extracted from reports would be useful. There may not be full agreement between the outcomes of interest identified in the scope/methods of a technology assessment (defining this as evidence synthesis and cost-effectiveness analysis) and the outcomes that are eventually included in the assessment due to the lack of data on the specific outcome; for example an outcome may be defined as of interest in the scope but no relevant data found and included in the synthesis. Also in the scope the outcomes may be more broadly expressed but in the included studies outcomes may be more specific reflecting what they could find. How was this approached in the data extraction? This may seem pedantic/theoretical but is there a possibility that the scope/methods sections could match well to a COS if it is a recent report, but the outcomes reported in the actual included studies may not if the data available are mainly older trials. So, there is another time lag issue here. Some further unpacking of this may be worth considering and whether there are any related recommendations to be made regarding future research on this topic. To ensure study replicability some additional information in the methods on how data were extracted would be helpful.\n\nAs the COSs were chosen initially and the HTAs identified that fit the COS it is possible these are not the most appropriate COS for each individual HTA. This is partially demonstrated in table 5 where almost half the HTAs have a narrower scope than the COS. It would be beneficial to have some discussion about whether these HTAs outcomes are specific to the narrower population or intervention.\n\nWe agree with the authors that a limitation of the study is that the majority of the included reports were published prior the introduction of the COS. In some parts of the paper e.g., the title and some parts of the discussion, we are not sure that the terminology ‘COS uptake’ is appropriate given that the COS was not necessarily available at the time a report was published. The terminology used for the study purpose and elsewhere “similarity between COS and outcomes included in HTAs” is more accurate and appropriate and this could be applied across the paper.\n\nThe conclusion refers to “improved uptake across NICE HTAs” – we are not convinced that the data presented warrant such a strong conclusion.\n\nUnderlying data – For the INHATA database only the general link to the site is available – is it possible to provide a list of the included reports?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7854", "date": "28 Feb 2022", "name": "Peter Cox", "role": "Author Response", "response": "Dear Dr McDaid and Mr Mott, Thank you for reviewing our manuscript “A pilot study assessing the uptake of core outcome sets in health technology assessments”. We hope that the corrections we have made capture your points. Yours sincerely, Peter Cox The study is described as a pilot in the title and the conclusions. In the discussion there is also reference to “the feasibility assessment in this pilot study”. However, this is not apparent from the study aim/purpose or the methods so we suggest the term pilot is not used unless further information can be presented to show that it was indeed intended as a pilot – in which case the objective and methods would need amended to reflect this. Author response: Thank you for raising this point. This study was conceptualised as a small scale study investigating the similarity between COS and HTA outcomes, given the presence of few prior studies assessing COS uptake. The results of this study provide backing for a similar study on a larger scale, to assess if these trends identified hold true given a larger sample size. This led to the description as a pilot study. The following line regards the feasibility assessment has been removed, “This was an important finding in terms of the feasibility assessment in this pilot study”. We were not totally convinced by the rationale for excluding oncology studies from the NICEcohort but not the INHATA cohort, when the stated purpose of including the former was tofill the gap identified in what was recorded on the INHATA database. It also looks as though a different process was used – as we interpret it, for the INHATA part of the study the COS were identified first then the reports and vice versa for the NICE cohort. We don’t think this will have introduced any important bias though possibly you could end up with the UK reports covering a different time period. You could consider adding some further descriptive information on the NICE cohort on range of publication year as you have for the INHATA cohort so that the similarity of the two datasets is clear. Author response: Thank you for this comment. NICE HTAs are of particular importance in the UK and following the discovery recent HTAs were not registered on the INAHTA database beyond 2011, they were searched for and identified from the NICE website to supplement our findings. Given COS vs oncology NICE HTAs has already been assessed in a previous project [Kalf et al.], non-oncology COS were assessed. From the original cohort of 10 COS, relevant NICE HTAs were only identified for 4 COS. Subsequently a further 6 non-oncology COS were selected to be assessed against NICE HTAs, providing the results in table 6. The methods text has been edited to include the following: “Within the UK, NICE HTAs are of particular importance. It was noted that no NICE HTAs had been registered on the INAHTA database beyond 2011, so in April 2021, the NICE website was searched for HTAs corresponding to each of the COS from the original cohort. Non-oncology conditions were selected given previous work has examined uptake across oncology HTAs (12, 13) and where no appropriate NICE HTAs were identified, alternative COS were selected, using the same criteria of publication between 2015 and 2019 with patient involvement. The most recently published reports which matched a COS were included, with outcomes compared to core outcomes from the corresponding COS. “The results are presented through tables, graphs and using descriptive statistics. The analysis was performed using Excel 2019. Comparison between the INAHTA and NICE results was not examined given they cover different time periods and utilised different COS.” The results section has been edited to include the following: “To assess the similarity between COS and recently published NICE HTA outcomes, the NICE website was searched for HTAs corresponding to each of the COS from the original cohort, except breast cancer. NICE HTAs were only identified for four COS, so six alternative COS were selected to include in the analysis. Ten non-oncology HTAs published between 2019 and 2021 were identified from the NICE website and compared against a relevant COS, shown in Table 6.” Selecting a subset of 10% of the breast cancer HTAs seems reasonable given the volume.Given that the most recent were selected it would be worth picking up in the discussionwhat effect this might have on the results as these are more likely to have been undertakenafter the COS were published than any of the other conditions. This might be of particular interest as it appears to be amongst the lower matching (median 38%) conditions. Author response: Thank you for this consideration. The following has been added to the discussion stating the dates of the HTAs included and to suggest why this COS had amongst the lowest outcome matching. “As a 10% subset of the most recent breast cancer HTAs were included in the analysis, it is intriguing to note it is amongst the lowest matching conditions. While reports published from 2016 onwards were solely analysed, it should be noted that the COS included 26 separate core outcomes. This was markedly greater compared to other COS in the analysis and may point to the importance of being selective when developing COS, as trial designers may find vast numbers of outcomes too overwhelming to take them all into consideration. Including too many outcomes may also serve to dilute the relevance of other more significant outcomes.” The methods state that the HTAs were taken from the INAHTA database. This database includes a variety of study designs and the term HTA can be defined in different ways. As reviewers we both interpreted differently whether or not trials were included so it would be useful to be explicit about what study designs were included under the term HTA. If all study designs were included some discussion around how the different designs may have affected the use of COS should be added. Author response: Thank you for this comment. The following has been added to the results section to clarify the different study designs included in the analysis. “Systematic reviews provided over 90% of studies included, the remainder being trials. To minimise similarity to COS being underestimated by reviews, through outcome reporting being limited to what data exists in the literature, outcomes stated in the methods were included in the analysis, independent of the findings.” If available, the date of searching the NICE website should be added as the content is regularly changing. Author response: The NICE website was searched in April 2021, this has been included in the methods section. The methods state that COS were selected from the COMET website based on year of publication (2015-2019) and whether they addressed a common disease or intervention. Is there any further information on how the decision about being common was applied? A study selection flow diagram would also be a helpful addition to the text in the first paragraph of the results. Author response: A flow chart has been added to the beginning of the results and the methods section edited to include the following line: “The selected COS were chosen as they were considered to be common conditions; examples of those excluded were: systemic sclerosis, malaria, thyroid eye disease, Hirschsprung's disease and twin–twin transfusion syndrome. This consideration heightened the likelihood sufficient HTAs were available for assessment” Further information in the methods on how outcomes were identified/established/extracted from reports would be useful. There may not be full agreement between the outcomes of interest identified in the scope/methods of a technology assessment (defining this as evidence synthesis and cost-effectiveness analysis) and the outcomes that are eventually included in the assessment due to the lack of data on the specific outcome; for example an outcome may be defined as of interest in the scope but no relevant data found and included in the synthesis. Also in the scope the outcomes may be more broadly expressed but in the included studies outcomes may be more specific reflecting what they could find. How was this approached in the data extraction? This may seem pedantic/theoretical but is there a possibility that the scope/methods sections could match well to a COS if it is a recent report, but the outcomes reported in the actual included studies may not if the data available are mainly older trials. So, there is another time lag issue here. Some further unpacking of this may be worth considering and whether there are any related recommendations to be made regarding future research on this topic. To ensure study replicability some additional information in the methods on how data were extracted would be helpful. Author response: Thank you for this consideration. Outcomes which were listed in the PICO or as a list of key research questions or equivalent were included and categorised as matches in our analysis. We have added the following line to the methods to clarify this and the paragraph to the discussion to give thought to the relevance of this: “Outcomes identified in the scope of an HTA which could not be reported due to a lack of available data were included in the analysis.” “There are instances where outcomes identified in the scope of an HTA cannot be included in the final assessment as sufficient available data is lacking. This may reflect appreciation and acknowledgement of the importance of these outcomes by those appraising the value of a technology which needs to be filtered down to those designing and conducting trials. Trial funders and researchers may need to consider the time taken to design, conduct and report research to build a sufficiently detailed literature base. This would suggest value in allowing an extended period of time before further research to allow publication of studies which have incorporated COS into their protocols.” As the COS were chosen initially and the HTAs identified that fit the COS it is possible these are not the most appropriate COS for each individual HTA. This is partially demonstrated in table 5 where almost half the HTAs have a narrower scope than the COS. It would be beneficial to have some discussion about whether these HTAs outcomes are specific to the narrower population or intervention. Author response: Thank you for this comment. The following has been included in the discussion addressing the scope of COS and relevance to narrower HTAs. “Likewise [to COS being broader than many of the HTAs], as many of the COS related to any individual with the disease, studies concerning specific populations would have fallen under their scope. While this could suggest that more COS are needed to cover the different subgroups, the value of COS are the uniformity they provide. If trials concerning subpopulations, such as pregnancy, advanced disease, or hormone responsive disease, recorded the outcomes designed for the greater population, it could reveal currently unknown trends, such as interventions experiencing improved efficacy in specific groups.” We agree with the authors that a limitation of the study is that the majority of the included reports were published prior the introduction of the COS. In some parts of the paper e.g., the title and some parts of the discussion, we are not sure that the terminology ‘COS uptake’ is appropriate given that the COS was not necessarily available at the time a report was published. The terminology used for the study purpose and elsewhere “similarity between COS and outcomes included in HTAs” is more accurate and appropriate and this could be applied across the paper. Author response: Thank you for this comment and suggestion. To reflect this point COS uptake has been replaced throughout the text with “similarity between COS and HTA outcomes” when referring to our findings. The title has been edited to “A pilot study assessing the similarity between core outcome sets and outcomes included in health technology assessments” The conclusion refers to “improved uptake across NICE HTAs” – we are not convinced that the data presented warrant such a strong conclusion. Author response: The conclusion has been edited to say “The degree of matching between COS and NICE HTA outcomes, albeit from a small sample, suggests this is both practical and achievable with the additional benefit of increasing patient representation, a particular challenge for HTA agencies” Underlying data – For the INHATA database only the general link to the site is available – is it possible to provide a list of the included reports? Author response: Links to individual HTA reports have been included in the data analysis on the Harvard Dataverse Repository under the extended data." } ] } ]	1	https://f1000research.com/articles/10-1084
https://f1000research.com/articles/9-1482/v1	18 Dec 20	{ "type": "Case Report", "title": "Case Report: Co-infection with SARS-CoV-2 and influenza H1N1 in a patient with acute respiratory distress syndrome", "authors": [ "Lekbir Baala", "Dalila Benzekri-Lefevre", "Laurent Bret", "Clémence Guillaume", "Laura Courtellemont", "Abdelkrim El Khalil", "Thomas Guery", "Sophie Iquel", "Olivier Perche", "Khalid Khadre", "Thomas Brungs", "Julien Decker", "Thomas Francia", "Julie Bois", "Benoit Delamare", "Jérôme Guinard", "Laurence Got", "Sylvain Briault", "Thierry Boulain", "Eric Legac", "Dalila Benzekri-Lefevre", "Laurent Bret", "Clémence Guillaume", "Laura Courtellemont", "Abdelkrim El Khalil", "Thomas Guery", "Sophie Iquel", "Olivier Perche", "Khalid Khadre", "Thomas Brungs", "Julien Decker", "Thomas Francia", "Julie Bois", "Benoit Delamare", "Jérôme Guinard", "Laurence Got", "Sylvain Briault", "Thierry Boulain", "Eric Legac" ], "abstract": "Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been a global public health concern. Co-infection of SARS-CoV-2 and other respiratory syndrome has been rarely reported. We report coinfection of SARS-CoV-2 and 2009 H1N1 Influenza strain in a French patient with pneumonia leading to acute respiratory distress syndrome. The patient also had a medical history of pulmonary sarcoidosis with a restrictive ventilatory syndrome, which would be a supplementary risk to develop a poor outcomes. This case highlights the possible coinfection of two severe SARS-CoV-2 and influenza H1N1 viruses, which presents a higher risk to extend the care duration. The overlapping clinical features of the two respiratory syndromes is a challenge, and awareness is required to recommend an early differential diagnosis.", "keywords": [ "Co-infection", "SARS-CoV-2", "Influenza H1N1" ], "content": "Introduction\n\nCoinfections involving SARS-CoV-2 and respiratory viruses influenza viruses (A or B) have been rarely reported1–6. To date, there is no published case with a co-infection between SARS-CoV2 and influenza H1N1.\n\n\nCase Report\n\nA 41-year-old man presented to the hospital’s emergency unit with fever and cough that has been progressing for several days. The SARS-CoV-2 RT-PCR test was positive on March 22nd 2020. On March 24th 2020, the patient had developed a dyspnea aggravation and was taken care of by a medical unity at home. He has a medical history of pulmonary sarcoidosis with a restrictive ventilatory syndrome, which was being treated with Methotrexate (15mg per week) and folinic acid (0,4mg one tablet per day). He also had malaria in 2004 from a trip to Central Africa.\n\nPhysical examination on the 24th March revealed a respiratory rate of 41 breaths/minute (normal range (NR) 12–20 breaths/minute) and oxygen saturation SpO2 of 75% (reference range (R-R) 95–100%) on ambient air. The SpO2 became at 96% when given mask flow oxygen at a rate of 12l/minute. The patient was transferred to the emergency room with 97% SpO2, body temperature 37.2°C, and respiratory rate of 30 breaths per minute. The patient presented with superficial polypnea, dyspnea with little effort, difficulty in speaking and bilateral “crackles”. Neurological and cardiovascular examinations were normal.\n\nOn supplemental oxygen (12l/min), arterial blood gas analysis revealed pH 7.50 (R-R 7.35–7.45), PCO2 35 mmHg (NR 35–45 mmHg), PO2 88 mmHg (NR 75–100 mmHg), HCO3- 27.3 mmol/l (R-R 22–26 mmol/l), and SaO2 94.4% (R-R 95–100%). The patient was then transferred to intensive care unit (ICU). Respiratory panel tests were negative for adenovirus (subtypes 2, 3, 6, 7.1 and 8), coronaviruses (229E, HKU1, NL63 and OC43), human metapneumovirus, rhinovirus, enterovirus, MERS-CoV, parainfluenza virus (1,2,3 and 4), respiratory syncytial virus, and Bordetella pertussis and parapertussis. However, influenza A, subtype influenza A-H1 variant 2009, was positive.\n\nA chest computed tomography scan revealed a predominant left interstitial lung condensation syndrome. Routine laboratory tests revealed higher parameters during patient hospitalization: creatine phosphokinase 2999 UI/l (R-R 30–200UI/l); gamma glutamyl transferase 119 Ui/l (R-R 12–64UI/l); D-Dimers, which has increased two fold in one week, 3620 ng/ml (April 6th) to 7520 (April 15th ) (R-R 40–500UI/l). Other parameters were also elevated: fibrinogen 8.58g/l (R-R 2–4g/l); aspartate-aminotransferase 55 UI/l (R-R 5–34UI/l); platelets 599x10e9/l (April 7th) (R-R 150–450x10e9/l); leukocytes 15.4x10e9/l (R-R 4–10x10e9/l) (Table 1).\n\nNA: data non available.\n\nOther parameters showed decreased values such as red cells, hemoglobins, hematocrits and mean corpuscular hemoglobin content (Table 1). The number of leukocytes and neutrophils underwent fluctuations with high rates between April 7th and 10th (Figure 1). In this period, bacteriological examination culture revealed infection by additional pathogens, with the presence of yeasts (Candidate albicans) and bacteria (Klebsiella pneumoniae) in bronchial sampling, probably with nosocomial origin.\n\nRdRp: RNA-dependent RNA polymerases; N: envelope protein N; S: Spike protein; CpK: creatine phosphokinase; SARS-CoV-2: severe acute respiratory syndrome coronavirus-2; Mar: March; Apr: April; #: Total count. The values between parentheses ‘()’ in the ordinate axis correspond to the reference range values.\n\nThe patient stayed at ICU for 26 days from March 24th to April 19th with orotracheal intubation and using Etomidate (40 mg intravenous dose) for sedation and 120mg of Celocurine for curarization. Enoxaparin (40mg /day) was administered by subcutaneous injection as a preventive anticoagulation up to April 1st and increased to 80 mg/day according to the patient being overweight (Body Mass Index 33.8) and to evolution of biological criteria (D-Dimers 1890ng/l, Fibrinogen 10.82g/l, platelets 528x10e9/l). Unfractionated heparin was used as relay according to the high probability of pulmonary embolism. Mechanical ventilation was used with several sessions of prone position and then oxygen therapy on April 19th. He was treated with hydroxychloroquine for 10 days (Plaquenil, 200mg every eight hours), and by Oseltamivir (4 days, oral suspension 6mg/ml: 75mg twice/day) for influenza H1N1 infection. Klebsiella pneumonia infection was treated using Meropenem (intravenous 1 g every four hours) from 6th to 15th April and Enterococcus faecalis infection was treated using Clamoxyl (intravenous 2 g every eight hours). Venous echodoppler performed on April 14th found no thrombosis and no pulmonary embolism.\n\nOn April 19th, the patient was transferred to the pulmonology department where he has a good respiratory evolution allowing oxygen weaning on April 23rd. He was discharged on April 28th, receiving kinesitherapy treatment, and taking a preventive anticoagulant therapy (Enoxaparine 4000 IU /0.4ml once daily by SC injection) for three weeks. The patient was integrated into the post COVID-19 rehabilitation program.\n\n\nDiscussion\n\nWe report, to the best of our knowledge, the first case of coinfection with SARS-CoV-2 and seasonal influenza H1N1. The low incidence rate of this co-infection reported in France may be explained by the late screening for COVID-19, which started in France in March 2020, which corresponds with the tapering off period for H1N17. The prolonged intensive care and detection of SARS-CoV-2 viral RNA on the bronchoalveolar sample for at least three weeks might be explained by patient immunosuppression caused by lung polyinfection (viral, bacterial and fungal) and probably by his medical history of pulmonary sarcoidosis with a restrictive ventilatory syndrome.\n\nBacterial coinfections in COVID-19 patients have been reported in nine studies with a rate of 8% (62/806) of bacterial/fungal co-infection cases8. A few cases have been reported with co-infection with SARS-CoV-2 and influenza viruses (A or B)1–6. Ding et al. has reported coinfected patients with SARS-CoV-2 and influenza virus and showed similar clinical characteristics as those patients with COVID-19 only, hence not all patients need ICU3. However, in a report by Cuadrado-Payán et al., all COVID-19 patients studied attended the emergency unit but had medical history of hypertension, end-stage kidney disease, or type 2 diabetes5.\n\nThe co-detection of SARS-CoV-2 and Influenza H1N1 in our case demonstrates the challenge to screen in the onset of the respiratory illness for a panel of viruses, which have overlapping clinical patterns and might exacerbate clinical symptoms, increase morbidity and prolong ICU stay. Hence, this case highlights the higher risk and poor outcomes caused by co-infection and the importance to achieve a differential diagnosis of respiratory distress syndromes, to limit contamination and adapt therapeutic strategies.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this article and any associated 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": "References\n\nWu D, Lu J, Ma X, et al.: Coinfection of Influenza Virus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS–COV–2). Pediatr Infect Dis J. 2020; 39(6): e79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu X, Cai Y, Huang X, et al.: Co-infection with SARS-CoV-2 and Influenza A Virus in Patient with Pneumonia, China. Emerg Infect Dis. 2020; 26(6): 1324–1326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDing Q, Lu P, Fan Y, et al.: The clinical characteristics of pneumonia patients coinfected with 2019 novel coronavirus and influenza virus in Wuhan, China. J Med Virol. 2020; 92(9): 1549–1555. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhodamoradi Z, Moghadami M, Lotfi M: Co-infection of Coronavirus Disease 2019 and Influenza A: A Report from Iran. Arch Iran Med. 2020; 23(4): 239–243. PubMed Abstract \| Publisher Full Text\n\nCuadrado-Payán E, Montagud-Marrahi E, Torres-Elorza M, et al.: SARS-CoV-2 and Influenza Virus Co-Infection. Lancet. 2020; 395(10236): e84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWehl G, Laible M, Rauchenzauner M: Co-infection of SARS CoV-2 and influenza A in a Pediatric Patient in Germany. Klin Padiatr. 2020. 232(4): 217–218. PubMed Abstract \| Publisher Full Text\n\nJhaveri R: Echoes of 2009 H1N1 Influenza Pandemic in the COVID Pandemic. Clin Ther. 2020; 42(5): 736–740. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRawson TM, Moore LSP, Zhu N, et al.: Bacterial and Fungal Coinfection in Individuals with Coronavirus: A Rapid Review To Support COVID-19 Antimicrobial Prescribing. Clin Infect Dis. 2020. 71(9): 2459–2468. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "86504", "date": "21 Jun 2021", "name": "Louise E. Lansbury", "expertise": [ "Reviewer Expertise Respiratory pathogens" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report, Baala et al. describe the clinical course of a patient with a history of pulmonary sarcoidosis who was co-infected with SARS-CoV-2 and influenza A (H1N1) virus and developed acute respiratory distress syndrome, requiring a prolonged ICU stay. The case is generally well-described and a useful reminder that the clinical presentation of SARS-CoV2 and influenza infections may overlap, and of the importance of the timely recognition of co-infection of SARS-CoV2 with other respiratory pathogens. However, the authors may wish to consider the following points:\nAbstract:\nPlease clarify the sentence: “Co-infection of SARS-CoV-2 and other respiratory syndrome has rarely been reported”. Do you mean: \"Co-infection with SARS-CoV2 and other respiratory pathogens\"?\n\nItalicisation of ‘respiratory distress syndrome’ is not required (also italicised in the title\n\nIntroduction:\n“To date, there is no published case with a co-infection between SARS-CoV-2 and influenza H1N1”. This can no longer be claimed as there are now several reports of co-infections with these viruses from several countries.\n\nCase Report:\n“The SARS-CoV-2 RT-PCR test was positive on March 22nd 2020.” Is there information on the date of onset of symptoms in relation to the timing of this test?\n\nDid the patient receive the seasonal influenza vaccine prior to the 2019-2020 influenza season?\n\nWould it be possible to present the chest CT-scan?\n\nHow many days had the patient been in ICU before Candida albicans and Klebsiella pneumoniae were first isolated?\n\nThe authors indicate that an infection with Enterococcus faecalis infection was also treated; what was believed to be the source of this infection?\n\nDiscussion:\nAs per the previous comment, this is no longer the first case report of a co-infection with SARS-CoV-2 and influenza A (H1N1). However, the fact that it occurred in a patient with a history of pulmonary sarcoidosis is perhaps more original and may be worth emphasising more in the discussion (and perhaps also in the title?).\n\nThe effect of SARS-CoV-2 and influenza co-infection on prognosis could perhaps be considered in a little more detail in the Discussion. The studies cited (Ding et al., Cuadrado-Payán et al.) indicated that the clinical and analytic courses of their patients did not differ from patients infected only with SARS-CoV-2. However, other studies have suggested that co-infection may be associated with worse outcomes (e.g. see Stowe et al.1 and Hashemi et al.2).\n\nOverall, I believe the case study contributes to the literature on patients who are co-infected with SARS-CoV-2 and influenza as there are many uncertainties regarding these patients. I support indexing but would recommend the clarifications I have suggested are considered.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "7981", "date": "03 May 2022", "name": "lekbir BAALA", "role": "Author Response", "response": "Dear All, I'm Dr. Lekbir BAALA, the author of this article. I thank Dr. Louise E. Lansbury for her comments and I'm pleased to give answers below in italics. Abstract: • Please clarify the sentence: “Co-infection of SARS-CoV-2 and other respiratory syndrome has rarely been reported”. Do you mean: \"Co-infection with SARS-CoV2 and other respiratory pathogens\"? Yes, we mean co-infection with SARS-CoV2 and other respiratory pathogens. it’s corrected. • Italicisation of ‘respiratory distress syndrome’ is not required (also italicised in the title Ok, it was corrected. Introduction: • “To date, there is no published case with a co-infection between SARS-CoV-2 and influenza H1N1”. This can no longer be claimed as there are now several reports of co-infections with these viruses from several countries. Yes, as we submitted our case report in December 2020, at that time no case with H1N1 and SARS-CoV-2 co-infection has been published in PUBMED, but now it is. We have changed the sentence. Case Report: • “The SARS-CoV-2 RT-PCR test was positive on March 22nd 2020.” Is there information on the date of onset of symptoms in relation to the timing of this test? Yes, the PCR was positive on 22nd March 2020. The cough and fever had already been evolving for three to four days, which is what motivated screening (clinical investigation). • Did the patient receive the seasonal influenza vaccine prior to the 2019-2020 influenza season? Not known as vaccinated against influenza. • Would it be possible to present the chest CT-scan? We have obtained the chest CT Scan for the patient. The conclusion was: “Appearance of more or less symmetrical, predominantly peripheral, bilateral pulmonary ground glass areas compatible with Covid 19 type pneumopathy in the context of the epidemic...”. • How many days had the patient been in ICU before Candida albicans and Klebsiella pneumoniae were first isolated? Klebsiella pneumonia was isolated on the deep bronchial sample of March 28, 2020, so five days after his admission and mechanical ventilation, he was on C3G (ceftriaxone) empirically at that time. On the 14th day of admission (06/04/2020), Klebsiella pneumonia was found once again at 10^6 in a bronchoalveolar lavage which means the failure of treatment with imipenem, meropenem is then introduced. Also found in this bronchoalveolar lavage candidas albicans 10^4. • The authors indicate that an infection with Enterococcus faecalis infection was also treated; what was believed to be the source of this infection? It is probably the digestive tract, the patient had presented abdominal pain, diarrhea, and vomiting which were attributed to poor tolerance of enteral nutrition. There was no specific digestive exploration because the evolution of this disorder was rapidly favorable. Discussion: • As per the previous comment, this is no longer the first case report of a co-infection with SARS-CoV-2 and influenza A (H1N1). However, the fact that it occurred in a patient with a history of pulmonary sarcoidosis is perhaps more original and may be worth emphasising more in the discussion (and perhaps also in the title?). We take into account this relevant remark on the prevalence. One meta-analysis (June 2021) of prevalence studies revealed that the frequency of influenza virus (A/B) co-infection among patients with COVID-19 was 4.5% in Asia, and 0.4 % from the American continent. This meta-analysis indicated that overall 1.2% of COVID-19 patients had influenza co-infection. We will add to the discussion the notion of the vulnerability of the immunocompromised (in our case sarcoidosis under methotrexate) • The effect of SARS-CoV-2 and influenza co-infection on prognosis could perhaps be considered in a little more detail in the Discussion. The studies cited (Ding et al., Cuadrado-Payán et al.) indicated that the clinical and analytic courses of their patients did not differ from patients infected only with SARS-CoV-2. However, other studies have suggested that co-infection may be associated with worse outcomes (e.g. see Stowe et al.1 and Hashemi et al.2). Indeed the data in the literature are divergent, but a large series of patients are needed or a review of the literature is probably necessary to conclude. The recent review meta-analyses (June 2021) indicated that the outcomes of COVID-19-Influenza co-infection were reported in 11 studies with 2 of 29 patients who died (6.9%), and 17 out of 29 patients recovered (58.6%), but more studies are needed to evaluate the exact effect of SARS-CoV-2 and influenza co-infection on prognosis and clinical outcomes (Masoud Dadashi et al. 2021). The impact of co-infection with SARS-CoV-2 and influenza (H1N1) have been recently developed in animal models. For example, the sequential infection with H1N1 and SARS-CoV-2 aggravated COVID-19 pathogenesis in a mammalian model (Linlin Bao Signal Transduct Target Ther 2021). They conclude that co-infection with H1N1 and SARS-CoV-2 extended the duration of clinical manifestation of COVID-19, and enhanced pulmonary damage." } ] }, { "id": "86503", "date": "20 Jul 2021", "name": "Khalid Sadki", "expertise": [ "Reviewer Expertise Immunology and immunogenetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 submitted by the authors is an interesting case report. They report a French patient co-infected by two severe viruses, SARS-CoV-2 and 2009 H1N1 Influenza strain.\nThe most important physical examination and diagnostic tests are reported in the case report. The methodology followed is well reported. However, data concerning the genetic investigations for both viruses is missed. It is recommended to discuss it, taking into consideration the specific phenotype observed in this case.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "8002", "date": "03 May 2022", "name": "lekbir BAALA", "role": "Author Response", "response": "I am Dr. BAALA, a co-author of this article, I thank Pr. SADKI for his review and I am pleased to respond to his comments: We didn't give more details of the molecular data used for the SARS-CoV-2 and H1N1 tests since we were limited by the number of words not to be exceeded in the article. However, we may include some Ct data for SARS-CoV2 to indirectly indicate viral load kinetics. As Pr Sadki and Pr Lansbury (first reviewer) suggested regarding the phenotype of the patient, the fact that the co-infection by SARS-Cov-2 and H1N1 viruses occurred in a patient with a history of pulmonary sarcoidosis, we will give more notice to the role of this immunosuppression history and poor outcome in the discussion." } ] } ]	1	https://f1000research.com/articles/9-1482
https://f1000research.com/articles/11-477/v1	29 Apr 22	{ "type": "Research Article", "title": "In vitro and in vivo study: Ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia and Philippines suppresses tumor growth of hepatocellular carcinoma by inhibiting IL-6/STAT3 signaling", "authors": [ "Ricadonna Raissa", "Wibi Riawan", "Anna Safitri", "Masruri Masruri", "Ma Asuncion Guiang Beltran", "Aulanniam Aulanniam", "Ricadonna Raissa", "Wibi Riawan", "Anna Safitri", "Masruri Masruri", "Ma Asuncion Guiang Beltran" ], "abstract": "Background: Azadirachta indica Juss. has been shown to suppress cancer progression through a variety of mechanisms. In order to treat cancer progression, cancer immunotherapy is used to stimulate the immune system where immunosuppression is present in tumor microenvironments. Many cancer cells produce a lot of interleukin-6 (IL-6) and signal transducer activator of transcription 3 (STAT3). STAT3 plays a key role in suppressing the expression of critical immune activation regulators. IL‐6‐mediated STAT3 activation is common in the tumor microenvironment. Inhibiting the IL-6/STAT3 signaling pathway has become a therapeutic option for cancer progression. As vimentin is also expressed in hepatic stellate cells boosting cancer survival. We focused on the precise effect of extract from leaves of Azadirachta indica Juss, on inhibiting the IL-6/STAT3 signaling cascade on hepatocellular carcinoma by in vitro and in vivo study. Methods: In the in vitro study, the effect of Azadirachta indica Juss. variant Indonesia and Philippines against the expression of IL-6 and STAT3 was examined in liver cancer cell line. In the in vivo study, 24 male rats (Rattus norvegicus) strain Wistar were induced by diethylnitrosamine and carbon tetrachloride (CCl4). Based on the therapy given, the groups were divided into negative control, positive control, Indonesia extract, and Philippine extract. Expression of IL-6, STAT3, and vimentin were tested using immunohistochemistry staining. The data were analyzed using analysis of variance, which was then followed by the Tukey test. Results: Statistically significant difference in IL-6 and STAT3 was observed between the treatment groups and positive control group by in vitro study and in vivo study. Generally, there is no significant difference between treatment using Indonesian and Philippine leaves. Conclusion: Both therapy doses of Azadirachta indica variant in Indonesia and Philippines were able to reduce IL-6, STAT3 and vimentin expression of hepatocellular carcinoma cell by in vitro and in vivo experiment.", "keywords": [ "Azadirachta indica", "cancer immunotherapy", "interleukin - 6 (IL - 6)", "signal transducer activator of transcription 3 (STAT3)", "vimentin", "hepatocellular carcinoma" ], "content": "Introduction\n\nHepatocellular carcinoma (HCC), primary liver cancer originating from hepatocytes, is the fourth most common tumor worldwide.1,2 From the report of statistics from the Global Burden of Cancer Study, approximately 906,000 new cases and 830,000 deaths of HCC cases were recorded in 2020.3 According to annual projections, more than one million people will die from liver cancer by 2030, according to the World Health Organization.4\n\nHCCs are complex ecosystems that include non-tumor cells, primarily immune-related cells, as well as tumor cells.4,5 Non-immune and immune cells, cytokines such interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) play central roles in inflammation cancer.6,7 IL-6 belongs to the cytokine family that signals via the Janus kinase-signal transducer and activator of transcription pathway.8–10 IL-6 has a predominant role in the tumor microenvironment which is found at high concentrations in cancer.11\n\nIL-6 is produced by an HCC cell which functions as a growth factor.12 In addition, it is also reported that hepar cancer produces IL-6, so an increase in IL-6 is a sign of excessive cell growth.13 HCC is caused by abnormal IL-6 signaling in liver progenitor cells with activated STAT3.14 The binding of IL-6 to the IL-6 receptor activates STAT3, a key oncogenic transcription factor. Due to STAT3 critical function in cell signaling, the protein STAT3 has become a popular target in tumor growth.15 Survival of cancer cells can be maintained by the complex IL-6 and STAT3 pathways.16\n\nA study by Iwahasi17 suggested that HCC tumor malignancy, through the IL-6/STAT3 pathway, is affected by the activation of hepatic stellate cells. These hepatic cells boosted cancer cell survival, and migratory ability. Vimentin, in particular, was substantially expressed in activated hepatocyte stellate cells (HSCs).18,19 As a result, inhibiting HSC cell growth and inhibiting the IL-6/STAT3 pathway could be a promising technique for inhibiting cancer cell progression.13,20,21\n\nCancer treatments that target the IL-6/STAT3 pathway contribute a therapeutic benefit for inhibiting tumor cell growth.22 A study by Kao et al.23 has shown that hepatic function, tumor development, and HCC patient survival were all impacted by IL-6 via the STAT3 signal pathway.\n\nCancer chemotherapy by natural agents is a promising therapy towards lowering cancer progression.24,25 Overcoming the immunosuppressive condition in tumor microenvironments is significant to improve the efficacy of cancer immunotherapy.26,27 Immune cells, non-immune cells and tissues, communicate through interleukins and related cytokines.28 Interleukins have a crucial role in the genesis, progression, and management of cancer. Interleukins can create an environment that promotes cancer growth while also being necessary for a successful tumor-directed immune response.28,29\n\nOver the last several decades, an increasing number of plant-derived products, including Azadirachta indica Juss., have been explored, as they are cheap and easy to grow in tropical countries, such as Indonesia and the Philippines.30 We found Azadirachta indica Juss. content in the leaf was more in Indonesia and Philippine during September–December, which is the component of the plant with the desired properties for cancer treatment. Azadirachta indica Juss. has been reported to promote many biological activities including anticancer. A study by Raissa31 suggested that Azadirachta indica Juss. variant Indonesia has more flavonoid content, and Azadirachta indica Juss. variant Philippines has more terpenoids content than flavonoid. However, the properties anticancer effect of Azadirachta indica Juss. from different region planting suppresses cancer progression by immunotherapy pathway of HCC has not yet been identified. This in vitro study uses HepG2 cells. Then, In vivo study uses a two weeks old male rat (Rattus norvegicus) as hepatocellular carcinoma animal model. Rat prefered for this experimental animal model due to their anatomical and physiological, and genetic similarity to humans.32 In this in vivo study prefers male than female rats. Female rats may bring variability results because of the changing hormonal state during the oestrous cycle.33 These animal model use two chemical step, once induction with diethylnitrosamine in neonatal age as agent genotoxic and CCl4 as promoter to improve the carcinogenesis in rats.34–36 This study aimed to evaluate the anticancer properties of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia and Philippines by inhibiting IL-6/STAT3 signaling.\n\n\nMethods\n\nAzadirachta indica leaves were collected from Madura, Indonesia and Camiling, Philippines, taxonomically identified by Laboratory of Taxonomy and Plant Development Structure, Brawijaya University with a certificate number 0238/UN10.F09.42/03/2018 and 0250/UN10.F09.42/03/2019. Air-dried powdered leaves (100 g) were extracted by cold maceration in 80% ethanol (300 ml) and filtered. The supernatant was concentrated with an under pressure rotary evaporator (IKA® HB 10 digital, Germany) at a temperature not exceeding 55°C. The extracts were stored in an air-tight box.\n\nHepG2 cell lines were obtained from American Type Culture Collection (ATCC, Cat# HB-8065, RRID:CVCL_0027). The HepG2 cells were grown in Dulbecco's modified eagle medium-high glucose medium (Gibco®, Cat#11965092) supplemented with 10% Fetal Bovine Serum qualified, US origin (Gibco®, Cat#26140087), along with 1% Penicillin-Streptomycin (5,000 U/mL) (Gibco®, Cat#15070063). These cells were maintained at 37°C in a humidified incubator under 95% air atmosphere plus 5% CO2 (Heracell™ 150i, Thermo Scientific, Cat#50116048).\n\nHepG2 cells with a density of 75 × 103 cells/well were grown on coverslips in 24-well plates until 80% confluent. Concentration of 170.105 g/ml ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia and 170.415 g/ml of ethanolic extract leaves of Azadirachta indica of variant Philippines were added to the cells in the media. After twenty-four hours post-exposure to both extracts, the media was removed and the plates containing the cells were aspirated with phosphate buffered saline (PBS) (Sigma-aldrich®, Cat#P4417). Cells were fixed with methanol for 10 minutes at 40°C and then washed again with PBS. Cells were added with protein block (Novocastra™, Leica, Cat#RE7102) and washed with PBS. Cells were added primary antibodies IL-6 (Santa Cruz Biotechnology, Cat# sc-57315, RRID:AB_2127596) and STAT3 (Santa Cruz Biotechnology, Cat# sc-8019, RRID:AB_628293) for one hour and washed again with PBS. After incubation with primary antibody, cells were added with Histofine® (Nichirei Biosciences Cat# 424144, RRID:AB_2868561) for one hour and washed again with PBS. After that, 3,3′-Diaminobenzidine (DAB) solution (Novolink™, Leica, Cat# RE7230-K) was added, and incubated for ten minutes. The DAB solution was washed with aquadest. Furthermore, the Mayer solution was added into the well and then incubated for 10 minutes and then discarded and washed with aquadest. In a light microscope, expressing IL-6 and STAT3 will give a brown color, while those that do not express IL-6 and STAT3 will give a purple/blue color. Immunohistochemistry staining was valued by counting the express protein (brown color) at 20 times field of view at every sample then the data is averaged and processed statistically.\n\nWhen the cells were 70% confluent, the cells were harvested and treated with ethanolic extract leaves of Azadirachta indica of variant Indonesia and Philippines for 24 hours. The medium was transferred to a centrifuge tube. The IL-6 levels were checked using the enzyme-linked immunosorbent assay (ELISA) technique with IL-6 ELISA kit (Elabscience®, Cat#E-EL-H6156) according to each manufacturer’s instructions.\n\nThe second part of this study is an in vivo laboratory study with 24 male rats (Rattus norvegicus) strain Wistar that induced with two step chemical hepatocellular carcinoma induction, using diethylnitrosamine (DEN) and carbon tetrachloride (CCl4). This research was conducted from January 2019 to December 2020 at the animal experimental laboratory in Institut Biosains, Universitas Brawijaya, Malang. The experimental protocols were approved by the Ethics Committee of the Institut Biosains, Universitas Brawijaya, Malang with protocol number 1138-KEP-38.\n\nThe experimental animals used in this study were male white rats aged two weeks old, with bodyweight (BW) ranging from 20-30 grams. During the experiment and the analysis, no rats were excluded due to illness or any other reasons. All samples were randomly allocated in each group. All samples (n = 24) were divided into four groups consisting of six rats each. The groups were divided into negative control, positive control, ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia treatment, and ethanolic extract leaves of Azadirachta indica Juss. variant of Philippines.\n\nEach rat was numbered 1-24 and each cage was marked (A-D) to indicate a different group. After that, randomization was carried out to group each rat by one researcher, and other researchers were blinded until the end of the experiment, so that there was no bias. The sample size was determined by Federer’s formula. A total of 24 rats were separated into four groups, each with six rats.\n\nT = Number of groups\n\nN = Number of rats\n\nSix-one month’s pregnant rats (Rattus norvegicus) (Institut Biosains, Malang, Indonesia) were purchased. After the rat mother gave birth, a total of 24-seven day old male rats were divided from the other seven day old female rats. As these rats were very young, they were kept with their mothers until the end of the weaning period. One mother rat for the six male neonatal rats was kept in one cage. After neonatal rats reach one months old, they are kept in individual cages. All these rats were maintained in Laboratory Animal at Institut Biosains, the University of Brawijaya (Malang, Indonesia). The animals were maintained under a controlled environment with temperature of 23°C ± 1°C, humidity of 55 ± 5% and a 12 h light/dark cycle during the experiment. Rats were fed with yellow corn, pollard, and water ad libitum. We provide a pipe for each rat to hide or play to reduce distress.\n\nHCC induction to these experimental animals was done by two step induction. First, the two weeks old male rats were induced by using DEN (Sigma-Aldrich Merck, Cat# N0258) at a dose of 50 mg/kg of body weight for single intraperitoneal injection. Second induction was done when the rats were eight weeks old for intraperitoneal injection of carbon tetrachloride (CCl4) (Sigma-Aldrich Merck, Cat# 289116) at a dose of 1 mL/kg of body weight (BW) three times a week for twelve weeks.\n\nThe rats in the positive group only received DEN-CCl4 induction. In the two curative group, DEN-CCl4-induced rats with elevated alpha fetoprotein (AFP) level were given ethanolic extract of Azadirachta indica of variant Indonesia (500 mg/kg BW) and ethanolic extract leave of Azadirachta indica of variant Philippines (500 mg/kg BW) orally for four weeks every day. After being anesthesia with ketamine (Merck, NMID686C) 75-100 mg/kg BW and xylazine (Xyla®, Interchemie, Cat#IX2) 10 mg/kg BW intraperitoneally, the hepar organ store in neutral buffered formalin solution 10% (Sigma-Aldrich Merck, Cat# HT501128). After being euthanized, paraffin blocks of hepar cancer tissue from each rat were made for immunohistochemistry (IHC) staining.\n\nAll efforts were made to ameliorate any suffering of animals.\n\nThe hepar organs were obtained and fixed in 10% buffered formaldehyde immediately. Then hepar organs were embedded in paraffin. The paraffin-embedded blocks were prepared into five-micrometer serial sections. For histopathological assessment, hepar tissue sections were deparaffinized with xylene. Hepar tissues were rehydrated with distilled water. After that, hepar tissues were added hydrogen peroxide 3% for ten minutes and washed with phosphate buffer saline (PBS). Hepar tissues were added protein block (Novocastra™, Leica, Cat#RE7102) for one hour and washed again with PBS. Then, hepar tissues were added with primary antibody IL-6 (Santa Cruz Biotechnology, Cat# sc-57315, RRID:AB_2127596), STAT3 (Santa Cruz Biotechnology, Cat# sc-8019, RRID:AB_628293), and vimentin (Novus Cat# NBP1-97524, RRID:AB_11190427) for one hour and washed with PBS, then added with Histofine ® (Nichirei Biosciences Cat# 424144, RRID:AB_2868561) for one hour and washed again with PBS. After that, DAB solution (Novolink™, Leica Cat# RE7230-K) was added, and the samples were incubated for ten minutes. DAB solution was discarded and then washed with aquadest. Furthermore, the mayer solution was added into the well and then incubated for 10 minutes and then discarded and washed with aquadest. In a light microscope, expressing IL-6, STAT3 and vimentin proteins will give a brown color, while those that do not express IL-6, STAT3 and vimentin proteins will give a purple/blue color. Immunohistochemistry staining was valued by counting the express protein (brown color) at 20 times field of view at one hepar tissue then the data is averaged and processed statistically. Immunohistochemistry staining was valued by counting the express protein (brown color) at 20 times field of view at every sample then the data is averaged and processed statistically.\n\nData are expressed as the mean ± standard deviation of each experiment. The values were analyzed by one-way analysis of variance followed by Tukey’s multiple comparison test using GraphPad Prism software version 9 for Windows (GraphPad Prism Software, RRID:SCR_002798, La Jolla, California, USA). Statistical differences were considered significant at the 0.05 levels of probability (p<0.05).\n\n\nResult\n\nThe representative image of immunohistochemistry staining for IL-6, STAT3 and vimentin in rat hepar tissue (Table 1) is shown in Figures 1, 2, and 3.37–39 Each image depicts a different expression based on the group and the biomarker (IL-6, STAT3 and vimentin).\n\np value<0.05 considered significant.\n\np value<0.05 considered significant.\n\np value<0.05 considered significant.\n\nThe representative image of immunocytochemistry staining for IL-6 and STAT3 in HepG2 is shown in Figures 4 and 5.40,41 The brown color intensity indicates that the related biomarker is expressed at a higher level. Additionally, the level of IL-6 was quantified by the ELISA method (Figure 6).\n\np value<0.05 considered significant.\n\np value<0.05 considered significant.\n\nThe level of IL-6 was analyzed by one-way analysis of variance followed by Tukey’s post hoc test. p value<0.05 considered significant.\n\nCytokines may contribute to cancer progression.42 In the present study, the IL-6, and STAT3 concentrations in HepG2 cell culture after treatment with Indonesia extract at 170.415 μg/mL and Philippines extract at 170.415 μg/mL were determined (Table 2). The highest level of inhibition on the release of cytokines was observed in HepG2 for IL-6 compared to untreated positive control cells (Figure 6). In contrast, there was a slight increase in Indonesia extract group (P1) the release of IL-6 in HepG2 cells than Philippines extract group (P2). In other words, the plant extract caused a significant change in the IL-6 levels and STAT3 expression in HepG2 hepar cancer cells.\n\n\nDiscussion\n\nOn the basis of ethno pharmacological utilization of Azadirachta indica in cancer treatment, Azadirachta indica variant Indonesia and Philippines was evaluated for its anticancer effect by inhibiting IL-6/STAT-3 signaling pathways. In vitro and in vivo approaches were used for the explication of possible underlying mechanisms to rationalize the Ayurveda ethno medical uses of the plant from different geographical locations.\n\nAzadirachta indica Juss. extract exhibited anticancer effects against DEN and CCl4-induced HCC and hepar cancer cell line (HepG2 cells), while its possible underlying mechanism was estimated through isolated tissue preparations and cells also associated with the reduction in cancer survival. DEN and CCl4 are used agents to induce two-step carcinogen protocol in cancer studies.36 Nitrosamine compounds are used as food additives to preserve meat products.43 Nitrosamines can form a genotoxic compound after it is metabolized in the human body by cytochrome P450 in liver. Metabolizing nitrosamine compounds can induce cancer in experimental animals.44–46 This experiment used the HCC rat model with DEN and CCl4-induction, Azadirachta indica Juss. leaf extract demonstrated potential of reducing cancer cell survival protective effect against DEN and CCl4-induced HCC and hepar cancer cell line (HepG2 cells). Azadirachta indica extracts contain an apoptotic constituent which mediates anticancer effect by inhibiting HSC cell growth and inhibiting the IL-6/STAT3 pathway. This study suggests that Azadirachta Indica extracts could be a promising technique for inhibiting cancer cell survival.\n\nIn this in vivo study by immunohistochemistry staining, every treatment group showed a lower expression of IL-6, STAT3 and vimentin in comparison with the positive control groups (p<0.05) (Table 2). The in vitro study by immunocytochemistry staining of expressed IL-6 and STAT3 in HepG2, in every treatment group showed a lower expression of IL-6 and STAT3 in comparison with the positive control groups (p<0.05) (Table 3). The Azadirachta indica Juss. variant Indonesia and Philippines groups exhibited effects on antitumor protein expression by inhibiting IL-6/STAT3 signaling pathway. The Azadirachta indica Juss. treatment group was able to significantly decrease IL-6 and STAT3 expression by immunohistochemistry and immunocytochemistry staining with both positive control groups. STAT3 has been shown to be involved in the development of human tumor malignancies.36 STAT3 inhibiting apoptosis or inducing cell proliferation, angiogenesis, invasion and metastasis result in promoting cancer initiation and progression. STAT3 is activated by IL-6-induced dimerization of the IL-6 receptor, which leads to cancer progression in an inflammatory environment.47 Dysregulation of the IL-6-mediated JAK/STAT3 signaling pathway is link to development of several human solid tumors.13,22,44,48\n\nIL-6 has been implicated as an autocrine stimulator of cancer growth.6,12,45,46 The development of effective therapeutic options requires an understanding of IL-6 survival signaling in malignancies.49,50 Apoptosis inhibition is a well-studied strategy for cell survival.51 The mechanisms involved in proliferation and acute phase protein synthesis have been thoroughly defined in most investigations of IL-6 signaling in hepatic.52 A study by Bregmann12 showed that one of the essential factors of the development of hepatocellular carcinoma (HCC) is IL-6 trans-signaling. In our study additional data of IL-6 level by ELISA, in every treatment group showed a lower expression IL-6 level in comparison with the positive control groups (p<0.05) (Table 3). Ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia and Philippines showed a significant decrease in HepG2 and IL-6 levels, when using ELISA method, compared to the positive control group (Figure 6). It may be said that Azadirachta indica leaf extract has a strong effect on reducing the inflammatory cytokine that plays a role in HCC.\n\nRelating to the decreased expression of vimentin protein by immunohistochemistry staining in hepar tissue of Azadirachta indica treatment group, it can be said that Azadirachta indica treatment decreases the HSCs by knowing the expression of vimentin. The microenvironment of HCC tumors is dominated by HSCs.48,53–55 The chronic inflammation is also related to HSC activation.50 HSCs are liver-specific pericytes that play an important role in fibrosis.48,56,57 During liver fibrogenesis, HSCs produce enormous amounts of extracellular matrix proteins.58,59 The most essential function of HSC is in the fibrosis (fibrogenesis) process. Besides that, HSCs also contribute to hepatic inflammation with their ability to secrete and respond to growth stimuli of HCC.60,61 HSC activation is marked by vimentin protein activation.18,62\n\nA study by Hu et al.63 used immunohistochemistry to investigate the relationship between vimentin overexpression and HCC metastasis utilizing a tissue microarray over 200 primary HCCs and 60 pairs of primary and matched metastatic HCC samples. Azadirachta indica Juss. variant Indonesia and Philippines treatment groups had lower expression of IL-6, STAT3 and vimentin that imply a decrease in cell cancer survival.\n\nTherefore, it can be assumed that the Azadirachta indica group showed apoptotic effect. The level production IL-6 in HepG2 by ELISA in every treatment group did not show significant difference with the negative control group. Inhibiting HSC cell growth and inhibiting the IL-6/STAT3 pathway could be a promising technique for inhibiting cancer cell survival.\n\nThe local growth environment has a strong influence on the quality of medicinal plants. Environmental elements such as precipitation, illumination, temperature, humidity, and soil would fluctuate depending on production location, which can lead to variation in the contents of active ingredients in this medicinal plant.64 This Azadirachta indica Juss. leaves variant Indonesia and Philippines were harvested in the same period of time, in November until December, and planted in the same tropical climate. A study by Raissa31 indicated Azadirachta indica Juss. variant Indonesia has more flavonoid content than the Philippines one, the Philippines variant has more terpenoids content than the Indonesia one. Despite this difference, the end result showed there is no significant difference of inhibiting STAT3/IL-6 signaling between ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia and Philippines.\n\nThere is evidence that bioactive neem chemicals including nimbolide, azadirachtin, and gedunin influence a wide range of biological processes in vitro and in vivo.56–59 Both variant have the same effect on inhibiting IL-6/STAT3 signaling, it may be caused by the same amount of main bioactive rather than the variation of bioactive groups such as flavonoid or terpenoids.\n\nThis study has limitations, such as administering one dose of the Azadirachta indica Juss. variant Indonesia and the Philippines, therefore, the effects could not be compared in a dose-dependent manner. As a result, research with a more variative dose is recommended. Furthermore, more study on other molecular pathways and biomarkers is required to explore the apoptosis potential of both variants in hepatocellular carcinoma.\n\n\nConclusion\n\nA preventive dose of Azadirachta indica Juss variant Indonesia and Philippines was able to reduce IL-6/STAT3 expression and decrease vimentin expression in hepar tissue and in HepG2. Our findings show that Azadirachta indica Juss variant Indonesia and Philippines, both have cancer-preventive effects in a well-characterized animal model of hepatocellular carcinoma and hepar cancer cell line. Further study including more molecular pathways and biomarkers, is required to explore the mechanism of action of this potential of anti-HCC carcinogenesis by ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia Philippines.\n\n\nData availability\n\nFigshare: Underlying data for ‘Expression of IL-6 in Rattus norvegicus hepar tissue by Immunohistochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2), control group (NC), and positive control group (PC)’, https://doi.org/10.6084/m9.figshare.1936708137\n\nThis project contains the following underlying data:\n\nIL-6 IHC NC 40x.tif\n\nIL-6 IHC NC 400x.tif\n\nIL-6 IHC NC 1000x.tif\n\nIL-6 IHC PC 40x.tif\n\nIL-6 IHC PC 400x.tif\n\nIL-6 IHC PC 1000x.tif\n\nIL-6 IHC P1 40x.tif\n\nIL-6 IHC P1 400x.tif\n\nIL-6 IHC P1 1000x.tif\n\nIL-6 IHC P2 40x.tif\n\nIL-6 IHC P2 400x.tif\n\nIL-6 IHC P2 1000x.tif\n\nFigshare: ‘Expression of STAT3 in Rattus norvegicus hepar tissue by Immunohistochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2), control group (NC), and positive control group (PC), https://doi.org/10.6084/m9.figshare.19366901.38\n\nThis project contains the following underlying data:\n\nSTAT3 IHC NC 40x.tif\n\nSTAT3 IHC NC 400x.tif\n\nSTAT3IHC NC 1000x.tif\n\nSTAT3 IHC PC 40x.tif\n\nSTAT3 IHC PC 400x.tif\n\nSTAT3 IHC PC 1000x.tif\n\nSTAT3 IHC P1 40x.tif\n\nSTAT3 IHC P1 400x.tif\n\nSTAT3 IHC P1 1000x.tif\n\nSTAT3 IHC P2 40x.tif\n\nSTAT3 IHC P2 400x.tif\n\nSTAT3 IHC P2 1000x.tif\n\nFigshare: Raissa, Ricadonna (2022): ‘Expression vimentin in Rattus norvegicus hepar tissue by Immunohistochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2), control group (NC), and positive control group (PC)’, https://doi.org/10.6084/m9.figshare.19367069.39\n\nThis project contains the following underlying data:\n\nVimentin IHC NC 40x.tif\n\nVimentin IHC NC 400x.tif\n\nVimentin IHC NC 1000x.tif\n\nVimentin IHC PC 40x.tif\n\nVimentin IHC PC 400x.tif\n\nVimentin IHC PC 1000x.tif\n\nVimentin IHC P1 40x.tif\n\nVimentin IHC P1 400x.tif\n\nVimentin IHC P1 1000x.tif\n\nVimentin IHC P2 40x.tif\n\nVimentin IHC P2 400x.tif\n\nVimentin IHC P2 1000x.tif\n\nFigshare: ‘Expression of IL-6 in HepG2 cell line by Immunocytochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2) and positive control group (PC)’, https://doi.org/10.6084/m9.figshare.19367081.40\n\nIL-6 ICC NC 40x.tif\n\nIL-6 ICC NC 400x.tif\n\nIL-6 ICC NC 1000x.tif\n\nIL-6 ICC PC 40x.tif\n\nIL-6 ICC PC 400x.tif\n\nIL-6 ICC PC 1000x.tifs\n\nIL-6 ICC P1 40x.tif\n\nIL-6 ICC P1 400x.tif\n\nIL-6 ICC P1 1000x.tif\n\nIL-6 ICC P2 40x.tif\n\nIL-6 ICC P2 400x.tif\n\nIL-6 ICC P2 1000x.tif\n\nFigshare: ‘Expression of STAT3 in HepG2 cell line by Immunocytochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2), and positive control group (PC)’, https://doi.org/10.6084/m9.figshare.19366460.41(p3)\n\nSTAT3 ICC NC 40x.tif\n\nSTAT3 ICC NC 400x.tif\n\nSTAT3 ICC NC 1000x.tif\n\nSTAT3 ICC PC 40x.tif\n\nSTAT3 ICC PC 400x.tif\n\nSTAT3 ICC PC 1000x.tif\n\nSTAT3 ICC P1 40x.tif\n\nSTAT3 ICC P1 400x.tif\n\nSTAT3 ICC P1 1000x.tif\n\nSTAT3 ICC P2 40x.tif\n\nSTAT3 ICC P2 400x.tif\n\nSTAT3 ICC P2 1000x.tif\n\nRepository name: ARRIVE checklist for ‘In vitro and in vivo study: Ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia and Philippines suppresses tumor growth of hepatocellular carcinoma by inhibiting STAT3-IL-6 signaling’, DOI: https://doi.org/10.6084/m9.figshare.19352474.v2.65\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor details\n\nRicadonna Raissa\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing\n\nWibi Riawan\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing\n\nAnna Safitri\n\nRoles: Conceptualization, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing\n\nMasruri Masruri\n\nRoles: Conceptualization, Investigation, Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing\n\nMa Asuncion Guiang Beltran\n\nRoles: Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing\n\nAulanni’am Aulanni’am\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing", "appendix": "Acknowledgment\n\nThe authors thank to Prof. Widodo, S.Si.,M.Si.,Ph.D.Med.Sc (Universitas Brawijaya) for providing HepG2 cells.\n\n\nReferences\n\nLlovet JM, Kelley RK, Villanueva A: Hepatocellular carcinoma. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaissa R: Expression of IL-6 in Rattus norvegicus hepar tissue by Immunohistochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2), control group (NC), and positive control group (PC). figshare.2022. Publisher Full Text\n\nRaissa R: Expression of STAT3 in Rattus norvegicus hepar tissue by Immunohistochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2), control group (NC), and positive control group (PC). figshare.2022. Publisher Full Text\n\nRaissa R: Expression vimentin in Rattus norvegicus hepar tissue by Immunohistochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2), control group (NC), and positive control group (PC). figshare.2022. Publisher Full Text\n\nRaissa R: Expression of IL-6 in HepG2 cell line by Immunocytochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2) and positive control group (PC). figshare.2022. Publisher Full Text\n\nRaissa R: Expression of STAT3 in HepG2 cell line by Immunocytochemistry staining in treatment group of ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia (P1), Philippines (P2), and positive control group (PC). figshare.2022. Publisher Full Text\n\nDranoff G: Cytokines in cancer pathogenesis and cancer therapy. Nat. Rev. Cancer. 2004; 4(1): 11–22. Publisher Full Text\n\nGrosse Y, Baan R, Straif K, et al.: Carcinogenicity of nitrate, nitrite, and cyanobacterial peptide toxins. Lancet Oncol. 2006; 7(8): 628–629. PubMed Abstract \| Publisher Full Text\n\nXu L, Qu YH, Chu XD, et al.: Urinary Levels of N-Nitroso Compounds in Relation to Risk of Gastric Cancer: Findings from the Shanghai Cohort Study. PloS One. 2015; 10(2): e0117326. PubMed Abstract \| Publisher Full Text\n\nZheng J, Daniel CR, Hatia RI, et al.: Dietary N-Nitroso Compounds and Risk of Hepatocellular Carcinoma: A USA-Based Study. Hepatology. 2021; 74(6): 3161–3173. PubMed Abstract \| Publisher Full Text\n\nBodakuntla S, Libi AV, Sural S, et al.: N-nitroso-N-ethylurea activates DNA damage surveillance pathways and induces transformation in mammalian cells. BMC Cancer. 2014; 14(1): 287. PubMed Abstract \| Publisher Full Text\n\nHuang Y, Yang X, Xu T: Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs. Int. J. Oncol. 2016; 49(1): 153–163. PubMed Abstract \| Publisher Full Text\n\nKitamura H, Ohno Y, Toyoshima Y: Interleukin-6/STAT3 signaling as a promising target to improve the efficacy of cancer immunotherapy. Cancer Sci. 2017; 108(10): 1947–1952. PubMed Abstract \| Publisher Full Text\n\nMasjedi A, Hashemi V, Hojjat-Farsangi M: The significant role of interleukin-6 and its signaling pathway in the immunopathogenesis and treatment of breast cancer. Biomed. Pharmacother. 2018; 108: 1415–1424. PubMed Abstract \| Publisher Full Text\n\nConlon KC, Miljkovic MD, Waldmann TA: Cytokines in the Treatment of Cancer. J. Interf. Cytokine Res. 2019; 39(1): 6–21. PubMed Abstract \| Publisher Full Text\n\nAbdelhafez ESMN, Ali SMNA, Hassan MRE, et al.: Apoptotic Inhibitors as Therapeutic Targets for Cell Survival. IntechOpen; 2019. Publisher Full Text\n\nServais FA, Kirchmeyer M, Hamdorf M: Modulation of the IL-6-Signaling Pathway in Liver Cells by miRNAs Targeting gp130, JAK1, and/or STAT3. Mol. Ther. - Nucleic Acids. 2019; 16: 419–433. PubMed Abstract \| Publisher Full Text\n\nHellerbrand, Wang SC, Tsukamoto H, et al.: Expression of intracellular adhesion molecule 1 by activated hepatic stellate cells. Hepatol. Baltim. Md. 1996; 24(3): 670–676. PubMed Abstract \| Publisher Full Text\n\nEfsen E, Bonacchi A, Pastacaldi S: Agonist-specific regulation of monocyte chemoattractant protein-1 expression by cyclooxygenase metabolites in hepatic stellate cells. Hepatol. Baltim. Md. 2001; 33(3): 713–721. PubMed Abstract \| Publisher Full Text\n\nSun B, Zhang X, Cheng X: Intratumoral Hepatic Stellate Cells as a Poor Prognostic Marker and a New Treatment Target for Hepatocellular Carcinoma. PloS One. 2013; 8(11): e80212. PubMed Abstract \| Publisher Full Text\n\nVeeraraghavan J, Natarajan M, Lagisetty P, et al.: Impact of curcumin, raspberry extract, and neem leaf extract on rel protein-regulated cell death/radiosensitization in pancreatic cancer cells. Pancreas. 2011; 40(7): 1107–1119. PubMed Abstract \| Publisher Full Text\n\nShakiba E, Ramezani M, Sadeghi M: Evaluation of serum interleukin-6 levels in hepatocellular carcinoma patients: a systematic review and meta-analysis. Clin. Exp. Hepatol. 2018; 4(3): 182–190. PubMed Abstract \| Publisher Full Text\n\nSchmidt-Arras D, Rose-John S: IL-6 pathway in the liver: From physiopathology to therapy. J. Hepatol. 2016; 64(6): 1403–1415. Publisher Full Text\n\nBarry AE, Baldeosingh R, Lamm R: Hepatic Stellate Cells and Hepatocarcinogenesis. Front. Cell Dev. Biol. 2020; 8. Accessed February 2, 2022. PubMed Abstract \| Publisher Full Text\n\nWang L, Tu L, Zhang J, et al.: Stellate Cell Activation and Imbalanced Expression of TGF-β1/TGF-β3 in Acute Autoimmune Liver Lesions Induced by ConA in Mice. Biomed. Res. Int. 2017; 2017: 1–12. PubMed Abstract \| Publisher Full Text\n\nFriedman SL: Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N. Engl. J. Med. 1993; 328(25): 1828–1835. PubMed Abstract \| Publisher Full Text\n\nMiyata E, Masuya M, Yoshida S: Hematopoietic origin of hepatic stellate cells in the adult liver. Blood. 2008; 111(4): 2427–2435. PubMed Abstract \| Publisher Full Text\n\nMilani S, Herbst H, Schuppan D, et al.: In situ hybridization for procollagen types I, III and IV mRNA in normal and fibrotic rat liver: evidence for predominant expression in nonparenchymal liver cells. Hepatol. Baltim. Md. 1989; 10(1): 84–92. PubMed Abstract \| Publisher Full Text\n\nRoskams T, Rosenbaum J, De Vos R, et al.: Heparan sulfate proteoglycan expression in chronic cholestatic human liver diseases. Hepatol. Baltim. Md. 1996; 24(3): 524–532. PubMed Abstract \| Publisher Full Text\n\nARRIVE checklist for “In vitro and in vivo study: Ethanolic extract leaves of Azadirachta indica Juss. variant of Indonesia and Philippines suppresses tumor growth of hepatocellular carcinoma by inhibiting STAT3-IL-6 signaling.”.Published online March 14, 2022. Publisher Full Text" }	[ { "id": "147588", "date": "02 Sep 2022", "name": "Fauzi Yusuf", "expertise": [ "Reviewer Expertise Gastroenterology", "Hepatology", "Microbiota", "Short Chain Fatty Acid", "Helicobacter pylori", "Covid 19", "Colorectal 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\nI think that this in vitro and in vivo study is well done. In the in vitro study with liver cancer cell line and in vivo study with male rats (Rattus novergicus) strain Wistar. The topics Hepatocelluler carcinoma pathways are also interesting and need further study including biomarker and molecular pathways to explore the mechanism of action of many cytokines in supressing immune activation regulator. I think this research article about plant-derived product (Azadiracta indica Juss) will benefit the further study to evaluate the anti cancer properties of ethanolic extract leaves by inhibiting IL-6/STAT3 signalling and another immune activation regulator.\nIt seems that the title of this manuscript too strong as the study did not measure the growth of the tumor, because determination of hepatocelluler carcinoma diagnosis in mice is only based on an increase in elevated alpha fetoprotein (AFP).\n\nIs the work 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": "198761", "date": "10 Oct 2023", "name": "Arif Ansori", "expertise": [ "Reviewer Expertise Molecular Biology", "Bioinformatics", "Cancer Biology", "Virology", "and Plant Extracts" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, this paper is clear and well-organized. However, I may require some comments on the following issues.\nTitle: The title is easy to follow and no mistakes.\nAbstract: This section was well-written and easy to understand. In addition, the objective of the study and the state of the art of the study are clear. It was mentioned by the authors. Furthermore, the keywords should represent the study (such as tumor growth, Azadirachta indica Juss., and cancer biology).\nIntroduction: Based on my review, I recommend that the authors should add various improvements, such as:\nHypothesis and objectives (It should be mentioned in the introduction). Add a statement or sentence about whether there are any similar studies that have been done before (It should be mentioned in the introduction). Add the important issues (It should be mentioned in the introduction).\nMaterial and Methods: This section should be a more detailed protocol or cite the reference. I cannot see it in the methods section. It is important to strengthen your study. Please kindly check all parts of your Methods section.\nResults: This section's part is easy to follow and has no mistakes. I agree about the results.\nDiscussion: The discussion section of the study is very comprehensive. But, please kindly add some references to enhance the discussion section (Paragraph 1-3).\nConclusion: In this part of the manuscript, the authors have successfully established the conclusion of this research. However, it might be helpful if the authors mention the suggestion for forthcoming research. It should make your conclusion more clear.\nReferences: The core references are acceptable.\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/11-477
https://f1000research.com/articles/9-1268/v1	23 Oct 20	{ "type": "Research Article", "title": "Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein", "authors": [ "Jennifer M. Mutisya", "Victor A. Mobegi", "Johnson K. Kinyua", "Martha N. Kivecu", "Raphael O. Okoth", "Gladys C. Chemwor", "Edwin W. Mwakio", "Agnes C. Cheruiyot", "Redempta A. Yeda", "Charles O. Okello", "Jackline A. Juma", "Benjamin H. Opot", "Dennis W. Juma", "Amanda L. Roth", "Hosea M. Akala", "Ben M. Andagalu", "Jennifer M. Mutisya", "Johnson K. Kinyua", "Martha N. Kivecu", "Raphael O. Okoth", "Gladys C. Chemwor", "Edwin W. Mwakio", "Agnes C. Cheruiyot", "Redempta A. Yeda", "Charles O. Okello", "Jackline A. Juma", "Benjamin H. Opot", "Dennis W. Juma", "Hosea M. Akala", "Ben M. Andagalu" ], "abstract": "Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds. Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modelled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: These interactions provide lead compounds for new anti-malarial molecules. Further in vivo testing is recommended.", "keywords": [ "Exported proteins", "Sulfated polysaccharides", "PHISTb/RLP1", "Interactions", "P. falciparum", "Anti-malarials" ], "content": "Introduction\n\nAfrican countries have 94% of malaria cases and the highest malaria-related death rates according to the 2019 World Health Organization (WHO) Malaria Report (WHO, 2019). Despite its prioritization in the Millennium Development Goals and other large scale global health initiatives, efforts and strategies to reduce the burden of malaria by 40% have stalled over the years due to different challenges (WHO, 2018). These challenges include emergence of Plasmodium falciparum resistance to first-line treatment, the lack of an efficacious vaccine, vector resistance to insecticides, the great diversity of malaria parasite, and insufficient funding towards the control of the disease. These challenges have created a gap that calls for quick intervention to reduce the burden of the disease (Dondorp et al., 2012). The latest drug resistance development of P. falciparum to artemisinin combination therapies in Southeast Asia (Takala-harrison et al., 2015), calls for the development of new drug compounds that can overcome parasite resistance to existing drugs.\n\nStudies have shown that sulfated polysaccharides have antimalarial activities (Mourão, 2015). Unlike most drugs that target the blood stages of the parasite, these compounds exploit newly identified pathways to interact with intracellular parasites. It has been established that merozoites perforate the erythrocyte membrane before egress (Boyle et al., 2010). Heparin enters the infected erythrocyte through these pores and prevents merozoite egress (Glushakova et al., 2017). These findings have led to therapies using heparin to treat severe malaria (Vogt et al., 2006). Moreover, heparin has been shown to reduce the cytoadherence of infected red blood cells and thus reduce parasitemia. However, heparin use was discontinued due to the induction of severe bleeding in patients (Leitgeb et al., 2011). Low concentrations of heparin were later used successfully in safety and efficacy trials to disrupt cytoadherence and rosette formation and additional research has been conducted to identify low anticoagulant sulfated polysaccharides with antimalarial activity. Modified heparin compounds and polysaccharide inhibitors were successfully profiled for activity against intracellular parasites (Boyle et al., 2017). These compounds have been identified in marine organisms and plants (Marques et al., 2016).\n\nThe interaction of sulfated polysaccharide molecules with merozoite proteins and the P. falciparum erythrocyte membrane protein family have been studied intensively. Members of the Duffy binding-like domain and reticulocyte binding-like domain were shown to have interaction with heparin at different affinities (Saiwaew et al., 2017). In addition, sulfated polysaccharides interacts with all P. falciparum reticulocyte binding homologues, including PfRH2, PfRH4 and PfRH5 (Somner et al., 2000). The P. falciparum parasite uses not only the merozoite surface proteins for invasion but also exported proteins that are found on the surface of the erythrocyte to form cross-linkers with erythrocytes (Tarr et al., 2014). The exported proteins have been classified to be essential for parasite survival and virulence, hence contribute to the pathogenesis of malaria (Maier et al., 2008). The Plasmodium-helical interspersed sub-telomeric domain (PHIST) family of exported proteins are expressed in most Plasmodium species but they are largely expanded in P. falciparum. The PHIST domain clusters into three subgroups across the Plasmodium genus, PHISTa, PHISTb, and PHISTc. PHISTa and PHISTb localize specifically in P. falciparum. PHISTb is found in P. vivax and P. knowlesi. One major virulent protein of the PHIST family is PHISTb containing ring-infected erythrocyte surface antigen (RESA)-like protein, abbreviated to PHISTb/RLP1 (PlasmoDB accession number, PF3D7_0201600). This protein has been involved in enabling attachment of infected erythrocytes to organs and blood vessels, as well as erythrocyte remodeling mechanism. Studies have associated this protein with controlling expression mechanism of PfEMP1 (P. falciparum erythrocyte membrane protein) (Moreira et al., 2016; Oberli et al., 2016; Warncke et al., 2016). As a drug target in the P. falciparum genome, PHISTb/RLP1 is identified in Tropical Disease Research Targets database (TDR Targets ID: 3288)\n\nIn this study, we profile specific protein-ligand interactions that give the first insight into drug compounds targeting exported proteins. Furthermore, we outline old and novel mutations found within PHISTb/protein sequences from whole blood samples collected from various malaria endemic sites in Kenya. We link the effects of these mutations on protein structure, active sites and interaction with the drug molecules.\n\n\nMethods\n\nThe study was conducted at the Malaria Drug Resistance laboratory in Kisumu, Kenya, under the United States Army Medical Research Directorate-Africa and Kenya Medical Research Institute. The study analyzed a subset of archived P. falciparum samples from ongoing approved research, studying the epidemiology of malaria drug resistance patterns in Kenya. Samples were collected from the following sites: Kisumu and Kombewa (endemic regions), Kericho and Kisii (highland epidemic areas), Marigat (seasonal transmission zone), and Malindi (coastal area with declining endemicity). The proportion of samples collected was roughly the same between the six collection sites\n\nSamples were collected from volunteers consenting to participate in the study, who were aged 6 months. 2.5 ml of venous blood was collected from participants presenting signs of uncomplicated Malaria. 0.5ml of the blood was transferred to the Acid Citrate Dextrose tubes for sterile culture. At least 1ml of the blood was transferred into an ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′tetraaceticacid (EDTA) tube for molecular assays which included DNA extraction. From the remaining blood sample, 3–5 drops of the syringe blood sample was transferred onto Whatman filter paper by blotting on three separate spots and archived at room temperature in a pouch.\n\nWritten informed consent was provided by participants and/or their legal guardians. The study was carried out in accordance to approved guidelines by the Ethical Review Committee of the Kenya Medical Research Institute (KEMRI), Nairobi, Kenya and Walter Reed Army Institute of Research (WRAIR) Institutional Review Board, Silver Spring, MD. The study was conducted under the approved study protocols KEMRI #1330/WRAIR #1384 and KEMRI #3628/WRAIR #2454.\n\nA total of 251 viable samples were used in this study. P. falciparum parasite DNA extraction was carried out using Qiagen DNA Mini extraction spin protocol (Qiagen, Valencia, CA) as per manufacturer’s instructions. Extracted DNA was stored at -20°C for preservation.\n\nFirst the samples were tested for the presence of Plasmodium parasite. Plasmodium genus was tested with primers F1 and R1 shown in Table 1, the probe was labeled with FAM (6-carboxyfluorescein) in 5’ and at the 3’ TAMRA (6-carboxytetramethyl-rhodamine). All the Plasmodium positive samples were then tested for P. falciparum species. P. falciparum species-specific assay was carried out with primers and probes designed with FAM reporter, as reported in previous work (Kamau et al., 2013; Veron et al., 2009). The samples were amplified in 0.1 milliliters 96-well plates. The kit used for this assay was Quantifast (QIAGEN). The components of each well were, 2µl DNA template, recommended amount of Quantifast master mix and recommended amount of primers and probes. The reaction mixture amounts were calculated according to the number of samples run per assay.\n\nThese are primers and probes were published in previous work by Kamau et al. (2013).\n\nThe real-time PCR assay was the carried out in Applied Biosystem Quantstudio 6 Flex (QuantstudioTm real-time PCR applied Biosystems by Thermos Fisher Scientific) with the following conditions: 95°C for 10 minutes, 40 cycles at 95°C for 15 minutes, and then 60°C for 1 minute, as previously published (Kamau et al., 2013). RNAse-P was used the housekeeping gene and Ct value was awarded by the equipment as the value for which the amplification was high enough to pass the detection threshold The results were exported into Excel worksheet showing the cycle threshold (CT) value for each sample. Only samples positive by P. falciparum confirmatory PCR with a cycle threshold value of at least 32 and below were considered for downstream assays (Table 1).\n\nGene-specific primers were designed using Primer3Plus, as described in the literature (Untergasser et al., 2012). Mapping of the primers to sequences to test the correct annealing on sequence was achieved using Sequence Manipulation Suite version 2. Primers are included in Table 2.\n\nThe table shows the regions on the gene where each primer should anneal as well as the expected PCR product. These are novel primers for this gene, designed and used in this study for the first time.\n\nPCR was carried out in Applied Biosystems thermocycler machine, the reaction conditions were as follows: the first pair of primers amplifying the first fragment were 94°C for 5 minutes during initial denaturation phase and 30 seconds in the normal denaturation, annealing 59°C for 30 seconds, and extension 72°C for 1 minute and 5 minutes in the final elongation stage. The second primer pair annealing temperature was 58°C for 30 seconds while the denaturation and extension phases were the same as primer pair one. We used the puRE Taq Ready-To-Go PCR beads standard master mix (GE Healthcare, CITY, STATE), as per manufacturer’s instructions.\n\nFollowing successful amplification, PCR amplicons were visualized in 2% agarose gel electrophoresis for confirmation. The 2% agarose gel was prepared using 2.25gm of agarose powder in 150 ml of 10% Tris Acetate EDTA (TAE) buffer. The gel was then heated in the microwave for 3 min to allow mixing. The gel was then cooled until it was lukewarm, then 15µl of gel red was added. Thereafter the gel was poured into the gel tank in which gel combs had been inserted. The gel was incubated at room temperature for 30 to 60 minutes to allow the gel to set. Upon setting, the gel was flooded with TAE running buffer just above the wells for loading of samples. Samples were then loaded into the wells on the gel wells by mixing 4µl of the sample with 4µl of loading dye. Additionally, 1.5µl each of 1kb hyper ladder 1 (Bioline) was loaded into the first and final wells on the gel. After loading all the samples, the gel was completely flooded with running buffer (10% TAE) and was run at 230 voltage for 50 min. The gel was then read on UVI save HD5 - Gel documentation (Uvitec Limited, United Kingdom).\n\nAmplification products were purified using ExoSAP-IT (Affymetrix, CITY, STATE) enzymatic cleanup procedure. The enzymatic reaction involves an exonuclease enzyme Shrimp Alkaline Phosphatase (SAP) enzyme, which cleaves phosphate group from unincorporated dNTPs. Using 0.2 ml 96 well plates, each well contained 2µl of ExoSAP-IT enzyme mixed with 8 µl of PCR product. The plates were incubated at 37°C for 20 minutes to allow the enzymes to work then for a further 20 minutes at 80°C to inactivate the ExoSAP-IT enzymes prior to sequencing. Big dye termination PCR was carried out using primary PCR primers and amplification conditions. Big dye terminator sequencing is a modification of Sanger sequencing where dideoxynucleotides (ddNTPs) labeled with a specific fluorescent dye corresponding to each nucleotide base are added to the reaction. The sequencing products were cleaned using Sephadex supplied by Sigma. 10 µl of HiDi formamide (Applied Biosystems) was added into the purified sequence products. The plates were then sealed and heated at 96°C for 3 minutes to denature the DNA and then analyzed using capillary electrophoresis ABI 3130/3500xL Genetic analyzer. Read assembling was done on Qiagen CLC main Workbench version 8.0.1 (CLC Bio, 2014).\n\nMultiple sequence alignment was achieved using Multiple Sequence Comparison by Log-Expectation (MUSCLE) (Edgar, 2004) in Jalview version 2.11.0 (Alzohairy, 2014; Waterhouse et al., 2009). Bioedit version 7.2.5 (Hall, 1999) was used to enable intron and gap deletions, as well as translation of the nucleotides to protein sequences. The alignment was copied to Microsoft Excel and frequencies of the observed single nucleotide polymorphisms (SNPs) counted, as well as generation of frequency bar graphs.‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬\n\nProtein structures of the PHIST/RLP1 reference (Pf3D7) and mutant proteins structure were predicted via ab initio multithreading tool LOMETS (Wu & Zhang, 2007; Zheng et al., 2019). The LOMETS tool has an internal selection of templates through in-built multiple sequence alignment and ranking the templates in descending order according to a normalized Z score. A Z score greater than or equal to one is considered a good alignment.\n\nProtein Data Bank (PDB) hits used as templates for the PHISTb/RLP1 reference structure were 4jle, 5ez3 and 6d03 all with a normalized Z-score ≥1. The templates selected for the PHISTb/RLP1 mutant structure modelling were 4jle, 2ziq and 6d03, which had a normalized z-score ≥ 1. Two templates were similar for the reference and mutant protein structure threading because of sequence similarities at the starting and ending domains of the the protein sequences. The middle template was different for both protein structure threading of mutant and reference structures due to presence of point mutations in this region of the mutant PHISTb/RLP1 protein sequence.\n\nThe models were submitted to ModRefiner (Xu & Zhang, 2011) for energy minimization. The energy minimized structure of PHISTb/RLP1 reference had a Template Model (TM) score of 0.97 to the initial model. The TM score is calculated between 0 and 1, whereby the higher the TM score the higher the perfect match between the two structures. The models were verified using Galaxy Refine web server tool (Ko et al., 2012) for correction of wrong rotamers. The protein model stability were validated through the parameter of percentage residues lying within the favored and allowed regions using Rampage tool (Lovell et al., 2003) and overall stability confirmed using PROCHECK web tool (Laskowski et al., 1993). Following model validations, the functional sites within the protein structure were predicted in the webserver tool FT Site (Ngan et al., 2012)(Brenke et al., 2009). FT SITE algorithm was reported to achieve near experimental accuracy of predicting druggable hotspots in 94% of apo-proteins used in evaluation of binding sites methods. For confirmation of our binding site clusters, COACH, a metaserver tool that uses comparisons of other servers prediction to profile highly accurate protein-ligand binding sites, was used (Yang et al., 2013).\n\nThe sulfated polysaccharides containing anti-malarial properties were identified through chemical modifications, as per previous research (Boyle et al., 2017). We searched for these compounds from the PubChem database, which stores chemical structures of identified chemical compounds and their biochemical activities (Kim et al., 2016). The drug likeness of the screened compounds were analyzed for the Lipinski Rule of Five, as follows: molecular mass <500 Dalton; high lipophilicity (expressed as LogP <5); <5 hydrogen bond donors; <10 hydrogen bond acceptors; and molar refractivity should be between 40–130. This was achieved using the Lipinski Rule of Five webserver (Gimenez et al., 2010)\n\nThe Standard Database Format (.SDF) files of the drug compounds obtained were converted to PDB format files using Open Babel tool version 2.3.1 on Linux command line (O’Boyle et al., 2011). Ligand files, receptor files (protein models), and grid parameters were prepared using MGL tools version 1.5.6 (Morris et al., 2009). Proteins (receptors) and the ligands (drug compounds) were converted from PDB format to PDBQT file format required by autodock tolls. Docking simulations were achieved using AutoDock Vina version 1.1.2 (Trott & Olson, 2010). The output of AutoDock Vina results was visualized using PyMOL version 2.3.5 (Schrodinger LLC).\n\n\nResults\n\nA total of 175 out of 251 (70%) samples tested positive for Plasmodium parasite. The 70% positive samples were further tested for P. falciparum species and 63% were positive. The positive samples (n=110) were used for the downstream experiments, i.e. they were used to sequence PHISTb/RLP1 gene.\n\n102 of the 110 samples processed for Sanger sequencing yielded sequences. Of these, 86 samples generated complete sequences; 16 samples had poor sequences.\n\nA total of 157 non-synonymous SNPs were observed across the full length of the protein sequences (485 amino acid in length). The SNPs occurred at different frequencies in the total sequenced samples. Only SNPs occurring at a frequency >50% were considered for protein structure analysis (n=20) (Table 3).\n\nThe total number of SNPs was 157 across the entire protein length, in all the 102 sequenced samples. 107 SNPs were only overserved in 1-10% of the samples and were excluded. 30 of the remaining SNPs occurred in 10-50% samples and this frequency was not high enough to be considered for downstream analysis, they were excluded as well. The final 20 codons of the total observed non-synonymous SNPS had a frequency >50% of the total samples sequenced. These were considered for protein structure analysis. The frequency was high thus the implications of these SNPs was analyzed further.\n\nWithin the 20 SNPs occurring at high frequency, codons F145L, D146R, Y147D, S156H, S208L and L219H were all novel mutations identified in our samples across all different sites where our samples were collected. We compared our data against SNPs recorded in PlasmoDB genetic variation tracks for the PHISTb/RLP1 3D7 reference (PF3D7_0201600). These six mutations were not present in the database; therefore, they could be newly identified mutations for PHISTb/RLP1 gene as observed in our samples. Analyzing the amino acids substitutions further, we observed that mutations at codons N108T, F145L, W209F, Y210N, V269A, V274I and M277L were all substitutions of amino acids within the same group. The F145L, W209F, V269A, V274I and M277L mutations were all substitutions within the non-polar group, while N108T and W209F were substitutions in the polar group. Amino acids within the same group play similar functional roles in the protein sequence and thus these SNPs were not considered for protein function analysis. On the other hand, I129T, T142V, Y147D, E154Q, S156H, T167I, S208L, M211T, L219H, D387N, D390N, and E403K were substitutions of amino acids across different functional groups. We postulated that these point mutations are most likely to change the folding of the protein structure and have an effect on the function of the protein. These codons were modelled in the mutant protein sequence for PHISTb/RLP1 structure. Their effect on the tertiary protein structure as well as effect on interaction with a drug compound were further investigated (Figure 1).\n\nNon-synonymous SNPs occurring in 50% and above of the total sample size. Codons I129T, T142V, Y147D, E154Q, S156H, T167I, S208L, M211T, L219H, D387N, D390N, and E403K were substitution of amino acids across different functional groups. These were modelled and their implications on protein structure and interactions with a drug compound investigated.\n\nPHISTb/RLP1 reference and mutant structures were modelled successfully. The LOMETs tool gave five models for the reference and mutant structure each. The fourth model in both reference and mutant modelling sessions was selected and submitted to ModRefiner for energy minimization. The energy minimized structure of PHISTb/RLP1 reference had a TM score of 0.97 to the initial model. The TM score is calculated between 0 and 1, whereby the higher the TM score the higher the perfect match between the two structures.The PHISTb/RLP1 mutant protein had a TM score of 0.98 following energy minimization. The models were submitted to Galaxy Refine for further model refinement and then we chose the first models with best scores. The Galaxy refined models were submitted to RAMPAGE software for Ramachandran plot assessment. The reference structure had 95% and the mutant structure 94% of the residues in the favored region. The PROCHECK results displayed 92% of the residues in the reference structure to be in the most favored region. The mutant structure had 90% of the residues to be in the favored region. Ramachandran plot analysis required a good model to have at least 90% of the amino acids in the favored region. These Ramachandran plot scores qualified the two models to be used for the docking experiments and were submitted to binding sites prediction tools.\n\nFT Site prediction outcome included Photoshop Element (.pse) files that were visualized in PyMol to visualize the active amino acids in the binding sites clusters for both reference and mutant PHISTb/RLP1 protein structures, as shown in Table 4. We also considered residues that were predicted by COACH software and had the highest confidence score (C-score) of 0.05 for PHISTb/RLP1 reference protein structure. COACH predicted residues in PHISTb/RLP1 protein structure had a C-score between 0.07 and 0.08. The C-score ranges between 0–1 where a higher C-score gives a more reliable prediction. In both the mutant and the reference structures, there were active residues predicted by both tools. This double prediction emphasized the accuracy of the binding sites as functional regions of the proteins (Figure 2 and Table 5).\n\nThe score considered included the poor rotamers scores in which they are very low in both inferring to correction in R chain conformations. The Rama favored scores were also used to consider the models. The scores above 90 for Rama favored shows that the models are good to be used for further analysis.\n\n(a) PHISTb/RLP1 reference protein structure shown in green; (b) structure of PHISTb/RLP1 mutant protein structure shown in cyan; (c) PHISTb/RLP1 reference protein binding site clusters, site 1 in red, site 2 in blue and site 3 in yellow; (d) PHISTb/RLP1 mutant protein binding sites clusters, site 1 in pink, site 2 in orange and site 3 in dark purple. All models visualized in PyMOL.\n\nThe highlighted residues are predicted by both softwares. This puts emphasis on the accuracy of the predictions.\n\nThe PubChem database search yielded the following compounds: beta carrageenan, alpha carrageenan, dextrin sulfate, amylopectin sulfate, zinc sulfate, ghatti sulfate, 2,4-diaminoanisole sulfate, cyclodextrin sulfate, Fucoidan, 3-aminophenylboronic acid and 3,6-Di-O-benzoyl-D-galactal. In this group of compounds, dextrin sulfate deviated from the Lipinski Rule of Five. Beta carrageenan has hydrogen bond acceptors of 12, the compound was considered for interaction experiments because the Rule of Five allows a window of one deviation of the physical properties. 3-aminophenylboronic acid PDB file contained atoms that could not be recognized by AutoDock Vina. The compound was left out for docking experiments. All the other drug compounds adhered to Lipinski Rule of Five hence passing the toxicity test of drug likeness and were tested for activity against the exported protein PHISTb/RLP1 (Table 6).\n\nAccession numbers, physical properties and Lipinski Rule of Five properties of each compound are curated. The Lipinski rule of five requires that a drug compound meets all the properties or deviates with at most one property. All the screened compound met the rules of a drug compound, except dextrine sulfate.\n\nThe identified compounds interacted with the target protein. Alpha carrageenan, amylopectin sulfate, cyclodextrin sulfate and Fucoidan exhibited optimum interactions with the PHISTb/RLP1 protein. The amino acids interacting with these compounds were identified in the binding sites. Of these interactions, amino acids S132, K84 and N80 were found within the PHISTb domain. These interactions significantly show the potential of interfering with the function of the exported protein. Beta carrageenan and dextrin sulfate compounds had specific interactions with the target protein. Amino acids K376, E402 and E403 were identified in the binding site clusters. 2, 4-Diaminoanisole sulfate and zinc sulfate interactions with the protein were not specific with the identified binding site clusters. However, we noted that the amino acids interacting with these compounds are within the PHIST domain which spans amino acids 1 to 167 of the protein sequence. Ghatti sulfate showed interaction with the reference protein as well, although not specific.\n\nThe same drug compounds were tested on the m odelled PHISTb/RLP1 mutant protein structure with the identified non-synonymous SNPs from the sequenced data. The aim was to test whether mutations found within the protein interfere with the interactions or not. The interactions of the drug compounds with mutant protein were not as strong as those of the reference protein. The interactions in the mutant protein occurred at different binding residues because the mutations within the proteins affected the folding of the protein structure. Due to these mutations, there was a shift of the binding sites clusters. Alpha carrageenan, ghatti sulfate and 3,6-Di-O-benzoyl-D-galactal depicted specific interactions with the mutant protein. Residues K35, N90, D17 and N18 were within the PHIST domain. The other compounds were interacting with different residues in the protein domains. The interactions were not optimized to the predicted binding site clusters. However, we noted that some of the interacting residues were within the PHISTb domain, which is the functional region of the protein. These interactions, therefore, gave insight to the action of the drug compounds against the mutant protein.\n\nWhen selecting the poses after docking experiments, we selected the first pose of the AutoDock Vina pdbqt output file. The docking energies of the screened reference and mutant targets were very low. The energies released therefore supported the drug likeness of these compounds. The resulting Root Mean Square Deviation (RMSD) was zero (first pose RMSD are always zero). There was a difference in docking energies of the drug compounds interacting with PHISTb/RLP1 reference and mutant proteins because of the difference in binding site clusters. The interactions with different residues caused by the shifting of binding sites due to mutations resulted in the slight difference in energy released during docking (Figure 3, Table 7–Table 9)\n\n(a and b) visualizations of alpha carrageenan (firebrick) and fucoidan (blue) drug compounds interacting with PHISTb/RLP1 reference protein structure (green) at specific binding sites. The interacting residues and polar contacts are shown in firebrick and blue PyMOL colours respectively. (c and d) are visualizations of alpha carrageenan (firebrick) and ghatti sulfate (purple) compounds interacting with PHISTb/RLP1 mutant protein structure binding pockets. The interacting residues and polar contacts are shown in firebrick and purple PyMOL colours, respectively. These specific interactions reveal potential inhibitory activity of these compounds against PHISTb/RLP1 proteins.\n\nThe highlighted residues show optimized interactions with amino acids that were predicted by the binding site predicting tools. Alpha carrageenan, beta carrageenan, amylopectin sulfate, cyclodextrin sulfate and fucoidan compounds interacted specifically with amino acids predicted in binding sites of the reference protein structure. They showed potential ability to clock functional domains of PHISTb/RLP1 protein.\n\nThe highlighted residues are specific amino acids predicted by the binding sites prediction tool. Alpha carrageenan, ghatti sulfate and 3,6-Di-O-benzoyl-D-galactal compounds interacted optimally with predicted amino acids in the mutant protein binding sites. The other compounds interacted with amino acids within the PHIST and RESA domains of the protein. These interactions revealed potential activity of these compounds as inhibitors of PHISTb/RLP1 protein from carrying out its functions.\n\nThe energies released were all very low, supporting the activity of the sulfated polysaccharides as drug compounds against the exported proteins. The low binding energies show high binding affinities. Generally, the docking energies in PHISTb/RLP1 mutant protein was slightly higher that in reference protein. As seen in the interaction results, the compounds are interacting with different residues in the two proteins hence the difference in the released energy.\n\n\nDiscussion\n\nIn P. falciparum, SNPs have been reported to cluster in subtelomeric regions of the chromosomes. A study comparing synteny of exported proteins in P. vivax and P. falciparum reported a large number of SNPs in chromosome 2 and 10 subtelomeric regions (Sargeant et al., 2006). The subtelomeric regions of the P. falciparum genome have been reported to be highly variable. The genes found in these regions have shown sequence diversity within and across different P. falciparum isolates. The drug target PHISTb/RLP1 is located in chromosome 2. Our genetic diversity analysis of this protein in Kenyan isolates depicted many SNPs, with many of the polymorphisms having been identified before. Novel SNPs were reported with high frequency across our sample size. Novel point mutations were identified in our sequences across different groups of amino acids. These included I129T, T142V, Y147D, E154Q, S156H, T167I, S208L, M211T, L219H, D387N, D390N, and E403K. These substitutions are hypothesized to affect the function of the protein and we considered them to analyze their effect on the structure of the protein, as well as interactions with drug compounds.\n\nThe homology modelling of full length PHISTb/RLP1 protein revealed that the 3D structure of this drug target is characterized by alpha helices. This was confirmed by the solved PHIST domain crystal structure PDB ID 4JLE. Upon comparing the reference and mutant structures, there was a difference in the folding of the alpha helices. This difference was clearly depicted by the difference in clustering of the binding sites. This can be accounted for by the point mutations, which changed the conformation of the protein structure. The tool used to model the two proteins selected one different template for threading, which explains the difference in the overall writing of the pdb file for the two structures. The PHIST domain spans amino acids residues 1 to 167 of the protein sequence. Both structures had a cluster of functional sites towards the C-terminal. This emphasizes crucial function of this part of the protein. Other clusters were distributed in the middle and towards the N-terminal of the structures. The PHISTb/RLP1 is a multi-domain protein, it contains the PRESAN domain (Plasmodium-RESA N-terminal) and the RESA domain, which is a fusion of other domains, namely the DnaJ domain and the DnaJ-associated X domain Pfam accession number PF0987. The distribution of these domains across the protein sequence explains the clustering of the binding sites in the protein structure of the PHISTb/RLP1 protein.\n\nSulfated polysaccharides are a wide group of biochemical molecules with therapeutic properties including anti-thrombotic, anti-viral and anti-plasmodial activities (Mourão, 2015). We narrowed down our search to sulfated polysaccharides with potential anti-malarial properties (Boyle et al., 2017). The database search yielded ten compounds that are curated in PubChem compounds: Beta carrageenan, alpha carrageenan, dextrin sulfate, amylopectin sulfate, zinc sulfate, ghatti sulfate, 2,4-Diaminoanisole sulfate, cyclodextrin sulfate, Fucoidan,3-Aminophenylboronic acid and 3,6-Di-O-benzoyl-D-galactal. The compounds were tested for activity against exported protein PHISTb/RLP1 protein except for 3-Aminophenylboronic acid. Despite dextrin sulfate deviating from Lipinski’s Rule of Five, it was still tested. Dextrin sulfate has been supported by previous research to contain antimalarial activity (Boyle et al., 2017). The drug activity of this compound supports its investigation as an antimalarial with further chemical modifications to suit rules of a drug compound. Carrageenan compounds antimalarial activity had previously been reported, however, the compound had to be further modified to reduce its toxicity (Adams et al., 2005; Recuenco et al., 2014). All these compounds interacted with the PHISTb/RLP1 at different affinities and this inferred their inhibitory activity against the PHIST family of proteins.\n\nThe identified drug compounds interacted with the exported protein PHISTb/RLP1. Alpha carrageenan compound interacted with both the reference and the mutant proteins. The compound shows potential inhibitory activity against exported proteins. Amylopectin sulfate, cyclodextrin sulfate, ghatti sulfate, Fucoidan and 3,6-Di-O-benzoyl-D-galactal compounds have specific interactions with the protein. The interactions of the drug compounds with specific amino acids found in binding site clusters depicted that these compounds have the potential to block the protein domains used to invade red blood cells. 2,4-Diaminoanisole sulfate and zinc sulfate showed weak interactions with the proteins. The interactions with the mutant protein are generally weaker. The mutations found in the PHISTb/RLP1 changed the tertiary folding of the protein thus interfering with the active sites. The identified mutations I129T, T142V, Y147D, E154Q, S156H and T167I are within the PHIST domain (PDB ID: 4JLE) (Oberli et al., 2014). I129T, T142V and T167I represent polar to non-polar and non-polar to polar substitutions. Y147D shows substitution of a polar amino acid to negatively charged aspartic acid residue. E154Q is a substitution from negatively charged to polar uncharged glutamine and S156H is a substitution from polar uncharged group to a positively charged amino acid. These changes affected the protein structure as well as enhancing the protein function. The binding affinity was different among the amino acids found in different structures and was shown by the difference in docking energies. The interactions of these compounds with exported proteins support the wide activity of sulfated polysaccharides against the P. falciparum parasite.\n\nP. falciparum parasite uses the mechanism of exporting proteins to the host erythrocyte to enhance its virulence. These changes induced by the exported proteins include changing the physical properties of the cell and giving the cell adhesive properties. These changes enhance the pathogenesis of malaria in humans (Boddey et al., 2016). Among the exported proteins, the PHIST family, which contains 89 proteins, has been identified to play a role in making the remodeled host cell more cytoadherent (Warncke et al., 2016). The key target we have studied in this research, PHISTb/RLP1 (Pf3D7_0201600) plays a key role in remodeling of the host erythrocyte to enhance malaria virulence through cytoadherence mechanism (Warncke et al., 2016). The PRESAN domain present in this protein contains the Plasmodium protein export element (PEXEL) motif that enables the export mechanism of the protein (Boddey et al., 2016; Moreira et al., 2016). The interactions of the screened drug compounds with amino acids found in the functional domains of this protein reveal novel chemical inhibitors targeting exported proteins. The compounds have the ability to inhibit the PRESAN and PHIST domains from carrying out the export functions of the proteins. In P. falciparum, the PHIST family of proteins are expressed in the early and late ring stages, as well as trophozoites. Using PHISTb/RLP1 as a representative of this protein family, the interactions of the protein with sulfated polysaccharides infers that these compounds can deactivate exported proteins. The PHISTb proteins have been linked with controlling the P. falciparum Erythrocyte Membrane Protein (PfEMP1) major virulent factor of the parasite (Tarr et al., 2014). The interactions of the screened drug molecules with the mutant protein of PHISTb/RLP1 show that even though the mutations change the protein folding, the functional domains are still being blocked by the chemicals. The screened sulfated polysaccharides adhere to the Lipinski Rule of Five. Their toxicity levels are very low and can be investigated further in in vitro analysis and clinical trials.\n\n\nConclusion\n\nThe interactions of specific sulfated polysaccharide compounds with PHISTb/RLP1 protein are the first findings showing compounds that can act against exported proteins of the Plasmodium parasite. These findings support further drug discovery downstream processes with these compounds as lead compounds for developing the next class of antimalarial agents. Crystal structures solving PHISTb/RLP1 and other exported proteins is recommended to enable more insight into the implications of structural variants on the protein structure and functions.\n\n\nData availability\n\nOpen Science Framework: Novel Characterization of Sulfated Polysaccharides Activity against Virulent Plasmodium falciparum PHIST Proteins, https://doi.org/10.17605/OSF.IO/YFQAZ (Mutisya, 2020).\n\nThis project contains the following underlying data:\n\n- Sequenced reads of PHISTb/RLP1 acquired through Sanger sequencing\n\n- Multiple Sequence Alignment files\n\n- Protein .pdb files (of both PHISTb/RLP1 reference and mutant protein structures)\n\n- Sulfated polysaccharides .sdf and .pdbqt files.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAdams Y, Smith SL, Schwartz-Albiez R, et al.: Carrageenans Inhibit the in Vitro Growth of Plasmodium Falciparum and Cytoadhesion to CD36. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "100719", "date": "31 Jan 2022", "name": "Eusébio Macete", "expertise": [ "Reviewer Expertise I am specialist in public health working mostly in malaria drug and 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\nMy first comment is regarding the selection of the sites, the authors did not mention how the sites were selected.\nWere the samples collected for this specific study? Or is this study a secondary analysis? If yes, what was the primary study?\nSecond, in the article the authors mentioned that the samples were tested for plasmodium falciparum, so, how were patients that had positive results treated? If that was the case?\nIn the method paragraph, the study design is not clear. How were the samples selected etc.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "7787", "date": "17 Feb 2022", "name": "Jennifer Mutisya", "role": "Author Response", "response": "Concerning the comments on sample sites selection and collection: The samples used in this study were not collected specifically for this secondary study but were used on basis of availability. The samples were collected for a bigger project that studies drug resistance patterns from the identified regions. The sampling for this study was hence non-randomly conducted from sample inventory of archived whole blood samples. This study was a sub-study in the Malaria Drug Resistance Laboratory and not the primary study of the institution. The sample sites were selected according to different Malaria endemic zones in Kenya and were guided by the proposal of the main study which studies epidemiology patterns of the disease in Kenya. The patients who tested positive were treated with recommended Artemesinin combined therapies at day zero of testing and follow up samples were collected after 7, 14 and 28 days. These were done according to the objectives of a different study. The current study did not need the treatment data." } ] }, { "id": "121686", "date": "01 Mar 2022", "name": "Abraham Madariaga", "expertise": [ "Reviewer Expertise Molecular modeling including docking and molecular dynamics simulations", "QSAR studies", "homology modeling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes the modeling of a protein and its mutant from Plasmodium falciparum, and the assessment of some ligands with these models by molecular docking analysis. The mutant was built based on experimental data from several samples. Both protein models were validated using online tools. Although the authors used several online tools for assessing the structural performance of the proteins, the mutant model is lacking confidence in its tridimensional structure. Furthermore, the docking protocol is not fully described. The results are too preliminary to suggest a potential activity of the selected ligands against PHISTb/RLP1 proteins. I would recommend major revision prior to indexing.\nThe modeled mutant protein resulted with a very different tridimensional configuration to the reference protein. For better prediction and assessment of a homology model, it is suggested to perform a molecular dynamics simulation of at least 50 ns in order to let the structure correctly minimize. Otherwise, the predicted protein may have a non-biological relevance.\nIt is not clear why the authors select the fourth model of each structure (reference and mutant) for energy minimization. Does LOMETs give a score? Furthermore, the TM score is significantly high for both structures, meaning a “perfect match” between the initial model and the generated model. It is surprising that the mutant model gets a TM as similar as the reference model, so the mutant has no apparent structural changes despite having a dozen different amino acids. Nevertheless we can see in Fig2a-b big differences among those structures. How do you explain this?\nRule of five is not synonymous with toxicity. Rule of five gives an empirical approximation of the pharmacokinetics of a molecule, in terms of absorption. Ro5 is a simple approach intended to select molecules with druggability properties. To examine toxicity of molecules, many other methods can be used (Ames mutagenicity prediction, ADME-Tox predictions, LD50, etc) in different online platforms.\nDocking protocol. Were the ligands used in their salt forms? For better results, the ligands should undergo a “washing” procedure that includes removing contra ions, adjusting charges, and geometric minimizing process. It is not described in the methodology.\n\nRegarding the gridbox, what size does it have? Depending on the size, the ligands (in this case the ligands are large molecules) are allowed to freely rotate or not. This may bias the docking results.\nOn the other hand, if a positive control is not used as a part of the docking experiments, it is difficult to argue that a molecule will have a potent activity, such in the case of the manuscript. It is preferable to suggest only a possible activity.\n\nFor completeness of the discussion, a figure depicting the ligand-protein interaction of the ligands with better affinity would be great.\nMinor Page 6, Section “Preparation of proteins and ligands, …”, line 8: Please change “autodock tolls” for “autodock tools”.\n\nIs the work 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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "8129", "date": "29 Apr 2022", "name": "Jennifer Mutisya", "role": "Author Response", "response": "Comment: The manuscript describes the modeling of a protein and its mutant from Plasmodium falciparum, and the assessment of some ligands with these models by molecular docking analysis. The mutant was built based on experimental data from several samples. Both protein models were validated using online tools. Although the authors used several online tools for assessing the structural performance of the proteins, the mutant model is lacking confidence in its tridimensional structure. Furthermore, the docking protocol is not fully described. The results are too preliminary to suggest a potential activity of the selected ligands against PHISTb/RLP1 proteins. I would recommend major revision prior to indexing. Response: All the tools used in modeling and assessment of the protein structures are open source tools with algorithms that are assessed and published. Furthermore, they have been used and cited in other scientific research prior to this work hence able to provide significant scientific data as documented in the provided links and cited publications for each tool used. The docking protocol details are included; that is preparation of ligand files in MGL tools, grid box parameters and python script used. Comment: The modeled mutant protein resulted with a very different tridimensional configuration to the reference protein. For better prediction and assessment of a homology model, it is suggested to perform a molecular dynamics simulation of at least 50 ns in order to let the structure correctly minimize. Otherwise, the predicted protein may have a non-biological relevance. Response: Energy minimization carried out using ModRefiner an algorithm for atomic-level, high-resolution protein structure refinement, which can start from either C-alpha trace. The models underwent energy minimization using this tool prior to docking experiments. Molecular dynamics simulation of at least 50ns were not possible to carry out due to minimal expertise in a tool that can do that. However, after energy minimization using ModRefiner the models quality improved and were fit for docking simulations. Comment: It is not clear why the authors select the fourth model of each structure (reference and mutant) for energy minimization. Does LOMETs give a score? Furthermore, the TM score is significantly high for both structures, meaning a “perfect match” between the initial model and the generated model. It is surprising that the mutant model gets a TM as similar as the reference model, so the mutant has no apparent structural changes despite having a dozen different amino acids. Nevertheless, we can see in Fig2a-b big differences among those structures. How do you explain this? Response: The fourth model was chosen after screening the Ramachandran score for the five suggested models hence the fourth model had the highest scores. Lomets does not provide scores for models since they are racked from number 1 to 5, but for the template selected. Structures match because the middle template selected by the algorithm was similar in both reference and mutant modelling jobs. The PHIST domain is the only present crystal structure for this protein family in PDB. This is why the TM score matches closely. The difference between the two structures can be seen in the alpha-helix folding which is significantly distinct and postulated to be an effect of the SNPs identified. Comment: Rule of five is not synonymous with toxicity. Rule of five gives an empirical approximation of the pharmacokinetics of a molecule, in terms of absorption. Ro5 is a simple approach intended to select molecules with draggability properties. To examine the toxicity of molecules, many other methods can be used (Ames mutagenicity prediction, ADME-Tox predictions, LD50, etc) in different online platforms. Response: ADMET analysis data was included using SwissADME tool as suggested. Comment: Docking protocol. Were the ligands used in their salt forms? For better results, the ligands should undergo a “washing” procedure that includes removing contra ions, adjusting charges, and geometric minimizing process. It is not described in the methodology. Response: The ligand files were converted using the Open Babel tool and prepared using MGL tools as described in the text. This editing enabled the removal of contradicting s ions prior to docking. Comment: Regarding the grid box, what size does it have? Depending on the size, the ligands (in this case the ligands are large molecules) are allowed to freely rotate or not. This may bias the docking results. Response: grid box dimensions were added in the main text. Comment: On the other hand, if positive control is not used as a part of the docking experiments, it is difficult to argue that a molecule will have a potent activity, such in the case of the manuscript. It is preferable to suggest only a possible activity. Response: Included low molecular weight heparin as the positive control compound since it is a well-researched sulfated polysaccharide with anti-malarial properties. Comment: For completeness of the discussion, a figure depicting the ligand-protein interaction of the ligands with better affinity would be great. Response: Included a figure in the discussion" } ] } ]	1	https://f1000research.com/articles/9-1268
https://f1000research.com/articles/10-542/v1	06 Jul 21	{ "type": "Research Article", "title": "Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production", "authors": [ "Hiroki Doi", "Taei Matsui", "Johannes M. Dijkstra", "Atsushi Ogasawara", "Yuki Higashimoto", "Seiji Imamura", "Tamae Ohye", "Hiromu Takematsu", "Itsuro Katsuda", "Hidehiko Akiyama", "Hiroki Doi", "Taei Matsui", "Johannes M. Dijkstra", "Atsushi Ogasawara", "Yuki Higashimoto", "Seiji Imamura", "Tamae Ohye", "Hiromu Takematsu", "Itsuro Katsuda" ], "abstract": "Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.\n\nMethods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors.\n\nResults: Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. The apoptotic activities of Andro were largely suppressed by N-acetyl-L-cysteine (NAC), an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Furthermore, whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression.\n\nConclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. Notably, the high sensitivity of H929 cells is encouraging for further studies on the use of Andro against multiple myeloma.", "keywords": [ "Andrographis paniculata", "andrographolide", "apoptosis", "reactive oxygen species", "monocytic leukemia cells", "multiple myeloma cells" ], "content": "Introduction\n\nMany plant-derived products possess a potential for use in chemotherapy. For example, vincristine (VCR) and vinblastine—two natural alkaloids isolated from Vinca rosea—inhibit cell division and are commonly used in anticancer medicine (Varma et al., 2011). Another example is andrographolide (Andro), a diterpenoid lactone isolated from the Asian herbal plant Andrographis paniculata, which has a variety of pharmacological effects including anti-tumor, anti-inflammatory, anti-viral, and anti-malarial activities (Hao et al., 2020; Kishore et al., 2017; Kumar et al., 2020; Sareer et al., 2014).\n\nAndro has been shown to have anti-tumor activities against solid and hematopoietic tumor cell lines, established from colon-, gastric-, liver-, breast-, and prostatic cancers, leukemia, and lymphoma (Banerjee et al., 2016; Chen et al., 2012; Cheung et al., 2005; Dai et al., 2017; Khan et al., 2018; Kim et al., 2005; Yang et al., 2010). Common observations in these studies were that Andro reduced the cell viability/proliferation, although the mechanisms were found to differ per cell type. In most cases, the reduced viability of the tumor cell lines could at least partially be explained by the induction of apoptosis (Banerjee et al., 2016; Cheung et al., 2005; Dai et al., 2017; Khan et al., 2018; Kim et al., 2005; Yang et al., 2010), but in several liver cancer cell lines the cell death caused by Andro was distinct from apoptosis (Chen et al., 2012). Moreover, among different tumor cell lines, Andro treatment showed a variable effect on the cell distribution among cell cycle phases (Banerjee et al., 2016; Cheung et al., 2005; Dai et al., 2017; Khan et al., 2018). From the perspective of its potential usage as an anti-cancer drug, the most important observations were that at concentrations at which Andro significantly reduced the viability of tumor cells, normal epithelial cells and lymphocytes were not noticeably affected (Banerjee et al., 2016; Khan et al., 2018; Yang et al., 2010). Equally important from a therapy point of view was that, at least in vitro, primary lymphoma cells were even more sensitive to Andro than lymphoma cell lines (Yang et al., 2010).\n\nTHP-1 (RRID:CVCL_0006) is a permanent human monocytic cell line derived from an acute monocytic leukemia patient (Abrink et al., 1994; Tsuchiya et al., 1980). Previously, preparations or modifications of Andro were found to be toxic for THP-1 cells (Habtemariam, 2003; Lee et al., 2012), to enhance the cells’ expressions of cytokine IFNγ and of stress-protein GRP-78 (Gupta et al., 2020), and to interfere with their functional properties such as the (immune-induced) activation and/or production of transcription factor NF-κB, matrix metalloproteinase-9, and various cytokines (Gupta et al., 2020; Lee et al., 2012; Low et al., 2015; Nie et al., 2017), and their migration in a chemotaxis assay (Zhang et al., 2019). An analogue of Andro,14-Deoxy-11,12-didehydroandrographolide (AND2), induced apoptosis in THP-1 cells (Raghavan et al., 2014), but—to the best of our knowledge—the present study is first to address how Andro itself induces apoptosis in these cells.\n\nH929 (aka “NCI-H929”) (RRID:CVCL_1600) is a permanent human IgA-kappa-producing multiple myeloma cell line (Gazdar et al., 1986) for which the sensitivity to Andro—as far as we know—has not been investigated. However, Andro was found to have inhibitory/cytotoxic/apoptotic effects on other multiple myeloma cell lines, even at low concentrations (Gao and Wang, 2016; Gunn et al., 2011). These high sensitivities led us to be interested in the effects of Andro treatment on H929 cells.\n\nIn the present study, we investigated the viability-reducing effects and their mechanisms of Andro on both THP-1 and H929 cells. The effects of Andro were compared with those of the common anti-cancer drugs VCR and Ara-C (aka cytarabine). VCR and Ara-C are widely used as chemotherapeutic agents against soft tissue tumors and hematopoietic tumors including acute leukemia, lymphoma, and multiple myeloma (Koharazawa et al., 2008; Lu et al., 2003; Short and Ravandi, 2016; Tsimberidou et al., 2014). Andro showed an excellent viability-reducing activity against both THP-1 and H929 cells, and in the case of H929 cells this effect was markedly superior to that of VCR or Ara-C. Unlike with Ara-C and VCR, the viability-reducing effect of Andro was found to be dependent on the enhanced production of reactive oxygen species (ROS).\n\n\nMethods\n\nAndro was purchased from Tokyo Chemical Industry (Tokyo, Japan), dissolved in ethanol at 10 mM, and used at 10-50 μM. Cytosin arabinoside (Ara-C) and vincristine (VCR) were purchased from SIGMA-ALDRICH (Missouri, USA), dissolved in phosphate-buffered saline (PBS; 150 mM NaCl, 10 mM phosphate-buffer, pH 7.2), and used at 40 μM and 0.1 μM, respectively. Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), a pan-caspase inhibitor was purchased from Promega (Tokyo, Japan) and used at 20 μM. As an antioxidant, N-acetyl-L-cysteine (NAC) was purchased from Funakoshi (Tokyo, Japan), dissolved in ultra-pure water at 1 M and used at 3 mM.\n\nTHP-1 cells (human monocytic leukemia cell line; EC88081201; RRID:CVCL_0006) and NCI-H929 cells (human IgA-kappa-producing multiple myeloma cell line; EC95050415; RRID:CVCL_1600) were obtained from DS PHARMA BIOMEDICAL (Osaka, Japan). They were grown in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio Inc, Kerrville, USA), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (GIBCO, Carlsbad, USA) at 37 °C with 5% CO2. For experiments, Andro, Ara-C, or VCR were added to the cell cultures at the appropriate concentrations. NAC was added to the cell culture one hour before the addition of Andro, Ara-C, or VCR. Control (untreated) cells were harvested at 24 h.\n\nCells were deposited on glass slides by the cytospin method at 40×g for 5 min (Cyto-Tek 2500 Cytocentrifuge, Sakura, Tokyo, Japan) (Tokunaga et al., 2017). The glass slides were fixed with Wright’s solution (Muto Pure Chemicals, Tokyo, Japan) and stained with Giemsa’s solution (Muto Pure Chemicals) to observe the morphological changes of the cells.\n\nCells were centrifuged at 300×g for 5 min and washed once with PBS. The cell pellet was suspended in 100 μL of cell lysis buffer (10 mM Tris–HCl buffer, pH 7.4 containing 10 mM EDTA and 0.5% Triton X-100), and kept at 4°C for 10 min. Cell lysate was centrifuged at 16,000×g for 20 min. The supernatants (100 μL) were incubated with 2 μL of RNase A (20 mg/mL; MACHEREY-NAGEL, USA) at 37°C for 60 min, and then with 2 μL of proteinase K solution (20 mg/mL; Wako, Japan) at 37°C for 60 min. After adding 20 μL of 5 M NaCl and 120 μL of isopropyl alcohol, these mixtures were kept at −30°C overnight. The precipitate was then collected by centrifugation at 16,000×g for 15 min and washed twice with 70% ethanol. After removal of ethanol, samples were allowed to stand for 5 min on a clean bench to volatilize the remaining ethanol. DNA samples were then dissolved in TE buffer (10 mM Tris–HCl, pH 7.4 and 1 mM EDTA), and subjected to 2% agarose gel electrophoresis at 100 V for 45 min. DNA was stained with 0.5 μg/mL ethidium bromide solution (Genesee Scientific, San Diego, USA).\n\nThe inhibition of cell proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Cayman Chemical Company, Ann Arbor, USA). The principle of this method relies on the production of purple pigments by living cells upon cleavage of tetrazolium salt to formazan by their intracellular NAD(P)H-oxidoreductase, whereas such pigmentation is not produced by dead cells. Cells were seeded in a 96-well plate (Becton and Dickinson) at a density of 3 × 104 cells/well in 100 μL of culture medium and incubated for 24 h at 37 °C with 5% CO2. Then, 10 μL of MTT reagent was added to each well. After mixing gently, the cells were incubated for 4 h at 37 °C with 5% CO2. After removal of the supernatant, 100 μL of crystal dissolving solution was added and mixed with the cell solution, and the sample was further incubated for 4 h at 37 °C with 5% CO2. Finally, the optical density at 550 nm was measured using a microplate reader (BIO-RAD, Benchmark, Hercules, USA).\n\nThe 50% inhibitory concentration (IC50) of Andro for each cell type was calculated using software ImageJ (ImageJ, RRID:SCR_003070).\n\nCells (2×105 cells) were collected by centrifugation (300×g at room temperature for 5 min), resuspended in 50 μL of PBS and fixed by 450 μL of 80% ethanol for more than 3 hours at -20°C. Cell pellets obtained by centrifugation (300×g, 5 min) were washed in 500 μL of PBS, incubated with 200 μL of Muse Cell Cycle Reagents (Merck Millipore Corporation, Darmstadt, Germany) in the dark for 30 min, and the cell cycle was measured by Muse Cell Analyzer (Merck Millipore Corporation) which uses miniaturized fluorescence detection and microcapillary cytometry to deliver single-cell analysis.\n\nApoptosis was detected using the MuseTM Annexin V and Dead Cell Assay Kit (Merck Millipore Corporation) in accordance with the manufacturer’s protocols. Briefly, cells were seeded in a 24-well plate dish (2×105 cells/well) for 24 h and collected by centrifugation (300×g at 4°C for 5 min), resuspended in 100 μL of RPMI 1640 medium and then incubated with 100 μL fluorescently labeled Annexin V reagent at room temperature for 20 min. Percentages of all cells (alive plus dead) labeled with Annexin V (a label of apoptotic cells) and/or 7-AAD (7-Aminoactinomycin D; a fluorescent chemical compound with a strong affinity for DNA which is used as a label of late-apoptotic/dead cells) were measured using the Muse Cell Analyzer and expressed by dot plots.\n\nCaspase-3/7 activity was analyzed using the MuseTM Caspase-3/7 Assay Kit (Merck Millipore Corporation) in accordance with the manufacturer’s protocols. Cells were seeded for 24 h at a concentration of 2×105 cells/mL in a 24-well plate dish (Falcon). Cells were collected by centrifugation (300×g at 4°C for 5 min) and suspended in 50 μL of RPMI 1640 medium. Then, 5 μL of caspase-3/7 Reagent working solution (1 μL of MuseTM Caspase3/7 Reagent and 7 μL of 1× PBS) was added, and cells were incubated for 30 min at room temperature in the dark. Finally, 150 μL of 7-AAD working solution was added, and Caspase-3/7 activity and cell viability were measured using a Muse Cell Analyzer.\n\nROS production was measured using the MuseTM Oxidative Stress Kit (Merck Millipore Corporation) according to the manufacturer’s protocols. Cells were collected by centrifugation (300×g at 4°C for 5 min), and then the supernatant was removed. Muse® Oxidative Stress Regent working solution (190 μL) was added into each tube containing 10 μL cell suspension. Cells were vortexed in the medium for 5 seconds and then incubated at 37°C for 30 min in the dark, and the percentage of ROS producing cells was determined by cytometry using the Muse Cell Analyzer.\n\nThe mitochondrial membrane depolarization was determined using the MuseTM MitoPotential Kit (Merck Millipore Corporation) according to the manufacturer’s protocols. Cells were collected by centrifugation (300×g at 20°C for 5 min) and then mixed with 100 μL of Assay Buffer, and 95 μL of MitoPotential working solution (MuseTM MitoPotential Dye diluted to 1:1000 in assay buffer). After incubating at 37°C for 20 min, 7-AAD reagent (5 μL) was added to each tube, and it was vortexed for 3 to 5 seconds. After incubation at room temperature for 5 min, percentages of all cells (alive plus dead cells) showing mitochondrial membrane depolarization and/or labeling with 7-AAD were measured using the Muse Cell Analyzer. The 7-AAD staining results of this experiment are not shown in the present study but were consistent with the 7-AAD staining results shown in Figure 4 and will be provided by the authors upon request.\n\nData were analyzed using Excel software (Microsoft Excel 365) and the Student’s t-test was used to assess statistical significance between the various treatments. Results were expressed as mean ± SD of three independent experiments. P < 0.05 was considered statistically significant.\n\n\nResults\n\nThe effects of Andro, Ara-C, and VCR on the viability of THP-1 and H929 cells were compared by incubating the cells for 24 h with or without an agent at the indicated concentrations, followed by an MTT assay (Figure 1). Treatment with Andro (50 μM) reduced the viability of THP-1 and H929 cells to 39.2% and 13.0%, respectively, compared with untreated cells. The viability-reducing effect by Andro was concentration-dependent (Figure 1) and its IC50 values for treating THP-1 and H929 cells were calculated as 31 μM and 8 μM, respectively.\n\nThe y-axis values of the cell viability histograms represent the optical density (550 nm) in comparison with the control (set as 100%) as measured by MTT assay. The optical density markedly decreased after treatment with Andro (10, 30, 50 μM), Ara-C (40 μM), or VCR (0.1 μM) compared with untreated cells in THP-1 (a) and H929 (b) cells. The results are expressed as mean ± SD of three independent experiments.\n\nBased on the therapeutic plasma concentrations of Ara-C and VCR for hematopoietic tumors (Capizzi et al., 1983; Nelson, 1982), Ara-C and VCR were used at 40 μM and 0.1 μM, respectively. They reduced the viability of THP-1 cells to 50-55%, whereas Ara-C only had a slight impact on H929 cells (Figure 1). The viability-reducing effect of Andro on THP-1 cells was similar to that of Ara-C and VCR (Figure 1a), whereas—at the concentrations used—Andro was markedly superior to Ara-C and VCR in reducing the viability of H929 cells (Figure 1b).\n\nCellular shrinkage and nuclear condensation were observed in both THP-1 and H929 cells after treatment for 24 h with either Andro, Ara-C, or VCR (Figure 2a,b). Andro induced both phenomena in almost all H929 cells (Figure 2b). Furthermore, DNA isolation followed by agarose gel electrophoresis revealed that these treatments with Andro, Ara-C, and VCR each had induced nuclear DNA fragmentation in both THP-1 and H929 cells (Figure 2c).\n\nMorphologies of THP-1 (a) and H929 (b) cells after 24 h of treatment with Andro, Ara-C, or VCR were compared with untreated cells after Wright-Giemsa staining. White arrows indicate cells showing nuclear condensation and black scale bars represent 20 μm. (c) Nuclear DNA fragmentation was revealed by agarose gel electrophoresis of DNA isolated after 24 h of treatment with Andro (50μM), Ara-C (40μM), or VCR (0.1μM) in THP-1 (lanes 2-5) and H929 cells (lanes 6-9). Lane 1, DNA size marker; lanes 2 and 6, untreated cells; lanes 3 and 7, cells treated with Andro; lanes 4 and 8, cells treated with Ara-C; lanes 5 and 9, cells treated with VCR.\n\nThe effects of 24 h treatment with Andro, Ara-C, or VCR on cell cycle progression were compared (Figure 3). In the case of Andro, the percentages of cells in the G0/G1, S, and G2/M phases were very similar to those in untreated THP-1 and H929 cells. On the other hand, Ara-C treatment significantly increased the percentage of cells in the G0/G1 phase, in agreement with its known inhibition of DNA synthesis (Li et al., 2017). Likewise as expected, VCR significantly increased the percentage of cells in the G2/M phase, in agreement with its known inhibition of mitotic spindle formation (Kothari et al., 2016).\n\nCell cycle phases of individual cells were measured after treatment for 24 h with Andro (50μM), Ara-C (40μM), or VCR (0.1μM) using the Muse Cell Analyzer. In contrast to Ara-C and VCR, treatment with Andro hardly affected the percentages of THP-1 or H929 cells found in the G0/G1, S, and G2/M phases. Percentages are expressed as mean of three independent experiments. For statistical analysis the percentages of cells in the G0/G1 phase were compared (P < 0.05, P < 0.01, *P < 0.001).\n\nPhosphatidylserine externalization from the inner to the outer cell membrane is a characteristic feature of apoptotic cell death which can be measured by annexin V-binding (Demchenko, 2013). Dual labeling with annexin V and 7-AAD (a label for cells with permeabilized membranes such as late-apoptotic cells and dead cells) of THP-1 and H929 cells was performed after they had been treated for 6~48 h with Andro, Ara-C, or VCR. The percentages of annexin V-positive cells among THP-1 and H929 cells increased depending on their time of treatment with either anti-tumor agent (Figure 4). Overall, higher percentages of annexin V-positive THP-1 cells were not found after treatment with Andro than with Ara-C or VCR (Figure 4a), whereas Andro was markedly superior to Ara-C and VCR in inducing apoptosis in H929 cells (Figure 4b). The 7-AAD-staining results, shown in the cell cytometry dot plots in the upper part of Figure 4, suggest that after 24 h treatment with Andro the majority of H929 cells were already dead, emphasizing the high toxicity of Andro for this cell type.\n\nLabeling with Annexin V and 7-AAD were analyzed by the Muse Cell Analyzer. The upper figures show representative dot plots in which the x-axis indicates Annexin V labeling and the y-axis indicates 7-AAD labeling. In the lower figures the Annexin V staining results are expressed as mean ± SD of three independent experiments.\n\nTreatment with Andro for 24 h increased the percentages of cells with caspase-3/7 activity from 4.3% to 81.7% in THP-1 cells (Figure 5a) and from 9.2% to 95.7% in H929 cells (Figure 5b). These increases were substantially higher than those induced with Ara-C or VCR treatments (Figure 5). In the presence of a caspase inhibitor, Z-VAD-FMK, the Andro-induced caspase-3/7 positive rates of THP-1 and H929 cells were significantly lower, namely only 25.9% and 56.7%, respectively (Figure 5). Z-VAD-FMK also significantly reduced, although not by as much, the enhancing effects of Ara-C and VCR on caspase 3/7 positive rates (Figure 5).\n\nThe results are expressed as mean ± SD of three independent experiments (P < 0.05, P < 0.01, P < 0.001, comparing with and without Z-VAD-FMK).\n\nTreatment with Andro (50 μM) for 24 h increased the percentage of ROS producing cells from 6.8% to 85.8% in THP-1 cells (Figure 6a-i) and from 4.8% to 91.1% in H929 cells (Figure 6b-i). Andro increased the ROS positive rates in a concentration-dependent manner, and in H929 cells even at 10 μM (the lowest concentration tested) the enhancing effect of Andro on ROS production was much higher than that of Ara-C or VCR. The ROS enhancing effect of Andro was largely abolished by the presence of ROS inhibitor NAC, whereas NAC only slightly reduced the ROS enhancing effects of Ara-C and VCR (Figure 6a-i, b-i).\n\nThe presence of the ROS production inhibitor NAC largely reduced the enhancing effects of Andro on both parameters in either cell type, whereas NAC had little or no impact on the effects of Ara-C and VCR. The results are expressed as mean ± SD of three independent experiments (P < 0.05, P < 0.01, P < 0.001, comparing with and without NAC).\n\nConsistent with the findings for ROS production, treatment with Andro (50 μM) for 24 h increased the percentages of cells with depolarized mitochondrial membranes from 12.3% to 80.5 % in THP-1 cells (Figure 6a-ii) and from 6.5% to 98.8 % in H929 cells (Figure 6b-ii). These Andro effects were concentration-dependent and even at 10 μM the effect of Andro on H929 cells was stronger than that of Ara-C or VCR. The presence of NAC significantly reduced the enhancement of mitochondrial membrane depolarization caused by Andro but hardly or not the effects of Ara-C or VCR (Figure 6a-ii, b-ii).\n\nFinally, we checked whether the presence of NAC interfered with the effects of 24 h incubation with Andro, Ara-C, or VCR on cell viability and the percentage of annexin V-positive cells. It was found that NAC largely abolished the effects of Andro on both properties, especially in H929 cells, but had little or no impact on the effects of Ara-C or VCR (Figures 7, 8).\n\nCell viability was measured after 24 h treatment with Andro, Ara-C, or VCR of THP-1 (a) and H929 (b) cells in the presence or absence of the ROS production inhibitor NAC. The y-axis values represent the optical density (550 nm) in comparison with the control (set as 100%) as measured by MTT assay.\n\nAnnexin V-positive rates were measured after 24 h treatment with Andro, Ara-C, or VCR of THP-1 (a) and H929 (b) cells in the presence or absence of the ROS production inhibitor NAC. The results are expressed as mean ± SD of three independent experiments (P < 0.05, P < 0.01, *P < 0.001, comparing with and without NAC).\n\n\nDiscussion\n\nThe herb Andrographis paniculate, called “king of bitters” because of its extremely bitter taste, has been used for centuries for various medicinal purposes. The primary bioactive component of this medicinal plant is andrographolide, which is bitter and present in all parts of the plants but maximally (>2 % of dry weight) in the leaves (Jarukamjorna and Nemoto, 2008; Sharma et al., 2018). The present study confirms that andrographolide can be toxic for tumor cell lines, and for the first time determins its toxicity for the human multiple myeloma cell line H929. Another novel observation is that Andro exerts its toxic effect on the human leukemia monocytic cell line THP-1, and also on H929 cells, via induction of ROS-dependent apoptosis.\n\nApoptosis is a form of programmed cell death involving cascades of interactions (Rossi and Gaidano, 2003; Schultz and Harrington, 2003). Andro-treated THP-1 and H929 cells showed typical symptoms of apoptosis, such as cellular shrinkage, nuclear condensation, DNA fragmentation, stainability with Annexin V, caspase 3/7 activation, and mitochondrial membrane depolarization. Notably, in the presence of NAC, an inhibitor of ROS production, the cytotoxic and apoptotic effects of Andro on THP-1 and H929 cells were largely abolished. The induction of ROS-dependent apoptosis by Andro has also been observed in other cancer cells such as a breast cancer cell line (Banerjee et al., 2016), a colon cancer cell line (Khan et al., 2018), and lymphoma cell lines and primary lymphoma (Yang et al., 2010). The levels of ROS production in THP-1 and H929 cells induced by Andro were much higher than induced by Ara-C and VCR, and—in sharp contrast to Andro—the cytotoxic/apoptotic effects of Ara-C and VCR were hardly sensitive to NAC. This implies a different mode of action and suggests that an additive anticancer therapeutic value might be achieved if Andro would be used in combination with agents such as Ara-C and/or VCR. While Ara-C is known to be a DNA polymerase inhibitor that inhibits DNA synthesis (Li et al., 2017), VCR inhibits mitosis by inhibiting microtubule polymerization (Kothari et al., 2016). Unfortunately, the mechanism by which Andro induces ROS-dependent apoptosis is still not understood (see below).\n\nThe (24 h) IC50 concentrations of Andro for reducing the cell viability of THP-1 and H929 cells were determined as 31 μM and 8 μM, respectively. These concentrations are far below the Andro concentrations at which normal cells are noticeably affected (Banerjee et al., 2016; Khan et al., 2018) and somewhat lower than the (24 h) IC50 concentrations determined as 52 μM for colon cancer MDA-MB-231 cells (Banerjee et al., 2016), 40 μM for acute myeloid leukemic HL-60 cells (Cheung et al., 2005), and 60 μM for colon cancer HT-29 cells (Khan et al., 2018). For THP-1 cells, previously, low concentrations of Andro, namely ≤3 μM, were found to affect functional properties (Gupta et al., 2020; Ji et al., 2005), but our current findings agree well with a report that the (72 h) LD50 concentration was ~20 μM (Habtemariam, 2003). The high sensitivity to Andro that we observed for H929 viability is reminiscent of observations for other types of lymphoma cell lines, considering the (48 h) IC50 values reported for Ramos (Burkitt lymphoma) (20 μM), Granta (mantle cell lymphoma) (40 μM), HF-1 (follicular lymphoma) (15 μM), and SUDHL4 (diffuse large B-cell lymphoma) (30 μM) (Yang et al., 2010). In primary follicular lymphoma cells strong apoptotic effects were induced after 24 h incubation with only 5 μM Andro (Yang et al., 2010). High sensitivities of multiple myeloma cell lines have also been reported, as for the cell lines RPMI-8226 and U266 the Andro (48 h) LC50 concentrations were determined as10 μM and 8 μM, respectively (Gunn et al., 2011). Furthermore, a 72 h incubation with only 1 μM Andro reduced the viability of the multiple myeloma cell line OPM1 (RRID:CVCL_5210) to less than 70% (Gao and Wang, 2016). In short, the Andro sensitivities that we observed for monocytic leukemia THP-1 and multiple myeloma H929 cells are in agreement with previous observations and emphasize that, in particular, multiple myeloma cells are very sensitive to Andro. From the viewpoint of potential therapeutic usage, this sensitivity is even more interesting given our finding that H929 cells are not very sensitive to Ara-C and VCR. Namely, this raises the hope that some tumor cells that are refractory to treatment with the common drugs Ara-C and VCR may be treated with Andro.\n\nAndrographolide is considered nontoxic even at high doses (Calabrese et al., 2000; Sattayasai et al., 2010), but its low aqueous solubility limits the plasma concentrations that can be readily achieved (Pandey and Rao, 2018). However, steady-state blood concentrations of ~1.9 μM have been reported in humans taking ~1 mg andrographolide per kg body weight per day (Panossian et al., 2000), and this is expected to be within the therapeutically effective concentration range for Andro against multiple myeloma cells (see above).\n\nSeveral studies found an effect of Andro on cell cycle phase distribution, and authors assumed that Andro induced cell cycle arrest (Banerjee et al., 2016; Cheung et al., 2005; Dai et al., 2017; Khan et al., 2018). For example, for the gastric cancer cell line SGC7901 (RRID:CVCL_0520), it was reported that higher concentrations of Andro caused cell cycle arrest in the G2/M phase (Dai et al., 2017). In contrast, for the colon cancer cell line HT-29 (RRID:CVCL_0320), it was reported that low concentration of Andro caused significant cell cycle arrest in the G2/M phase, while higher Andro concentrations caused arrest in the G0/G1 phase (Khan et al., 2018). Meanwhile, for the acute myeloid leukemic cell line HL-60, Andro reportedly arrested the cells in G0/G1 phase (Cheung et al., 2005). In contrast to those studies, the present study did not find a notable effect of Andro on the cell cycle phase distribution of THP-1 and H929 cells. Our findings combined with the inconsistent cell cycle distribution effect of Andro reported for other tumor cell lines (Banerjee et al., 2016; Cheung et al., 2005; Dai et al., 2017; Khan et al., 2018), and the fact that in those studies a true arrest probably cannot be considered as proven, suggest that Andro does not specifically target a specific step in cell cycle progression.\n\nAn important question is why cancer cells, compared to normal cells, can be more sensitive to the induction of ROS-mediated apoptosis. It may be related to mitochondria being the major source of reactive oxygen species (ROS) (Orrenius, 2007; Vyas et al., 2016) and the unusual properties of typical cancer cell mitochondria. Most cancer cells show an increased reliance on aerobic glycolysis (Warburg effect) (Warburg, 1956) and many cancers, including multiple myeloma, show an enhanced biogenesis of mitochondria compared to normal cells (Zhan et al., 2017). Many cancers, including multiple myeloma (MM), retain more cytosolic iron to promote tumor cell growth, and higher cytosolic iron promotes oxidative damage due to its interaction with reactive oxygen species generated by mitochondria (Zhan et al., 2017).\n\nThe possibility to specifically target multiple myeloma cells for the induction of ROS-dependent apoptosis has already been shown for a number of agents. For example, ex vivo analysis showed that pharmacological-dosed ascorbic acid (PAA; ultra-high doses of vitamin C) selectively induced apoptosis in primary multiple myeloma cells while not significantly harming other bone marrow cells, and PAA-induced apoptosis in the multiple myeloma cell line OCI-MY5 could be inhibited by NAC (Xia et al., 2017). Furthermore, treatment with a mitochondrial-targeting agent decyl-triphenylphosphonium (10-TPP) increased intracellular steady-state pro-oxidant levels and apoptosis in multiple myeloma cell lines (Schibler et al., 2016); 10-TPP is a lipophilic agent that associates directly with mitochondria, likely with the inner membrane (Murphy, 2008; Ross et al., 2008; Schibler et al., 2016). Dexamethasone, a glucocorticoid, is another hydrophobic lipophilic molecule that induced apoptotic cell death in multiple myeloma cell lines, and this effect could also be reduced by NAC (Bera et al., 2010); in sharp contrast, in normal cells dexamethasone was found to inhibit ROS generation (Dandona et al., 1999). As with Andro, the mechanism for the induction of apoptosis in tumor cells is likely not fully understood for any of the above three agents.\n\nWe speculate that the main effect of Andro involves a—yet to be identified—direct interaction with mitochondrial membranes, and that the end-effect of this interaction on the cell depends on the condition of the mitochondria and the redox status of the cell. Such a model would make it easier to explain why we and others find that Andro can induce apoptotic cell death (see above), whereas in other cell systems Andro has been proven to protect against oxidative stress and apoptosis (reviewed by Kishore et al., 2017; Mussard et al., 2019). A direct interaction of Andro—which is a lipophilic molecule (Pandey and Rao, 2018)—with the mitochondrial membranes might also explain a protective effect of Andro against mitochondrial fission (Geng et al., 2019). Selective disruptive/apoptotic effects against only some mitochondria, such as in THP-1 and H929 cells, may also help explain why Andrographis paniculate can have Andro stored in different tissues without the plant itself being harmed. In plants, Andro appears to have defensive roles against bacteria (Zhang et al., 2020) and herbivores (Edwin et al., 2016), although the mechanisms are not yet well understood. Possibly, the same features of Andro that evolved in plants to distinguish between self and enemy cells may also determine its different effects on cancerous and non-cancerous cells. The enormous medicinal potential of Andro means that future research to better clarify its functions and mechanisms is imperative.\n\n\nConclusion\n\nAndro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells. This cytotoxic effect is mechanistically different from that of Ara-C and VCR, suggesting that these agents could have supplementary effects if used in combination therapies. H929 cells, in particular, are very sensitive to Andro while they are not very sensitive to Ara-C and VCR, underscoring Andro’s promise as a potential drug against multiple myeloma. Future studies must unravel the mechanisms of Andro’s anti-tumor effect in more detail. Our study supports that Andro may be a valuable addition to the growing palette of drugs that are available for chemotherapy against hematopoietic tumors.\n\n\nData availability\n\nHarvard Dataverse: Doi et al. Table with individual data. https://doi.org/10.7910/DVN/W7UJMD (Doi, 2021).\n\nThis project contains the following underlying data.\n\n• Doi et al. data (this file lists the individual data that underlie the figures).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThis study was supported by Institute of Health and Immunology Science and Fujita Health University.\n\n\nReferences\n\nAbrink M, Gobl AE, Huang R, et al.: Human cell lines U-937, THP-1 and Mono Mac 6 represent relatively immature cells of the monocyte-macrophage cell lineage. Leukemia. 1994; 8(9): 1579–1584. PubMed Abstract\n\nBanerjee M, Chattopadhyay S, Choudhuri T, et al.: Cytotoxicity and cell cycle arrest induced by andrographolide lead to programmed cell death of MDA-MB-231 breast cancer cell line. J Biomed Sci. 2016; 23(40): 1–16. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang G, Jiang C, Xie N, et al.: Treatment with andrographolide sulfonate provides additional benefits to imipenem in a mouse model of Klebsiella pneumoniae pneumonia. Biomed Pharmacother. 2019; 117: 109065. PubMed Abstract \| Publisher Full Text\n\nZhang L, Bao M, Liu B, et al.: Effect of Andrographolide and Its Analogs on Bacterial Infection: A Review. Pharmacology. 2020; 105(3-4): 123–134. PubMed Abstract \| Publisher Full Text" }	[ { "id": "90836", "date": "19 Aug 2021", "name": "Christian Bailly", "expertise": [ "Reviewer Expertise Anticancer pharmacology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is another study of the effect of the diterpenoid lactone andrographolide on the growth of cancer cells and the induction of cell death. Two cell lines (THP-1 and H929) were used and the effects of Andro compared to those of the Vinca alkaloid vincristine and cytarabine (Ara-C). The data are correctly presented but there is nothing really new or exciting in this study. The ROS-dependent pro-apoptotic activity of Andro has been already reported in several studies with other cancer cell lines. Showing the same type of data/effects with additional cell lines is not a major discovery. There is a lack of innovation and originality, despite the quality of the data.\nMoreover, the conclusion is excessively optimistic and excessive. Andro is known for a long time and the development of this compound has failed, due to an unfavorable benefit/risk ratio (low bioavailability, toxicity, etc.). The limitations should be more clearly indicated.\nThe literature coverage could be improved, to cite recent reviews about Andro and cancer.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-542
https://f1000research.com/articles/11-374/v1	31 Mar 22	{ "type": "Research Article", "title": "The relationship between parents' oral hygiene knowledge and children with Down Syndrome's oral hygiene via OHI-S", "authors": [ "Agung Sosiawan", "Dian Agustin Wahjuningrum", "Dini Setyowati", "Michelle Suhartono", "Natasha Winona Audrey", "Tata Prasantat Mawantari", "Fery Setiawan", "Ajinkya M. Pawar", "Dian Agustin Wahjuningrum", "Dini Setyowati", "Michelle Suhartono", "Natasha Winona Audrey", "Tata Prasantat Mawantari", "Fery Setiawan", "Ajinkya M. Pawar" ], "abstract": "Background: Down Syndrome (DS) is a genetic disorder responsible for mental and development retardation. DS occurs when a person has an extra chromosome (47 instead of 46), the third copy of the Trisomy 21 chromosome. This causes structural and functional anomalies in the human body and some degree of intellectual disability. Children with DS have poor oral hygiene as they are unable to understand and are unaware of its importance. Oral hygiene problems commonly found in children with DS are gingivitis, periodontal problems, pain, infection, and problems with the masticatory system. This study explored the relationship between parents' knowledge and maintenance of the oral hygiene of children with DS through the OHI-S (Oral Hygiene Index Simplified) Index Score. Method: This study was conducted by distributing 25 questions via Google Form to 100 subjects that belong to POTADS (Down Syndrome Parents Association. The participants were children diagnosed with Down Syndrome and their parents. Questionnaires were used to assess parents' knowledge about their child's oral hygiene. To assess the children’s oral hygiene, dental exams were performed with the OHI-S on six tooth surfaces. To calculate the OHI-S score for each individual, the debris and calculus scores from the 6 surfaces of the teeth were totalled and divided by six. Results: The relationship between parents' knowledge and the maintenance of oral hygiene of children with DS was found by conducting a linear regression analysis and hypothetical test. The equation of Y = 77.734 + (-7.377) X was achieved through linear regression analysis, and indicated that a 1% increase in parents' knowledge caused a reduction in OHI-S score to 7.377. The hypothetical test showed that parents' knowledge affected their children's OHI-S score significantly. Conclusion: There was a significant contradictive relationship between parents' knowledge and the maintenance of oral hygiene of children with DS.", "keywords": [ "Primary care", "Mental Disease", "Down Syndrome", "Index Score OHI-S", "Oral Hygiene" ], "content": "Introduction\n\nChildren with special needs require more care than those without. Special care is given to these children as they carry specific physical, mental, intellectual, social, and emotional disabilities or abnormalities.1 Mentally disabled children are one of many examples of special needs children as they possess a below-average intellect with mild, severe, and very severe abnormalities (IQ between 25 and 70).2\n\nDown Syndrome or DS is a genetic disorder that affects a child's development and causes mental retardation. Down Syndrome occurs when a person has a third copy of the Trisomy 21 Chromosome. The third copy of Trisomy 21 causes structural and functional anomalies in the human body and some degree of intellectual disability.3 Down Syndrome occurs in one of 700 births and is present in all ethnicities. The chance of someone having a baby with DS is higher when she is 35 years old or older, and baby boys are more likely to have DS rather than baby girls.4 Children with DS tend to have poor oral hygiene as they are unable to understand and are unaware of the importance of oral hygiene.5 Oral hygiene problems commonly found in children with DS are gingivitis, periodontal problems, pain, infection, and problems with the masticatory system.6 Mental retardation or MR, developmental delay, physical disability, and other problems could affect disabled children's daily activities and are reasons why they could not maintain their oral hygiene. Therefore, it is necessary for parents, nannies, and people close to these children to help them maintain their oral hygiene.7\n\nChildren's oral hygiene has become a particular concern in this era. Knowledge is the result of curiosity and occurs because someone senses particular objects through human senses. Education regarding children's oral hygiene should be obligatory for parents as it could significantly help their children's teeth development and growth. The parents’ knowledge and ability in maintaining their children's oral hygiene could be influenced by several aspects such as age, education, economic and social status, experience, mass media, and environment. The parents' ability to maintain their children's health will significantly impact their children’s attitude and behavior.8\n\nTo boost awareness and effort towards the maintenance of the oral hygiene of children with special needs, this study analyzed parents' knowledge levels in maintaining their children's oral hygiene at POTADS (Down Syndrome Parents Association) Surabaya.\n\n\nMethods\n\nWe obtained ethical approval for this study from Universitas Airlangga Faculty of Dental Medicine Health Research Ethical Clearance Commission on 21-2-2020 (058/HRECC.FODM/II/2020). Due to the COVID-19 pandemic, ethical approval was also re-issued on 24-02-2021 (082/HRECC.FODM/II/2021).\n\nWritten informed consent was obtained from the parents for their own and for their children’s involvement in this study as children were not able to consent. Participants were informed of their right to withdraw and that they could refuse to answer any questions, end the survey, or refuse the child’s dental exam at any point.\n\nAn analytical-observational study was conducted in Surabaya, 2020-2021, via Google Forms to 100 parents whose children require special care (Down Syndrome) in POTADS Surabaya. The sample size was based on the Lemeshow formulas. The dental examination procedure is further explained below.\n\nThe populations of this study are children who require special care (Down Syndrome) and their parents in POTADS Surabaya. Inclusion criteria are parents whose children were diagnosed with Down Syndrome, children aged 7-9 years old, and parents or guardians who agreed to be a part of this study. The samples used in this study were purposive as they were taken based on specific considerations of the researcher.\n\nThe potential participants were recruited from the POTADS. We also used social media, such as Twitter, WhatsApp, Instagram, and Line, as our recruitment strategy to identify the POTADS’ members and distribute the questionnaire. To be eligible for participation in this study, the participants had to be parents whose children were aged 7 to 9 years old and were diagnosed with Down Syndrome, and be a POTADS’ member. Participants were first contacted to give their consent to participate. Those who did not consent did not progress to the questionnaire. The questionnaire had been tested for its validity and reliability.\n\nWe conducted a self-administered questionnaire survey using Google Forms to collect data about parents' knowledge. After the participants completed the questionnaire, we contacted them to arrange a time to do their child’s oral hygiene exams at the place most convenient to them. All participants who had completed the questionnaire had their child participate in the oral hygiene examinations. To assess their child’s oral hygiene, we used a basic dental diagnostic kit comprising two dental mirrors, one dental explorer, and one dental cotton tweezer. The principal researcher performed the child’s oral hygiene examinations using the oral hygiene index simplified (OHI-S). There were six tooth surfaces examined: the buccal surface of the upper right first molar (16), the labial surface of the upper right central incisor (11), the buccal surface of the upper left first molar (26), the lingual surface of the lower left first molar (36), the labial surface of the lower left central incisor (31), and the lingual surface of the lower right first molar (46). The OHI-S score consists of the Debris Index (DI) scores and the Calculus Index (CI) scores. The DI and CI scores represent the amount of debris and calculus, respectively, on the six tooth surfaces. To calculate the OHI-S score for each individual, the DI and CI scores were totalled and divided by six (the number of tooth surfaces examined). During the oral hygiene examination, the challenge we faced was the child’s resistant behaviors, such as pushing the hand or the dental instrument away, moving the head, and refusing to open their mouth. With the help of the parents, we made a few distractions to comfort the child and gain their cooperation.\n\nData were compiled in Excel 2016 before being added to SPSS v25.0 for analysis.\n\nThe debris score was assessed through the existence of debris found by using OHI-S. The score 0 was chosen if there was not any debris or stain, score 1 was chosen if soft debris or extrinsic stains were found covering one-third of a tooth surface, score 2 was chosen if soft debris was found covering more than one-third of a tooth surface but not more than two-thirds of a tooth surface, and score 3 was chosen if soft debris were found covering more than two-thirds of a tooth surface.\n\nThe calculus score was assessed through the existence of calculus by using OHI-S. Score 0 was chosen if no calculus was found, score 1 was chosen if supragingival calculus were found covering not more than one-third of a tooth surface, score 2 was chosen if supragingival calculus or individual subgingival calculus spots were found to cover one-third but not more than two-thirds of a tooth surface, and score 3 was chosen if supragingival calculus was found covering more than two-thirds of a tooth surface.\n\nParents' knowledge was assessed through questionnaires that contained 25 questions (see extended data) and were distributed via social media. The ordinal scale was used to measure the parents' knowledge and was categorized as low and high. The low category was chosen if parents' knowledge levels were between 0-71.5, and the high category was chosen if parents' knowledge levels were between 72-100. We decided to use this level based on the median score as the cut-off point to categorize parents' knowledge.\n\nThe collected data was analyzed using Statistical Package for the Social Science Software or SPSS (IBM SPSS Statistics 25.0). The questionnaire validity test searched for the correlation between individual questionnaire scores and the total score (bivariate). The reliability test was conducted using a reliability re-test, and the normality test was conducted using the Shapiro-Wilk method. The linear regression method was chosen if the data distribution was standard, and the ordinal regression method was chosen if the data distribution was not expected.\n\nWe used STROBE's cross-sectional reporting guidelines to ensure research meets international standards for peer-reviewed articles. The checklist is completed by entering the page number of the manuscript where the reader can easily find each item listed. If we believe that an item is not valid, we will write “N/A” and provide a brief explanation in the STROBE cross-sectional reporting guidelines.9\n\n\nResults\n\nThe characteristics of the samples were reported collectively with the findings of every factor, respectively (debris and calculus score via OHI-S and parents' knowledge level).\n\nStudy subject characteristics consisted of the gender, age, education, and profession of each subject. The subject characteristics are shown in Table 1.\n\nBased on Table 1, the parent participants were mostly female (80%). Respondents who participated in the study were mostly 30-39 and 50-59 years old (30% and 40% each respectively). The respondents of the study were primarily high school graduates (70%). Most of the respondents were housewives (55%).\n\nBased on Table 2, it could be seen that 55.6% of all respondents possessed enough knowledge in maintaining children' oral hygiene, with a median score of ≥ 72. Scores below 72 are classed as low, meaning that the parents’ knowledge in maintaining children’s oral hygiene is low. Scores above 72 from the questionnaire are high, meaning that the parents’ knowledge is high.\n\nIn Table 3, the frequency of OHI-S score of children with Down Syndrome (7-9 years old) in POTADS Surabaya could be seen. Children with Down Syndrome were mainly found to have good oral hygiene (65% of the total respondents). The average respondents were rated ≤ 1.2 on the OHI-S score.\n\nBased on Table 4, the average parents' knowledge and children with Down Syndrome’s OHI-S score could be seen (67.64 and 1.3683, respectively).\n\nTable 5 exhibits the results of the simple regression analysis test. The test was conducted after the data significance (p > 0.05) was found by using the data normality test of Shapiro-Wilk. Furthermore, linearity, heteroscedasticity, and autocorrelation tests were also conducted, resulting in the simple regression analysis. The simple regression result between parents' knowledge toward OHI-S score was as follows:\n\nThe interpretation of the linear equation above is as follows:\n\na) The A constant of 77.734 means that if the OHI-s score is equal to zero or constant, the value of knowledge consistency to the OHI-s score is 77.734.\n\nb) The knowledge variable regression coefficient is – 20.001, meaning that if parents' knowledge increases by 1%, the OHI-S score will decrease by 20.001.\n\nc) The significance value in Table 5 is 0.000 (p < 0.05), meaning that parental knowledge significantly affects children's OHI-S score.\n\nTable 6 shows the data analysis value of R = 0.896a. The value indicates that the relationship between the dependent and independent variables is strong, as R > 0.5. The number of R square or the coefficient of determination is 0.803, which means that the independent variable could explain 0.803 or 80.3% of the variation of the dependent variable. At the same time, the remaining 19.7% is the result of other unexamined causes.\n\n\nDiscussion\n\nThis study was conducted to understand the influence of parents' knowledge in maintaining children with special care’s oral hygiene in POTADS Surabaya. Parents' knowledge levels were obtained through an online survey which consisted of 25 questions about basic knowledge of oral hygiene for children with special care. The study's data found that the average parents' knowledge value was 67.64. Furthermore, the average OHI-S score of 7-9 years old children was 1.3683. This score indicates that they have well-maintained oral hygiene.10\n\nOHI-S measures a person's oral hygiene based on Debris Index (DI) and Calculus Index (CI). A person's oral hygiene could be measured by valuing the calculus and plaques.11 The Sig. value of 0.000 was found as the result of this study. This value suggests that the parents' knowledge has significantly influenced children with special care’s oral hygiene in POTADS Surabaya. The hypothetical test was conducted by comparing the value of t-count with t-table, and the result was -20.001 of the t-table values. The value is greater than the t-count value, which is 2.365 (a negative value). If the value of t-count is more significant than t-table and is negative, it would be concluded that a contradictive influence existed in the relationship between parents' knowledge and children with special need's hygiene in POTADS Surabaya. The study conducted by Guswan and Yandi (2017) has proven that a higher level of parents' knowledge would mean a lower children's OHI-S score.12\n\nKnowledge is the foundation for the creation of action. A person is said to lack knowledge if he/she cannot recognize, explain, and analyze a situation. Proper knowledge could affect someone's action in improving health, especially in maintaining oral hygiene. On the other hand, a lack of knowledge could cause problems in maintaining oral hygiene, such as caries.12 Parents are obligated to have a proper education in maintaining oral hygiene and should exhibit more concern in children's oral hygiene, especially if they need special care. Therefore, information regarding the significance of oral hygiene for parents is necessary.13 Special training and adaptations for children with Down Syndrome are necessary to ensure that they brush their teeth effectively. This is due to their lack of motor skills compared to other children, and parents play a role in supervising and training children to brush their teeth effectively.14\n\nMoreover, as the response of Down Syndrome children to a given stimulus is much different from children without Down Syndrome in general, parents are required to be more creative and active when providing learning activities.15 Based on the determinative coefficient data analysis result, it could be understood that the relationship between children with special needs’ OHI-S score and parents' knowledge level in maintaining oral hygiene is strong (R = 0.896a). The R square value of 0.803 or 80.3% means that the variation of the dependent variable, the OHI-S score of children with special needs, could be explained by the independent variable, namely the value of parents' knowledge about children's oral hygiene. Meanwhile, the rest were not investigated because we only focused on parents’ knowledge and the child’s oral hygiene.\n\nThe oral hygiene of children with Down Syndrome could be affected by various factors such as predisposing factors, enabling factors, and reinforcing factors. Predisposing factors are triggering factors or antecedents of behavior that provide reasons or motivation for the said behavior.16,17 Enabling factors are factors that could facilitate behavior or actions conducted by individuals in pain. Furthermore, other factors included infrastructure and health facilities.18 Resources were gathered from promotional media (booklet, leaflet, flyer, flipchart, and posters), electronic media (television, radio, video, slide, and filmstrip), and billboards.18,19\n\nReinforcing factors could strengthen a person's motivation to change his/her action based on a rule or policy.20 A family is one of many examples of reinforcing factors. In a family, various factors could influence health behavior in addition to parents' knowledge, such as parents' age, occupation, education, attitudes, and family support.21 Parents whose children are diagnosed with Down Syndrome should have sufficient oral hygiene knowledge, such as appropriate usage of a toothbrush, appropriate nutritional provision, and appropriate methods in elevating children's psychomotor ability. Moreover, the effort to lift children with Down Syndrome's life quality through oral hygiene could avoid malnutrition and improve their nutritional intake and growth speed.13 Therefore, it is important that parents' have sufficient oral hygiene knowledge as it could significantly influence their children's oral hygiene. Nevertheless, other factors could also affect children with Down Syndrome's oral hygiene.\n\nOne of the limitations of the study was that the self-administered questionnaire may cause bias in the participants’ responses. Another drawback was related to the selection of a place for the oral hygiene examination that was based on the parent's preference. Some of them did not have a good light to perform oral hygiene examination causing the potential bias during the examination.\n\n\nConclusions\n\nContradictive influences were significant in the relationship between parents' knowledge about oral hygiene and children with special needs' oral hygiene in POTADS Surabaya. Further studies could be conducted to determine other factors that could affect children with special needs' oral hygiene.\n\n\nData availability\n\nMendeley: The Relationship Between Parents' Oral Hygiene Knowledge and Children with Down Syndrome's Oral Hygiene via OHI-S. https://doi.org/10.17632/329zrbpkc8.4.22\n\nThis project contains the following files:\n\n- Gender Age and Profession 100 Sampels.xlsx\n\n- Raw Data Responses (Before and After COVID-19).xlsx\n\nMendeley: The Relationship Between Parents' Oral Hygiene Knowledge and Children with Down Syndrome's Oral Hygiene via OHI-S. https://doi.org/10.17632/329zrbpkc8.4.22\n\nThis project contains the following files:\n\n- List questionnaires.docx\n\n- Table 1. xlsx\n\n- Table 2. xlsx\n\n- Table 3. xlsx\n\n- Table 4. xlsx\n\n- Table 5. xlsx\n\n- Table 6. xlsx\n\n- STROBE-checklist-v4-cross-sectional.doc\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor contributions\n\nAS: designed, wrote, and made the firs design of manuscript, DAW: checked and involved in the correction of the manuscript, MS, NWA, TPM: helped AS in analyzing, conducting, and counting, the data DS: checked and helped AS in making, designing, and writing the whole manuscript, FS: helped AS in writing the manuscript, AMP: helped in submitting the manuscript.", "appendix": "Acknowledgements\n\nThe authors acknowledge the Indonesian Ministry of Education and Faculty of Dental Medicine, Universitas Airlangga, Surabaya-Indonesia.\n\n\nReferences\n\nSoetjiningsih: Tumbuh Kembang Anak. Jakarta: EGC; 2010.\n\nSomantri S: Psikologi anak luar biasa. Bandung: Refika Aditama; 2007.\n\nWajuihian SO: Down syndrome: An overview. African Vision and Eye Health. 2016; 75(1): 11–16. Publisher Full Text\n\nSelikowitz M: Down Syndrome. 3rd ed.Sydney: Oxford University Press. 2008.\n\nAlJameel AH, Watt RG, Tsakos G, et al.: Down syndrome and oral health: mothers’ perception on their children’s oral health and its impact. Journal of Patient-Reported Outcomes. 2020; 4(45): 45–48. PubMed Abstract \| Publisher Full Text\n\nPorovic S, Zukanovic A, Juric H, et al.: Oral Health of Down Syndrome Children in Bosnia and Herzegovina. Materia Socio Medica. 2016; 28(5): 370–372. Publisher Full Text\n\nLira A, Silva C, Rebelo S: Dentists' actions about the oral health of individuals with Down Syndrome. Brazilian Journal of Oral Sciences. 2015; 14(4): 256–261. Publisher Full Text\n\nSjarif WS: Pencegahan penyakit Gigi dan Mulut Penyandang Sindrom Down. Jurnal Pengabdian Kepada Masyarakat. 2019; 4(3): 55–58.\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.\n\nRompis C, Pangemanan D, Gunawan P: Hubungan tingkat pengetahuan ibu tentang kesehatan gigi anak dengan tingkat keparahan karies anak TK di Kota Tahuna. Jurnal e-Gigi. 2016; 4(1): 46–52. Publisher Full Text\n\nMarya C: A Textbook of Public health dentistry. 1st edn,Public Health.New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2011. Publisher Full Text\n\nSherlyta M, Wardani R, Susilawati S: Tingkat kebersihan gigi dan mulut siswa Sekolah Dasar Negeri di desa tertinggal Kabupaten Bandung. Jurnal Kedokteran Gigi Universitas Padjadjaran. 2017; 29(1): 69–76. Publisher Full Text\n\nGuswan G, Yandi S: Hubungan pengetahuan dan tindakan ibu terhadap indeks plak anak di Taman Kanak-Kanak Ibnu Akbar Kota Padang. Jurnal Kedokteran Gigi Unpad. 2017; 29(3): 164–167. Publisher Full Text\n\nAl-Bader D, Al-Athel L, Wyne AH, et al.: Oral health knowledge and sources of information in parents of Saudi disabled children. Pak Oral Dent J. 2006; 26: 101–108.\n\nKalyoncu IÖ, Girai FE, Tanboga I: Parent’s attitudes and knowledge on oral health in a group of individual with Down syndrome in Turkey. KAP Study. 2018; 68(9): 1368–1372.\n\nSoewondo W: Pendidikan Kesehatan Gigi untuk Penyandang Sindrom Down. Jurnal Pengabdian Kepada Masyarakat. 2019; 4(3): 55–58.\n\nSiswantoro T: Analisis Pengaruh Predisposing, Enabling, dan Reinforcing Factors terhadap Kepatuhan Pengobatan TB Paru di Kabupaten Bojonegoro. Jurnal Administrasi Kebijakan Kesehatan. 2012; 10(3): 152–159.\n\nZurhayati Z, Ashar T, Tarigan L: Faktor Predisposing, Enabling, Reinforcing Terhadap Kualitas Pengendalian Nyeri Pada Remaja Mengalami Dismenorea. Jurnal Endurance. 2018; 3(2) Publisher Full Text\n\nNotoatmodjo S: Ilmu Perilaku Kesehatan. Jakarta: Rineka Cipta; 2014.\n\nPrawati DD, Haqi DN: Faktor yang Mempengaruhi Kejadian Diare di Tambaksari, Kota Surabaya. Jurnal Promkes: The Indonesian Journal of Health Promotion and Health Education. 2019; 7(1): 34–45. Publisher Full Text\n\nDarmawan AAKN: Faktor-Faktor yang Mempengaruhi Perilaku Kunjungan Masyarakat terhadap Pemanfaatan Pelayanan Posyandu di Desa Pemecutan Kelod Kecamatan Denpasar Barat. Jurnal Dunia Kesehatan. 2016; 5(2): 29–39.\n\nWahjuningrum DA, Sosiawan A, Setyowati D, et al.: The Relationship Between Parents' Oral Hygiene Knowledge and Children with Down Syndrome's Oral Hygiene via OHI-S. Mendeley Data. 2022; V4. Publisher Full Text" }	[ { "id": "129463", "date": "11 Apr 2022", "name": "Nor Azlida Mohd Nor", "expertise": [ "Reviewer Expertise dental public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract:\nBackground section is too long, suggest to shorten it and make it concise. If word count permits, authors may want to expand the results and conclusion sections.\n\nMethods: Page 3:\nSample size calculation is not very clear. Authors can add explanation on sample size calculation, and how they get (n=100).\nPage 4:\nFor a clinical examiner, is there any training and calibration exercise conducted? What is the examiner reliability score? Where is the clinical examination conducted? Is it at the dental school or at the children’s school? Is the examination performed under natural light or portable light?\n\nThe principal researcher performed the child’s oral hygiene examinations using the oral hygiene index simplified (OHI-S) - please add reference.\n\nSentences related to description about the index for clinical examination require reference.\n\nParental questionnaire - how did you develop the questionnaire? Was the questionnaire validated? There is a lack of clear explanation on the survey instrument used. Authors need to add this information to make it clearer to reader.\n\nParents’ knowledge score – how do you assign a score for each question. Is there any reference for the cut-off category for high and low knowledge score?\n\nResults: Page 5:\nTable 1 caption: suggest change the word “each subject” to “study participants”\n\nTable 3: The categorisation of good, moderate and poor OHI-S score should also be explained in the methods section.\n\nDiscussion: Page 6:\nLast paragraph - in statistics, the p value is not 0.00 but can be written as (p<0.001) instead.\nPage 7:\nFirst paragraph - authors can interpret the findings and provide clearer explanation, rather than using statistical jargon.\n\nParagraph 4 & 5- how can the authors relate the factors stated in paragraph 4 with the demographic characteristics of the participants in your study?\n\nLimitation:\nDo you think the small sample size is part of the study limitation?\n\nConclusion:\nI found the term “contradictory relationship” is rather confusing. Do you mean inverse relationship?\n\nIs the work 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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-374
https://f1000research.com/articles/11-473/v1	29 Apr 22	{ "type": "Research Article", "title": "Associations among smoking, IDH mutations, MGMT promoter methylation, and grading in glioma: a cross-sectional study", "authors": [ "Rusdy Ghazali Malueka", "Rachmat Andi Hartanto", "Maria Alethea", "Christina Megawimanti Sianipar", "Adiguno Suryo Wicaksono", "Endro Basuki", "Kusumo Dananjoyo", "Ahmad Asmedi", "Ery Kus Dwianingsih", "Rusdy Ghazali Malueka", "Rachmat Andi Hartanto", "Maria Alethea", "Christina Megawimanti Sianipar", "Adiguno Suryo Wicaksono", "Endro Basuki", "Kusumo Dananjoyo", "Ahmad Asmedi" ], "abstract": "Background Several molecular markers have important roles in glioma management. Mutations in the isocitrate dehydrogenase (IDH) gene are associated with the grading and prognosis of glioma. Methylation in the promoter region of the O (6)-methylguanine-DNA methyltransferase (MGMT) gene is an important determinant of glioma sensitivity to alkylating agents. Studies in various cancers indicated that IDH1 mutations and MGMT promoter methylations were associated with smoking habits. However, these associations in gliomas are still unclear. Accordingly, this study aimed to examine the association among smoking, IDH1 mutations, MGMT promoter methylation, and grading in glioma patients. Methods Patients were recruited from Dr. Sardjito General Hospital (a referral hospital in Yogyakarta and Central Java region) and its network hospitals. Genomic DNA was extracted from formalin-fixed paraffin-embedded samples or fresh glioma tissues. Identification of IDH1 mutation was performed using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) or DNA sequencing. Methylation-specific real-time PCR was performed to identify MGMT promoter methylation status. Smoking status was obtained by history taken from the patient or family members. Results In total, 122 patients were included in this study. As many as 35 patients (28.7%) had a smoking history. Most smokers (57.1%) smoke less than ten cigarettes per day. However, most of them (68.8%) have been smoking for more than 20 years. Smoking patients have a significantly higher proportion of high-grade glioma than non-smokers (80% vs. 55.2%, p=0.01). Among 122 patients, 24 (19.7%) of them carried IDH1 mutation. Smoking patients have a significantly higher proportion of IDH1 mutation compared with non-smokers (31.4% vs. 14.9%, p<0.001). No significant association was found between intensity and duration of smoking with IDH1 mutations and glioma grading. No significant association was found between smoking and MGMT promoter methylation.\n\nConclusions In glioma patients, smoking is associated with IDH1 mutations and grading but not with MGMT promoter methylation.", "keywords": [ "smoking", "IDH1 mutation", "MGMT methylation", "glioma", "grading" ], "content": "Introduction\n\nGlioma is one of the most common central nervous system (CNS) tumors in adults, accounting for about 60% of primary brain tumors (Huang et al., 2019). The annual incidence for Caucasians and Asians is about 6 cases per 100,000 people (Hofer et al., 2014). Currently, gliomas are classified by the World Health Organization (WHO) based on histological and molecular characteristics. Histologically, gliomas can be classified into astrocytomas, oligodendrogliomas, and ependymomas. However, the WHO Classification of Tumors of the Central Nervous System in 2016, and then in 2021, combined the molecular characteristics into this classification, with one of the most important molecular characteristics used being IDH gene mutations (Louis et al., 2016, 2021). Another important marker is MGMT promoter methylation which determines patients’ response to alkylating agents such as temozolomide (Hegi et al., 2005).\n\nIDH mutations are quite common in malignancies. In glioblastoma (GBM) itself, the role of IDH mutations was first shown by Parsons et al. (2008). Since then, many studies have shown the significance of IDH mutations in the management of gliomas due to their diagnostic, prognostic, and predictive implications (Picca et al., 2018). In fact, IDH1 mutations have been shown to be an early event in the development of glioma (Watanabe et al., 2009). The most commonly found IDH1 mutation was IDH1 R132 mutation, a missense heterozygous mutation at codon 132 of the IDH1 gene. This mutation has mostly been found in grade II and III astrocytomas and oligodendrogliomas (55–80%). IDH1 mutations are more common in secondary GBM (>80%) and much rarer in primary GBM (<10%). IDH1 mutations are associated with significantly better outcomes, with patients with IDH-mutated glioma having a longer overall survival than wild-type patients (Uno et al., 2011).\n\nAssociation between smoking and glioma is not clear. Several studies showed a significant association between smoking and glioma formation, while other studies failed to show this association. A large meta-analysis involving seven cohorts and 17 case-control studies with more than 2.3 million subjects found no significant association between cigarette smoking and glioma. The metanalysis, however, showed a statistically significant increase of glioma cases in past smokers in females (RR: 1.13, p=0.046) but not in males (Li et al., 2016). Another study in Korea showed that cigarette smoking might be associated with developing malignant glioma in a dose-dependent manner (Ahn et al., 2020). This study, however, only involved malignant glioma. The association of smoking with glioma grades, in general, remains unknown.\n\nOne possible mechanism by which smoking can cause gliomas is through its effect on IDH gene mutations. An association between smoking and IDH1 mutation has been shown in several cancers. For example, smoking history is associated with IDH1 mutations in chronic myelomonocytic leukemia (CMML), myelodysplastic syndromes (MDS), and lung adenocarcinoma (Madanat et al., 2017; Toth et al., 2018). IDH1 mutations play a significant role in gliomagenesis, so knowing the association between smoking and IDH1 mutations in gliomas is crucial. Unfortunately, this association is still unclear.\n\nPrevious studies also showed the association of smoking with MGMT methylation. Studies in head and neck squamous cell carcinoma and colorectal cancer showed that methylation of MGMT was suppressed by heavy smoking (Matsuda et al., 2020). This is thought to be part of a biological defense mechanism to suppress various genetic mutations caused by smoking (Matsuda et al., 2020).\n\nAs mentioned above, smoking is associated with IDH mutations and MGMT promoter methylation in various cancers. However, these associations have never been proven in glioma. Therefore, this study aimed to examine the association between smoking, IDH mutations, and MGMT promoter methylation in patients with glioma. In addition, because IDH mutations are known to be associated with glioma grade, we also assessed the association of smoking with glioma grade.\n\n\nMethods\n\nThis study used a cross-sectional design. All patients diagnosed with glioma based on pathology examination who were treated at Dr. Sardjito General Hospital (a referral hospital in Yogyakarta and Central Java region) and its network hospitals in 2010-2020 were included in this study. Only patients with complete data regarding smoking status and IDH mutations were recruited (See Underlying Data) (Dwianingsih et al., 2022). Written and verbal informed consents were obtained from all patients or family members. Glioma tissue in the form of fresh tissue and formalin-fixed paraffin-embedded (FFPE) samples were taken from the anatomical pathology laboratory and tissue bank at the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada.\n\nAn experienced neuropathologist performed the histopathology examinations. Gliomas were classified according to the 2016 WHO classification of CNS tumors. Smoking status was obtained by history taken from the patients or their family members at the time of consent. To avoid potential bias, neither the neuropathologist analyzing the patient sample nor the physician interviewing the patient and family members were made aware of the IDH1 mutation status. Demographic and clinical data were obtained from medical records. This study was approved by the Medical and Health Research Ethics Committee, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia (KE/FK/0115/EC).\n\nSmoking status was classified as previously reported (Ahn et al., 2020). Current smokers were patients who had smoked more than five packs (100 cigarettes) in their lifetime and were still smoking when diagnosed with glioma. Former smokers were patients who smoked more than five packs (100 cigarettes) in their lifetime but had stopped smoking when diagnosed with glioma. Never smokers were patients who had never smoked at all or had smoked but less than five packs (100 cigarettes) in their lifetime. In addition, data on total smoking duration in years and the average number of daily cigarettes consumption were collected. Total cigarette smoke was classified as < 10 cigarettes per day, 10–19 cigarettes per day, and ≥ 20 cigarettes per day (Ahn et al., 2020).\n\nIsolation of genomic DNA from FFPE samples or fresh glioma tissue was performed. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was then performed to determine IDH1 mutation status. Finally, samples that did not show conclusive results on PCR-RFLP were subjected to DNA sequencing. Our previous publication described all methods (Malueka et al., 2020a).\n\nMethylation-specific qRT-PCR was performed to identify MGMT promoter methylation (See Underlying Data) (Dwianingsih et al., 2022). First, bisulfite conversions of genomic DNA were performed, followed by quantitative real-time PCRs as previously described (Malueka et al., 2020b; Rivera et al., 2010).\n\nStatistical tests were performed to identify the association between smoking with IDH1 gene mutation, MGMT promoter methylation, and grading in glioma. T-test or Mann-Whitney test was used to analyze numerical data using IBM SPSS Statistics version 22 (SPSS, RRID:SCR_002865). Meanwhile, for categorical variables, Chi-square or Fisher’s exact test was used. The subjects who had incomplete or missing data were not included in the analysis, as mentioned before in the inclusion criteria.\n\n\nResults\n\nThis study recruited a total of 122 patients. Detailed patients’ characteristics are shown in Table 1. According to gender groups, most patients were male (69, 56.6%). The average age at glioma diagnosis was 38.61±18.6 years old. The smoking patients were significantly older (49.09±11.5 vs. 34.4±19.3 years old, p<0.001). As many as 35 patients (28.7%) had a smoking history. Of these, only two patients were former smokers, while the rest were current smokers. For further analysis, these two groups were combined in one group (ever smokers). Most smokers were male (male vs. female smokers, 88.6% vs. 11.4%, p<0.001). In terms of intensity, most smokers (57.1%) smoke less than ten cigarettes per day (Table 2). However, most of them (68.8%) have been smoking for more than 20 years.\n\n\n\n• Male\n\n\n\n• Female\n\n\n\n• Astrocytic\n\n\n\n• Mixed\n\n\n\n• Oligodendroglial\n\n\n\n• Ependymal\n\n\n\n• Grade I\n\n\n\n• Grade II\n\n\n\n• Grade III\n\n\n\n• Grade IV\n\n\n\n• Low grade (Grade I & II)\n\n\n\n• High grade (Grade III & IV)\n\n\n\n• Methylated\n\n\n\n• Unmethylated\n\n* Mann Whitney test.\n\n** Fisher’s exact test, other tests use chi-square test.\n\n\n\n• Yes (current or former smoker)\n\n\n\n• No (never smoker)\n\n\n\n• <10\n\n\n\n• 10–19\n\n\n\n• ≥20\n\n\n\n• <10\n\n\n\n• 10–19\n\n\n\n• ≥20\n\n* Chi-square test.\n\n** Fisher’s exact test.\n\nMGMT methylation analysis using methylation-specific real-time PCR was performed in 81 patients. Methylation of MGMT promoters was found in 24 patients (29.6%). No significant difference was found in the proportion of patients with MGMT methylation between the smoking and non-smoking group (39.3 vs. 24.5%, p=0.167).\n\nAccording to the WHO grading, the most commonly found glioma was grade IV (41.8%), followed by grade II (32.8%), grade III (17.2%), and grade I (8.2%). Smoking history was significantly associated with glioma grading. Smoking patients have a significantly higher proportion of high-grade glioma than non-smokers (80% vs. 55.2%, p=0.01).\n\nAmong 122 patients, 24 (19.7%) of them carried IDH1 mutation. Smoking patients have a significantly higher proportion of IDH1 mutation compared with non-smokers (31.4 vs. 14.9 %, p<0.001). However, no significant association was found between intensity and duration of smoking with IDH1 mutation and glioma grade (Tables 2 and 3).\n\n\n\n• <10\n\n\n\n• 10–19\n\n\n\n• ≥20\n\n\n\n• <10\n\n\n\n• 10–19\n\n\n\n• ≥20\n\n* Fisher’s exact test.\n\n\nDiscussion\n\nThis study showed a significant association between smoking and IDH1 mutation in glioma. A higher proportion of IDH1 mutation was found in smokers compared to non-smokers. The association of smoking with IDH1 mutation in gliomas was previously unclear. Several studies have reported a possible association between smoking and IDH mutations in other types of cancers. For example, smoking has been associated with IDH1 mutations in patients with lung adenocarcinoma, MDS, and CMML (Madanat et al., 2017; Rodriguez et al., 2020; Toth et al., 2018). In these studies, smoking intensity seemed to play an important role. In the case of MDS and CMML, an association of smoking with IDH1 mutations was identified in smokers who smoke more than two packs per day or more than 40 pack-years (Madanat et al., 2017). Our study, however, did not find this association between smoking intensity and IDH1 mutation (Table 2).\n\nThe association between smoking and glioma grade is unclear. A study from Korea reported a higher risk of malignant glioma formation in smokers (Hazard ratio (HR) 1.22) compared to never smokers. This risk was greater for those who smoked ≥20 cigarettes a day (HR = 1.50) (Ahn et al., 2020). These results suggest that cigarette smoking may be associated with the development of malignant glioma in a dose-dependent manner (Ahn et al., 2020). Our study also showed a significant association of smoking with a higher glioma grade. Smoking patients in our study have a significantly higher proportion of high-grade glioma than non-smokers (80% vs. 55.2%, p= 0.01). However, we did not find a significant association between smoking intensity and glioma grading (Table 3).\n\nSeveral mechanisms have been proposed to explain the association between smoking and glioma. Smoking can induce several mutational signatures, including the IDH mutation. The mutations can occur in tissues exposed to smoking directly or indirectly (Ahn et al., 2020; Madanat et al., 2017). Because the IDH mutations are early events in gliomagenesis, the finding of an association between smoking and IDH mutations suggests a possible role for smoking in glioma formation (Turkalp et al., 2014). Recent studies showed that smoking could breach the blood-brain barrier by damaging endothelial tight junctions. This may facilitate the penetration of carcinogens into the brain (Ahn et al., 2020). Tobacco products have been shown in an animal experiment to produce exogenous N-nitroso compounds, which have been shown to induce glioma. More than 60 known carcinogens have been found in cigarette smoke, including polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and nitrosamines; all play a key role in tumorigenesis. Nicotine has also been shown to increase the proliferation and migration of human glioma cells (Grando, 2014). Indeed, a recent experimental study has shown that nicotine-induced stimulation of malignancy in glioma cells (Kalita et al., 2021). Therefore, nicotine is not only the main addictive compound that keeps smokers in their habits but also makes a genotoxic contribution to cancer pathogenesis (Grando, 2014).\n\nPrevious studies have shown an association between smoking and methylation of MGMT promoters. A study by Pulling et al. in lung adenocarcinoma showed that smokers had decreased frequency of having a methylated MGMT than non-smokers (Pulling et al., 2003). Another study in head and neck squamous cell carcinoma showed that methylation of MGMT was suppressed by heavy smoking (Matsuda et al., 2020). A similar study in colorectal cancer showed that smoking is associated with a lower level of MGMT hypermethylation (Matsuda et al., 2020). These studies indicated that methylation of MGMT was suppressed by smoking. This is thought to be part of a biological defense mechanism. Several mutagens in tobacco, namely, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N′-nitrosonornicotine, and N-dimethylnitrosamine attack DNA at the O6-position of guanine. As a defense mechanism, the resulting O6-alkylguanine adducts will be repaired by the MGMT enzyme (Christmann & Kaina, 2012). Therefore, upregulation of MGMT through suppression of methylation occurred to facilitate this repair mechanism (Matsuda et al., 2020). However, our study did not show an association between smoking and MGMT methylation status. Another study in patients with squamous cell carcinoma of head and neck showed a similar result with our study, namely no association between smoking and MGMT promoter methylation was found (Puri et al., 2005). Further research is needed to determine the cause of this difference in results.\n\nThis study has several limitations. The first limitation is the small number of former smokers (2 patients) which required us to analyze current and former smokers in the same group, namely ever smokers. The second limitation is that because the patients included in this study were only patients who had undergone surgery, the available data may not represent the general glioma population. Finally, the third limitation is the possibility of recall bias when the patient or family was asked about smoking behavior.\n\nIn conclusion, this study showed that smoking is associated with IDH1 mutations and higher grades in glioma patients. This finding underlined the importance of smoking as a possible risk factor for glioma.\n\n\nData availability\n\nFigshare: Underlying data for ‘Associations among smoking, IDH mutations, MGMT promoter methylation, and grading in glioma a cross-sectional study’, https://www.doi.org/10.6084/m9.figshare.19337141 (Dwianingsih et al., 2022).\n\nThis project contains the following underlying data:\n\n• Demographic, clinical and IDH mutation and MGMT methylation of Glioma patient in Dr. Sardjito General Hospital\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAuthor contributions\n\nConceptualization: RGM, RAH, EKD. Data Curation: RAH, ASW, EB. Formal Analysis: MA, CMS. Funding Acquisition: RGM, EKD. Investigation: MA, CMS. Methodology: KD, AA, MA, CMS. Project Administration: EKD, RGM. Resources: KD, AA, RAH, ASW, EB. Software: ASW, EB. Supervision: RGM, RAH, EKD. Validation: EKD, RGM. Visualization: KD, AA. Writing – Original Draft Preparation: RGM, MA, CMS. Writing – Review & Editing: RGM, EKD.", "appendix": "Acknowledgement\n\nWe would like to thank Klinik Bahasa of Faculty of Medicine, Public Health and Nursing, UGM for assistance in language editing of this manuscript.\n\n\nReferences\n\nAhn S, Han KD, Park YM, et al.: Cigarette smoking is associated with increased risk of malignant gliomas: a nationwide population-based cohort study. Cancers (Basel). 2020; 12(5). PubMed Abstract \| Publisher Full Text\n\nChristmann M, Kaina B: O(6)-methylguanine-DNA methyltransferase (MGMT): impact on cancer risk in response to tobacco smoke. Mutat. Res. 2012; 736(1-2): 64–74. PubMed Abstract \| Publisher Full Text\n\nDwianingsih EK, Malueka RG, Hartanto RA, et al.: Associations among smoking, IDH mutations, MGMT promoter methylation, and grading in glioma a cross-sectional study. Figshare. Dataset. 2022. Publisher Full Text\n\nGrando S: Connections of nicotine to cancer. Nat. Rev. Cancer. 2014; 14: 419–429. PubMed Abstract \| Publisher Full Text\n\nHegi ME, Diserens A-C, Gorlia T, et al.: MGMT gene silencing and benefit from temozolomide in glioblastoma. N. Engl. J. Med. 2005; 352(10): 997–1003. Publisher Full Text\n\nHofer S, Rushing E, Preusser M, et al.: Molecular biology of high-grade gliomas: what should the clinician know?. Chin. J. Cancer. 2014; 33(1): 4–7. PubMed Abstract \| Publisher Full Text\n\nHuang J, Yu J, Tu L, et al.: Isocitrate dehydrogenase mutations in glioma: from basic discovery to therapeutics development. Front. Oncol. 2019; 9: 506. PubMed Abstract \| Publisher Full Text\n\nKalita O, Sporikova Z, Hajduch M, et al.: The influence of gene aberrations on survival in resected IDH wildtype glioblastoma patients: a single-institution study. Curr. Oncol. 2021; 28(2): 1280–1293. PubMed Abstract \| Publisher Full Text\n\nLi H-X, Peng X-X, Zong Q, et al.: Cigarette smoking and risk of adult glioma: a meta-analysis of 24 observational studies involving more than 2.3 million individuals. Onco. Targets. Ther. 2016; 9: 3511–3523. PubMed Abstract \| Publisher Full Text\n\nLouis DN, Perry A, Reifenberger G, et al.: The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 2016; 131(6): 803–820. PubMed Abstract \| Publisher Full Text\n\nLouis DN, Perry A, Wesseling P, et al.: The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro-Oncology. 2021; 23(8): 1231–1251. PubMed Abstract \| Publisher Full Text\n\nMadanat YF, Radivoyevitch T, Nazha A, et al.: Mutational signatures associated with intensity and duration of smoking in Myelodysplastic Syndromes (MDS). Blood. 2017; 130: 424. Publisher Full Text\n\nMalueka RG, Dwianingsih EK, Bayuangga HF, et al.: Clinicopathological features and prognosis of Indonesian patients with gliomas with IDH mutation: insights into its significance in a Southeast Asian population. Asian Pac. J. Cancer Prev. 2020a; 21(8): 2287–2295. PubMed Abstract \| Publisher Full Text\n\nMalueka RG, Theresia E, Fitria F, et al.: Comparison of polymerase chain reaction-restriction fragment length polymorphism, immunohistochemistry, and DNA sequencing for the detection of IDH1 mutations in gliomas. Asian Pac. J. Cancer Prev. 2020b; 21(11): 3229–3234. PubMed Abstract \| Publisher Full Text\n\nMatsuda S, Mafune A, Kohda N, et al.: Associations among smoking, MGMT hypermethylation, TP53-mutations, and relapse in head and neck squamous cell carcinoma. PLOS ONE. 2020; 15(4): e0231932. PubMed Abstract \| Publisher Full Text\n\nParsons DW, Jones SN, Zhang X, et al.: An integrated genomic analysis of human glioblastoma multiforme. Science. 2008; 321(5897); 1807–1812. PubMed Abstract \| Publisher Full Text\n\nPicca A, Berzero G, Di Stefano AL, et al.: The clinical use of IDH1 and IDH2 mutations in gliomas. Expert. Rev. Mol. Diagn. 2018; 18(12): 1041–1051. Publisher Full Text\n\nPulling LC, Divine KK, Klinge DM, et al.: Promoter hypermethylation of the O6-methylguanine-DNA methyltransferase gene: more common in lung adenocarcinomas from never-smokers than smokers and associated with tumor progression. Cancer Res. 2003; 63(16): 4842–4848. PubMed Abstract\n\nPuri SK, Si L, Fan CY, et al.: Aberrant promoter hypermethylation of multiple genes in head and neck squamous cell carcinoma. Am. J. Otolaryngol. 2005; 26(1): 12–17. PubMed Abstract \| Publisher Full Text\n\nRivera AL, Pelloski CE, Gilbert MR, et al.: MGMT promoter methylation is predictive of response to radiotherapy and prognostic in the absence of adjuvant alkylating chemotherapy for glioblastoma. Neuro-Oncology. 2010; 12(2): 116–121. PubMed Abstract \| Publisher Full Text\n\nRodriguez EF, De Marchi F, Lokhandwala PM, et al.: IDH1 and IDH2 mutations in lung adenocarcinomas: Evidences of subclonal evolution. Cancer Med. 2020; 9(12): 4386–4394. PubMed Abstract \| Publisher Full Text\n\nToth LN, de Abreu FB , Tafe LJ: Non-small cell lung cancers with isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations. Hum. Pathol. 2018; 78: 138–143. Publisher Full Text\n\nTurkalp Z, Karamchandani J, Das S: IDH mutation in glioma: new insights and promises for the future. JAMA Neurol. 2014; 71(10): 1319–1325. Publisher Full Text\n\nUno M, Oba-Shinjo SM, Silva R, et al.: IDH1 mutations in a Brazilian series of glioblastoma. Clinics (Sao Paulo). 2011; 66(1): 163–165. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatanabe T, Nobusawa S, Kleihues P, et al.: IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. Am. J. Pathol. 2009; 174(4): 1149–1153. PubMed Abstract \| Publisher Full Text" }	[ { "id": "136340", "date": "04 May 2022", "name": "I. Putu Eka Widyadharma", "expertise": [ "Reviewer Expertise Pain management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research has some interesting ideas to discuss, but there are some things that should be added, such as the points below.\nIn the introduction, data on the incidence of gliomas should use more up-to-date literatures.\n\nIn the method section of the abstract, the research design, research time period should be added.\n\nIn the method section, please explain in more detail about the inclusion criteria and exclusion criteria in this study, for example whether the use of e-cigarettes was included in this study or not, whether there were certain comorbid diseases that became the exclusion criteria and other confounding variables.\n\nIn the section on smoking status, clarify again for the definition of former smokers, how long it took the patient to stop smoking when glioma was diagnosed.\n\nIn the section on identification of IDH mutation and MGMT methylation, it is better to explain the method again, not just to mention that it has been explained in previous publications.\n\nIt is better to add a figure regarding the relationship between smoking and glioma, so that the reader can understand better.\n\nAre these risk factors explained at any age? Are the results the same for young and old?\n\nIn table 2, the non-smoker population actually has more wild-type IDH. Can you explain why that is? Or can you explain the difference between IDH mutation and IDH wild type.\n\nIn the conclusion section, it should be made in its own sub-chapter, not combined in a discussion sub-chapter.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "139699", "date": "22 Jun 2022", "name": "Alberto Picca", "expertise": [ "Reviewer Expertise Neuro-oncology" ], "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 short paper, Malueka and colleagues perform a case-control analysis to answer an old question in the field of neuro-oncology, namely the association between glioma and potential environmental risk factors, in this case tobacco smoking.\nDespite I recognize the effort done, the paper oversimplifies several aspects of the analysis. In first, the distribution of either glioma grading, IDH mutations, and MGMT promoter methylation is age-and sex-related, as smoking habit is. Without an adequate correction for these potential confounding factors, all result is not interpretable (this is the main limitation of the paper, but it is not mentioned in the discussion). Nevertheless, I am afraid that the cohort is too small to perform this correction. Furthermore, the cohort is too small to stratify for and perform statistical analysis according to smoke intensity (cigarettes/day) or duration. Grade I gliomas and ependymomas should not be included in the analysis as they are molecularly distinct pathologies with their own tumorigenesis.\nOverall, the presented data are insufficiently powered to analyse a complex question; bigger cohorts (PMID: 243359211) and a meta-analysis (PMC49135392) did not find any correlation between glioma occurrence and smoking habit. In conclusion, in my opinion the paper does not contain sufficient quality data warranting indexing.\nMinor comments regarding the different paragraphs:\nIntroduction:\nI would suggest to replace or implement some ref (Huang et al. 2019, Hofer et al. 2014) with more appropriate major papers with epidemiological data (ex., annual CBTRUS report: PMID 346089453; the Molinaro and colleagues review on epidemiology of gliomas: PMID 312277924); also Uno et al 2011 seems inappropriate as it is a minor paper, consider the seminal Yan & Parsons (2009)5 or Sanson et al. 20096 for the prognostic role of IDH mutation in gliomas\n\nI disagree with the sentence “IDH mutations are quite common in malignancies”. IDH mutations remain restricted to specific tumor subtypes, and even among diffuse gliomas, IDH mutant ones represent a minority of cases. Maybe it would be more appropriate to say that IDH mutations can be found in several types of malignancies.\n\nI would remove the sentence “IDH1 mutations are more common in secondary GBM (>80%) and much rarer in primary GBM (<10%).” As it is outdated; the secondary GBM is not an entity anymore, and the new 2021 WHO classification reserves the GBM nosology to IDHwt gliomas only. IDHm grade IV gliomas are now called Grade 4 astrocytomas, IDH mutant. Please rewrite.\nMethods:\nThe inclusion criteria used for patients are unclear. Were ALL patients diagnosed with a glioma included in the analysis? Including children? Or only adult patients? This is of evident importance but is not specified. Please, clarify.\n\nThe definition of “former smokers” is unclear. You should give the minimum time range from smoke cessation to glioma diagnosis utilized to consider a patient a former smoker. Please add this.\nDiscussion:\nThe sentence “N-nitroso compounds, which have been shown to induce glioma.” needs a supporting reference (this association is unknown to me).\n\nIn the sentence “Nicotine has also been shown to increase the proliferation and migration of human glioma cells”, the according reference (Grando et al. 2014) is an opinion letter that does not provide experimental evidence nor talks about glioma. Please, modify.\n\nSimilarly, in the sentence “a recent experimental study has shown that nicotine-induced stimulation of malignancy in glioma cells”, the provided reference does not correspond to the mentioned experimental data (?).\nOverall, be careful in citing articles. Directly cite the evidence supporting your hypotheses and not only other papers that mentioned them.\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?\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/11-473
https://f1000research.com/articles/10-489/v1	22 Jun 21	{ "type": "Research Article", "title": "Silencing of Carboxypeptidase E expression inhibits proliferation and invasion of Panc-1 pancreatic cancer cells", "authors": [ "Hong Lou", "Y Peng Loh", "Hong Lou" ], "abstract": "Background: Pancreatic cancer is one of the leading cause of cancer-related death globally. The molecular basis of this disease is complex and not fully understood. Previous studies have indicated that carboxypeptidase E (CPE) plays a role in promoting tumorigenesis in many cancer types. Here we have investigated the effect of carboxypeptidase E (CPE), including its isoform, in regulating the proliferation, migration and invasion of Panc-1 cells, a pancreatic cell line. Methods: Panc-1 cells were transfected with CPE siRNA which targets both CPE-wild type and its isoform, or scrambled siRNA, for 24 h and then assayed for proliferation by the MTT and colony formation assays, and migration and invasion by wound healing and matrigel assays, respectively. Results: CPE siRNA treatment of Panc-1 cells down-regulated the expression of CPE mRNA by 94.8%. Silencing of CPE mRNA expression resulted in a significant decrease in proliferation as revealed by the MTT assay and a 62.8% decrease in colony formation. Western blot analysis of expression of Cyclin D1 in Panc-1 cells treated with CPE siRNA showed a decrease of 32.5% compared to scr siRNA treated cells, indicating that CPE regulates proliferation through modulating this cell cycle protein. Additionally, suppression of CPE expression in Panc-1 cells significantly decreased migration and invasion. Conclusions: Our findings indicate that CPE may play an important role in regulating cell proliferation, migration and invasion to promote pancreatic cancer tumorigenesis.", "keywords": [ "Carboxypeptidase E", "pancreatic cancer", "cell proliferation", "cell migration", "cell invasion" ], "content": "Abbreviations\n\nCPE, carboxypeptidase E\n\nHCC, hepatocellular carcinoma\n\nWT, wild type\n\n\n1. Introduction\n\nPancreatic cancer is an intractable malignancy which has one of the highest mortality rates world -wide. Patients with pancreatic cancer seldom display any symptoms until an advanced stage. It is one of the most lethal malignant carcinomas, with a survival rate of 9% over five years. The incidence of pancreatic cancer is projected to increase worldwide, despite the availability of better methods for early diagnosis. Generally, surgery, chemotherapy, and radiotherapy are used to prolong survival, but there is no cure for advanced stage patients.1 Hence, it is necessary to search for new molecular targets in order to develop novel therapeutic approaches to treating pancreatic cancer.\n\nCarboxypeptidase E (CPE) is an enzyme that processes prohormones2,3 and has also been shown to be a trophic factor mediating neuroprotection, stem cell differentiation, and the regulation of bone mass, independent of its enzymatic activity.4–6 CPE is expressed in the brain and endocrine organs,7 but also in epithelial-derived cancers such as colorectal, liver, cervical, and lung cancers.8–11 Clinical investigations have revealed that elevated CPE levels are correlated with poor prognosis in colorectal, cervical, and liver cancers, as well as lung adenomas.8,9,11,12 Additionally, CPE is a tumor survival factor under hypoxic conditions.13 Recently, two major forms of CPE have been cloned and identified in human hepatocellular carcinoma (HCC) cell line: wild-type CPE (50-53 kD in size) and an N-terminal truncated splice variant named 40 kD CPE-ΔN.10\n\nIn the present study, we investigated the effect of silencing the expression of CPE by siRNA on proliferation, migration, and invasion in Panc-1 cells. We also studied the mechanism by which CPE regulates Panc-1 cell proliferation.\n\n\n2. Methods\n\nThe human pancreatic cell line Panc-1 was purchased from ATCC (Manassas, VA, RRID:CVCL_0480). The cells were cultured in DMEM media (Millipore Sigma, Burlington, MA, USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a humidified 5% CO2 incubator.\n\nProteins from cells were extracted with RIPA lysis and extraction buffer (Thermo Fisher, Waltham, MA) supplemented with Complete Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN). 20 μg of the protein from cell lysate was loaded per lane on SDS-PAGE gel and subjected to Western blotting according to our procedure published previously.13 Antibodies against GAPDH (1:5000 dilution) and anti-cyclin D1 (1: 500 dilution) were purchased from Cell Signaling Technology (Danvers, MA).\n\nPanc-1 cells plated at 40–50% density were grown to ~75% confluency overnight as described above. The next day, cells were treated with 50-60 pmols of three CPE siRNAs custom synthesized by Invitrogen, (Carlsbad, CA) which target both CPE-WT and CPE-ΔN mRNAs:\n\nsiCPE Seq1#: GAU UUG UCC GAG ACC UUC AAG GUA A;\n\nsiCPE Seq2#: UUA CCU UGA AGG UCU CGG ACA AAU C;\n\nsiCPE Seq3#: GAU CCU GAG AGU UCC GAA CGU UUA A,\n\nor scrambled siRNA, which is a non-targeting 20-25nt siRNA (Santa Cruz Biotechnology, Dallas, TX), using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) according to manufacturers’ protocol. After 24h incubation, the cells were dissociated by trypsinization, cell count were obtained, and seeded in various plates for cell invasion, cell migration, colony formation assay, and cell proliferation assays.\n\nPanc-1 cells were treated for 48h with siRNA. RNA was then extracted from the cells using the RNeasy Mini Kit (Qiagen, Germantown, MD). First-strand cDNA was synthesized with 200 ng of total RNA using the SensiFAST cDNA Synthesis Kit (Bioline Reagents Ltd, United Kingdom) for assay of CPE expression. Quantitative PCR was performed using 1μl first strand cDNA and SYBR Green Master Mix (Invitrogen) under the conditions of 95 °C for 15 s, annealing at 62 °C for 60 s, extension at 72 °C for 30 s for 40 cycles, and a final extension at 72 °C for 10 min. 18 s rRNA was used as a normalizing control. Primer sequences were: for amplifying CPE (generic), fwd: 5′- CCATCTCCGTGGAAGGAATA and rev: 5′-CTT ACA GCC TCA GCT CCA GG; 18S RNA, fwd: 5′-CTCTTAGCTGAGTGTCCCGC and rev: 5′-CTGATCGTCTTCGAACCTCC.\n\nPanc-1 cells treated with siRNA for 24 h were seeded in a 96-well plate at a density of 2000 cells/well in 200μl media and incubated for four to five days. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed daily from day one to four. 25 μl of MTT reagent (5 mg/ml) (Sigma-Aldrich, St. Louis, MO) added to each well were then incubated in a CO2 incubator at 37°C for 4 h. The supernatant was then removed and DMSO (150 μl) was added to each well. Absorbance value at 490 nm was then measured in a microplate reader (BioTek, Winooski, VT) after 5 min. Experiments were performed in five different wells for each condition. Two independent experiments were performed on separate days.\n\nPanc-1 cells treated with CPE siRNA or scrambled siRNA were trypsinized and single cell suspensions were obtained. Viable cells, (2000/well) were seeded in a 6-well plate and cultured for 15-21 days. Culture medium was changed every five days. Cells were then gently washed twice with PBS and fixed in 100% methanol, before staining with 1% crystal violet solution for 10 minutes. Excess stain was removed by washing with PBS. The number of colonies containing at least 50 cells were counted with Image J software from the images captured under a light microscope. The experiment was repeated three times and each experiment was done in triplicates.\n\nApproximately 1 × 105 Panc-1cells were plated in the well of culture insert in non-coated 35 mm culture dish (Ibidi, Martinsried, Germany) and allowed to form a monolayer. A ~500 μm wound gap was created when the culture insert was removed, and the cells were immersed in the complete media following manufacturer’s instructions. Images of the wound were captured at time 0, and then at 12, 24, and 36 h after incubation in a CO2 incubator at 37°C. Wound closure was evaluated by measuring the areas of the wound at different time points using Image J software. Three experiments were done on separate days and each experiment was done in triplicates.\n\nA 24-well Corning Matrigel invasion chamber (Corning, NY) with 8-micron pores was used for cell invasion assay. 500 μl of cell suspension (1 × 105 cells/ml) in serum free media was added to the top chamber. Then 500 μl of FBS supplemented media was added to the lower chamber to serve as a chemoattractant. 24 h. later, cells that did not invade through the pores were carefully removed with a cotton swab. Cells on the lower surface of the membrane were fixed with 100% methanol and stained with 1% crystal violet solution for 10 min. Excess stain was removed with water and images from five different fields/well were captured. Cells were counted with ImageJ software. Experiments were done in triplicates.\n\nThe data reported are the mean ± SD (standard deviation) or mean ± SE (standard error) of at least triplicate values (n) in each experiment. Number of independent experiments (N) is stated in the figure legends. Statistical significance was determined by Student’s t-test and p values are indicated as p < 0.05, p < 0.01, p < 0.001.\n\n\n3. Results\n\nFigure 1A shows a schematic of the two forms of CPE: wild type CPE and 40 kD CPE-ΔN expressed by Panc-1 cells reported previously.14 Moreover, CPE-WT protein was detected mainly in the secretion medium, while CPE-ΔN was found in the nucleus and cytoplasm of Panc-1 cells.14 To evaluate the function of CPE, Panc-1 cells were treated with CPE si-RNA targeting the silencing of expression of both forms of CPE (see materials and methods). Figure 1B shows that treatment of Panc-1 cells with CPE siRNA down-regulated expression of CPE-transcripts by 94.8 ± 2.2%. Down-regulation of CPE with siRNA treatment resulted in significant inhibition of Panc-1 cell proliferation from day two to day four as observed in the MTT cell proliferation assay (Figure1C). Additionally, CPE siRNA treatment reduced colony formation in Panc-1 cells by 62.8%, versus cells treated with scrambled (scr) siRNA (Figure 1D and E).\n\nA. Schematic showing the structure of 53 kDa CPE-WT and 40 kDa CPE-ΔN protein. SP, signal peptide; P, pro-region; TM, transmembrane domain.\n\nB. Bar graph showing CPE mRNA expression detected by qRT-PCR in Panc-1 cells treated with CPE siRNA (siCPE) or scrambled siRNA (scr). (p < 0.01, N = 4). Error bars denote SD.\n\nC. Line graphs of MTT assay data showing the time course of cell proliferation of Panc-1 cells treated with CPE siRNA or scr siRNA, (day 2 - day4: *p < 0.01, n = 5). This is representative of two independent experiments with similar results. Error bars denote SEM.\n\nD. Image of colony formation assay of Panc-1 cells treated with CPE siRNA or scr si RNA.\n\nE. Bar graphs showing decrease in number of colonies/well formed after CPE siRNA (siCPE) treatment compared with scr siRNA controls (p < 0.05, N = 3). Error bars denote SEM.\n\nTo determine if the inhibition of proliferation of Panc-1 cells is mediated by down-regulation of Cyclin D1, a protein necessary for cell cycle progression, Western blot analysis of Panc-1 cells treated with CPE-siRNA was carried out. Figure 2A and B show that Cyclin D1 expression was significantly inhibited by 32.5% in CPE siRNA treated cells compared to scr siRNA treated cells.\n\nA. Western blot showing expression of CyclinD1 in Panc-1 cells with CPE siRNA or scrambled (scr) siRNA treatment. Full-length blots are deposited in the data repository.\n\nB. Bar graph showing reduced expression of Cyclin D1 in Panc-1 cells after CPE siRNA compared to scr siRNA treatment (n = 3, p = 0.016). GAPDH served as loading control. Error bars denote SEM. Similar results were obtained in a second experiment.\n\nWound healing assay was carried out to study the effect of down-regulation of CPE expression on migration of Panc-1 cells. Treatment of Panc-1 cells with CPE siRNA significantly inhibited migration of these cells. At the 24-hour time point, the wound healing area of siCPE RNA treated cells was ~ 8-fold larger than scr siRNA treated cells (Figure 3A and B). In addition, suppression of CPE expression by CPE siRNA inhibited invasion of these cells, as revealed by the matrigel invasion assay. The number of cells which invaded across the matrigel membrane was ~three-fold greater in scr siRNA treated versus CPE siRNA treated cells (Figure 3C and D).\n\nA. Representative images of wound healing assay showing wound recovery in Panc-1 cells of CPE siRNA or scrambled siRNA treated group at 0, 12 and 24 hours.\n\nB. Bar graph showing reduced recovery, represented by larger wound area in Panc-1 cells treated with CPE siRNA (siCPE) at 24-h versus scrambled siRNA (n = 3, p < 0.01). Representative of three experiments performed on separate days. Error bars denote SEM.\n\nC. Representative image of cells in Matrigel invasion assay of Panc-1 cells treated with CPE siRNA or scrambled siRNA (scr, control) for 24h.\n\nD. Bar graph showing reduced number of invaded Panc-1 cells after CPE siRNA compared to scr siRNA treatment (N = 3, *p < 0.01). Error bars denote SEM.\n\n\n4. Discussion\n\nPrevious studies have detected little or no WT-CPE protein present in Panc-1 and BXPC-3 pancreatic cancer cell extracts, although there was a high amount of WT-CPE mRNA in these cells.14 Instead, WT-CPE was found primarily secreted into the medium in both these pancreatic cancer cell lines.14 This is similar to that observed in liver cancer (HCC97H),10 ovarian cancer (COAV3)10 and glioblastoma cell lines.15 The splice variant, 40 kD CPE-ΔN was found in the cytoplasm and the nucleus in Panc-1 cells,14 similar to that found in other cancer cell lines from liver (HCC97H) and ovarian (CAOV3) cancers.10 Suppression of CPE expression by CPE si-RNA which does not distinguish between WT-CPE and CPE-ΔN resulted in inhibition of proliferation of Panc-1 cells, as revealed by MTT and colony formation assay. Additionally, migration and invasion of Panc-1 cells were inhibited after CPE siRNA treatment. These findings suggest that CPE plays a significant role in promoting pancreatic tumor growth and metastasis. This conclusion corroborates with another study showing that down-regulation of CPE expression in the BXPC-3 pancreatic tumor cell line inhibited proliferation and invasion in vitro.16 Moreover, inoculation of these CPE-siRNA treated cells into null mice, prevented pancreatic tumor formation in vivo.16 Hence loss of function studies from two pancreatic cancer cell lines (Panc-1 and BX-PC3) support the importance of CPE in promoting pancreatic cancer progression.\n\nThe mechanism of action of CPE on proliferation has been examined in Panc-1 cells in our study. CPE siRNA treatment of Panc-1 cells showed down-regulated expression of Cyclin D1 and decreased proliferation, indicating that CPE acts through regulation of cell cycle in these cells. This is similar to another study where silencing of CPE expression caused decreased Cyclin D1 expression, cell cycle arrest and inhibition of proliferation of osteosarcoma cells.17 In BX-PC3 cells, it was suggested that CPE may exert its tumorigenic effect via NF-κB since CPE-regulated NF-κB expression, and NF-κB-siRNA inhibited invasion in these cells.\n\nA recent bioinformatic network analysis of transcriptional and epigenetic profiles of Panc-1 cells treated with CPE-siRNA, showed differentially expressed RNAs versus control, which included mRNA, miRNA, circRNA and IncRNA, that were correlated with cancer onset and/or progression. Furthermore, the analysis showed that certain RNAs such as HUWE1, hsa-miR-6780b-5p, has_circ_0058208 and lnc-G3BP1-3:8 were found to be in central positions of the network, suggesting their importance in promoting pancreatic cancer progression on many levels.18\n\nIn gain of function studies, overexpression of CPE-ΔN in Panc-1 cells increased CXCR2, a pro metastatic gene.14 Likewise, in a hepatocellular carcinoma cell line, (HCC97L cells), transfection of 40 kD CPE-ΔN resulted in >3-fold increase in expression of several tumor metastasis-related genes, CCXCR2, CXCR4, and CCL12.10\n\nTaken together, secreted WT-CPE which acts extracellularly and CPE-ΔN which is translocated to the nucleus10,14 are able to regulate a large number of genes that promote proliferation and metastasis, through different pathways, in pancreatic cancer cell. Some of these mechanisms mediated by CPE may be functioning in other types of cancers as well, to promote cancer progression.10,13,14 Our study together with others suggest that CPE is a potential novel therapeutic target for treating pancreatic cancer. Inhibiting CPE expression through delivery of CPE siRNA/shRNA into tumor cells via exosomes could represent a new therapeutic approach in suppressing pancreatic tumor growth. Indeed, it has been demonstrated that CPE-shRNA loaded HEK293 cell exosomes can inhibit proliferation when taken up by highly malignant recipient HCC97H cells.19 As proof of concept, exosomes carrying KRAS specific siRNA injected into orthotopic pancreatic cancer mouse models have been shown to suppress tumor growth, inhibit metastasis and enhance overall survival of these animals.20 Thus, future studies could focus on injecting CPE sh-RNA loaded exosomes into orthotopic pancreatic cancer mouse models to test for efficacy in improving survival of these animals.\n\n\nData availability\n\nHarvard Dataverse: Silencing of Carboxypeptidase E expression inhibits proliferation and invasion of Panc-1 pancreatic cancer cells. https://doi.org/10.7910/DVN/TUFG9M.21\n\nThis project contains the following underlying data\n\n• Figure 1B. tab (CPE qRT-PCR data in Panc-1 cells treated with CPE siRNA (siCPE) or scrambled siRNA (scr).)\n\n• Figure 2 source data.tab (Quantification of Western Blot of CyclinD1 in Panc-1 cells treated with siCPE or scr RNA)\n\n• Figure 1C source data.tab (MTT assay of Panc-1 cells treated with siCPE or scr RNA)\n\n• Figure 1D source data.tab (Quantification of the number of colonies formed in Panc-1 cells treated with siCPE or scr RNA)\n\n• Figure 3 invasion data.tab (Quantification of Panc-1 cells treated with siCPE or scr RNA in invasion assay)\n\n• Figure 3 wounding data.tab (Quantification of wound area formed by Panc-1 cells treated with siCPE or scr RNA)", "appendix": "Acknowledgements\n\nWe thank Dr. Lin Cong, Peking Union Medical College, Beijing, China and Guest Researcher, Eunice Kennedy Shriver National Institutes of Child health and Human Development, National Institutes of Health, USA. for his contribution to some of the data. Dr. Ashley Xiao for her help in preparing Figures and Drs. Xuyu Yang and Sangeetha Hareendran for critical reading of the manuscript.\n\n\nAuthorship contributions\n\nConceptualization, Y.P.L.; methodology, H.L; investigation, H.L; resources, Y.P.L.; data curation, H.L. and Y.P.L; writing—original draft preparation, Y.P.L.; writing—review and editing, H.L. Y.P.L.; visualization, Y.P.L.; supervision, Y.P.L.; project administration, Y.P.L.; funding acquisition, Y.P.L.\n\n\nReferences\n\nRawla P, Sunkara T, Gaduputi V: Epidemiology of Pancreatic Cancer: Global Trends, Etiology and Risk Factors. World J Oncol. 2019; 10(1): 10–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHook VY, Eiden LE, Brownstein MJ: A carboxypeptidase processing enzyme for enkephalin precursors. Nature. 1982; 295(5847): 341–342. PubMed Abstract \| Publisher Full Text\n\nFricker LD, Carboxypeptidase E: Annu. Rev. Physiol. 1988; 50: 309–321. Publisher Full Text\n\nXiao L, Yang X, Sharma VK, et al.: Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-alphal variants in embryonic mouse brain. FASEB J. 2019; 33: 808–820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSelvaraj P, Xiao L, Lee C, et al.: Neurotrophic Factor-alpha1: A Key Wnt-beta-Catenin Dependent Anti-Proliferation Factor and ERK-Sox9 Activated Inducer of Embryonic Neural Stem Cell Differentiation to Astrocytes in Neurodevelopment. Stem Cells. 2017; 35: 557–571. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChougule A, Kolli V, Baroi S, et al.: Nonenzymatic and Trophic Activities of Carboxypeptidase E Regulate Bone Mass and Bioenergetics of Skeletal Stem Cells in Mice. JBMR Plus. 2020; 4: e10392. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCawley NX, Wetsel WC, Murthy SR, et al.: New roles of carboxypeptidase E in endocrine and neural function and cancer. Endocr. Rev. 2012; 33: 216–253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiang XH, Li LL, Wu GG, et al.: Upregulation of CPE promotes cell proliferation and tumorigenicity in colorectal cancer. BMC Cancer. 2013; 13: 412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen HW, Tan JF, Shang JH, et al.: CPE overexpression is correlated with pelvic lymph node metastasis and poor prognosis in patients with early-stage cervical cancer. Arch. Gynecol. Obstet. 2016; 294: 333–342. PubMed Abstract \| Publisher Full Text\n\nYang X, Lou H, Chen YT, et al.: A novel 40kDa N-terminal truncated Carboxypeptidase E splice variant: Cloning, cDNA sequence analysis and role in regulation of metastatic genes in human cancers. Genes Cancer. 2019; 10(5-6): 160–170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun J, Meng D, Li L, et al.: N-terminal truncated carboxypeptidase E expression is associated with poor prognosis of lung adenocarcinoma. Oncol. Lett. 2016; 12: 4659–4664. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang SF, Wu HD, Chen YT, et al.: Carboxypeptidase E is a prediction marker for tumor recurrence in early-stage hepatocellular carcinoma. Tumour Biol. 2016; 37: 9745–9753. PubMed Abstract \| Publisher Full Text\n\nMurthy SRK, Dupart E, Al-Sweel N, et al.: Carboxypeptidase E promotes cancer cell survival, but inhibits migration and invasion. Cancer Lett. 2013; 341: 204–213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHareendran S, Yang X, Lou H, et al.: Carboxypeptidase E-ΔN promotes proliferation and invasion of pancreatic cancer cells via up-regulation of CXCR2 gene expression. Int J Mol Sci. 2019 Nov 15; 20(22): 5725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoring E, Harter PN, Seznec J, et al.: The “go or grow” potential of gliomas is linked to the neuropeptide processing enzyme carboxypeptidase E and mediated by metabolic stress. Acta Neuropathol. 2012; 124: 83–97. PubMed Abstract \| Publisher Full Text\n\nLiu A, Shao C, Jin G, et al.: Downregulation of CPE regulates cell proliferation and chemosensitivity in pancreatic cancer. Tumour Biol. 2014; 35: 12459–12465. PubMed Abstract \| Publisher Full Text\n\nFan S, Li X, Li L, et al.: Silencing of carboxypeptidase E inhibits cell proliferation, tumorigenicity, and metastasis of osteosarcoma cells. Onco Targets Ther. 2016; 9: 2795–2803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBai Z, Feng M, Du Y, et al.: Carboxypeptidase E down regulation regulates transcriptional and epigenetic profiles in pancreatic cancer cell line: A network analysis. Cancer Biomark. 2020; 29(1): 79–88. PubMed Abstract \| Publisher Full Text\n\nHareendran S, Albraidy B, Yang X, et al.: Exosomal Carboxypeptidase E (CPE) confers, and CPE-shRNA loaded exosomes inhibit tumorigenesis in liver cancer cells. Extracell. Vesicles & Circ. Nucleic Acids. 2020; 1: 23.\n\nKamerkar S, LeBleu VS, Sugimoto H, et al.: Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature. 2017; 546: 498–503. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLou H, Peng Loh Y: Silencing of Carboxypeptidase E expression inhibits proliferation and invasion of Panc-1 pancreatic cancer cells. Harvard Dataverse, V3. 2021. Publisher Full Text" }	[ { "id": "88445", "date": "08 Jul 2021", "name": "Vinayak C. Palve", "expertise": [ "Reviewer Expertise Cancer cell biology", "cell signaling", "drug resistance", "mechanism of acquired resistance", "drug discovery", "pancreatic cancer", "lung cancer", "molecular biology", "cell biology", "biochemistry", "off-targets of drugs", "cancer cell signaling", "etc." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present manuscript, the author's aims to understand the role of Carboxypeptidase E in regulating proliferation, migration and invasion to promote pancreatic cancer tumorigenesis. The authors present some data but I believe it needs further evidence to support the conclusion made in the current manuscript. I would recommend implementing the following suggestions would improve the overall quality of the manuscript.\n\nMajor comments:\n\nThe authors have used only one pancreatic cell line (Panc-1) throughout the manuscript. Generating data for most of the experiment in 2-3 pancreatic cell lines would strengthen the findings.\n\nMinor comments:\nIt would be interesting to describe, how the different variants of CPE works?\n\nWestern blots in Figure 2A need to be cleaner. The GAPDH blot is not cropped well. I suggest to add better quality of blots and load the lysates taking control at first followed by CPEsi RNA.\n\nPlease provide the good quality and resolution of images (especially for Figure 3).\n\nThe authors can blot for downstream targets of Cyclin D1 to address how depletion of Cyclin D1 helps the pancreatic cells to stop proliferation in a more mechanistic approach.\n\nI believe addressing these comments shall help improve the quality of the manuscript. All the best.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7856", "date": "24 Feb 2022", "name": "Y Peng Loh", "role": "Author Response F1000Research Advisory Board Member", "response": "Major comments We agree with the reviewer that generally, studies should be done on 2 cell lines . However since there is a publication already with similar results using the BXPC3 pancreatic cell line , we feel that our findings are supported by another pancreatic cell line already and there was no need to do it on an additional cell line. Minor comments: We have published a paper (ref14) showing that CPE -WT promotes proliferation while the CPE-DN promotes both proliferation and invasion via up-regulation of CXCR2 , a pro-metastatic gene in Panc-1 cells. This was reported in the Discussion section. 2,3. Thanks for these comments. We have provided cleaner Western Blots and have asked F1000research to help exchange the figures in the first version for the new figures. 4. We agree that additional work to investigate the mechanism of how downstream of depleting Cyclin D1 could stop proliferation is a good idea. However, to blot for and identify potential downstream targets of Cyclin D that control proliferation via signaling that regulates cell cycle progression requires extensive work and beyond the scope of this paper. We hope that this reviewer can now approve this paper. Thank you." } ] }, { "id": "92019", "date": "01 Sep 2021", "name": "Lata Singh", "expertise": [ "Reviewer Expertise Oncology", "Pediatrics", "Ocular cancers", "Immunotherapy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled 'Silencing of Carboxypeptidase E expression inhibits proliferation and invasion of Panc-1 pancreatic cancer cells ' is well written and planned study. This is the study how silenced CPE enzyme plays an important role in tumorigenesis of pancreatic cancer.\n\nThere are some minor concerns regarding this manuscript - Please provide the full blot image of western blotting of cyclin D1 and GAPDH.\n\nIs the work 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": "7857", "date": "24 Feb 2022", "name": "Y Peng Loh", "role": "Author Response F1000Research Advisory Board Member", "response": "Response to reviewer 2 Thank you for the comment. We had already provided a full blot image of western blot of cyclin D1 and GAPDH in the Harvard website where the raw data are saved and publicly available. Please click on the link in the paper, or for your convenience here is the link below and you can see the full length gel. https://doi.org/10.7910/DVN/TUFG9M," } ] } ]	1	https://f1000research.com/articles/10-489
https://f1000research.com/articles/11-265/v1	02 Mar 22	{ "type": "Software Tool Article", "title": "METASnake: a Snakemake workflow to facilitate automated processing of metagenomic data through the metaWRAP pipeline", "authors": [ "John Krapohl", "Brett E. Pickett", "John Krapohl" ], "abstract": "Generating high-quality genome assemblies of complex microbial populations from shotgun metagenomics data is often a manually intensive task involving many computational steps. METASnake is a novel tool, implemented in the Snakemake workflow language, to automate multiple metaWRAP modules. Specifically, it wraps the shell scripts provided within the original metaWRAP software, within Snakemake. This approach enables high-throughput simultaneous assembly and analysis of multiple shotgun metagenomic datasets using the robust modular metaWRAP software. We expect this advancement to be of import in institutions where high-performance computing infrastructure is available, especially in the context of big data. This software tool is publicly available at https://github.com/jkrapohl/METASnake.", "keywords": [ "metaWRAP", "shotgun metagenomics", "high-performance computing", "big data", "Snakemake" ], "content": "Introduction\n\nAs sequencing technology has become cheaper and more readily accessible, the need for the increased computational capacity to process these data has become apparent. In particular, high-throughput sequencing has been particularly useful when applied to the field of metagenomics. Substantial effort has been devoted to developing software and computational pipelines, such as MetaWRAP, which cater to this growing area of research. MetaWRAP combines many of the necessary tools to process reads, create bins, and visualize data within a robust modular design (Uritskiy, DiRuggiero, & Taylor, 2018). The primary limitation arising from this design is the inability to automatically scale its usage to massive datasets. Snakemake is a widely-used Python based workflow management system that automates repetitive tasks, allowing software processes to be both scalable and reproducible (Mölder et al., 2021). By integrating the original MetaWRAP processes into Snakemake, the customizable modular nature of MetaWRAP can be preserved even when automatically processing large datasets through a single workflow. Our METASnake software automates the tasks performed within MetaWRAP, allowing for individual modules to be toggled on and off using Snakemake-defined “rules”.\n\n\nMethods\n\nAs this tool makes use of Snakemake, individual steps of the workflow are broken into rules, many of which can be toggled (Mölder et al., 2021). To do this, Snakemake requires a yaml configuration file as an input to determine which steps are specified for the run, as well detailed parameters such as which assembly tool to use. This configuration file also specifies the location of a metadata file, which contains a list of files associated with the run. An example of both the yaml and metadata file are included in the Github repository. METASnake can be submitted for scheduling from the command line by using the submission script also found within the repository. Running the script requires the complete installation of both Snakemake and METAWrap, as well as all of their dependencies. It is recommended that Snakemake and METAWrap be installed using the latest version of Miniconda, following instructions found on their corresponding Github repositories (Mölder et al., 2021; Orjuela, Huang, Hembach, Robinson, & Soneson, 2019).\n\nThe advantage of using METASnake over the base METAWrap program is the ability to appropriately support large-scale job sizes while requiring less user input by automating the entire process. This enables jobs to be better organized and allows improved consistency in outputs. We recommended that this tool be run on cluster computing or within a high-performance infrastructure for larger jobs. In such cases, we have found that a minimum of 100 GB of RAM across 24 CPUs is sufficient, with larger jobs requiring additional resources.\n\nThe rules found within the Snakemake file correspond to different steps within the original METAWrap software. Some initial required steps include read quality control, assembly, binning, and bin refinement and reassembly. However, we have provided toggle settings for memory-heavy modules that generate figures, which are Blobology, Kraken, bin quantification, bin annotation, and bin classification. This design reduces resource usage by only generating the desired figures, and allows the modification of this setting to create these figures at a later time by toggling the module on in the yaml file, deleting any outputs created for that module, and resubmitting the job. Snakemake will skip any rules already completed and only run the required rule(s).\n\nAs stated above, this tool works best with a high-performance computing infrastructure. We anticipate that the most effective use of this tool is applying it to big metagenomics data, such as meta-analyses of existing datasets or datasets with many samples. To assess the capabilities of this tool, the publicly available samples SRR13296364 and SRR13296365, accessed from the NCBI Sequence Read Archive from the study SRP299130, were run together successfully. We then ran a larger set of files from the SRP257563 study through the metaWRAP pipeline using this software. We found that this tool will run as expected as long as sufficient memory is provided and all dependencies are installed correctly. Inputs include sequencing files in fastq format, a metadata file listing all fastq filenames, and a yaml file describing paths for all file sources and destinations. Outputs include high-quality genomic bins, as wells as a number of generated figures such as a blobplot, heatmap, and kronogram.\n\n\nDiscussion\n\nWhile the processing of metagenomics datasets is untractable for most personal computers, researchers with access to high-performance computing infrastructure can take full advantage of this software. The core functions found within the original MetaWRAP include read quality control, assembly, and binning are required for the analysis and cannot be toggled off. These functions include several refinement steps that are unique to MetaWRAP, which allow it to create higher-quality bins than existing stand-alone programs (Uritskiy et al., 2018). Our design enables the user to decide whether computationally-expensive modules, such as Kraken and Blobology, that assist with figure generation can be skipped.\n\nSnakemake automatically generates a directed acyclic graph (DAG) to order tasks, track the progress of each task for each sample, and eliminate duplicate tasks for the same sample (Mölder et al., 2021). This is vital to efficiently processing and analyzing large datasets, as jobs can fail due to insufficient memory or timing out. An advantage of implementing this workflow in Snakemake is the ability to resume the workflow from the same step that was underway if a process fails. The input paths, output paths, and parameters for each job are assigned by the end-user within a configuration file. This file is read by Snakemake to prevent data from being incorrectly assigned or lost and to facilitate reuse and customization of the workflow. We anticipate that this advance in automating metagenomics data processing will facilitate the generation, analysis, and re-analysis of larger datasets in the future.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nSoftware availability\n\nSource code available from: https://github.com/jkrapohl/METASnake.\n\nArchived DOI at time of publication: https://doi.org/10.5281/zenodo.5719960.\n\nLicense: Open.", "appendix": "Acknowledgements\n\nWe would like to thank the Brigham Young University (BYU) Office of Research Computing as well as BYU for providing the facilities for this work.\n\n\nReferences\n\nMölder F, Jablonski KP, Letcher B, et al.: Sustainable data analysis with Snakemake. F1000Res. 2021; 10: 33. PubMed Abstract \| Publisher Full Text\n\nOrjuela S, Huang R, Hembach KM, et al.: ARMOR: An Automated Reproducible MOdular Workflow for Preprocessing and Differential Analysis of RNA-seq Data. G3 (Bethesda). 2019; 9(7): 2089–2096. PubMed Abstract \| Publisher Full Text\n\nUritskiy GV, DiRuggiero J, Taylor J: MetaWRAP—a flexible pipeline for genome-resolved metagenomic data analysis. Microbiome. 2018; 6(1): 158. PubMed Abstract \| Publisher Full Text" }	[ { "id": "126391", "date": "29 Mar 2022", "name": "Tobias Jakobi", "expertise": [ "Reviewer Expertise Bioinformatics", "High Performance Computing", "Computational RNA Biology", "Cardiovascular Disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript submitted by Krapohl et al. outlines a Snakemake-based framework for the metaWRAP software. Given complex workflows like metaWRAP it makes sense to streamline operations by using workflow managers such as Snakemake. The authors provide a good overview of the feature of their software, but the addition of more details in different parts of the manuscript might help readers and potential users to get a better picture.\nThe abstract mentions the metaWRAP software, but does not really introduce what metaWRAP does. Although there is clearly a connections to metagenomics, one sentence to introduce the software would make the link more direct.\n\nThe introduction only focuses on metaWRAP; here, mentioning other, similar tools would make the introduction more inclusive. Why did the authors choose metaWRAP as basis for their software and not another tool?\n\nThe snakemake-defined rules probably do not require quotes.\n\nYaml should be capitalized to YAML and spelled out when it’s first used.\n\nThe authors state that 100GB RAM and 24 CPUs are sufficient for “normal” jobs. Here, it would be very helpful to give readers and potentials user an idea what “normal” and “larger” jobs correspond to, e.g. number of reads, or libraries?\n\nThe authors refer to the suitability of the approach for HPC environment. However, are any interfaces to specific scheduling systems included? The most time consuming step of setting up new HPC pipelines usually is the scheduling setup.\n\nThe authors write that two different datasets completed successfully, but how long did the run take? Of course that’s dependent on the hardware used, but it should be quantifiable.\n\nThere seems is another, unrelated software on GitHub that uses the same name: https://github.com/EthanHolleman/metasnake.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-265
https://f1000research.com/articles/10-421/v1	26 May 21	{ "type": "Research Article", "title": "Concomitant chronic subdural hematomas and arachnoid cysts in young adults", "authors": [ "Huseyin Berk Benek", "Emrah Akcay", "Emrah Akcay" ], "abstract": "Objective: This study aimed to evaluate the correlation between arachnoid cysts and chronic subdural hematomas in young adults. Methods: This retrospective study evaluated ten patients having concomitant chronic subdural hematomas and arachnoid cysts. Patients were evaluated with the data of age and gender, location of hematoma and arachnoid cyst, trauma history, symptoms at admission, maximum hematoma diameter, contiguity between arachnoid cyst and hematoma, and treatment methods. Results: We treated 285 patients who were diagnosed with cSDH between January 2013 and December 2019. 22 patients were under the age of 40 years. Ten of them had both cSDH and arachnoid cysts. The mean age of patients was 24.8±3.9 years. Patients with only chronic subdural hematoma had higher mean age than the patients with arachnoid cyst-related chronic subdural hematoma. In four patients, the onset of chronic subdural hematoma was reported after arachnoid cyst diagnosis. Four of the patients did not have causative trauma history, and two patients suffered minor sports-related traumas. All patients had headache, and only two patients had hemiparesis. The location of arachnoid cysts were in the middle fossa in eight patients. All patients had chronic subdural hematomas on the ipsilateral side of arachnoid cyst. Four patients who had smaller than 10 mm maximal cSDH diameter underwent conservative management. They were followed by serial neuroimaging studies and it was noted that the hematoma disappered and the size of the arachnoid cysts decreased over time without any neurological complication. In six cases, craniotomy was required, and all recovered completely. cSDH did not recur during 5–60 months of follow-up period (median 12 months). Conclusions: It seems that presence of an arachnoid cyst in young adults is a predisposing factor for the formation of chronic subdural hematoma. Coincidentally diagnosed arachnoid cyst patients may be followed up with periodical clinical examinations and neuroimaging studies.", "keywords": [ "Arachnoid cyst", "chronic subdural hematoma", "headache", "middle fossa", "young adult" ], "content": "Abbreviations\n\nAC: arachnoid cyst\n\ncSDH: chronic subdural hematoma\n\nCSF: cerebrospinal fluid\n\nCT: computed tomography\n\nMRI: magnetic resonance imaging\n\n\nIntroduction\n\nArachnoid cysts (ACs) are intracranial extra-parenchymal cavities filled with cerebrospinal fluid (CSF) between the arachnoid and pia mater or within the arachnoid.1 ACs are benign congenital lesions with a prevalence of 0.7% to 1,7% of the population and seen three-times more in males than females.2,3 They are commonly observed in the middle fossa, mainly on the left side.2,4 Most ACs are asymptomatic, but they could become clinically obvious if the cyst grows and causes a cerebral parenchyma mass.1 However, the most common symptom of arachnoid cyst-associated chronic subdural hematoma (cSDH) is headache.3,5 Paresis is usually also seen in cSDH patients; however, headache is more frequent symptom than paresis in young cSDH patients.6 In this study, we present ten cases and aimed to investigate if ACs could predispose to cSDH in young adults. Although there are several previous manuscripts, our study is one of the most comprehensive assessments of the combination of ACs and cSDH.2-5,7-9 Since there is still gap of knowledge about this subject today, we tried to contribute to the body of literature.\n\n\nMethods\n\nThis study retrospectively reviwed the data of the patient files diagnosed with chronic subdural hematoma who were admitted to University of Health Sciences Izmir Bozyaka Education and Research Hospital Department of Neurosurgery between January 2013 and December 2019. Patients having both a chronic subdural hematoma and an arachnoid cysts were determined and included. We also included young adult cSDH patients under the age of 40 years for comparison. Patients under the age of 18 were not included the study. The same neurosurgeons evaluated all of the patients.\n\nThe patient data evaluated were age of patients and gender, location of the hematoma and arachnoid cyst, trauma history, symptoms at admission, cSDH maximum diameter, contiguity between AC and cSDH and treatment methods. Patients aged 18–40 years were accepted as young adults. Magnetic resonance images (MRIs) and computed tomographies (CTs) of patients were assessed. The maximum cSDH diameter measurements were performed on axial MRI slices. In six cases, we performed open cranitomy with the evacuation of the hematoma and arachnoid cyst.\n\nStatistical Package for the Social Science 20.0 (IBM SPSS Statistics, RRID:SCR_019096) was used for analysis of parameters; JASP (RRID:SCR_015823) is an open access alternative which can perform the same function. Univariate analyses were performed using the Mann–Whitney U-test for non-normally distributed scale parameters. Median and ranges were used for description of scale parameters. All analysis were performed at 95% confidence interval and 0.05 significance level.\n\n\nResults\n\nWe treated 285 patients who were diagnosed with cSDHs at the University of Health Sciences Izmir Bozyaka Education and Research Hospital Department of Neurosurgery between January 2013 and December 2019. 22 (7.7%) of the 285 cSDH patients were aged under 40 years, and the other 263 patients (92.3 %) were more than 40 years old. 10 (45.5%) of the 22 young adult patients were diagnosed with both AC and cSDH. The mean age of these ten patients was 24.8 ± 3.9 years and the range was 19–36 years. Eight patients (80%) were male and two patients (20%) were female. Patients with both AC and cSDH had lower mean age than the patients having only a cSDH (p < 0.0001).\n\nThe characteristics of the ten patients with AC and concomitant cSDH were shown in the Table 1. In four patients, the onset of cSDH diagnosed after AC diagnosis (Cases 1,5,6 and 9) (Figures 1,2,3). Four patients did not have causative trauma history (Cases 1,3,5,8), two patients sufferred minor sports-related traumas (Cases 2,10). All patients had headache and only two patients had hemiparesis. AC locations were found in the middle fossa in eight patients (Cases 2,3,4,5,6,7,9,10) and two patients had the convexity of the Sylvian fissure location (Cases 1,8). The middle fossa located ACs were Galassi Tip I in seven patients and Galassi Tip II in one patient.10 All patients had cSDHs on the ipsilateral side of AC. None of the patients had cSDH on the contralateral side of AC. In eight patients, cSDH was close to ACs on neuroimaging studies, and the remaining two patients had a cSDH apart from an AC. Surgical intervention was performed in patients with a larger than 10 mm maximal diameter of cSDH. We evacuated the hematoma and arachnoid cyst following open craniotomy in six cases (Table 1: Cases 1,4,5,6,7,9). We also performed cyst fenestration and tried to remove the cyst membranes as completely as possible. The four patients who had smaller than 10 mm maximal cSDH diameter underwent conservative management (Table 1: Cases 2,3,8,10). They were followed by serial neuroimaging studies and it was noted that the hematoma disappered and the size of AC decreased over time without any neurological complication (Figure 4). All six operated patients recovered completely (Figures 1 and 3). cSDH recurrence was not reported during the period of follow-up of 5-60 months (median 12 months).\n\nArachnoid cyst had diagnosed before the onset of subdural hematoma and there was no history of a trauma. Postoperative axial T1W MRI (E) shows the total removal of both the arachnoid cyst and the subdural hematoma.\n\nAxial T1W MRI(C), T2W MRI (D,E) and sagittal MRI (F) images shows that they are apart from each other. He had conservative treatment with a complete recovery.\n\nAxial T1W MRI (C) shows ipsilateral subdural hematoma (black arrows). Postoperative axial T1W MRI (D,E) demonstrates that the hematoma and the arachnoid cyst was evacuated with a tiny fluid in the subdural place.\n\nAfter two years follow-up, the patient’s AC size decreased as seen on axial T1W MRI (F), and cSDH disappeared thereafter the conservative treatment as seen on axial T1W (G), coronal MRI (H).\n\nTable 2 shows the comparison of the young adult patients who were between 18–40 years old and had chronic subdural hematomas; ten patients having arachnoid cysts and the twelve patients without arachnoid cysts. The gender, frequency of trauma history, frequency of sports-related trauma, headache incidence, paresis incidence and outcomes distribution differences between patient groups were statistically insignificant (p > 0.05).\n\n\nDiscussion\n\nIn this study, we present ten cases and aimed to investigate the correlation between the chronic subdural hematomas and the arachnoid cysts in young adults. We tried to contribute to the body of literature as there is still a lack of information on this subject today. cSDHs are the pathologies that often seen in elderly patients, but uncommonly in young patients.11 When we compared the patients at the same period in our clinic, AC and concomitant cSDH patients had lower age mean than the patients with only cSDH In our study, all ten patients were under age of 36. In our present study, which included 285 cSDH patients, 22 patients (7.7%) were under the age of 40 years. Previously reported rates were 2.4–8.8% and were consistent with the rate of this study.6,8 10 young adult patients under the age of 40 years with chronic subdural hematoma were found to have arachnoid cysts (45%). Approximately half of the 18–40-year-old patients had cSDH with AC. Considering that the prevalence of arachnoid cyst is 0.7% to 1.7% of the population2,3, we can conclude that ACs may cause cSDH in young patients with or without trauma.\n\nBut what is the mechanism of that? Various previous researches have conferred the risk of cSDH related with ACs.3,4,7,8,12,13 The exact mechanism is still unclear. However, theories suggest that 1) veins within the wall could be hurt because of decreased compliance 2) veins without structural support of cyst wall are vulnerable 3) a slit-valve mechanism is formed, lead to increased pressure within the AC and vein rupture.9,12-14 The compliance of the cyst is less than the normal brain. An increase in ICP causes rupture of these bridging veins. AC-related symptoms usually start with the cyst enlargement.9,12 In our study, in four patients without evident trauma, these theories explaining the spontaneous rupture of the AC into subdural space may be valid.\n\nAccording to the ACs and cSDHs locations on neuroimaging studies; three types were defined: 1) a close cSDH to AC, 2) a separate cSDH from an AC on the ipsilateral side, 3) a contralateral side located cSDH to an AC.9,13 Wester et al.3 observed that there are small bridging veins between the dura mater and the outer membrane of the AC. They propounded that the bridging Sylvian veins may cause blood leakage into the subdural place. Rupture of an AC outer wall after head trauma is suggested to cause subdural effusion that could enlarge the cSDH.12,17 Especially in young adults, spontaneous tearing of the AC wall leads to leakage of CSF and blood into the subdural space.4 Page et al.12 declared that ACs are less flexible than the normal without AC brain with reduced intracranial buffering after trauma.3,12 Thus, hematoma could grow up on the ipsilateral hemispheric subdural space other than AC. The subdural hematoma in Cases 3 and 8 can be explained by this mechanism. The association between cSDH and AC could not be insidental. None of our cases had cSDH on the contralateral side of AC. In previous series, almost half of the 18–40-year-old patients with cSDHs had ACs.14,15 Young adults with arachnoid cysts tends to be more susceptible to the development chronic subdural hematomas.\n\nHeadache is one of the most common syptoms in the patients having both cSDH and AC. In our study, headache was observed in all patients and paresis was observed in two patients who were related with an accident. Headache may be due to increased intracranial pressure.18,19 Since the subarachnoid space is smaller in young patients than in the elderly, it is possible that they are more affected by increased intracranial pressure, in cSDH enlargement cases. In our study, sports-related cSDH was found in two of the young patients with ACs. Several case reports of cSDH associated with ACs have been reported after head injury in sports.20-23 Sports is an important factor of cSDHs in young patients.\n\nThe surgical management of a cSDH related with an AC is a debated subject. Open craniotomy including the membranes of the AC and the cSDH removement, drainage of the hematoma using a burr hole, cyst fenestration or cystoperitoneal shunt could be chosen. The most common surgical method is open craniotomy alone.13,15 Although some of the recent studies have argued against burr hole irrigation method, we prefered to remove part of the membranes with open craniotomy and to perform cyst fenestration as a safe procedure. Complex dissection interventions could be necessary in such cases in which a burr hole is insufficient in the management. Initial observation may be considered in patients with a smaller than 10 mm maximal cSDH diameter without symtoms of intracranial hypertension.\n\n\nConclusion\n\nIt seems to be likely that presence of an arachnoid cyst in young adults is a predisposing factor for the formation of a chronic subdural hematoma with or without a head trauma. Headache is one of the most common symptom in the patients with both arachnoid cyst and chronic subdural hematoma. Coincidentally diagnosed arachnoid cyst patients must be followed up with periodical clinical examinations and neuroimaging studies. Young adults with an arachnoid cyst should be informed of the potential risk of developing a chronic subdural hematoma with forced physical exercises or even spontaneously.\n\n\nEthical approval\n\nThis study was approved by the institutional ethics review committee at the University of Helth Sciences Izmir Bozyaka Education and Research Hospital (Date: 13,01,2021, Issue No: 07) in accordance with the World Medical Association Declaration of Helsinki and its most recent amendments. Formal consent was not obligatory for this research.\n\n\nData avaibility\n\nOpen Science Framework: Underlying data for ‘Concomitant chronic subdural hematomas and arachnoid cysts in young adults’. https://doi.org/10.17605/OSF.IO/XM3GW.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAl-Holou WN, Terman S, Kilburg C, et al.: Prevalence and natural history of arachnoid cysts in adults. J Neurosurg. 2013; 118: 222–231. PubMed Abstract \| Publisher Full Text\n\nParsch CS, Krauss J, Hofmann E, et al.: Arachnoid cysts associated with subdural hematomas and hygromas: analysis of 16 cases, long-term follow-up, and review of the literature. Neurosurgery. 1997; 40: 483–490. PubMed Abstract \| Publisher Full Text\n\nWester K, Helland CA: How often do chronic extra-cerebral haematomas occur in patients with intracranial arachnoid cysts? J Neurol Neurosurg Psychiatry. 2008; 79: 72–75. PubMed Abstract \| Publisher Full Text\n\nMori K, Yamamoto T, Horinaka N, et al.: Arachnoid cyst is a risk factor for chronic subdural hematoma in juveniles: twelve cases of chronic subdural hematoma associated with arachnoid cyst. J Neurotrauma. 2002; 19: 1017–1027. PubMed Abstract \| Publisher Full Text\n\nDomenicucci M, Russo N, Giugni E, et al.: Relationship between supratentorial arachnoid cyst and chronic subdural hematoma: neuroradiological evidence and surgical treatment. J Neurosurg. 2009; 110: 1250–1255. PubMed Abstract \| Publisher Full Text\n\nAlbanese A, Tuttolomondo A, Anile C, et al.: Spontaneous chronic subdural hematomas in young adults with a deficiency in coagulation factor XIII. Report of three cases. J Neurosurg. 2005; 102: 1130–1132. PubMed Abstract \| Publisher Full Text\n\nKwak YS, Hwang SK, Park SH, et al.: Chronic subdural hematoma associated with the middle fossa arachnoid cyst: pathogenesis and review of its management. Childs Nerv Syst. 2013; 29: 77–82. PubMed Abstract \| Publisher Full Text\n\nTakizawa K, Sorimachi T, Honda Y, et al.: Chronic Subdural Hematomas Associated with Arachnoid Cysts: Significance in Young Patients with Chronic Subdural Hematomas. Neurol Med Chir (Tokyo). 2015; 55: 727–734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu X, Li G, Zhao J, et al.: Arachnoid Cyst-Associated Chronic Subdural Hematoma: Report of 14 Cases and a Systematic Literature Review. World Neurosurg. 2018; 109: e118–e130. PubMed Abstract \| Publisher Full Text\n\nGalassi E, Tognetti F, Gaist G, et al.: CT scan and metrizamide CT cisternography inarachnoid cysts of the middle cranial fossa: classification and pathophysiological aspects. Surg Neurol. 1982; 17: 363–369. PubMed Abstract \| Publisher Full Text\n\nOu Y, Dong J, Wu L, et al.: The Clinical Characteristics, Treatment, and Outcomes of Chronic Subdural Hematoma in Young Patients. World Neurosurg. 2019; 125: e1241–e1246. PubMed Abstract \| Publisher Full Text\n\nPage A, Paxton RM, Mohan D: A reappraisal of the relationship between arachnoid cysts of the middle fossa and chronic subdural haematoma. J Neurol Neurosurg Psychiatr. 1987; 50: 1001–1007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZuckerman SL, Prather CT, Yengo-Kahn AM, et al.: Sport-related structural brain injury associated with arachnoid cysts: a systematic review and quantitative analysis. Neurosurg Focus. 2016; 40(4): E9. PubMed Abstract \| Publisher Full Text\n\nPark SH, Lee SH, Park J, et al.: Chronic subdural hematoma preceded by traumatic subdural hygroma. J Clin Neurosci. 2008; 15: 868–872. PubMed Abstract \| Publisher Full Text\n\nTakayasu T, Harada K, Nishimura S, et al.: Chronic subdural hematoma associated with arachnoid cyst: two case histories with pathological observations. Neurol Med Chir (Tokyo). 2012; 52: 113–117. PubMed Abstract \| Publisher Full Text\n\nWeber F, Knopf H: Incidental findings in magnetic resonance imaging of the brains of healthy young men. J Neurol. Sci .2006; 240: 81–84. PubMed Abstract \| Publisher Full Text\n\nBilginer B, Onal MB, Oguz KK, et al.: Arachnoid cyst associated with subdural hematoma: report of three cases and review of the literature. Childs Nerv Syst. 2009; 25: 119–124. PubMed Abstract \| Publisher Full Text\n\nSprung C, Armbruster B, Koeppen D, et al.: Arachnoid cysts of the middle cranial fossa accompanied by subdural effusions: experience with 60 consecutive cases. Acta Neurochir (Wien). 2011; 153: 75–84. PubMed Abstract \| Publisher Full Text\n\nPascoe HM, Phal PM, King JA: Progressive posttraumatic tearing of an arachnoid cyst membrane resulting in intracystic and subdural haemorrhage. J Clin Neurosci. 2015; 22: 897–899. PubMed Abstract \| Publisher Full Text\n\nTsuzuki N, Katoh H, Ohtani N: Chronic subdural hematoma complicating arachnoid cyst secondary to soccer-related head injury: case report. Neurosurgery. 2003; 53: 242–243. PubMed Abstract \| Publisher Full Text\n\nDemetriades AK, McEvoy AW, Kitchen ND: Subdural haematoma associated with an arachnoid cyst after repetitive minor heading injury in ball games. Br J Sports Med. 2004; 38: E8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKertmen H, Gürer B, Yilmaz ER, et al.: Chronic subdural hematoma associated with an arachnoid cyst in a juvenile taekwondo athlete: a case report and review of the literature. Pediatr Neurosurg. 2012; 48: 55–58. PubMed Abstract \| Publisher Full Text\n\nPrabhu VC, Bailes JE: Chronic subdural hematoma complicating arachnoid cyst secondary to soccer-related head injury: case report. Neurosurgery. 2002; 50: 195–197. PubMed Abstract \| Publisher Full Text" }	[ { "id": "99264", "date": "01 Dec 2021", "name": "Roberto Colasanti", "expertise": [ "Reviewer Expertise Neurosurgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors report a series of 10 patients with concomitant chronic subdural hematomas and arachnoid cysts.\nThe authors could briefly summarize and underline the novelty of their findings.\n\nAs the authors stated, various treatment modalities have been proposed for the management of patients with CSDH and arachnoid cysts (open craniotomy, burr hole and drainage of the hematoma, cyst fenestration or cystoperitoneal shunt). They could briefly report about the pros and cons of the different treatments as well as of the associated recurrence rates.\n\nCould embolization be helpful to increase the complete cure rate and avoid recurrences? What about medical therapy, e.g. corticosteroids?\n\nIn the introduction section, the topic of the manuscript (concomitant chronic subdural hematomas and arachnoid cysts) could be better introduced. What about the incidence of CSDH in patients with arachnoid cysts?\n\nA clinical deterioration due to hemorrhage into the cyst or subdural compartment represents a typical presentation of an arachnoid cyst. I believe it is quite obvious that “presence of an arachnoid cyst in young adults is a predisposing factor for the formation of a chronic subdural hematoma with or without a head trauma”.\n\nEnglish needs to be revised.\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": "7533", "date": "07 Feb 2022", "name": "Huseyin Berk Benek", "role": "Author Response", "response": "The authors could briefly summarize and underline the novelty of their findings. Response: We accepted the suggestion and summarized it. Although there are several previous manuscripts, our study is one of the most comprehensive assessments of the combination of ACs and cSDH. Since there is still a gap of knowledge about this subject today, we tried to contribute to the body of literature. As the authors stated, various treatment modalities have been proposed for the management of patients with CSDH and arachnoid cysts (open craniotomy, burr hole and drainage of the hematoma, cyst fenestration or cystoperitoneal shunt). They could briefly report about the pros and the cons of the different treatments as well as the associated recurrence rates. Response: Complex dissection interventions could be necessary in such cases in which a burr hole is insufficient in the management. A single burr-hole-mini craniotomy and hematoma evacuation followed by endoscopic inspection of the surgical cavity could be a preferred choice. Initial observation may be considered in patients with a thickness of 10 mm or less cSDH diameter without symptoms of intracranial hypertension. Recurrence can occur in 10%-20% of patients and is associated with several clinical and radiographic predictors. (Feghali J, Yang W, Huang J. Updates in Chronic Subdural Hematoma: Epidemiology, Etiology, Pathogenesis, Treatment, and Outcome. World Neurosurg. 2020 Sep;141:339-345). Postoperative CSDH volume and the Nakaguchi classification subtypes proved the most powerful predictors of recurrence and cure. (separated or trabecular subtypes and postoperative CSDH volume ≥35.0 mL) Could embolization be helpful to increase the complete cure rate and avoid recurrences? What about medical therapy, e.g. corticosteroids? Response: We mentioned about middle meningeal artery embolization and medical therapy, e.g. corticosteroids as recommended. Middle meningeal artery embolization may be represented as a minimally-invazive alternative to surgery for new or recurrent chronic subdural hematomas, or as prophylaxis to reduce the risk of recurrence after surgery. [24] Link TW, Boddu S, Paine SM, Kamel H, Knopman J. Middle Meningeal Artery Embolization for Chronic Subdural Hematoma: A Series of 60 Cases. Neurosurgery. 85(6) (2019), 801-807 [25] Ban SP, Hwang G, Byoun HS, Kim T, Lee SU, Bang JS, Han JH, Kim CY, Kwon OK, Oh CW. Middle Meningeal Artery Embolization for Chronic Subdural Hematoma. Radiology. 286(3) (2018), 992-999 Some studies suggest that corticosteroids might be beneficial in the treatment of CSDH; however, there is a lack of well-designed trials that support or refute the use of corticosteroids in CSDH. [26] Berghauser Pont LM, Dirven CM, Dippel DW, Verweij BH, Dammers R. The role of corticosteroids in the management of chronic subdural hematoma: a systematic review. Eur J Neurol. 19(11) (2012), 1397-403 In the introduction section, the topic of the manuscript (concomitant chronic subdural hematomas and arachnoid cysts) could be better introduced. What about the incidence of CSDH in patients with arachnoid cysts? Response: The introduction section has been revised as suggested. Chronic subdural hematoma (CSDH) is a complex disease with an overall incidence of 1.7-20.6 per 100,000 persons per year and is more commonly encountered in the elderly population. ACs are benign congenital lesions with a prevalence of 0.7% to 1,7% of the population. Since CSDH in arachnoid cysts patients is a rare phenomenon, several papers have been reported in the literature. A clinical deterioration due to hemorrhage into the cyst or subdural compartment represents a typical presentation of an arachnoid cyst. I believe it is quite obvious that “ presence of an arachnoid cyst in young adults is a predisposing factor in the formation of a chronic subdural hematoma with or without a head trauma”. Response: Previously reported rates of cSDH in young adults were 2.4–8.8% of all cSDH. Young adults with arachnoid cysts tend to be more susceptible to the development of chronic subdural hematomas. English needs to be revised. Response: As you suggested, we have revised our English draft once again. Also, our paper has been edited and certificated for English language and spelling by Enago (www.enago.com), an editing brand of Crimson Interactive Inc." }, { "c_id": "7547", "date": "07 Feb 2022", "name": "Huseyin Berk Benek", "role": "Author Response", "response": "Additional response to the questions of reviewer 1: What about the incidence of CSDH in patients with arachnoid cysts? A clinical deterioration due to hemorrhage into the cyst or subdural compartment represents a typical presentation of an arachnoid cyst. I believe it is quite obvious that “ presence of an arachnoid cyst in young adults is a predisposing factor the formation of a chronic subdural hematoma with or without a head trauma”. Response: cSDH is a rare complication in patients with intracranial AC. In the case series of Wu et al., the incidence of AC-associated cSDH was 1.9% (5 of 266). [Wu X, Li G, Zhao J, Zhu X, Zhang Y, Hou K. Arachnoid Cyst-Associated Chronic Subdural Hematoma: Report of 14 Cases and a Systematic Literature Review. World Neurosurg. 109 (2018), e118-e130] Wester and Helland reported that 4.6% (11 of 241) of their patients admitted for AC were identified with cSDH. [Wester K, Helland CA.How often do chronic extra-cerebral haematomas occur in patients with intracranial arachnoid cysts? J Neurol Neurosurg Psychiatry. 79 (2008), 72–75] Parsch et al. identified AC in 16 of 658 patients (2.4%) with cSDH. They stated that subdural hematomas are infrequently encountered complications of arachnoid cysts. [Parsch CS, Krauss J, Hofmann E, Meixensberger J, Roosen K.Arachnoid cysts associated with subdural hematomas and hygromas: analysis of 16 cases, long-term follow-up, and review of the literature. Neurosurgery. 40 (1997), 483–490]" } ] }, { "id": "98792", "date": "13 Dec 2021", "name": "Jehuda Soleman", "expertise": [ "Reviewer Expertise Neurosurgery", "Pediatric Neurosurgery", "Endoscopy", "Vaskular Neurosurgery" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for allowing me to review this manuscript which I read with interest.\nAlthough the authors claim they add to the literature with their 10 cases and that information on the topic is lacking. In my opinion it would make more sense to analyze the rate of bleeding in young adults with known AC and maybe analyze which patients are at risk for a bleeding. The comparison to cSDH patients does not add much information.\n\nThe authors treat these patients by craniotomy, which is a rather aggressive approach. There is no literature showing that craniotomy, hematoma evacuation, and cyst resection/fenestration is superior to burr hole and treatment of the hematoma alone. An additional option would be burr hole hematoma fenestration and endoscopic cyst fenestration (which is nowadays the more minimal invasive approach to AC leading to good results as well).\n\nThe conclusion that AC bleeding may be caused by minor trauma or sport activities is not supported by the literature, and also not something that can be concluded for the authors data. There are studies showing that sport is not contraindicated in these patients since the risk for bleeding is similar for patient with AC whether they do sport or not (even contact sport). The conclusion that these patients “must” be followed is not supported by the data presented, it is merely a subjective impression of the authors. If the authors had analyzed how many patients with AC have bled they could recommend whether follow up is indicated in these patients. Most of these patients do not bleed and therefore follow up is not a “must”. In addition, as described by the authors most patients show (mild) symptoms, meaning they should be informed that if symptoms arise they should seek medical attention, but not necessarily need to be followed routinely.\n\nThe conclusions drawn by the authors are somewhat flawed. The methodology comparing CSDH with AC to other CSDH patients is not ideal as mentioned above. For these reasons I unfortunately do not recommend the indexing of this work.\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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "7717", "date": "17 Jan 2022", "name": "Huseyin Berk Benek", "role": "Author Response", "response": "Although the authors claim they add to the literature with their 10 cases and that information on the topic is lacking. In my opinion it would make more sense to analyze the rate of bleeding in young adults with known AC and maybe analyze which patients are at risk for a bleeding. The comparison to cSDH patients does not add much information. Response: Most of the studies about this subject analyzed the complicated ACs, from a total of patients with cSDH. The references of our study indicate all the studies and reviews. Parsch et al. identified AC in 16 of 658 patients (2.4%) with cSDH. They stated that subdural hematomas are infrequently encountered complications of arachnoid cysts. [2] Parsch CS, Krauss J, Hofmann E, Meixensberger J, Roosen K.Arachnoid cysts associated with subdural hematomas and hygromas: analysis of 16 cases, long-term follow-up, and review of the literature. Neurosurgery. 40 (1997), 483–490 [8] Takizawa K, Sorimachi T, Honda Y, Ishizaka H, Baba T, Osada T, et al. Chronic Subdural Hematomas Associated with Arachnoid Cysts: Significance in Young Patients with Chronic Subdural Hematomas. Neurol Med Chir (Tokyo). 55 (2015), 727-34. Analyzing the rate of bleeding in young adults with known AC is really difficult, as most ACs do not have symptoms and do not undergo surgery. And it’s hard to find the proportion of ACs of the population and the AC patients coming to outpatient clinics. The authors treat these patients by craniotomy, which is a rather aggressive approach. There is no literature showing that craniotomy, hematoma evacuation, and cyst resection/fenestration is superior to burr hole and treatment of the hematoma alone. An additional option would be burr hole hematoma fenestration and endoscopic cyst fenestration (which is nowadays the more minimal invasive approach to AC leading to good results as well). Response: Open craniotomy including the membranes of the AC and the cSDH removement, drainage of the hematoma using a burr hole, cyst fenestration or cystoperitoneal shunt could be chosen. The most common surgical method is open craniotomy alone.13,15 Zuckermann et al stated that open craniotomy was the most common form of surgical treatment (%49). In their systematic review in Neurosurgical Focus , they analysed 65 cases in Table 4, and nearly half of all cases mentioned cyst fenestration after open craniotomy. [13] Zuckerman SL, Prather CT, Yengo-Kahn AM, Solomon GS, Sills AK, Bonfield CM. Sport-related structural brain injury associated with arachnoid cysts: a systematic review and quantitative analysis. Neurosurg Focus. 40(4) (2016), E9 [15] Takayasu T, Harada K, Nishimura S, Onda J, Nishi T, Takagaki H. Chronic subdural hematoma associated with arachnoid cyst: two case histories with pathological observations. Neurol Med Chir (Tokyo). 52 (2012), 113-117 The conclusion that AC bleeding may be caused by minor trauma or sport activities is not supported by the literature, and also not something that can be concluded for the authors data. There are studies showing that sport is not contraindicated in these patients since the risk for bleeding is similar for patient with AC whether they do sport or not (even contact sport). The conclusion that these patients “must” be followed is not supported by the data presented, it is merely a subjective impression of the authors. If the authors had analyzed how many patients with AC have bled they could recommend whether follow up is indicated in these patients. Most of these patients do not bleed and therefore follow up is not a “must”. In addition, as described by the authors most patients show (mild) symptoms, meaning they should be informed that if symptoms arise they should seek medical attention, but not necessarily need to be followed routinely. Response: Several case reports of cSDH associated with ACs have been reported in the literature after head injury in sports.20,21,22,23 Sport is an important factor of cSDHs in young patients. [20] Tsuzuki N, Katoh H, Ohtani N. Chronic subdural hematoma complicating arachnoid cyst secondary to soccer-related head injury: case report. Neurosurgery. 53 (2003), 242-243 [21] Demetriades AK, McEvoy AW, Kitchen ND. Subdural haematoma associated with an arachnoid cyst after repetitive minor heading injury in ball games. Br J Sports Med. 38 (2004), E8 [22] Kertmen H, Gürer B, Yilmaz ER, Sekerci Z. Chronic subdural hematoma associated with an arachnoid cyst in a juvenile taekwondo athlete: a case report and review of the literature. Pediatr Neurosurg. 48 (2012), 55-58 [23] Prabhu VC, Bailes JE. Chronic subdural hematoma complicating arachnoid cyst secondary to soccer-related head injury: case report. Neurosurgery. 50 (2002), 195–197 Also Mori et al stated in a study in Journal of Neurotrauma that AC is a risk factor for chronic subdural hematoma in juveniles. We changed “must” to “should” as recommended. [4] Mori K, Yamamoto T, Horinaka N, Maeda M. Arachnoid cyst is a risk factor for chronic subdural hematoma in juveniles: twelve cases of chronic subdural hematoma associated with arachnoid cyst. J Neurotrauma. 19 (2002), 1017–1027 The conclusions drawn by the authors are somewhat flawed. The methodology comparing CSDH with AC to other CSDH patients is not ideal as mentioned above. For these reasons I unfortunately do not recommend the indexing of this work. Response: cSDH is a rare complication in patients with intracranial AC. In the case series of Wu et al., the incidence of AC-associated cSDH was 1.9% (5 of 266). Wu X, Li G, Zhao J, Zhu X, Zhang Y, Hou K. Arachnoid Cyst-Associated Chronic Subdural Hematoma: Report of 14 Cases and a Systematic Literature Review. World Neurosurg. 109 (2018), e118-e130. It would be better to do a search in literature of our references." } ] } ]	1	https://f1000research.com/articles/10-421
https://f1000research.com/articles/11-469/v1	28 Apr 22	{ "type": "Research Article", "title": "Preparedness regarding first response to emergencies in the community among graduate students: A cross-sectional study in India", "authors": [ "Prithvishree Ravindra", "Rachana Bhat", "William Wilson", "Jayaraj Mymbilly Balakrishnan", "Jeffrey Pradeep Raj", "Avhishek Bhattacharya", "Prithvishree Ravindra", "Rachana Bhat", "Jayaraj Mymbilly Balakrishnan", "Jeffrey Pradeep Raj", "Avhishek Bhattacharya" ], "abstract": "Background: The key to successful outcomes in time-sensitive emergencies involves a pivotal role played by bystanders in identifying and initiating first response. This in turn depends on their awareness and perspective regarding the same. Objective: This study aimed to assess the existing awareness and attitude regarding first response to emergencies in students pursuing graduation, in an Indian University. Methods: The students were administered a pilot-tested questionnaire regarding first response, cardiopulmonary resuscitation (CPR) and automated external defibrillators (AEDs). A total of 1851 students were included in the study from various disciplines- both health care professional training (HCPT) students and non-HCPT students. Results: Poor knowledge was noticed across several themes including awareness of emergency helpline number (61.48%), recognising cardiac arrest (33.98%), awareness about hands-only CPR (12.26%), knowledge about rate of chest compressions in adults (25.01%) and infants (19.67%), and steps of using an AED (17.45%). Only 29.17% of the participants had undergone previous training in CPR. Only 57% of the participants were willing to initiate CPR in cardiac arrest victims. Some of the major knowledge barriers identified included no prior training (62.4%) and perceived harm to victims (26.9%). The mean knowledge score was low and 69% of the participants scored less than 50%. The significant predictors of low knowledge score were lower age, male gender, non-HCPT students, no previous training in CPR and having not witnessed a cardiac arrest before. Conclusions: There is low preparedness regarding the first response and low awareness regarding crucial links in the chain of survival of cardiac arrest. However, willingness among the students to learn CPR was an encouraging finding.", "keywords": [ "Automated External Defibrillator (AED)", "Bystander CPR", "Cardiac arrest", "Cardiopulmonary resuscitation (CPR)", "First-aid" ], "content": "Introduction\n\nThe care for the acutely ill has evolved over the last few decades.1 Advances in emergency medicine, prehospital care systems and evidence-based medicine culminating into standard protocols for care has led to an improvement in outcomes.1 Despite all advancements, the key step for optimal outcome of the patient in time-sensitive emergencies remains early identification of the illness, immediate activation of the emergency response system and often the initial care delivered by the bystander till further help arrives.2 This is reflected in the American Heart Association's chain of survival for out of hospital cardiac arrest (OHCA), where the onus of the first three links in the chain are dependent on the community.2 Hence, preparedness among the community is essential- more so in scenarios that require timely interventions by bystanders, such as OHCA, choking, stroke and myocardial infarction.\n\nThe global incidence of out of hospital cardiac arrest (OHCA) is estimated to be 55 per 100,000 person-years,3 making it an important public health challenge. The outcome of these patients depends on early recognition of cardiac arrest, immediate bystander cardiopulmonary resuscitation (CPR) and early defibrillation.4,5 Bystander CPR initiated in witnessed ventricular fibrillation arrests has shown to improve survival from 42 to 65%.5 There is a wide global variation in the rate of bystander CPR. Bystander CPR rate in countries like the USA (45.7% in adults and 61.4% in children)6 and Sweden (68.2%)7 are much higher than in India (9.8%)8 The International Liaison Committee on Resuscitation Advisory has a hypothetical formula called the “Utstein formula for survival from cardiac arrest”, which suggests, survival = guideline quality × education efficiency × local implementation.9 The studies done in India are mainly focused on medical students, and they show inadequate knowledge regarding CPR.10 Studies regarding knowledge of automated external defibrillator (AED) in India are lacking as well.\n\nThus, this study aimed to assess the preparedness regarding the first response to emergencies among the graduate students at a university in India across various disciplines and explore their perspective on CPR and AED. This study will provide us with crucial information that will enable us to appeal for and plan nationwide programs for emergency first response training in India.\n\n\nMethods\n\nThis was an online questionnaire-based study using Google forms (Publisher: Google LLC, California, USA, 2018) involving undergraduate students at a University in South India, that offers both medical and non-medical courses. The University has five colleges under health sciences and six colleges under other streams. This university was selected as it has multidisciplinary teaching institutes in the campus and students from various parts of country, giving pan-national representation. It is also in the vicinity of the study authors, making it accessible for them to conduct the large-scale study. All consenting students pursuing graduation were included in the study.\n\nThe questionnaire was first constructed in the online form portal and was pilot tested. Pilot testing was done by administering the questionnaire to a small group of undergraduate students (twenty-five) and taking their feedback (Beta testing). Feedback showed that questions were easy to understand and found relevant and no changes were necessary. This data from pilot testing was excluded from final analysis. Using Delphi method, nine experts from the fields of Emergency Medicine, Anaesthesiology, Critical Care and Internal Medicine were approached to give score to the knowledge questions according to their importance. The 30 questions pertaining to knowledge were sent to the experts, and they were either asked to give zero/one/two scores as per the importance they attach to the question. Then we looked at the scores allotted, and the score which was the mode (most experts suggested) was selected. These scores were allotted to questions pertaining to knowledge, which added to total score of 50. After allotting, the experts were again sent the final knowledge score sheet, all of them agreed that the scoring was appropriate. The questions with scores are available online.11 There was consensus that <50% would be considered as poor knowledge score. After obtaining permission from the relevant authorities (KMC and KH Institutional Ethics Committee IEC no. 29/2018 and heads of all the participating institutes), students were approached as batches and the study was explained to them in detail. The sampling technique used was cluster random sampling. The list of various class batches from the institutes were noted and allotted numbers. Then using online available randomisation software,12 class batches were randomly selected. The selected batches were approached to participate in study. Among the batch, students consenting to participate in the study were enrolled after informed consent. By using cluster random sampling, we reduced selection bias. In order to estimate the level of knowledge regarding cardiopulmonary resuscitation among undergraduate students at 10%, with a relative precision of 20% at 95% confidence level, and a design effect of 2, minimum of 1730 students needed to be recruited for our study. We approached 1930 students. Among them, 1851 students were willing to take part in the study and answered the questionnaire. Using such a large sample size also helped to reduce any unknown bias.\n\nData from the online forms were exported into Microsoft Excel (Publisher: Microsoft Corporation, Redmond, Washington, USA, 2016) (RRID: SCR_016137) for consolidation [28]. Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) Statistics for Windows, Version 20.0 (Publisher: IBM Corp., USA, 2011) (RRID: SCR_002865).\n\nThe minimum number of participants needed to estimate the level of knowledge regarding first response and CPR was decided by assuming a good knowledge level among 10% students, with a relative precision of 20% at 95% confidence level, and a design effect of two. These parameters mean that 1730 students needed to be recruited for our study. The demographic characteristics and responses in the questionnaire were summarized using descriptive characteristics. The proportion of participants giving the correct responses were compared between health care professional training (HCPT) students and non-HPCT students using the Chi-square test, Fisher's exact test and odds ratio (OR). The hypothesized predictors of knowledge score were subjected to univariate analysis using simple linear regression. Those whose p < 0.2 in the univariate analysis were subjected to multivariate analysis using the linear regression model. The statistical significance was set at p < 0.05.\n\n\nResults\n\nUsing cluster random sampling, classroom batches were randomly selected. A total of 1930 students were approached. Among them 1851 students agreed to participate in study. All students who agreed to participate, completed the questionnaire with no missing data. The total number of responses received were N = 1851. The Cronbach’s alpha of the knowledge assessment questionnaire was 0.880 suggesting good internal consistency. The median (interquartile range) age of the study participants was 20 (19-21) years. The study included both HCPT students and non-HCPT students. Non-HCPT students formed the vast majority (n = 1187/1851; 64.13%). The demographic characteristics of the study participants are tabulated in Table 1.\n\n* Health care professional training (HCPT) students were n = 411/664 (61.89%) whereas non-HCPT students were n = 129/1187 (10.87%); p < 0.001.\n\n# HCPT students were n=166/664 (25%) and non-HCPT students were n = 221/1187 (18.62%); p = 0.001.\n\n^ As per the six administrative zones of India recognized under Part-III of the States Reorganisation Act, 1956.\n\nThe proportion of participants who gave the right responses to various knowledge assessment questions and a comparison between non-HCPT and HCPT students are summarized in Table 2. Some of the significant observations we found were that only 61.48% (n = 1138) knew the emergency helpline number. Some themes were associated with poor knowledge, even among the HCPT students, including recognising cardiac arrest and correct initial response (33.98%), awareness about hands-only CPR (12.26%), rate of chest compressions in adults (25.01%) and infants (19.67%), steps while using an AED (17.45%), and resuscitation of an unresponsive choking infant (15.94%). Very few participants were aware that AEDs are available at airports (n = 750/1851; 40.52%) and railway stations (n = 377/1851; 20.37%).\n\nIn terms of CPR, 29.17% (n = 540/1851) had undergone previous training in CPR and 20.91% (n = 387/1851) had previously witnessed a cardiac arrest. When data from HCPT students was compared with non-HCPT students, the proportion of HCPT students who either underwent CPR training (63.82% versus 10.87% respectively; p < 0.001) or witnessed a cardiac arrest (25.78% versus 18.62% respectively; p = 0.001) was significantly higher. Among the students who had witnessed a cardiac arrest (n = 387) 28.68%, with a close to equal proportion in both non-HCPT (n = 62/221, 28.05%) and HCPT (n = 49/166; n = 29.52%) groups, felt that the situation could have been handled better. Also, 85.27% (n = 330/387) felt that they could have helped if they were previously trained.\n\nTable 3 summarizes the participants’ responses on their perspective towards providing CPR and the use of AED. Only 57% (n = 1055) of participants were willing to initiate CPR in cardiac arrest victims. Some of the barriers identified included no prior training (62.4%), perceived harm to the victim (26.9%), belief that CPR may lead to contraction of disease (17.88%), harm to the provider due to AED use (17.94%), and religious and cultural barriers (12.91%). Further, non-HCPT students believed that they lack the ability to use an AED (OR = 11.803, p < 0.001) and were unwilling to use an AED even after training (OR = 2.466, p < 0.001). On average, non-HCPT students had increased odds (approximately two times) for unfavourable attitudes towards CPR when compared to HCPT students.\n\nThe mean knowledge score was 20.12 (11.26) out of 50 [15.66 (7.74) in non-HCPT students versus 28.10 (12.13) in HCPT students; p < 0.001]. The proportion of participants who scored less than 50% was 1278 (69.04%) of which n = 1026/1278 (80.28%) were non-HCPT students (OR = 10.419; p < 0.001). The details of the univariate and multivariate analysis evaluating predictors for the knowledge score are summarized in Table 4. The significant predictors (ß coefficient of multivariate analysis; 95% CI; p value) of knowledge score were lower age (0.374; 0.140, 0.608; 0.002), male gender (1.561; 0.699, 2.332; <0.001), non-HCPT students (6.159; 5.162, 7.155; <0.001), No previous training in CPR (10.594; 9.560, 11.628; <0.001) and not witnessed a cardiac arrest before (2.588; 1.619, 3.556; <0.001)\n\n* Adjusted R square = 0.444.\n\n\nDiscussion\n\nSuccessful outcomes in time-sensitive emergencies involve a pivotal role played by people in the community in identifying and initiating emergency response.2 Our study was done among students pursuing graduation in a university and had a pan-India representation of participants, this formed a window for us to understand the community awareness and perspective regarding first response.\n\nActivation of the emergency response system is a crucial step in initiating emergency care. In our study, we noted that only 33.9% of participants knew how to recognise cardiac arrest and only 61.48% knew the helpline number for activation of the emergency response system. Aroor et al and Modi et al also found low awareness (56.3% and 76.2% respectively) of the emergency helpline number in India.10,13 A study done by Schuffelen et al in the Netherlands, among the school students, found 81.3% were aware of the emergency helpline number.14 This forms the first link in the chain of survival and hence is essential. This dismal number of educated graduates who know this number in India, highlights the importance to scale up efforts towards creating awareness in the community.\n\nIn our study, we noted that only 29.1% of the students pursuing graduation had previous training in CPR, For those outside HCPT a mere 10.87% had previous training. Qara et al from Saudi Arabia had reported that only 28.7% of the 600 non-medical professionals interviewed had prior training in CPR.15 In contrast, the training rates are higher in countries such as the USA, Canada, and South Korea.16–18\n\nOne of the potential solutions to improve community training rates is school CPR training programs. Unlike in some countries, India does not have a robust school CPR training program. The World Health Organisation recommends CPR training in secondary schools and to include it as part of national legislation.19 This will ensure a large population is trained and has been listed as a key component to raise bystander CPR rates.20,21\n\nThe knowledge score was considered poor (<50%) in 69% of the participants. A study from Karnataka state, India conducted among school teachers revealed that 87% (n = 127/146) had moderate knowledge but none were in the good knowledge category.22 A similar study conducted among HCPT students in South India found that the mean score was approximately 42% (poor knowledge).10 However, all these studies have used different tools to assess knowledge so these numbers may not be directly comparable. The awareness and knowledge has been found to be low in other developing countries as well.23–25\n\nWe noted that the participants in our study lacked knowledge regarding aspects of CPR such as hand placement during CPR (47.43%), rate and depth of compression (25.01% and 43.81% respectively) and hands-only CPR. In India, another study found that only 5.47% (n = 8/146) knew the correct procedure of CPR.22 Similarly, a study by Urban et al had found that only 23.3% had knowledge regarding hands-only CPR.26 In contrast, a nationwide survey in Taiwan found that 57.5% of participants knew how to perform CPR.27\n\nWe found 51% of our participants knew the function of an AED and 17.45% of respondents knew the steps of using an AED. However, participants had low awareness of the availability of AED at railway stations and airports. Another Indian study among HCPT students found that only 11.3% (n = 59/520) knew what an AED stands for10 Similarly, a study in Hong Kong also revealed that 77.6% did not know the location of AEDs in their vicinity/workplace.28\n\nIn our study, although only 57% were willing to start CPR, 80.33% of respondents believed CPR could save lives and 83.3% were willing to undergo CPR training. The most common barrier for the willingness to performing CPR was the lack of prior training, followed by a belief that CPR may harm the victim. A Chinese study revealed 76.3% were willing to perform CPR on strangers and 53.2% were worried about legal issues.29 A survey among adults in the UK reported that people who received prior CPR training were 3.4 times more likely to be willing to perform CPR compared to people with no training. Similarly, people trained in AED use were 2.62 times more likely to go, get or use an AED.30 Hence, training the participants in CPR and dispelling the myths regarding CPR is essential to increase the bystander CPR rates in the community. The positive attitude of the students towards the usefulness of CPR and readiness to undergo training is encouraging and was the silver lining of our study.\n\nAnalysis for predictors of knowledge score revealed that with every year of increased age, the average score is likely to increase by 0.374 units. HCPT students are most likely to have their mean knowledge scores higher by 10% compared to their non-HCPT peers. On a similar note, those who underwent prior training or who have witnessed a cardiac arrest before are likely to score better by an average of 9.5 and 1.5 units respectively. These findings once again stress the importance of formal training and hands-on experience as the sole driver of better awareness and knowledge.\n\nOur study was a questionnaire-based survey, so there is a possibility of self-selection bias where individuals with poor knowledge or attitude did not consent to participate in the study. By taking a huge sample size, and considering that out of approached 1930 students, 1851 consented to participate, this bias may not have strongly affected our results. Secondly, since it was done on students pursuing graduation, it will mainly reflect awareness and attitude among the educated population, and not represent the population as a whole.\n\n\nConclusions\n\nThere is a lack of preparedness regarding the first response to emergencies. The knowledge regarding the first three links of the chain of survival- activation of emergency response, high quality CPR and defibrillation in cardiac arrest was lacking among our participants. Only 61.48% knew emergency response helpline number, 33.98% knew to recognise cardiac arrest and initiate response, 25.01% knew rate of compressions and 43.81% knew depth of compressions which form components of high quality CPR, 17.45% knew steps of using an automated external defibrillator. Each link is integral to improve the survival of patients with OHCA. These alarming statistics warrant immediate acceleration of CPR training programs among the community, as well as have training and refresher programs among the HCPT students and graduates. We recommend that methods such as inclusion of school CPR training programs in curriculum, use of social media to disseminate information, innovative contests to attract community participation,31 CPR training for receptive population such as new parents and relatives of cardiac patients prior to discharge,32 in addition to the existing community CPR training programs could be used to increase the awareness about CPR and AED.\n\nWillingness to learn CPR among the participants was the silver lining in the study.\n\n\nData availability\n\nOSF: Preparedness regarding first response to emergencies in the community among graduate students: A cross-sectional study in India. https://doi.org/10.17605/OSF.IO/S8NGV11\n\nThis project contains the following underlying data:\n\n- Master chart.xlsx (raw data from questionnaires)\n\nOSF: Preparedness regarding first response to emergencies in the community among graduate students: A cross-sectional study in India. https://doi.org/10.17605/OSF.IO/S8NGV11\n\nThis project contains the following extended data:\n\n- Code sheet.docx (key for understanding raw data)\n\n- Data summary.docx\n\n- Knowledge score.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to acknowledge the support received from the Department of Emergency Medicine and the University for the study.\n\n\nReferences\n\nPek JH, Lim SH, Ho HF, et al.: Emergency medicine as a specialty in Asia. Acute Med Surg. 2015 Aug 27; 3(2): 65–73. PubMed Abstract \| Publisher Full Text\n\nKleinman ME, Brennan EE, Goldberger ZD, et al.: Part 5: Adult Basic Life Support and Cardiopulmonary Resuscitation Quality. Circulation. 2015 Nov 3; 132(18_suppl_2): S414–S435. PubMed Abstract \| Publisher Full Text\n\nBerdowski J, Berg RA, Tijssen JGP, et al.: Global incidences of out-of-hospital cardiac arrest and survival rates: Systematic review of 67 prospective studies. Resuscitation. 2010 Nov 1 [cited 2020 May 16]; 81(11): 1479–1487. PubMed Abstract \| Publisher Full Text Reference Source\n\nEisenberg MS, Bergner L, Hallstrom A: Cardiac resuscitation in the community. Importance of rapid provision and implications for program planning. JAMA. 1979 May 4; 241(18): 1905–1907. Publisher Full Text\n\nEisenberg MS, Cummins RO, Larsen MP: Numerators, denominators, and survival rates: reporting survival from out-of-hospital cardiac arrest. Am. J. Emerg. Med. 1991 Nov; 9(6): 544–546. 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Health Care. 2015 Dec 1; 7(4): 274–281. PubMed Abstract \| Publisher Full Text\n\nMajid A, Jamali M, Ashrafi MM, et al.: Knowledge and Attitude Towards Cardiopulmonary Resuscitation Among Doctors of a Tertiary Care Hospital in Karachi. Cureus. [cited 2020 May 25]; 11(3). Reference Source\n\nOlajumoke T, Afolayan J, Raji S, et al.: Cardiopulmonary Resuscitation - Knowledge, Attitude & Practices in Osun State, Nigeria. J. West Afr. Coll. Surg. 2012 [cited 2020 May 25]; 2(2): 23–32. PubMed Abstract Reference Source\n\nOteir AO, Almhdawi KA, Kanaan SF, et al.: Cardiopulmonary resuscitation level of knowledge among allied health university students in Jordan: a cross-sectional study. BMJ Open. 2019 Nov 19 [cited 2020 May 25]; 9(11). e031725. PubMed Abstract \| Publisher Full Text Reference Source\n\nUrban J, Thode H, Stapleton E, et al.: Current knowledge of and willingness to perform Hands-Only CPR in laypersons. Resuscitation. 2013 Nov; 84(11): 1574–1578. PubMed Abstract \| Publisher Full Text\n\nPei-Chuan Huang E, Chiang W-C, Hsieh M-J, et al.: Public knowledge, attitudes and willingness regarding bystander cardiopulmonary resuscitation: A nationwide survey in Taiwan. J. Formos. Med. Assoc. 2019 Feb 1 [cited 2020 May 25]; 118(2): 572–581. PubMed Abstract \| Publisher Full Text Reference Source\n\nEmergency Medicine Unit, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong KongFan K, Leung L, et al.: Public knowledge of how to use an automatic external defibrillator in out-of-hospital cardiac arrest in Hong Kong. Hong Kong Med. J. 2016 Oct 31 [cited 2020 May 25]. Reference Source\n\nChen M, Wang Y, Li X, et al.: Public Knowledge and Attitudes towards Bystander Cardiopulmonary Resuscitation in China. Hindawi: BioMed Research International; 2017 [cited 2020 May 25]; vol. 2017. : e3250485. Reference Source\n\nHawkes CA, Brown TP, Scott B, et al.: Attitudes to Cardiopulmonary Resuscitation and Defibrillator Use: A Survey of UK Adults in 2017. J. Am. Heart Assoc. 2019 Apr 2 [cited 2020 May 25]; 8(7): e008267. PubMed Abstract \| Publisher Full Text\n\nMerchant RM, Griffis HM, Ha YP, et al.: Hidden in Plain Sight: A Crowdsourced Public Art Contest to Make Automated External Defibrillators More Visible. Am. J. Public Health. 2014 Dec [cited 2020 May 25]; 104(12): 2306–2312. PubMed Abstract \| Publisher Full Text Reference Source\n\nVaillancourt C, Stiell IG, Wells GA: Understanding and improving low bystander CPR rates: a systematic review of the literature. CJEM. 2008 Jan; 10(1): 51–65. PubMed Abstract \| Publisher Full Text" }	[ { "id": "255541", "date": "26 Mar 2024", "name": "Shajitha Thekke Veettil", "expertise": [ "Reviewer Expertise Epidemiology", "primary care research", "life science research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper titled \"Preparedness regarding first response to emergencies in the community among graduate students: A cross-sectional study in India\" provides valuable insights into the awareness and attitudes of graduate students towards first response to emergencies, particularly focusing on cardiopulmonary resuscitation (CPR) and automated external defibrillators (AEDs) in India. Here are some feedback and suggestions for improvement: Abstract: The abstract provides a clear overview of the study objectives, methods, and key findings. It effectively summarizes the main points of the paper. Introduction:\nThe introduction effectively sets the context by discussing the importance of bystander response in time-sensitive emergencies, particularly focusing on the role of bystanders in initiating CPR and AED use. It provides relevant background information on the global incidence of out-of-hospital cardiac arrest (OHCA) and the significance of early recognition and intervention. Consider providing a brief overview of previous research on bystander CPR rates and knowledge levels in India to provide more context for the current study.\nMethods:\nThe methods section provides a detailed description of the study design, eligibility criteria, study procedures, and data management. Consider providing more information on the development and validation of the questionnaire used in the study, including the specific knowledge questions and their scoring criteria. Provide more details on the cluster random sampling technique used to recruit participants, including how classroom batches were randomly selected and approached. Clarify how the sample size was determined and why the specific parameters were chosen for estimating the level of knowledge regarding first response and CPR. Provide more information on the statistical analysis plan, including the rationale for the chosen statistical tests and significance level.\nResults:\nThe results section presents the main findings of the study in a clear and organized manner. Consider presenting the demographic characteristics of the study participants in a more concise format, such as a table or figure. Provide more context for some of the findings, such as the comparison of knowledge scores between HCPT and non-HCPT students. Consider discussing the implications of the findings in more detail, including potential factors contributing to low knowledge levels and attitudes towards CPR and AED use. Provide more information on the limitations of the study, including potential sources of bias and implications for generalizability.\nDiscussion:\nThe discussion effectively summarizes the key findings of the study and provides insightful interpretations. Consider discussing the implications of the findings in relation to existing literature on bystander CPR rates and knowledge levels in India and other countries. Provide recommendations for future research and interventions aimed at improving bystander response to emergencies, particularly focusing on increasing CPR training programs and awareness campaigns.\nOverall, the paper provides valuable insights into the awareness and attitudes of graduate students towards first response to emergencies in India. Consider incorporating the suggested improvements to enhance the clarity, rigor, and relevance of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "136243", "date": "25 May 2024", "name": "Sumanth Mallikarjuna Majgi", "expertise": [ "Reviewer Expertise epidemiology", "epidemiology of diabetes/hypertension/ accidents (NCD)", "tribal health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study has addressed an important issue in first response during any emergency, particularly in public area/space. even though study is done only once university/city, this situation wont be quiet different from rest of India, though being one the developed/educated district- (higher HDI), the situation may be better in this study compared to rest of the country.\n\nThe fact that there is low awareness of response, gives opportunity to provide the training on these aspects/ life skills. also, attitudes also are not conducive to save life during emergency, which needs to be catered.\n\nMost important part of recognizing the arrest itself is very low ( 30%), than the help number , hands only CPR is very essential part. The belief that by doing CPR, one can get disease from the victim and perceived utility of CPR both are same in HCPT and non-HCPT students, which is surprising and indicate the need for the better knowledge and attitude changes even in HCPT.\n\nThe study has yielded higher beta coefficients for those with health care training and previous CPR training, witnessing arrest indicating, the training on CPR would improve the knowledge, and in life time witnessing cardiac arrest would improve knowledge and in turn can save life.\n\nAlthough the authors have recommended for CPR training for school children, however, there is also dire need for CPR training to HCPT also, as their knowledge and attitude also need to be upgraded.\n\nIs the work 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/11-469
https://f1000research.com/articles/11-271/v1	03 Mar 22	{ "type": "Research Article", "title": "Temporomandibular joints disorders (TMDs) prevalence and their relation to anxiety in dental students", "authors": [ "Lujain Homeida", "Emtenan Felemban", "Wed Kassar", "Mazen Ameen", "Salwa Aldahlawi", "Emtenan Felemban", "Wed Kassar", "Mazen Ameen", "Salwa Aldahlawi" ], "abstract": "Background: Temporomandibular joint disorders (TMDs) are very common disorders affecting the population and causing pain. Researchers have reported a high prevalence of TMDs among university students due to increased distress. The aims of this study were to determine the frequency of TMDs in Umm al-Qura University (UQU) dental students using the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD), and to examine the relationship between anxiety, bruxism, and TMDs among those students. Methods: The DC/TMD pain screener questionnaire was completed by dental students. Students who reported TMD pain or had at least one positive TMD symptom were invited to the dental clinic for a full TMJ evaluation. History of bruxism was documented and an ultra-brief tool for detecting anxiety and depression (Patient Health Questionnaire-4 PHQ) was completed by all students. Results: A total of 240 students responded to the TMD pain screener in which 119 reported at least one TMJ symptom. Only 93 dental students presented to clinical examination in which 64.5% (n=60) of them had temporomandibular joint disorders. Disc displacement with reduction and local myalgia (38.7% & 32.25%, respectively) were the most frequent diagnosis. A total of 29% (n=27) of students had more than one diagnosis. Overall, 41 participants (44.09%) reported a positive response to the anxiety scale and (n=38) 40% of participants reported parafunctional habits. Both the history of bruxism and anxiety were significantly related to TMDs (P=0.0002) and also significantly higher in women of higher academic years (P≤0.01). Conclusions: This study found a high prevalence of TMDs among UQU dental students. Disc displacement with reduction was the most prevalent disorder. Bruxism and anxiety were associated with painful TMDs.", "keywords": [ "TMD", "DENTAL", "STUDENTS", "PAIN", "STRESS", "PARAFUNCTIONAL HABITS" ], "content": "Introduction\n\nTemporomandibular joint disorders (TMDs) are a very common group of musculoskeletal disorders affecting the temporomandibular joint (TMJ) and the face causing pain. They are considered a significant public health burden in approximately 5% to 12% of the general population (National Institute of Dental and Craniofacial Research 2018, July). Painful TMD has a direct impact on the person’s quality of life and daily activity (Schiffman, Ohrbach et al. 2014). The TMD has a multifactorial pathogenesis in which it involves physiological and/or psychological factors like emotional distresses. Chronic parafunctional habits can cause repetitive trauma to the masticatory system, which may result in painful TMD episodes (Schiffman, Ohrbach et al. 2014). Parafunctional habits including but not limited to bruxing and clenching are known to have a critical role in aggravation and progression of TMD (Chisnoiu, Picos et al. 2015). Furthermore, psychosocial distress is also considered an important comorbidity contributing to TMD (Schiffman, Ohrbach et al. 2014). Some individuals, when exposed to stressful situations, tend to activate the stomatognathic system by clenching or grinding their teeth and increasing masticatory muscle contraction in order to relieve their stress. This increased masseter activation during stress and decrease in a relaxing situation was highly associated with the presence of TMD in individuals under more stress (Calixtre, Gruninger et al. 2014).\n\nMany studies have looked into the psychological stress among university students and its impact on student’s academic achievement and well-being. High prevalence of mental issues between university students was reported (Adlaf, Gliksman et al. 2001). Stallman et al evaluated mental stress among Australian universities students and found a high prevalence of mental health problems (19.2 %) and subsyndromal symptoms (67.4%) which were significantly higher than those of the general population (Stallman 2010). This supports that university student population live under more stress than the general population. Thus, the prevalence of TMDs is relatively high among university students of different specialties.\n\nBinoleil et al, assessed the prevalence of headaches and painful TMDs and examined the relationship between TMDs, headaches, and depression rates among dental and medical students. They reported higher depression scores in patients with painful TMD compared to patients without TMD (Benoliel, Sela et al. 2011). Furthermore, the relationship between stress level and painful TMD in students of health science was supported in few more studies locally (Alkhudhairy, Al Ramel et al. 2018) and internationally where stress played an important role in TMD progression (Monteiro, Zuim et al. 2011, Wieckiewicz, Grychowska et al. 2014).\n\nThe Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) was first proposed in 1992 and has been used widely as diagnostic protocol for TMD research (Dworkin and LeResche 1992). However, more research was done over the years to improve its validity and clinical utility. In 2014, an evidence-based new Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) was published and was considered a valid and reliable screening tool for use in clinical and research settings and included important modifications to the original RDC/TMD (Schiffman, Ohrbach et al. 2014). An acceptable sensitivity and specificity for a definitive diagnosis are considered as sensitivity ≥ 70% and specificity ≥ 95%. DC/TMD has diagnostic algorithms used to diagnose the most common pain-related TMD and most common intra-articular disorders (Schiffman, Ohrbach et al. 2014). The Axis I diagnostic algorithm consists of two parts; a self-report instrument where it is used for pain screening. The second part is used for TMJ clinical examination. The DC/TMD Axis II protocol included instruments to evaluate pain behavior, psychological status, and psychosocial functioning (Schiffman, Ohrbach et al. 2014).\n\nTo the best of our knowledge, the prevalence of TMDs among Umm al-Qura University (UQU) dental students has not been evaluated. In this study, we aimed to determine the prevalence of TMDs in UQU dental students using Diagnostic Criteria for Temporomandibular Disorders (DC/TMD). Also, to examine the relation between anxiety, self-reported bruxism and TMDs among UQU dental students.\n\n\nMethods\n\nThis cross-sectional study was approved from Umm Al-Qura University (UQU), College of Dentistry Institutional Review Board (IRB) # (98-18). This study took place at collage of Dental Medicine Umm al Qura University. Data were collected between July 2019 and December 2019.\n\nThe UQU dental collage program starts at 2nd level and it is involves 5 academic years followed by internship year. All UQU dental students (year 2 - internship year) were included in this study and were invited by email to fill The Research Diagnostic Criteria (RDC) three-items pain screener on TMD symptoms (Schiffman, Ohrbach et al. 2014). Non-dental students from UQU and dental student from other universities were excluded. All participants have given their consent to participate in the study. Demographic data were collected including age, gender, marital status and year of study. In addition, history of TMD diagnosis and history of parafunctional habits (bruxism), and the use of chronic medication was included. The response to each of the three questions was documented.\n\nThe respondents who reported TMD pain or had at least one positive answer to one of the 3-items questionnaire were invited to have a full TMJ clinical examination in the specialty clinic at UQU teaching hospital. The TMJ clinical examination included detailed assessment of the TMJ position and structure, range of motion measurements, and palpation of muscle of mastication following DC/TMD protocol. The clinical examination provided to all students was performed by one oral medicine/TMD specialist. Written informed consent was obtained from all participants prior to clinical examination.\n\nThe DC/TMD diagnostic criteria algorithms were followed to reach a TMD diagnosis. This included pain-related temporomandibular disorders (local myalgia, myofascial pain, myofascial pain with referral, arthralgia, and headache attributed to TMD) and intra-articular disorders (disc displacement with reduction, disc displacement with reduction with intermittent locking, disc displacement without reduction with limited opening, disc displacement without reduction without limited opening). History of bruxism was obtained and those who had painful TMD were referred to an oral medicine specialist clinic for further treatment.\n\nDuring the clinical examination, a valid and ultra-brief tool for detecting anxiety and depression (Patient Health Questionnaire-4 PHQ) was completed by all participants (Kroenke, Spitzer et al. 2009). This four-items questionnaire (PHQ-4) consisted of two core anxiety and two core depression items. The total score of this scale ranges from 0-12 and categorized as normal (0 –2), mild (3–5), moderate (6 – 8), and severe (9–12). This instrument is not diagnostic, however, is indicator for further assessment of possible clinical disorder warranting treatment.\n\nThe data analysis was performed using Statistical Package for the Social Sciences version 22 (SPSS Inc., Chicago, IL, USA, RRID:SCR_019096). Student T-test and chi-square analysis were used to relate the existence of TMD problem to age, gender, academic year, history of bruxism and anxiety level. Also, to compare the anxiety scores among male and female groups. Statistical significance was set at P ≤ 0.05.\n\n\nResults\n\nA total of 304 electronic questionnaires were sent via email to all dental students at Umm al-Qura university who were between 2nd year to the internship with a total of 6 academic years. A total of 240 questionnaires were completed and returned with a compliance rate of 78.9% (Homeida 2022). The demographics of respondents from the pain screening questionnaire can be found in Table 1.\n\nOut of the 240 responders, 49.5% (n=119) reported either TMD pain or other TMD symptoms in the past 30 days. And 20.4% (n=49) reported TMJ pain that comes and goes. The majority of students 30% (n=72) reported jaw habits with 25% (n=60) had pain on opening, 16% (n=40) reported pain on chewing hard or tough food and 14% (n=35) had pain with jaw activities such as talking and yawning (Table 2).\n\nAll the responders who reported TMD pain or symptoms (n=119) were invited for a TMJ examination. A total of 93 (78%) subjects presented for clinical assessment. The demographics of the subjects received the clinical examination are presented in Table 1. During TMJ clinical examination, more than half of the subjects 64.5% (n=60) had temporomandibular disorders (TMDs), while only 35.4% (n=33) students had normal TMJ findings at the time of the examination.\n\nDisc displacement with reduction was the most frequent diagnosis 38.7%(n=36) followed by local myalgia 32.25% (n=30) and arthralgia 16.1% (n=15) (Figure 1). Interestingly, 29% (n=27) students had more than one diagnosis and coexistence of disc displacement with reduction and local myalgia was found to be the most frequent combination in 19.3% (n=18) students (Table 3). Overall, the diagnosis of TMD was significantly higher in the female students compared to the male students (P≤0.022). Both, pain-related disorders and intra-articular disorders were significantly higher in females with (P=0.027) and (P=0.024), respectively. Also, disc displacement with reduction showed a significant increase with the higher academic year (P≤0.01).\n\nDC/TMD: Diagnostic Criteria for Temporomandibular Disorders.\n\nIn total, 41 participants (44.09%) reported a positive response to the anxiety and depression scale. In which, 48.7% (n=20) had a mild score and 51% (n=21) had moderate to severe anxiety score (Table 3). Of the 41 subjects with a positive response to PHQ-4, 31 (73%) had been diagnosed with a painful TMDs. Moderate to severe anxiety was significantly associated with TMDs (P=0.006).\n\nOverall, 40% of the participants (n=38) reported a parafunctional habit. Out of 38 (71%) with a history of bruxism, 27 had TMDs. Self-reported history of bruxism was significantly associated with TMDs in all students (P≤0.01).\n\nBoth the history of bruxism and the level of anxiety were significantly related (P=0.0002) and also significantly higher in females than males (P≤0.01). Besides, the anxiety level and history of bruxism significantly increased with higher academic years (P≤0.05). Those high scores of anxieties and bruxism were reversed in the internship year (Figure 2).\n\n\nDiscussion\n\nThe present study found that half of UQU dental students reported at least one TMD symptom. Moreover, the clinical examination found that 64% were diagnosed with at least one TMJ disorder and about 30% (one- third) had multiple diagnoses. Bruxism and high anxiety levels were related to TMDs in this student population.\n\nEvidence suggests that TMD is a common complain among students. The prevalence of TMD reported by this study is higher than what has been reported by other studies in Saudi universities which ranged between 25-39% (Alkhudhairy, Al Ramel et al. 2018, Srivastava, Shrivastava et al. 2021). Studies that addressed general university Saudi students also reported TMD prevalence of 20 to 50% (Zulqarnain, Khan et al. 1998, Habib, Al Rifaiy et al. 2015, Zwiri and Al-Omiri 2016, Srivastava, Shrivastava et al. 2021). All the above-mentioned studies with the exception of Srivastava et al. relied on self-administered questioners to identify subjects with TMDs and did not include TMJ clinical examination to confirm the diagnosis as the present study which may explain the difference in prevalence. When RDC/TMD algorithm was used to estimate the prevalence of TMD, 30 to 36% of dental students were found to have TMD (Fernandes Azevedo, Camara-Souza et al. 2018, Lövgren, Österlund et al. 2018). The present study used RDC/TMD pain screener to identify the subject with possible TMJ symptoms first, then, only those with positive responses to the pain screener were examined clinically for TMD diagnosis and that may explain the higher prevalence of TMDs.\n\nDisc displacement with reduction was the most prevalent TMD disorder followed by myalgia. This finding concurs with a systematic review in which disc displacement of TMJ was highly prevalent TMD in the general population with prevalence ranging from 18 to 35% (Naeije, Te Veldhuis et al. 2013). Also, the finding of the increasing prevalence of disc displacement with age in this study is in alignment with other studies were disc displacement with reduction develops during childhood and adolescence and it’s prevalence levels off towards adulthood (Marpaung, van Selms et al. 2019, Sankuratri, Verma et al. 2021). Although myalgia was the reported as the commonest diagnosed condition in some studies (Srivastava, Shrivastava et al. 2021), it was the second most prevalence condition in this study. On the other hand, females had a higher prevalence of TMDs compared to males. This findings was in alignment with other studies (Bagis, Ayaz et al. 2012, Naeije, Te Veldhuis et al. 2013, Wieckiewicz, Grychowska et al. 2014, de Melo Junior, Aroucha et al. 2019, Xie, Lin et al. 2019, Sankuratri, Verma et al. 2021, Srivastava, Shrivastava et al. 2021). Interestingly, 30% of the subjects had more than one TMDs diagnosis. Similar finding were reported by Azevedo 2018 et al (Fernandes Azevedo, Camara-Souza et al. 2018) which highlights the importance of diagnosis and early management of TMDs.\n\nPrevious cross-sectional studies reported a significant increase in anxiety and depression scores among medical and dental students in different Saudi universities (Inam 2007, Aboalshamat, Hou et al. 2015, Basudan, Binanzan et al. 2017). In this study, 44% of the students with TMD symptoms reported a positive response to the PHQ-4 with half of them classified as having moderate to severe anxiety. Female dental students had a higher PHQ-4 mean score than male students which was in agreement with similar studies done on Saudi dental students (Inam 2007, Al-Saleh, Al-Madi et al. 2010, Benoliel, Sela et al. 2011, Al-Sowygh 2013). Overall, the rate of anxiety in women has been reported to be higher than men (Kessler, Sonnega et al. 1995, Steel, Marnane et al. 2014, Xie, Lin et al. 2019). This could be explained by the slower processing in neurotransmitter serotonin which has a critical role in anxiety and depression. Besides, women are more sensitive to specific hormone such as corticotropin-releasing factor which has an important role in stress response (Bangasser, Curtis et al. 2010).\n\nThe majority of those students who showed moderate to severe levels of anxiety and depression were diagnosed with painful TMDs on clinical examination and therefore, anxiety was found to be related to TMDs. Other studies have conflicting results. stress and anxiety were positively associated with TMD in university students in general and dental students in particular (Ton, Mota et al. 2020, Jaiswal and Deshpande 2021). While Azevedo et al found no association between anxiety and TMD (Fernandes Azevedo, Camara-Souza et al. 2018). Anxiety and stress in dental students can be caused by many external factors like exams, clinical requirements and academic assignments. This study was conducted during the academic year which could contribute to the higher prevalence of anxiety and TMDs.\n\nIn general, oral parafunctional habits are known as a major contributor to TMDs and play an important role in its progression (Chisnoiu, Picos et al. 2015). In the present study, self-reported bruxism was significantly associated with TMDs. This finding concurs similar findings in Swedish dental students in which participants with TMD reported significantly higher oral parafunctional habits (Lövgren, Österlund et al. 2018). Moreover, the results of this study are in accordance with Jaiswal et al, where they reported significant relationship between TMDs and parafunctional habits in Indian dental students (Jaiswal and Deshpande 2021).\n\nHistory of bruxism and anxiety levels were found to be higher among the senior dental students at UQU. Similar findings were reported by other studies in which a statistical significant relationship between anxiety and the para-functional habit was revealed (Paterson, Lamb et al. 1995, Ahlberg, Lobbezoo et al. 2013). Most individuals who have anxiety disorders tend to relieve their stress by clenching and/or grinding their teeth and contracting masticatory muscle which leads to activation of the stomatognathic system (Calixtre, Gruninger et al. 2014). This increase in stress levels among final-years students can be due to more participants from those two academic years. However, it can be also explained by the higher clinical demands and more workload during these final clinical years. Declining of both bruxism and anxiety parameters was noted in participants from the internship year where there is a significant decrease in academic load and clinical requirements. Similar findings were reported by Ahuja (Ahuja, Ranjan et al. 2018).\n\n\nLimitation of the study\n\nThis is a cross-sectional study in which only association between variables is detected. Longitudinal studies are needed to prove causation. The absence of a control group for anxiety screening was regarded as a limitation for this study, as no clinical examination nor depression score was done for the participants who denied TMD pain or symptoms. Also, a comparison of TMDs prevalence with age-matched general population (non-dental students) was not considered in this study and it is recommended for future research projects.\n\n\nConclusions\n\nTMDs is highly prevalent among dental students. Disc displacement with reduction was the most predominant one. Greater prevalence was observed among females and higher academic years. Bruxism and anxiety were associated with painful TMDs.\n\n\nData availability\n\nDryad: Temporomandibular Joints Disorders TMDs Prevalence and Its Relation to Anxiety in Dental Students. https://doi.org/10.5061/dryad.kkwh70s62 (Homeida 2022).\n\nThis project contains the following underlying data:\n\n- Pain screener results for 240 participants.xlsx\n\n- TMD diagnosis results of 93 participants.xlsx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAboalshamat K, Hou XY, Strodl E: Psychological well-being status among medical and dental students in Makkah, Saudi Arabia: a cross-sectional study. Med. Teach. 2015; 37(Suppl 1): S75–S81. PubMed Abstract \| Publisher Full Text\n\nAdlaf EM, Gliksman L, Demers A, et al.: The prevalence of elevated psychological distress among Canadian undergraduates: findings from the 1998 Canadian Campus Survey. J. Am. Coll. Heal. 2001; 50(2): 67–72. PubMed Abstract \| Publisher Full Text\n\nAhlberg J, Lobbezoo F, Ahlberg K, et al.: Self-reported bruxism mirrors anxiety and stress in adults. Med. Oral Patol. Oral Cir. Bucal. 2013; 18(1): e7–e11.\n\nAhuja V, Ranjan V, Passi D, et al.: Study of stress-induced temporomandibular disorders among dental students: An institutional study. Natl. J. Maxillofac. Surg. 2018; 9(2): 147–154. PubMed Abstract \| Publisher Full Text\n\nAl-Saleh SA, Al-Madi EM, Al-Angari NS, et al.: Survey of perceived stress-inducing problems among dental students, Saudi Arabia. Saudi. Dent. J. 2010; 22(2): 83–88. PubMed Abstract \| Publisher Full Text\n\nAl-Sowygh ZH: Academic distress, perceived stress and coping strategies among dental students in Saudi Arabia. Saudi. Dent. J. 2013; 25(3): 97–105. PubMed Abstract \| Publisher Full Text\n\nAlkhudhairy MW, Al Ramel F, Al Jader G, et al.: A Self-Reported Association between Temporomandibular Joint Disorders, Headaches, and Stress. J. Int. Soc. Prev. Community Dent. 2018; 8(4): 371–380. PubMed Abstract \| Publisher Full Text\n\nBagis B, Ayaz EA, Turgut S, et al.: Gender difference in prevalence of signs and symptoms of temporomandibular joint disorders: a retrospective study on 243 consecutive patients. Int. J. Med. Sci. 2012; 9(7): 539–544. PubMed Abstract \| Publisher Full Text\n\nBangasser DA, Curtis A, Reyes BA, et al.: Sex differences in corticotropin-releasing factor receptor signaling and trafficking: potential role in female vulnerability to stress-related psychopathology. Mol. Psychiatry 2010; 15(9): 877. 896-904. Publisher Full Text\n\nBasudan S, Binanzan N, Alhassan A: Depression, anxiety and stress in dental students. Int. J. Med. Educ. 2017; 8: 179–186. PubMed Abstract \| Publisher Full Text\n\nBenoliel R, Sela G, Teich S, et al.: Painful temporomandibular disorders and headaches in 359 dental and medical students. Quintessence Int. 2011; 42(1): 73–78. PubMed Abstract\n\nCalixtre LB, Gruninger BL, Chaves TC, et al.: Is there an association between anxiety/depression and temporomandibular disorders in college students? J. Appl. Oral Sci. 2014; 22(1): 15–21. PubMed Abstract \| Publisher Full Text\n\nChisnoiu AM, Picos AM, Popa S, et al.: Factors involved in the etiology of temporomandibular disorders - a literature review. Clujul. Med. 2015; 88(4): 473–478. PubMed Abstract \| Publisher Full Text\n\nde Melo Junior PC , Aroucha J, Arnaud M, et al.: Prevalence of TMD and level of chronic pain in a group of Brazilian adolescents. PLoS One. 2019; 14(2): e0205874. PubMed Abstract \| Publisher Full Text\n\nDworkin SF, LeResche L: Research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specifications, critique. J. Craniomandib. Disord. 1992; 6(4): 301–355. PubMed Abstract\n\nFernandes Azevedo AB, Camara-Souza MB, Dantas IS, et al.: Relationship between anxiety and temporomandibular disorders in dental students. Cranio. 2018; 36(5): 300–303. PubMed Abstract \| Publisher Full Text\n\nHabib SR, Al Rifaiy MQ, Awan KH, et al.: Prevalence and severity of temporomandibular disorders among university students in Riyadh. Saudi. Dent. J. 2015; 27(3): 125–130. PubMed Abstract \| Publisher Full Text\n\nHomeida L: Temporomandibular joints disorders TMDs prevalence and its relation to anxiety in dental students. Dryad, Dataset 2022. Publisher Full Text\n\nInam SB: Anxiety and Depression among Students of a Medical College in Saudi Arabia. Int. J. Health Sci. (Qassim) 2007; 1(2): 295–300. PubMed Abstract\n\nJaiswal E, Deshpande S: Temporomandibular Disorders in Dental Students ofIndia: Prevalence, Severity and Associated Factors. Clin. Dent. 2021; XV(4): 12–17.\n\nKessler RC, Sonnega A, Bromet E, et al.: Posttraumatic stress disorder in the National Comorbidity Survey. Arch. Gen. Psychiatry 1995; 52(12): 1048–1060. Publisher Full Text\n\nKroenke K, Spitzer RL, Williams JB, et al.: An ultra-brief screening scale for anxiety and depression: the PHQ-4. Psychosomatics 2009; 50(6): 613–621. PubMed Abstract \| Publisher Full Text\n\nLövgren A, Österlund C, Ilgunas A, et al.: A high prevalence of TMD is related to somatic awareness and pain intensity among healthy dental students. Acta Odontol. Scand. 2018; 76(6): 387–393. PubMed Abstract \| Publisher Full Text\n\nMarpaung C, van Selms MKA , Lobbezoo F: Temporomandibular joint anterior disc displacement with reduction in a young population: Prevalence and risk indicators. Int. J. Paediatr. Dent. 2019; 29(1): 66–73. PubMed Abstract \| Publisher Full Text\n\nMonteiro DR, Zuim PR, Pesqueira AA, et al.: Relationship between anxiety and chronic orofacial pain of temporomandibular disorder in a group of university students. J. Prosthodont. Res. 2011; 55(3): 154–158. PubMed Abstract \| Publisher Full Text\n\nNaeije M, Te Veldhuis AH, Te Veldhuis EC, et al.: Disc displacement within the human temporomandibular joint: a systematic review of a 'noisy annoyance'. J. Oral Rehabil. 2013; 40(2): 139–158. PubMed Abstract \| Publisher Full Text\n\nNational Institute of Dental and Craniofacial Research: Facial Pain.2018,July, July 2018. Retrieved 28. March, 2020. Reference Source\n\nPaterson AJ, Lamb AB, Clifford TJ, et al.: Burning mouth syndrome: the relationship between the HAD scale and parafunctional habits. J. Oral Pathol. Med. 1995; 24(7): 289–292. PubMed Abstract \| Publisher Full Text\n\nSankuratri B, Verma RK, Iqbal A, et al.: Assessment of Incidence of TMJ Disorders in Dental Graduates Competing for Post Graduation Exam: Original Research. Ann. Rom. Soc. Cell Biol. 2021; 25(6): 10837–10842.\n\nSchiffman E, Ohrbach R, Truelove E, et al.: Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) for Clinical and Research Applications: recommendations of the International RDC/TMD Consortium Network* and Orofacial Pain Special Interest Groupdagger. J. Oral Facial Pain Headache. 2014; 28(1): 6–27. PubMed Abstract \| Publisher Full Text\n\nSrivastava KC, Shrivastava D, Khan ZA, et al.: Evaluation of temporomandibular disorders among dental students of Saudi Arabia using Diagnostic Criteria for Temporomandibular Disorders (DC/TMD): a cross-sectional study. BMC Oral Health. 2021; 21(1): 211. PubMed Abstract \| Publisher Full Text\n\nStallman H: Psychological distress in university students: A comparison with general population data. Aust. Psychol. 2010; 45(4): 249–257. Publisher Full Text\n\nSteel Z, Marnane C, Iranpour C, et al.: The global prevalence of common mental disorders: a systematic review and meta-analysis 1980-2013. Int. J. Epidemiol. 2014; 43(2): 476–493. PubMed Abstract \| Publisher Full Text\n\nTon LAB, Mota IG, Martins APVB, et al.: Prevalence of temporomandibular disorder and its association with stress and anxiety among university students. Brazilian Dent. Sci. 2020; 23(1). Publisher Full Text\n\nWieckiewicz M, Grychowska N, Wojciechowski K, et al.: Prevalence and correlation between TMD based on RDC/TMD diagnoses, oral parafunctions and psychoemotional stress in Polish university students. Biomed. Res. Int. 2014; 2014: 472346.\n\nXie C, Lin M, Yang H, et al.: Prevalence of temporomandibular disorders and its clinical signs in Chinese students, 1979-2017: A systematic review and meta-analysis. Oral Dis. 2019; 25(7): 1697–1706. PubMed Abstract \| Publisher Full Text\n\nZulqarnain BJ, Khan N, Khattab S: Self-reported symptoms of temporomandibular dysfunction in a female university student population in Saudi Arabia. J. Oral Rehabil. 1998; 25(12): 946–953. PubMed Abstract \| Publisher Full Text\n\nZwiri AM, Al-Omiri MK: Prevalence of temporomandibular joint disorder among North Saudi University students. Cranio. 2016; 34(3): 176–181. PubMed Abstract \| Publisher Full Text" }	[ { "id": "126222", "date": "07 Apr 2022", "name": "Arwa Farag", "expertise": [ "Reviewer Expertise OFP and OM" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear author,\nThank you for conducting this investigation. The study aimed to estimate the prevalence of TMDs among dental student and characterize the most common TMD diagnoses along with associated symptoms/conditions. The methodology followed was optimal, starting from screening to comprehensive clinical assessment and self-reported questionnaires. Recruitment and inclusion of the study participants were done using the gold standard diagnostic criteria (RDC-TMD). All the utilized tools of assessment were reliable and previously validated for TMD population.\nBelow, I’m including minor points/comments, that I hope will further optimize this well-put-together manuscript.\nMethodology:\nIn the exclusion criteria, author should specify that health history was obtained and those with underlying rheumatoid conditions, generalized pain symptoms, connective tissue disorders were excluded from the study.\nDiscussion:\nIt would great if the author can provide some statistics/citations for prevalence of TMD (disc displacement/myalgia) along with stress and anxiety in the general population and compare them to this special population (dental students). This will provide insight, and clearly highlight, the increased prevalence of these conditions among dental students.\nThank you once again for allowing me the opportunity to review this 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?\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": "8139", "date": "27 Apr 2022", "name": "Lujain Homeida", "role": "Author Response", "response": "Response to TMD reviewer comments Thank you for the comments and the review, below are our responses 1 - in the exclusion criteria, author should specify that health history was obtained and those with underlying rheumatoid conditions, generalized pain symptoms, connective tissue disorders were excluded from the study. Thank you for the comment, the material and method section was amended as follows: under TMJ clinical examination and diagnosis the following sentence was added: “Detailed medical history was obtained and subjects with underlying rheumatoid conditions, generalized pain symptoms, connective tissue disorders were excluded from the study”. 2- It would great if the author can provide some statistics/citations for prevalence of TMD (disc displacement/myalgia) along with stress and anxiety in the general population and compare them to this special population (dental students). This will provide insight, and clearly highlight, the increased prevalence of these conditions among dental students. Thank you for this point. Most of the literature on TMD addressed specific population (patients, medical or dental students, or university students) very few literature addressed general population or included non TMD general population as a comparison group. The following paragraph was added to the discussion. 'In comparison to the general population, TMD prevalence ranges between 5 – 35 % (Schiffman, Ohrbach et al. 2014, Nadershah 2019). However, none of those studies reported on the association of TMD with stress, anxiety or phycological factors in general population. In general, anxiety disordered have been reported in 35 % of chronic pain patients compared to only 18 % of the general population (Poleshuck, Bair et al. 2009). in addition, Reissmann et al. compared patients diagnosed with TMD and general population without TMD related pain and reported that Trait anxiety is significantly associated with diagnoses of TMD pain. One point increase in the State-Trait Anxiety Inventory score related to an increase of the odds for pain-related TMD by the factor 1.04 (Reissmann, John et al. 2014). A similar finding reported by kmeid et al. were TMDs was significantly associated with depression, anxiety, and stress among Lebanese population (Kmeid, Nacouzi et al. 2020)'" } ] } ]	1	https://f1000research.com/articles/11-271
https://f1000research.com/articles/11-10/v1	06 Jan 22	{ "type": "Research Article", "title": "Comorbidities and laboratory parameters associated with SARS-CoV-2 infection severity in patients from the southeast of Mexico: a cross-sectional study", "authors": [ "Eduardo De la Cruz-Cano", "Cristina del C Jiménez–González", "José A Díaz-Gandarilla", "Carlos J López–Victorio", "Adelma Escobar-Ramírez", "Sheila A Uribe-López", "Elizabeth Huerta-García", "Jorge-Tonatiuh Ayala-Sumuano", "Vicente Morales-García", "Liliana Gútierrez-López", "José A González-Garrido", "Eduardo De la Cruz-Cano", "Cristina del C Jiménez–González", "José A Díaz-Gandarilla", "Carlos J López–Victorio", "Adelma Escobar-Ramírez", "Sheila A Uribe-López", "Elizabeth Huerta-García", "Jorge-Tonatiuh Ayala-Sumuano", "Vicente Morales-García", "Liliana Gútierrez-López" ], "abstract": "Background. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic. Among the risk factors associated with the severity of this disease is the presence of several metabolic disorders. For this reason, the aim of this research was to identify the comorbidities and laboratory parameters among COVID-19 patients admitted to the intensive care unit (ICU), comparing the patients who required invasive mechanical ventilation (IMV) with those who did not require IMV, in order to determine the clinical characteristics associated with the COVID-19 severity. Methods. We carried out a cross-sectional study among 152 patients who were admitted to the ICU from April 1st to July 31st, 2021, in whom the comorbidities and laboratory parameters associated with the SARS-CoV-2 infection severity were identified. The data of these patients was grouped into two main groups: “patients who required IMV” and “patients who did not require IMV”. The nonparametric Mann–Whitney U test for continuous data and the χ2 test for categorical data were used to compare the variables between both groups. Results. Of the 152 COVID-19 patients who were admitted to the ICU, 66 required IMV and 86 did not require IMV. Regarding the comorbidities found in these patients, a higher prevalence of type 2 diabetes mellitus (T2DM), hypertension and obesity was observed among patients who required IMV vs. those who did not require IMV (p<0.05). Concerning laboratory parameters, only glucose, Interleukin 6 (IL-6), lactate dehydrogenase (LDH) and C-reactive protein (CRP) were significantly higher among patients who required IMV than in those who did not require IMV (p<0.05). Conclusion. This study performed in a Mexican population indicates that comorbidities such as: T2DM, hypertension and obesity, as well as elevated levels of glucose, IL-6, LDH and CRP are associated with the COVID-19 severity.", "keywords": [ "SARS-CoV-2", "type 2 diabetes mellitus", "hypertension", "obesity", "laboratory parameters." ], "content": "Abbreviations\n\nACE-2: angiotensin-converting enzyme 2\n\nALT: alanine transaminase\n\nAST: aspartate transaminase\n\nCOPD: chronic obstructive pulmonary disease\n\nCOVID-19: coronavirus disease-2019\n\nCKD: chronic kidney disease\n\nCRP: C-reactive protein\n\nEWS: Early Warning Score\n\nFiO2: fraction of inspired oxygen\n\nICU: intensive care unit\n\nIL-1β: Interleukin 1 beta\n\nIL-6: interleukin 6\n\nIMV: invasive mechanical ventilation\n\nMSQ: Medical Symptom Questionnaire\n\nPaO2: partial pressure of oxygen\n\nPCR: polymerase chain reaction\n\nRAAS: Renin-Angiotensin-Aldosterone System\n\nSARS-CoV-2: severe acute respiratory syndrome-coronavirus 2\n\nSCQ: Self-administered Comorbidity Questionnaire\n\nspO2: blood oxygen saturation\n\nT2DM: type 2 diabetes mellitus\n\nTNFα: tumor necrosis factor alpha\n\nWHO: World Health Organization.\n\n\n1. Introduction\n\nWithout a doubt, the current pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) represents one of the greatest public health challenges, which has led to extensive worldwide research efforts to identify individuals at greatest risk of developing critical illness.1–3 The clinical manifestations of the disease caused by SARS-CoV-2, known as coronavirus disease 2019 (COVID-19), are highly variable and range from asymptomatic forms, moderate manifestations and even severe complications, such as: pneumonia, respiratory failure, septic shock, multiple organ dysfunction and death.4,5 Unfortunately, the molecular mechanisms involved with COVID-19 severity seem to be particularly complex, due to important immunopathological changes induced by SARS-CoV-2;6–9 as well as metabolic conditions (e.g. obesity, diabetes, hypertension, heart diseases, among others) that underlie the clinical presentation in these patients.10–14 Regarding latter, a growing body of evidence has suggested that these comorbidities contribute significantly to increased COVID-19 severity and fatal outcomes.13,15–18 For instance, several studies have reported that obesity is a comorbidity that increases the risk of complications in SARS-CoV-2 infection, for the following reasons: (a) it has been suggested that the ACE-2 receptor expression (target of SARS-CoV-2) is higher in adipose tissue than in the lung parenchyma, which makes adipose tissue an important viral reservoir (see Figure 1);19,20 (b) the Renin-Angiotensin-Aldosterone System (RAAS), a hormonal cascade which regulates blood pressure and is generally overactive in obese patients, has been linked to SARS-CoV-2 cellular infection as well as myocardial and lung injury;21,22 and (c) it is well known that obesity is related to an increase in circulating levels of many adipokines and pro-inflammatory mediators released by adipocytes.10 Therefore, obesity-induced adipose tissue inflammation generates important metabolic abnormalities and disproportionate effects on the immune system, which are relevant pathophysiological aspects in COVID-19 severity.2,23,24 On the other hand, in comorbidities like hypertension and type 2 diabetes mellitus (T2DM), besides the previously mentioned points, severe metabolic dysfunctions and several coagulation system alterations take place.25–27 For example: (a) it has reported that the endothelial dysfunction in obese patients with hypertension promotes the development of a hypercoagulable pro-thrombotic state (by exposure of tissue factor and other pathways), which contributes markedly to life-threatening complications of COVID-19, such as venous thromboembolic disease, systemic vasculitis, endothelial cell apoptosis and multiple organ involvement;27,28 and (b) it is also clear that insulin resistance contributes substantially to the more severe phenotype associated with obesity and T2DM in COVID-19.29,30 In fact, it has been suggested that the SARS-CoV-2 infection could cause disturbances in glucose metabolism, therefore the acute hyperinflammatory state itself could worsen insulin resistance in these patients.10,30 In this context, these important pathophysiological alterations in COVID-19 patients have led to an urgent necessity in identifying clinical laboratory predictors, which could provide relevant information in determination of prognosis, patient follow-up, and therapeutic monitoring, as well as differentiate severe patients from the mild/moderate form of COVID-19.31,32 For instance, biomarkers of an overactive innate immune system, such as markedly elevated neutrophil count, IL-6, C-reactive protein and serum ferritin, could help recognize a potential severe SARS-CoV-2 infection during triage, while biomarkers of organ failure could be helpful in monitoring evolution of COVID-19 patients admitted to the intensive care unit (ICU).33,34 Thus, both the etiological diagnosis of SARS-CoV-2 and the classification of these patients are the most obvious scenarios in the current health crisis, in which the clinical laboratory plays a fundamental role.31,32,35 Because of all the above described, the early identification of comorbid conditions and laboratory predictors associated with the SARS-CoV-2 infection severity, as well as the rapid application of measures to control this infection are currently the main strategies to prevent and reduce the risk of the virus spreading. For this reason, the present study aimed to identify the comorbidities and laboratory parameters among COVID-19 patients admitted to the ICU, comparing those patients who required invasive mechanical ventilation (IMV) to those who did not require IMV, in order to determine the clinical characteristics associated with the COVID-19 severity.\n\nAbbreviations. ACE-2: angiotensin-converting enzyme-2; ER: endoplasmic reticulum; ERGIC: endoplasmic reticulum Golgi intermediate compartment; ORF1a: open reading frames 1a; ORF1ab: open reading frames 1ab; pp1a: polyprotein 1a; pp1ab: polyprotein 1ab; RNA: Ribonucleic Acid; SARS-CoV-2: severe acute respiratory syndrome-coronavirus 2; T2DM: type 2 diabetes mellitus; TMPRSS2: Transmembrane protease, serine 2.\n\nWhat are the comorbidities and laboratory parameters associated with SARS-CoV-2 infection severity in patients from the southeast of Mexico?\n\n\n2. Methods\n\nThis research used a cross-sectional design.\n\nThe present study enrolled 152 patients diagnosed with COVID-19, who were admitted to the ICU of the General Hospital “Dr. Desiderio G. Rosado Carbajal\" from April 1st to July 31st, 2021. For the confirmatory diagnosis of this disease, nasopharyngeal and throat swab specimens were collected upon admission, which were subsequently analyzed by real-time polymerase chain reaction (qPCR) for SARS-CoV-2 RNA detection. Case definitions for these patients were in accordance with the interim guidance of the World Health Organization (WHO), which includes: fever, cough, dyspnea, respiratory frequency ≥ 30/min, SpO2 ≤93%, PaO2/FiO2 ratio <300 and lung infiltrates >50%, as severe clinical manifestations of COVID-19.36 It is necessary to highlight that this is one of the most important hospitals in the southeast of Mexico and has been designated by the Federal Secretary of Health for the hospitalization of COVID-19 patients since February 2020. Identification of these patients was achieved by reviewing and analyzing admission records and clinical histories from all available electronic medical records. Therefore, patients with clinical data of pneumonia, but with negative SARS-CoV-2 test results were excluded from this study. Additionally, since it has been documented that both phenotypic and genotypic characteristics could contribute substantially to the development of specific comorbidities,37–39 COVID-19 patients from other ethnicities (e.g. Asian, African and Caucasian individuals) were also excluded from the present study.\n\nThis cross-sectional study was conducted according to the guidelines laid down in the Declaration of Helsinki40 and all procedures involving research study participants were requested and verbally approved by the Ethics Commission of the hospital on March 16th, 2021. Written informed consent was waived by the Ethics Commission of the designated hospital due to the rapid onset of this public health emergency. Verbal consent of the patients was witnessed by a medical professional assigned to the hospital (D.C.E.) and formally documented in the medical record. However, regarding patients who were unable to provide this consent due to their severe clinical condition, a patient's family member provided it, which was then confirmed once the patient was found lucid.\n\nConcerning this point, the data collected at the time of admission was the following. 1) Demographic data, including age and gender. 2) Comorbidities, such as T2DM, hypertension, dyslipidemia, chronic kidney disease, heart disease, chronic obstructive pulmonary disease, obesity and malignancy, which were chosen and determined according to the self-administered comorbidity questionnaire (SCQ),41 an instrument that asks about the presence, treatment and functional limitations of 12 common comorbidities and three additional non-specified medical problems.41 3) Clinical symptoms, which included: fever, cough, sore throat, nasal congestion, breathing difficulties, headache, myalgia, diarrhea, vomiting and nausea. These were measured according to the medical symptom questionnaire (MSQ) that identifies several symptoms which help to find the underlying causes of illness, by using 15 categories in which the patients rate a particular symptom from 0 (never experienced) to 4 (frequently experienced and severe) (available from Lake Travis Integrative Medicine). Here it is important to note that in those cases where the patient was unable to provide the information described above (i.e., comorbidities and clinical symptoms) due to their severe clinical condition (including confused and unconscious states), a patient's family member provided it. 4) Vital signs, such as: temperature, spO2, respiratory and heart rate, which were measured using a monitoring equipment and chosen according to the early warning score (EWS),42 a physiological scoring system based on the individual values of multiple vital signs to quickly evaluate the level of clinical deterioration, in both emergency and general care conditions.42 Approximately 15 minutes after admission, the blood samples for laboratory tests were collected, which included: complete blood count (CBC), blood chemistry, serum electrolytes, liver function test as well as C-reactive protein (CRP), interleukin 6 (IL-6) and lactate dehydrogenase (LDH) as inflammation-related biomarkers, which were part of the standard of medical care. Finally, all the data mentioned in this section was recorded in an electronic database by two independent researchers (D.C.E. and J.G.C.) and verified by two experienced doctors (G.G.J.A. and L.V.C.J.).\n\nWith the purpose of understanding the comorbidities and laboratory parameters associated with the SARS-CoV-2 infection severity in patients from the Mexican southeast, the data collected was grouped into two main groups: patients who required invasive mechanical ventilation (IMV) and patients who did not require IMV. The continuous data was described as mean and standard error, while categorical data was described as percentages. The nonparametric Mann–Whitney U test for continuous data and χ2 test for categorical data were used to compare variables between both groups. On the other hand, in order to evaluate the laboratory parameters in predicting the COVID-19 severity, the Receiver Operating Characteristic (ROC) curves were plotted corresponding to the variables found to show significance, with the corresponding areas under the curve (AUC), sensitivity, specificity, 95% confidence intervals (95%CI), as well as the optimal cutoff, which was defined as the value maximizing the Youden index. A p value <0.05 was considered statistically significant. All statistical analyses were performed using SPSS Statistics version 23.0 software, and figures created with SPSS and CorelDRAW graphics suite 2020, or Inkscape 0.92 could also be used as an alternative. Lastly, it is important to mention that a large number of patients who were included in this research (n=127) had incomplete data in their medical records concerning socioeconomic status, sanitary conditions, physical activity, nutritional habits, household income and access to healthcare services, so it was decided not to capture this information in the database, in order to reduce biases in the interpretation of the results.\n\n\n3. Results\n\nAs shown in Table 1, 152 COVID-19 patients (men n=92; mean age=59.62) were admitted to the ICU, of whom 66 required IMV (men n=43; mean age=59.80) and 86 did not require IMV (men n=49; mean age=59.48). Regarding comorbidities observed, the ICU-admitted patients who required IMV showed a higher prevalence of T2DM, hypertension and obesity compared to those who did not require IMV (p<0.05). In contrast, here it should be emphasized that while dyslipidemia was a prevalent condition among the COVID-19 patients admitted to the ICU (n=67), this did not show significant differences when both patient groups were compared. Finally, the least prevalent comorbidities in the whole sample were as follows: chronic kidney disease (n=8), heart disease (n=5), chronic obstructive pulmonary disease (n=12) and malignancy (n=1), in which no significant differences were found either.\n\nRegarding the clinical symptoms, we observed that the COVID-19 patients admitted to the ICU showed common symptoms of acute respiratory infection (see Table 1), such as: fever (88.15%), cough (83.55%), sore throat (60.78%), nasal congestion (59.86%), breathing difficulty (78.94%), headache (51.97%) and myalgia (28.94%); however, of all these symptoms, only breathing difficulty was significantly higher in those patients who required IMV (p< 0.05). On the other hand, digestive symptoms such as: diarrhea, vomit and nausea were less frequent (5.26%, 3.94% and 7.23%, respectively), and no significant differences were observed either. According to vital signs, as was expected, a lower oxygen saturation and a higher respiratory rate were observed among patients who required IMV than those who did not require IMV (p<0.05). Finally, no significant differences in temperature and heart rate were observed between both patient groups.\n\nAs observed in Table 2, several hematological and biochemical alterations were found among the ICU-admitted patients. For example, regarding the complete blood count, (CBC) an accentuated lymphopenia (11.76%) and a high neutrophil count (81.22%) were observed in these patients; however, when both patient groups were compared, no significant differences were found in these hematological parameters. Likewise, the results of blood chemistry showed elevated levels of glucose, cholesterol and triglycerides among these patients (169.87mg/dL, 202.05 mg/dL and 155.22 mg/dL, respectively); nevertheless, only glucose levels were significantly higher in those patients who required IMV than those who did not require IMV (p<0.05). With regard to liver function test performed among the ICU-admitted patients, only a slight increase in transaminases levels (ALT=51.44 U/L; AST=68.07 U/L) was observed; however, no significant differences were found in these enzymatic parameters either. Concerning the inflammation related-biomarkers, a marked elevation in IL-6, CRP and LDH levels was observed among the ICU-admitted patients (183.59 pg/mL, 267.79 mg/L and 481.32 U/L, respectively), which were significantly higher in those patients who required IMV than those who did not require IMV (p<0.05). Finally, no significant changes in electrolyte levels were observed among the COVID-19 patients admitted to the ICU.\n\n* Determined by Mann-Whitney U test for independent samples.\n\nWe performed ROC curves on the above laboratory parameters with significant differences to assess their value predictive in the COVID-19 severity (Figure 2). For instance, the IL-6 was the most specific predictor (specificity 95.3%) with a high sensitivity (97.0 %) for COVID-19 severity, based on a cut-off of 84.1 pg/mL and an area under the curve (AUC) of 0.675 (95% CI: 0.588-0.761). Similarly, the CRP levels showed a 98.5% sensitivity and 94.2% specificity for predicting severe COVID-19, based on an AUC of 0.651 (95% CI: 0.564-0.738) and a cut-off of 154.6 mg/L. Likewise, LDH levels showed a 95.5 % sensitivity and 84.9 % specificity for predicting severe COVID-19, based on an AUC of 0.610 (95% CI: 0.520-0.700) and a cut-off of 325.0 U/L. In contrast, the glucose levels showed a high sensitivity (86.4%) but a very poor specificity (40.7%) for the COVID-19 severity, based on an AUC of 0.736 (95% CI: 0.653-0.818) and a cut-off of 116.0 mg/dL. All the above data are described in Table 3.\n\nAbbreviations: CRP: C-reactive protein; IL-6: interleukin-6; LDH: lactate dehydrogenase.\n\n* Defined as the value maximizing the Youden index.\n\n\n4. Discussion\n\nIn this paper, the comorbidities and laboratory parameters associated with SARS-CoV-2 infection severity in patients from the Mexican southeast were analyzed. Regarding the comorbidities found in our study, a higher prevalence of obesity, T2DM and hypertension in those patients who required IMV was observed (p<0.05). These findings attract a lot of attention, since unfortunately Mexico is among the highest places in terms of prevalence of these comorbidities,43,44 which could partially explain the high hospital mortality rate related to COVID-19 in this country; in fact, according to data compiled by John Hopkins University, until October 2021, more than 284,381 deaths had been registered in Mexico. In this context, several studies support our findings, in which it has been documented that COVID-19 patients suffering from these metabolic disorders increase the need for critical care and particularly for IMV requirement.45–50 For instance, Simonnet et al.47 and Costa Monteiro et al.48 reported an elevated prevalence of obesity and T2DM among ICU-admitted patients infected with SARS-CoV-2. These studies indicated that these comorbidities could serve as clinical predictors for risk stratification models. Likewise, these studies concluded that early measurement of anthropometric and metabolic parameters in these patients could be crucial to avoid unfavorable clinical outcomes.47,48 Similarly, Busetto et al.,45 Cummings et al.46 and Borobia et al.50 evaluated the clinical course of critically ill patients with COVID-19. In summary, these studies reported that COVID-19 patients with obesity, T2DM and hypertension showed a higher risk of more severe clinical symptoms and extrapulmonary organ dysfunction during SARS-CoV-2 infection, thus requiring a more frequent need for ICU admission and IMV.45,46 Likewise, Petrilli et al.49 conducted a study including more than 4000 COVID-19 cases, in which obesity was the strongest predictor of critical illness, substantially higher than pulmonary or cardiovascular diseases.49 In this regard, precise pathophysiological mechanisms related to a higher prevalence of these comorbidities among COVID-19 patients requiring IMV are not completely understood. However, recently several studies have reported that these metabolic disorders are multifactorial conditions that are closely associated with severe respiratory dysfunctions as well as impaired molecular mechanisms that could worsen the course of SARS-CoV-2 infection.51,52 For example, (a) it is clear that obesity causes mechanical compression of the diaphragm, thoracic cavity and lungs, which could lead to a restrictive pulmonary damage and consequently to an impaired respiratory ventilation.53,54 Moreover, several studies have indicated that an excessive adipose tissue amount in the abdominal area reduces the strength of the respiratory muscles, decreases the total compliance of the respiratory system and increases pulmonary resistance.54–56 (b) It has been reported that obese patients with hypertension are more likely to develop severe respiratory diseases, such as: asthma,57 chronic obstructive pulmonary disease,58,59 obesity hypoventilation syndrome,60,61 pulmonary hypertension and obstructive sleep apnea,62,63 which predisposes them to low levels of blood oxygenation and evidently to fatal respiratory outcomes in severe SARS-CoV-2 infection.64,65 (c) T2DM could negatively impact clinical outcomes in COVID-19 patients admitted to the ICU, since it has been documented that hyperglycemia in diabetic patients could increase SARS-CoV-2 replication, at the same time aerobic glycolysis could facilitate SARS-CoV-2 replication via synthesis of mitochondrial reactive oxygen species and activation of hypoxia-inducible factor 1α.66,67 Thus, alterations in glucose metabolism could also have influenced a greater need for IMV as well in the poor prognosis in these patients.66,67 On the other hand, several hypotheses have emerged suggesting that SARS-CoV-2 could also be a key contributor in the worsening of metabolic status in comorbid patients requiring IMV, for example: (a) acute inflammatory state induced by SARS-COV-2 could alter the lipid and glucose metabolism. This hypothesis is supported by the fact that pro-inflammatory cytokines (e.g. IL-1β, IL-6 and TNF-α) modulate the metabolism of these biomolecules; hence, dyslipidemia and hyperglycemia observed in the ICU-admitted patients could also be due to an inadequate cellular secretion of cytokines and/or an inappropriate immune response induced by SARS-CoV-2.68,69 (b) Oxidative stress promoted by SARS-COV-2 infection could exacerbate dyslipidemia in COVID-19 patients with underlying metabolic disorders. This argument arises from the fact that most viral infections manipulate antioxidant systems in several chronic conditions, leading to abnormalities in cellular metabolism.70–72 (c) SARS-CoV-2 could directly affect liver function and thus alter the lipid biosynthesis. This hypothesis could partially explain the biochemical changes found in our study, in which a slight increase in serum aspartate transaminase (AST) and alanine transaminase (ALT) levels was observed. However, these slight changes likely do not contribute significantly to the increased levels of cholesterol and triglycerides in the COVID-19 patients admitted to the ICU. Finally, in our study the elevated levels of CRP, IL-6 and LDH were the most specific and statistically significant parameters in both groups of patients, which suggests that these molecules could play a key role during the progression and the prognosis of fatal outcomes in COVID-19 patients who required IMV.5,73,74 In fact, these biomarkers have previously been associated with the severity and mortality of COVID-19 in most cases defined by the Chinese National Health Commission.75–77 Moreover, recent publications have provided additional information that strengthen the role of CRP, IL-6 and LDH as predictive markers of SARS-CoV-2 infection severity, especially in critically ill patients.75,77,78\n\nA strength of this research is that it provides scientific evidence indicating that comorbidities such as obesity, T2DM, and hypertension, as well as elevated levels of glucose, IL-6, LDH and CRP are associated with the COVID-19 severity among ICU-admitted patients. Moreover, the clinical and laboratory data was collected from one of the most important hospitals in the Mexican southeast, which concentrates a large part of COVID-19 patients in that region. Finally, our study has some limitations inherent to methodological design that could affect the interpretation of results. First, since our study included only COVID-19 patients from the southeast of Mexico, one needs to be careful to extrapolate our findings to those who reside in other geographical areas of the country and the world. Second, only the basal clinical findings were included in this research, while the clinical changes induced by disease progression, pharmacological treatment and invasive mechanical ventilation were not addressed in this paper, which could lead to important biases of clinical observation and interpretation in these patients. Third, the small number of patients included in this study limit the ability to determine causal inferences linked to the COVID-19 severity. Therefore, randomized clinical trials and observational studies (with a larger number of patients) addressing the factors underlying the severe conditions of COVID-19 in patients with obesity, hypertension, T2DM and/or metabolic dysfunction could contribute to determine the causes associated with the clinical progression and severity of this disease. Fourth, the retrospective design of the present study is also an important limitation, since a large number of cases included in this study had incomplete data in their medical records (e.g. information on physical activity, socioeconomic status, nutritional habits, hygienic conditions, access to healthcare services as well as household income); thus, it was not possible to adjust the risks associated with the COVID-19 severity in these patients. Besides, it is likely that the categorical stratification used in our study was not the most appropriate method, since a validated severity scale for COVID-19 patients admitted to ICU was not used; hence, the results presented in this research should be viewed with caution.\n\n\n5. Conclusion\n\nIn conclusion, the results of this retrospective case study performed in a Mexican population indicates that metabolic disorders such as: obesity, T2DM, and hypertension, as well as elevated levels glucose, IL-6, LDH and CRP are associated with the SARS-CoV-2 infection severity. Therefore, patients suffering from these conditions should take additional measures to avoid COVID-19 infection by enforcing prevention during the current pandemic. Likewise, public health policies and social support services should focus on disadvantaged communities with high rates of obesity, T2DM, hypertension and nutritional disorders to promote healthy lifestyle choices and preventive strategies that help minimize the risks and health consequences of these diseases, including COVID-19 complications. Moreover, as further waves of the pandemic and new variants of faster spread than early forms of SARS-CoV-2 are expected, improvement of guidelines for individuals with these comorbidities is strongly recommended. Finally, our characterization provides a quick clinical guidance to stratify high susceptibility patients in SARS- CoV-2 infections.\n\n\nData availability\n\nHarvard Dataverse: Comorbidities and laboratory parameters associated with SARS-CoV-2 infection severity in patients from the southeast of Mexico: A cross-sectional study. https://doi.org/10.7910/DVN/DFALL683\n\nThis project contains the following files:\n\n- COVID19_Database (v1).tab (Data on clinical features and laboratory parameters of COVID-19 patients).\n\n- Data key.docx (Data key for variables and abbreviations in the tab file)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors thank the doctors and nurses assigned to the COVID-19 area by the data provided to carry out this research.\n\nAuthor roles: De la Cruz Cano E: Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Resources, Writing – Original Draft Preparation, Writing – Review & Editing; Jiménez González C: Data Curation, Investigation, Writing – Review & Editing; Díaz Gandarilla JA: Data Curation, Investigation, Writing – Review & Editing; Lopez Victorio CJ: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing; Escobar Ramírez A: Data Curation, Investigation, Writing – Review & Editing; Uribe López SA: Data Curation, Investigation, Writing – Review & Editing; Huerta García E: Data Curation, Investigation, Writing – Review & Editing; Ayala Sumuano JT: Data Curation, Investigation, Writing – Review & Editing; Morales García V: Data Curation, Investigation, Writing – Review & Editing; Gutiérrez López L: Data Curation, Investigation, Writing – Review & Editing; Gonzalez Garrido JA: Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Resources, Writing – Original Draft Preparation, Writing – Review & Editing.\n\n\nReferences\n\nGuan WJ, Liang WH, Zhao Y, et al.: Comorbidity and its impact on 1590 patients with COVID-19 in China: a nationwide analysis. 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PubMed Abstract \| Publisher Full Text\n\nCodo AC, Davanzo GG, Monteiro LB, et al.: Elevated Glucose Levels Favor SARS-CoV-2 Infection and Monocyte Response through a HIF-1α/Glycolysis-Dependent Axis. Cell metabolism. 2020; 32(3): 437–446.e5. PubMed Abstract \| Publisher Full Text\n\nCavounidis A, Mann EH: SARS-CoV-2 has a sweet tooth. Nature reviews Immunology. 2020; 20(8): 460. PubMed Abstract \| Publisher Full Text\n\nChen Y, Yu CY, Deng WM: The role of pro-inflammatory cytokines in lipid metabolism of metabolic diseases. International Reviews of Immunology. 2019; 38(6): 249–266. Publisher Full Text\n\nCoppack SW: Pro-inflammatory cytokines and adipose tissue. The Proceedings of the Nutrition Society. 2001; 60(3): 349–356. Publisher Full Text\n\nDelgado-Roche L, Mesta F: Oxidative Stress as Key Player in Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Infection. Archives of Medical Research. 2020; 51: 384–387. PubMed Abstract \| Publisher Full Text\n\nYang RL, Shi YH, Hao G, et al.: Increasing Oxidative Stress with Progressive Hyperlipidemia in Human: Relation between Malondialdehyde and Atherogenic Index. Journal of Clinical Biochemistry and Nutrition. 2008; 43(3): 154–158. PubMed Abstract \| Publisher Full Text\n\nSchwarz KB: Oxidative stress during viral infection: a review. Free Radical Biology & Medicine. 1996; 21(5): 641–649. Publisher Full Text\n\nLiu Y, Du X, Chen J, et al.: Neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with COVID-19. The Journal of Infection. 2020.\n\nQin C, Zhou L, Hu Z, et al.: Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. 2020; 71: 762–768. Publisher Full Text\n\nLiu F, Li L, Xu M, et al.: Prognostic value of interleukin-6, C-reactive protein, and procalcitonin in patients with COVID-19. Journal of Clinical Virology. 2020; 127: 104370. PubMed Abstract \| Publisher Full Text\n\nZhu Z, Cai T, Fan L, et al.: Clinical value of immune-inflammatory parameters to assess the severity of coronavirus disease 2019. International Journal of Infectious Diseases. 2020; 95: 332–339. PubMed Abstract \| Publisher Full Text\n\nVelavan TP, Meyer CG: Mild versus severe COVID-19: Laboratory markers. International Journal of Infectious Diseases. 2020; 95: 304–307. PubMed Abstract \| Publisher Full Text\n\nHerold T, Jurinovic V, Arnreich C, et al.: Elevated levels of IL-6 and CRP predict the need for mechanical ventilation in COVID-19. The Journal of Allergy and Clinical Immunology. 2020; 146: 128–136.e4. PubMed Abstract \| Publisher Full Text\n\nKhailany RA, Safdar M, Ozaslan M: Genomic characterization of a novel SARS-CoV-2. Gene Rep. 2020; 19: 100682. PubMed Abstract \| Publisher Full Text\n\nMalik YA: Properties of Coronavirus and SARS-CoV-2. The Malaysian Journal of Pathology. 2020; 42(1): 3–11. PubMed Abstract\n\nValencia DN: Brief Review on COVID-19: The 2020 Pandemic Caused by SARS-CoV-2. Cureus. 2020; 12(3): e7386. PubMed Abstract \| Publisher Full Text\n\nDu L, He Y, Zhou Y, et al.: The spike protein of SARS-CoV--a target for vaccine and therapeutic development. Nature Reviews. Microbiology. 2009; 7(3): 226–236. PubMed Abstract \| Publisher Full Text\n\nDe la cruz-Cano E, Jiménez-González Cristina d C, Díaz-Gandarilla José A, et al.: Comorbidities and laboratory parameters associated with SARS-CoV-2 infection severity in patients from the southeast of Mexico: A cross-sectional study. Harvard Dataverse. 2021; V2. Publisher Full Text" }	[ { "id": "119989", "date": "11 Feb 2022", "name": "Mohammad Safiqul Islam", "expertise": [ "Reviewer Expertise Pharmacology", "Clinical Pharmacy", "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\nThe manuscript entitled “Comorbidities and laboratory parameters associated with SARS-CoV-2 infection severity in patients from the southeast of Mexico: a cross-sectional study” submitted by Cruz-Cano et al. evaluated the connection of comorbidities and laboratory parameters with the severity of SARS-CoV-2 infection. The authors recruited 152 ICU admitted COVID-19 patients, and among them, 66 required invasive mechanical ventilation (IMV), whereas 86 did not require IMV. Among the comorbidities, the authors found a connection of diabetes mellitus (T2DM), hypertension, and obesity with COVID-19 severity, whereas, among the laboratory parameters, glucose, Interleukin 6 (IL-6), lactate dehydrogenase (LDH), and C-reactive protein (CRP) were associated with the severity of COVID-19 considering the requirement of IMV. The manuscript has substantial merit for indexing. However, some points should be clarified from the authors' side.\n\nNo details of the analysis procedures of blood laboratory parameters were included, making the study replicable. Authors may add the detailed methodology at least for glucose, Interleukin 6, lactate dehydrogenase, and C-reactive protein or add the general method of the overall analysis.\n\nThe blood parameters were detected on the blood samples collected after 15min of admission of the patients, which makes it difficult to study to connect with the severity of COVID-19. If there is an opportunity to add the follow-up blood sample parameters like after 15 days of admission, the comparison with severity will be recognizable.\n\nSome grammatical errors were found (e.g., it has reported that: correct form: it has been…., conclusion section: indicates- correct form: indicate, etc.) that should be corrected.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8101", "date": "27 Apr 2022", "name": "JOSE ARNOLD GONZALEZ-GARRIDO", "role": "Author Response", "response": "Reviewer 2 wrote: “A small sample size of 152 patients. Many studies looking at the same question have been done on much larger populations.” Reply: Thank you very much for your kind comment. For this reason, the small sample size has been included in our study as an important limitation (see third limitation at the end of the discussion section). “I agree with Invasive Mechanical Ventilation as the criteria for severity--however, there is some subjectivity in deciding who will be intubated. Therefore, this is a second but less critical limitation.” Reply: We deeply appreciate your kind comment. Although it is true that in our paper we did not use a validated severity scale (as specified in the fifth and last limitation at the end of the discussion section), the criteria for invasive mechanical ventilation were based on clinical characteristics and laboratory parameters of these patients (e.g. blood oxygen saturation, respiratory rate, IL-6, CRP, etc), which have also been used as severity predictors in similar studies, and recently included in validated scales as severity criteria for COVID-19. Reviewer 2 wrote: “The question is not novel--many findings are well known to us. It is well known that patients with obesity, type-2 diabetes, and those with higher CRP and IL-6 have more severe diseases.” Reply: Thank you very much for your kind comment. Although this study shows many findings that are well known to the scientific community, we believe that this small research could modestly contribute to the reinforcement of the current evidence." } ] }, { "id": "125705", "date": "12 Apr 2022", "name": "Perminder Gulani", "expertise": [ "Reviewer Expertise COVID-19", "Sepsis", "Cardiomyopathy." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI enjoyed reading this article evaluating comorbidities and laboratory parameters associated with Severe COVID-19 disease.\nThis study re-enforces many findings that are already well known in COVID-19. The study enrolled 152 COVID-19 patients admitted to ICU. RT-PCR was used to confirm the diagnosis of COVID. Patients requiring Invasive Mechanical Ventilation were classified as Severe COVID-19 cases. The comparison was made between patients who required invasive mechanical ventilation with those who did not.\n\nLimitations of the study:\n\nA small sample size of 152 patients. Many studies looking at the same question have been done on much larger populations.\n\nI agree with Invasive Mechanical Ventilation as the criteria for severity--however, there is some subjectivity in deciding who will be intubated. Therefore, this is a second but less critical limitation.\n\nThe question is not novel--many findings are well known to us. It is well known that patients with obesity, type-2 diabetes, and those with higher CRP and IL-6 have more severe diseases.\n\nIs the work 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": "8100", "date": "27 Apr 2022", "name": "JOSE ARNOLD GONZALEZ-GARRIDO", "role": "Author Response", "response": "Reviewer 1 wrote: “No details of the analysis procedures of blood laboratory parameters were included, making the study replicable. Authors may add the detailed methodology at least for glucose, Interleukin 6, lactate dehydrogenase, and C-reactive protein or add the general method of the overall analysis.” Reply: Thank you very much for this important observation. Consequently, in this version of the manuscript the analyzers and methods used to determine the laboratory parameters have been mentioned at the end of the section of clinical data and sample collection. “The blood parameters were detected on the blood samples collected after 15min of admission of the patients, which makes it difficult to study to connect with the severity of COVID-19. If there is an opportunity to add the follow-up blood sample parameters like after 15 days of admission, the comparison with severity will be recognizable.” Reply: We appreciate your valuable comment. However, due to the nature of this research (cross-sectional study), a follow-up study of these patients was not considered (see second limitation at the end of the discussion section). 3. “Some grammatical errors were found (e.g., it has reported that: correct form: it has been…., conclusion section: indicates- correct form: indicate, etc.) that should be corrected.” Reply: Thank you very much for your kind observation. Consequently, these grammatical errors have been corrected." } ] } ]	1	https://f1000research.com/articles/11-10
https://f1000research.com/articles/11-465/v1	27 Apr 22	{ "type": "Review", "title": "Molecular mediators involved in skin healing: a narrative review", "authors": [ "Camila dos Santos Leite", "Oscar César Pires", "Pedro Henrique Avi", "Maria Luiza M. Soto", "Ariane Ribeiro Martins", "Thalita Rocha", "Oscar César Pires", "Pedro Henrique Avi", "Maria Luiza M. Soto", "Ariane Ribeiro Martins", "Thalita Rocha" ], "abstract": "Background: The expression of biological mediators, such as growth factors and cytokines, after skin damage, and their balance, is important to guarantee proliferation, differentiation and migration of cells and extracellular matrix, as well as to the homeostasis during tissue remodeling. The present review means to clarify their functions over inflammation, proliferation and remodeling stages of skin regeneration. Methods: Reviews, experimental studies and clinical trials included in this paper were search on PubMed database using the following terms: platelet-derived growth factor, interleukin 1, tumor necrosis factor alpha, keratinocyte growth factor, transforming growth factor beta, endothelial vascular growth factor, matrix metalloproteinase 9, tissue metalloproteinase inhibitor 1, interferon gamma, transcription nuclear factor kappa B, skin inflammation, skin cell proliferation, skin extracellular matrix and skin regeneration. Results and Discussion: Several studies on the signaling mechanism of these mediators in normal and impaired healing have already been developed and have shown promising results. Currently, some of these mediators are already direct focuses of potential clinical therapies that address the treatment of acute and chronic skin wounds. These mediators, basically synthetized by platelets, macrophages, vascular endothelial cells, fibroblasts and keratinocytes, act on epidermis and dermis proliferation, hypertrophy and cell migration, resulting in formation of granulation tissue, reepithelization and extracellular matrix remodeling and angiogenesis. A better understanding of the action of these mediators can provide greater knowledge not only of their roles in the natural healing process but also in the presence of disorders that affect skin repair. Conclusion: Future studies aimed to understand the mechanism of action of these mediators in the different types of cells involved in wound healing may lead to the discovery of new therapeutics to optimize the treatment of skin pathologies.", "keywords": [ "olecular mediators", "biomarkers", "inflammation", "extracellular matrix", "cell proliferation", "skin regeneration", "skin healing", "wounds." ], "content": "Introduction\n\nWound healing involves three basic phases: inflammation, proliferation and remodeling.1 Immediately after an injury, platelets are activated and degranulate releasing chemokines and growth factors (e.g. platelet-derived growth factor – PDGF), which act forming a fibrin clot and promoting local.2,3\n\nIn the inflammatory phase neutrophils, monocytes and macrophages are recruited to remove cell’s debris and possible microorganisms.1–3\n\nIn the proliferative phase, from 72 hours up to 2-3 weeks, there is proliferation and migration of quiescent cells (fibroblasts, keratinocytes, endothelial cells) aiming at reepithelization.3,4\n\nCytokines (e.g. interleukin 1 – IL-1, tumor necrosis factor alpha – TNF-α) and growth factors (e.g. keratinocyte growth factor – KGF, transforming growth factor beta – TGF-β and vascular endothelial growth factor – VEGF) are also present in this phase.2,5,6\n\nVEGF mediates angiogenesis, ensuring nutrition to the new formed tissue, and reestablishes the extracellular matrix (ECM),5 formed by proteoglycans, collagen III, elastin and laminin.3\n\nIn the remodeling phase, which can last for months or even years, there is a gradual degradation of extracellular matrix and type III collagen, formation of type I collagen and reorganization of these collagen fibers in the dermis.3 This event is controlled by matrix metalloproteinase (MMPs) and their inhibitors (TIMPs), balancing apoptosis and new cell differentiation.2,3,7\n\nBiological mediators, such as growth factors and cytokines, are crucial to the process of wound healing. Improving our knowledge in the role of growth factors and cytokines involved in the regulation of inflammatory, proliferative and remodeling responses during skin healing may have a significant impact on wound therapy.\n\nGenerally, these mediators were able to induce keratinocytes and fibroblast proliferation, hypertrophy and migration, resulting in a regenerated skin with a new epidermis and dermis.\n\nThus, this review aims to address the role of some molecular mediators, such as: PDGF, IL-1, TNF-α, KGF, TGF-β, VEGF, MMP-9, TIMP-1, IFN-γ and NF-kB active in three phases of wound healing (Figure 1).\n\n\nMethods\n\nReviews, experimental studies and clinical trials included in this paper were searched on PUBMED database using the following terms: platelet-derived growth factor (PDGF), interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-α), keratinocyte growth factor (KGF), transforming growth factor beta (TGF-β), endothelial vascular growth factor (VEGF), matrix metalloproteinase 9 (MMP-9), tissue metalloproteinase inhibitor 1 (TIMP-1), interferon gamma (IFN-γ), transcription nuclear factor kappa B (NF-kB), skin inflammation, skin cell proliferation, skin extracellular matrix and skin regeneration. A total of 121 references, from 1980 to 2020, were incorporated.\n\n\nResults\n\nThe role of PDGF includes all stages of healing, from the mediation of inflammation, angiogenesis, proliferation of fibroblasts and formation of granulation tissue.2\n\nPDGF family consists of five distinct homodimeric isoforms of glycoproteins (PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD and PDGF-AB), which: PDGF-AA is synthesized by epithelial, muscle and neural cells; PDGF-BB by endothelial cells and megakaryocytes; PDGF-CC by epithelial, endothelial cells and neurons and PDGF-DD by fibroblasts and vascular smooth muscle.8\n\nThe five isoforms are also synthesized by platelets, macrophages, vascular endothelial cells, fibroblasts and keratinocytes, and has a mitogenic effects over mesenchymal cells through proteolytic reactions mediated by receptors platelet-derived growth factor alpha and beta (PDGFR-α and PDGFR-β).8–10\n\nIn normal skin or chronic wounds, PDGF levels are almost absent.11 However, during healing, PDGF act on fibroblast proliferation and chemotaxis9,12 mediate inflammation, angiogenesis and form the granulation tissue.8,9,13\n\nIL-1 is a pro-inflammatory cytokine found in two forms: interleukin 1 alpha (IL-1α) and interleukin 1 beta (IL-1β). Both act on keratinocytes proliferation and migration, angiogenesis control and MECs remodeling.14–16 During the inflammatory phase, IL-1 is highly produced by keratinocytes, in the epidermis15,16; high levels of IL-1α and IL-1β are related to negative changes in the prognosis of skin inflammatory and proliferative events17 and to the synthesis and regulation of other inflammatory mediators.18\n\nAntagonists receptors recombinant human (IL-1Ra) and mouse (IL-36Ra) inhibit the expression of high levels of IL-1 and other cytokines during intense skin inflammatory and fibrotic responses.19 Inhibition of IL-1 by IL-1Ra results in a decrease in pro-inflammatory cytokines (IL-1α, IL-1β, IL-12 and IFN-γ) and increase of IL-10, M2 macrophages, endothelial cells and granulation tissue, confirming their role over cell differentiation and proliferation.20\n\nRegarding, IL-1β Inhibition signaling pathway leads to a decrease in the pro-inflammatory M1 macrophages, an increase in the anti-inflammatory macrophages M2 and growth factors resulting in optimization of skin wounds healing.21\n\nTNF-α is a pro-inflammatory cytokine synthesized by several cells (fibroblasts, keratinocytes, monocytes, macrophages, eosinophils and T cells), especially in the initial phase of healing,22 which acts on the regulation of immune, inflammatory and proliferative responses (fibroblasts and keratinocytes) through type 1 (TNFR1 - p55) and type 2 (TNFR2 - p75) receptors, which trigger internal signals in target cells, leading to NF-kB activation and these responses.23\n\nRapid skin healing occurred in the absence of the TNFR1-p55, with reduction of cytokines and inflammatory infiltrates, and increase of angiogenesis and collagen I24; suggesting that TNFR1-p55 can negatively affect repair by inducing leukocyte infiltration at the wound site and decreasing reepithelization.23–25\n\nLow levels of TNF-α stimulate the inflammatory process and synthesis of growth factors by macrophages, optimizing healing. However, at high levels, TNF-α impairs repair by increasing synthesis and activity of MMPs and decreasing synthesis of their inhibitors (TIMPs), which results in degradation of the ECM, inhibition of cell migration and collagen deposition and, consequent, delay in the process of healing, that is, chronification of the wound.14\n\nThe TNF-α deficiency leads to less granulation tissue formation and slower reepithelization.26\n\nKeratinocyte growth factor is poorly synthesized in intact skin tissue but becomes highly active in fibroblasts and keratinocytes after epithelial injury. Both, KGF-1 and KGF-2, can modulate the proliferation and migration of keratinocytes, resulting in reepithelization and epidermis regeneration.27\n\nKGF-1, highly synthesized by fibroblasts at the beginning of healing, binds to the fibroblast growth factor receptor 2 (FGFR2-IIIb) in keratinocytes, regulating migration, proliferation and differentiation.28–32\n\nKGF-2 has a mitogenic effect similar to KGF-1, but binding to the specific KGFR receptor.33,34 During wound healing, KGF also acts in the formation of granulation tissue in addition to protecting cells from toxicity induced by active oxygen species.35\n\nTGF-β family consists of the TGF-β1, TGF-β2 and TGF-β3 isoforms36,37 and is synthesized by platelets, macrophages, fibroblasts and keratinocytes, and constitute an important antiproliferative mediator to control the different stages of healing,14,37 by recruiting inflammatory cells, inhibiting proliferation and migration of endothelial cells and, consequently, angiogenesis, inhibiting fibroblasts and keratinocytes migration and ECM production (collagen and fibronectin), promoting remodeling and adequate repair of epidermis.36–42\n\nTGF-β1 is synthesized at high levels prior to reepithelialization and is related to cellular hypertrophy36,43; it can later inhibit the MMPs synthesis leading to an increase in collagen fibers.36 TGF-β3 is synthesized during the initial stage of healing and can improve repair, preventing fibroplasia.39,43\n\nThe main mechanism by which TGF-β inhibits and/or controls cell growing occurs through the interaction between both type 1 (TGF-βRI) and type 2 (TGF-βRII) receptors, promoting specific pathway signaling (TGF-β/SMAD), phosphorylation of R-Smads 2 or 3 and binding to Smad4, and, consequently, growth inhibition by regulation of target genes.42,44,45\n\nNeovascularization, mediated by the VEGF family, is an important event in the skin repair and can occur mediated by endothelial progenitors (EPCs).46,47\n\nThe VEGF family consists of six isoforms of pro-angiogenic glycoproteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PIGF), VEGF-A121, VEGF-A165, VEGF-A189 and VEGF-A206.41,48,49\n\nVEGF-A is the main pro-angiogenic growth factor related to wound healing,41,46,49 being expressed mainly by endothelial cells and platelets, but also by fibroblasts, keratinocytes and macrophages,50,51 once their specific tyrosine kinase 1 receptor Flt-1 (VEGFR-1) and Flk/KDR-1 (VEGFR-2) are also present in these cells.46,52,53\n\nAccording to Ref. 54 an increase in angiogenesis can be mediated by VEGF-A synthesized by monocytes and macrophages in chronic venous wounds. Such a process is possible because of the interactions between endothelial and inflammatory cells.\n\nVEGF-A increases vascular permeability in the early stages of skin repair51 and stimulates quiescent endothelial cells to interact with adjacent cells in full state of proliferation and migration, promoting vascular growth.46,55 The balance in VEGF-A levels is also important to promote normal or delayed dermis’ healing, re-epithelialization and wound contraction.46,56,57\n\nVEGFR-2 receptor is also quite important to control angiogenesis, since binding to VEGF activates the signaling pathways of protein kinase B, resulting in inhibition of apoptosis and induction of cell proliferation.58\n\nDifferent types of matrix metalloproteinases (MMPs), such as collagenases, gelatinases, stromelysins, are present in several tissues performing specific functions regulated by different metalloproteinase inhibitors.59,60\n\nMMP-9 or gelatinase B, one of the 25 existing isoforms, is synthesized by basal cells and keratinocytes60,61 and has an important role in the skin healing,62 regulating the acute and chronic inflammation63,64 through the activation and control of cytokines and chemokines,65,66 controlling the release of growth factors,59 angiogenesis67 and migration/proliferation of keratinocytes68; regulating communication cells-ECM; promoting ECM reorganization and, consequently, tissue reepithelization.63,64,66\n\nThe deficiency of MMP-9 in rat skin results in an inflammatory process, with a high deposit of IL-1α and disorganization of basement membrane and ECM64; as well as the absence of MMP-9 results in decrease of angiogenesis and delay in reepithelization, with scarcity of keratinocyte migration at the edges of the wound.68,69\n\nMMP-9 is inhibited by tissue metalloproteinase inhibitor-1 (TIMP-1) and the balance between them promotes adequate skin repair63; high levels of MMP-9 and decrease in TIMP-1 result in slow healing in wounds of diabetic rats, for example.69\n\nTIMPs (tissue inhibitor of metalloproteinase), TIMP-1, TIMP-2, TIMP-3 and TIMP-4, regulate several cellular mechanisms, depending or not on MMPs.70–72\n\nIn wound healing, TIMP-1 plays an important role in inflammation by modulating the expression of cytokines and growth factors (PDGF and TGF-β1); regulating cell differentiation, angiogenesis and apoptosis.71–76 It also acts in the remodeling of the ECM, stimulating synthesis and degradation of protein.72,73\n\nTIMP-1 increases proliferation of fibroblasts,74 protects quiescent endothelial cells75 and epithelial cells76 against TNF-α induced apoptosis through activation of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways and Akt mitogen-activated kinase (ERK/MAPK), respectively.\n\nTogether with Glycosylphosphatidylinositol (TIMP-1-GPI), TIMP-1 stimulates migration and proliferation of epidermal keratinocytes, reducing dermal myofibroblasts and the secretion of TGF-β, improving skin repair.76\n\nIFN-γ is a pro-inflammatory cytokines (Type I: IFN-α and IFN-β; Type II: IFN-γ) produced by several cells, mainly by T-lymphocytes (Th1) and natural killer cells in response to viral infections and events involving inflammatory and mitogenic activities.77,78 IFN-γ responds to type 1 (IFNGR1) and type 2 (IFNGR2) receptors79 and acts by signaling the JAK1/2 STAT pathway and tyrosine kinase 2 (TYK2).80\n\nThis glycoprotein has anti-proliferative and anti-fibrotic effects, inhibiting fibroblast proliferation, collagen, cytokines IL-4 and TGF-β synthesis77,81; acts in the activation of macrophages and control the collagenase synthesis by these cells81; regulates the immune activity of stem cells of different tissues.82\n\nThe presence of IFN-γ, at high levels, can delay or impair skin repair,80,83–85 by decreasing angiogenesis, synthesis, deposition, organization and stability of collagen in ECM, and controlling cell proliferation.86,87\n\nHowever, reduction or absence of IFN-γ leds to early angiogenesis, by increasing VEGF and decreasing cytokine CXCL10 levels, attenuating inflammation, proliferating keratinocytes and fibroblasts, increasing expression of FGF-7, IGF and EGF, improving reepithelization, collagen deposition and synthesis of TGF-β1, accelerating, consequently, healing and wound contraction.82,88,89\n\nNF-kB family (p50/p105, p65/RelA, c-Rel/RelB and p52/p100) regulates genes involved in a variety of cellular processes, including skin healing, by activating and controlling the inflammatory response, the proliferative and migratory cellular activities, the expression of MMPs, the release and activity of growth factors, TGF-β and collagen, resulting in reepithelization90–92 and ECM remodeling.93\n\nThe NF-kB can be activated through several cellular stimuli, such as the phosphorylation of the IkB kinase complex (IKK), decreasing the secretion of cytokines (TNF-α, IL-I and IL-6) in human dermal fibroblasts.93\n\nHowever, the inhibition of the NF-kB signaling pathway by the IkB kinase inhibitor (IKK) complex resulted in marked tissue inflammation due to high expression of cytokines. In keratinocytes, the inhibition of NF-kB leads to skin inflammation, through TNF-α high expression, and epidermal hyperplasia.94\n\n\nDiscussion\n\nGrowth factors and cytokines are essential mediators to initiate the skin healing process, which starts by an inflammatory phase, followed by epidermis and dermis proliferation and remodeling.1–6\n\nDuring the inflammatory phase keratinocytes, in the epidermis,15,16 produce IL-1. Like a positive-feedback, IL-1α and IL-1β act in the proliferation, differentiation and migration of keratinocytes,14–16 resulting in reepithelization.90–92\n\nAntagonists receptors IL-1Ra and IL-36Ra, generally act by inhibiting IL-1 in the presence of intense inflammation and/or cutaneous fibrosis,19 accelerating the inflammatory phase and consequently closing the wound.95 IL-1Ra accelerated reepithelialization,96 leading to earlier skin healing in rats.95,97 IL-1 also plays an important role in this phase of healing activating neutrophils and macrophages infiltration.95\n\nAs well described by the literature, PDGFs activate some inflammatory mediators,9,12 induce8,9,13 and control98 the granulation tissue formation.\n\nAssociated to KGF-1, PDGF-BB mediate the proliferation of keratinocytes from mesenchymal cells derived from adipose tissue during healing, inducing faster reepithelialization, resulting in an epidermis similar to that found in normal skin.99\n\nPDGF-BB is also important for fibroblasts growing and collagen deposition,100 suggesting its effectiveness in repairing acute and chronic wounds,98 promoting ECM remodeling.94 In addition, PDGF-BB acts in the oxidative balance, decreasing (3rd day) and increasing (7th day) the levels of nitric oxide,101 controlling the rate of wound contraction.102\n\nThe synthesis of KGF by fibroblasts is essential for modulating the proliferation and differentiation of keratinocytes. These cells interfere with the fibroblast response by modulating genes linked to ECM, changing phenotypes, synthesis of IL-1 and MMPs during healing. The balance between keratinocyte synthesis and apoptosis mediated by KGF is fundamental for the prevention of cutaneous fibrosis.103\n\nKerotinocytes and fibroblasts also activate TGF-β family,36–42 VEGF and EPCs47,48,98 improving formation of granulation tissue.103\n\nTGF-β1 contributes to epidermis and dermis thickness,104 acting as an antiproliferative mediator in the healing phases, inhibiting migratory events and controlling cellular hypertrophy, usually caused by myofibroblast clusters. The suppression of myofibroblast synthesis and consequent inhibition of smooth muscle α-actin and collagen deposition is important in the control of hypertrophic healing.105\n\nVEGF promotes angiogenesis, increases rate of wound contraction and, consequently, early skin healing.106 Low levels of VEGF and PDGF were found both in healing and in the intact skin of mice.107 Both VEGFR-1 and VEGFR-2 receptors58 are targets for VEGF binding, which induces cell proliferation.108\n\nDuring healing, the persistent presence of pro-inflammatory M1 macrophages in the granulation tissue impairs the repair, however this event can be regulated by the signaling of the VEGFR-1 receptor, which acts on the balance of the levels of pro-inflammatory M1 and anti-inflammatory macrophages M2 to promote angiogenesis.108\n\nThe activation of VEGFR-2 increases expression of IL-10, which reduces macrophages and, consequently, inflammation and differentiation of myofibroblasts in cutaneous wounds, as well as increases density of angiogenesis, improving the quality of repair.109\n\nOther pro-inflammatory cytokines synthesis, such as TNF-α22 and NF-kB23 were activated by IL-1 and fibroblasts. TNF-α acts on the inflammatory process and on the proliferative and remodeling events of ECM during healing.14,26 Its imbalance interferes in tissue repair, since at low levels TNF-α positively modulates inflammation, inducing proliferation of keratinocytes110 and reepithelization14; at high levels TNF-α inhibits cell migration, increases MMPs synthesis and ECM degradation.14 Persistent inflammation, with M1 macrophage and neutrophil infiltration, TNF-α and TIMP-1 were observed in chronic skin wounds.111–113\n\nNF-kB also acts on inflammation, proliferation and cell migration during skin repair, through the mediation of growth factors, collagen synthesis and MMPs, promoting re-epithelialization91 and tissue remodeling.93\n\nKeratinocytes also contribute to the regulation of TIMPs/MMPs activities, modulating ECM by inactivating MMPs, increasing TIMP-1.114 Myofibroblasts also contribute to the regulation of TIMPs/MMPs during skin repair.115\n\nMMP-9 is involved in the inflammatory,65,66 proliferative67,68 and remodeling phases during skin healing.63,64,66 Therefore, the balance between MMP-9 synthesis and degradation by TIMP-1 is essential for adequate repair.63 Both high levels of MMP-9 and very low levels impair the migratory activity of keratinocytes and consequently the reepithelialization and wound remodeling of epidermis.116\n\nExogenous MMP-9 decreases collagen IV, delaying wound healing in rats, suggesting that MMP-9 interferes with the composition of basement membrane proteins, which prevent keratinocyte migration, accession and restructuring of the epidermis.117\n\nJust like the MMP-9, TIMP-1 also acts during the healing phases by modulating events related to ECM inflammation, proliferation, migration and remodeling73–76; and the balance in their expression levels contributes to the prevention of fibrosis and to a better skin repair.114\n\nAn increase in TIMP-1 and a decrease in MMP-9,115,118 improved angiogenesis, wound contraction115 and reduced inflammation.118 TIMP-1-GPI blocked the secretion of MMPs by altering the association of MMPs with the cell surface, improving the proliferation of dermal fibroblasts and reducing the expression of fibrotic genes, suggesting that this complex may help control fibrosis during cutaneous healing.119\n\nAs the proliferative phase is established, IFN-γ regulates the neutrophilic inflammatory responses120 and the immune activity of skin stem cells,82 initiating the remodeling phase,81–83,85–88 and the anti-proliferative and anti-fibrotic events,78,83 as inhibits the proliferation of fibroblasts and, consequently, the synthesis of collagen,120 cytokines and TGF-β.77,81 In complete absence of IFN-γ, collagen deposition and wound resistance to traction were reduced.121\n\n\nConclusion\n\nThe complete healing of a wound is essential to restore the structure and function and aesthetics of the injured tissue. Biological mediators play an important role in the healing process. A better understanding of the role of these mediators can improve knowledge not only about physiological healing, but also about tissue healing and regeneration in the presence of skin and/or systemic disorders or pathologies.\n\nGenerally, these mediators were able to induce fibroblast and keratinocytes proliferation, hypertrophy and migration, resulting in a regenerated skin with a new epidermis and dermis. Future studies aimed to understand the mechanism of action of these mediators in the different types of cells involved in wound healing may lead to the discovery of new therapeutics to optimize the treatment of skin pathologies.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nKomi DEA, Khomtchouk K, Maria PLS: A Review of the Contribution of Mast Cells in Wound Healing: Involved Molecular and Cellular Mechanisms. Clinic Rev. Allerg. Immunol. 2020; 58(3): 298–312. 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PubMed Abstract \| Publisher Full Text\n\nMi B, Liu J, Liu G, et al.: Icariin promotes wound healing by enhancing the migration and proliferation of keratinocytes via the AKT and ERK signaling pathway. Int. J. Mol. Med. 2018; 42(2): 831–838. PubMed Abstract \| Publisher Full Text\n\nElliott CG, Forbes TL, Leask A, et al.: Inflammatory microenvironment and tumor necrosis factor alpha as modulators of periostin and CCN2 expression in human non-healing skin wounds and dermal fibroblasts. Matrix Biol. 2015; 43: 71–84. PubMed Abstract \| Publisher Full Text\n\nNunomura S, Nanri Y, Ogawa M, et al.: Constitutive overexpression of periostin delays wound healing in mouse skin. Wound Repair Regen. 2018; 26(1): 6–15. PubMed Abstract \| Publisher Full Text\n\nHuang SM, Wu CS, Chiu MS: High glucose environment induces M1 macrophage polarization that impairs keratinocyte migration via TNF-α: An important mechanism to delay the diabetic wound healing. J. Dermatol. Sci. 2019; 96(3): 159–167. PubMed Abstract \| Publisher Full Text\n\nSimon F, Bergeron D, Larochelle S, et al.: Enhanced secretion of TIMP-1 by human hypertrophic scar keratinocytes could contribute to fibrosis. Burns. 2012; 38(3): 421–427. PubMed Abstract \| Publisher Full Text\n\nMayrand D, Lavoie AL, Larochelle S, et al.: Angiogenic properties of myofibroblasts isolated from normal human skin wounds. Angiogenesis. 2012; 15(2): 199–212. Publisher Full Text\n\nZhang C, Lim J, Jeon HH, et al.: FOXO1 deletion in keratinocytes improves diabetic wound healing through MMP9 regulation. Sci. Rep. 2017; 7(1): 10565. PubMed Abstract \| Publisher Full Text\n\nReiss M, Han YP, Garcia E, et al.: Matrix metalloproteinase-9 delays wound healing in a murine wound model. Surgery. 2010; 147(2): 295–302. PubMed Abstract \| Publisher Full Text\n\nBabaei S, Bayat M, Norouzian M, et al.: Pentoxifylline improves cutaneous wound healing in streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 2013; 700(1-3): 165–172. Publisher Full Text\n\nDjafarzadeh R, Notohamiprodjo S, Rieth N, et al.: Treatment of dermal fibroblasts with GPI-anchored human TIMP-1 protein moderates processes linked to scar formation. J. Invest. Dermatol. 2013; 133(3): 803–811. Publisher Full Text\n\nKanno E, Tanno H, Masaki A, et al.: Defect of Interferon γ Leads to Impaired Wound Healing through Prolonged Neutrophilic Inflammatory Response and Enhanced MMP-2 Activation. Int. J. Mol. Sci. 2019; 20(22): 5657. PubMed Abstract \| Publisher Full Text\n\nSchaffer M, Bongartz M, Hoffmann W, et al.: Regulation of nitric oxide synthesis in wounds by IFN-gamma depends on TNF-alpha. J. Investig. Surg. 2006; 19(6): 371–379. PubMed Abstract \| Publisher Full Text" }	[ { "id": "149095", "date": "06 Oct 2022", "name": "David Leavesley", "expertise": [ "Reviewer Expertise Cutaneous wound healing and tissue repair", "human epithelial cell physiology", "pericellular interface", "extracellular matrix" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 dos Santos Leite et al, presents a review of the “expression of biological mediators, such as growth factors and cytokines, after skin damage, and their balance… to guarantee proliferation, differentiation and migration of cells and extracellular matrix, as well as to the homeostasis during tissue remodeling”. The stated objective is to clarify the functions of ‘biological mediators’ over inflammation, proliferation, and remodeling stages of skin regeneration. It is assumed that the reader will know that this means mammalian skin tissue. Clearly, mammals are not the only organism possessing skin tissue that is subject to injury necessitating skin repair.\nIt is notable that mammalian skin does not ‘regenerate’; mammalian skin ‘heals’ and is ‘repaired’. The mechanism of 'repair' is distinct to ‘regeneration’; while it restores tissue form, it does not fully restore tissue function.\nThis subtle distinction has direct and substantial impact on the results returned from the described PubMed search strategy. It is also notable that few of the many known cytokines, lymphokines, chemokines, fibrotic, pro-inflammatory, and anti-inflammatory mediators were not included in the search strategy, and presumably, have been overlooked. Consequently, this review should not be considered to be ‘comprehensive’.\nSo what value does this review offer, that is not already covered by other recent reviews of this topic? In my opinion, this review adds little to an already well-provided literature in cutaneous wound healing.\nI further interpret that the authors limited perspective of cutaneous wound healing is (mis)informed by literature other than those highly-cited works by authors from the cutaneous wound healing community. Why are so few recently published works (e.g. 3 of 121 are ≥2020) included among the cited works?\nWhat is the keyword “olecular mediators”? (Obviously an editing widow)\nWhy is the text formatted as a newspaper article (each sentence as a paragraph)? This is inappropriate.\n[Introduction]\nThe opening sentence “Wound healing involves three basic phases…” is controversial! The most widely respected publication “Cutaneous Wound Healing” by Singer & Clark (1999)1 recognises four phases contribute to the healing of cutaneous tissue (i.e., skin). I believe the majority of practitioners in this field accept coagulation as the first of four phases of mammalian cutaneous wound healing, these being: blood clotting (hemostasis); inflammation; proliferation (tissue growth), and remodeling (tissue maturation, restoration of function).\n\nSo what are the “biological mediators” involved in the early (initiating) events of tissue trauma and wounding? These have been well-described by others (see for example: 2,3), yet they are absent here.\n\nI am confused by the antithetical statement; “quiescent cells” proliferate and migrate? (sic) How can quiescent cells proliferate and migrate?\n[Methods]\nWhat is the rationale for limiting this review to “the role of some molecular mediators: PDGF, IL-1, TNF-α, KGF, TGF-β, VEGF, MMP-9, TIMP-1, IFN-γ and NF-kB”? Ironically, nearly every one of these mediators requires interaction with glycosaminoglycans to realize their biological activity; however, this critical aspect is not mentioned.\n\nWhat is the rationale for limiting searches of the PubMed databases to “references from 1980 to 2020”? The most significant recent advance in cutaneous wound healing occurred in 1962, when George D. Winter published evidence that re-epithelisation (aka wound closure) proceeded twice as fast in a moist environment than in a dry environment4. This is outside the author’s PubMed search terms.\n\nThe literature cited in support of the PDGF synthesis is inappropriate. These data are from mesenchymal cells (aka. fibroblasts). Cutaneous tissue comprises epidermis, dermis and hypodermis. In humans, the epidermis is dominated by keratinocytes, cells of epithelial origin. What is the evidence keratinocytes secrete, or more importantly, respond to PDGF isoforms? Should the reader presume that PDGF synthesised in the dermis somehow traverses the basement membrane into the epidermis to act as a paracrine mediator? Critically, the key event in humans that determines the speed and quality of cutaneous wound healing is wound closure (aka ‘re-epithelialisation’). What is the evidence PDGF regulates re-epithelialisation? Insufficient detail is provided that might allow the non-expert reader to comprehend the role of PDGF in human cutaneous wound healing.\n[Results]\nThe description of the role of IL-1 is superficial, and incomplete. What are M2 macrophages? What is the origin of M2 macrophages? Is there no role for IL-1 regulating the activity of M1 macrophages? What of polymorphoneutrophils (PMNs, neutrophils), the first inflammatory cells to infiltrate the site? I understand that there is evidence that neutrophils also differentiate into N1 and N2 phenotype subpopulations? What of these cells?\n\nThe statement: “Keratinocyte growth factor is poorly synthesized in intact skin tissue but becomes highly active in fibroblasts and keratinocytes after epithelial injury” (sic) suggests that “synthesis” and “activity” are synonymous. This statement is inaccurate. Many, probably ‘most’, “biological mediators” are synthesised in a latent (inactive) form, and require activation (e.g. by proteolysis, co-factor binding / cations, pH) before biological activity can be measured.\n\nI suggest that including “Transcription nuclear factor kappa B” under its own sub-heading confers it with equivalence to the aforementioned glycoprotein mediators. This is misleading; the “NF-kB family” (acronym undefined) are nuclear transcription factors and function via a mechanism that is unlike the mechanism of glycoprotein mediators. This needs to be revised.\n\nNo mention is included of recent discoveries and characterizations of specialized pro-resolving lipid mediators (SPMs), micro-RNAs (miRs) nor of long non-coding RNAs (lncRNAs). SPMs are synthesized at the site of tissue injury via metabolism of 3-omega fatty acids. See 5,6,7. LncRNA and miRs are synthesised by all cells and regulate many physiological events, including wound healing and tissue repair. See 8,9,10)\n[Discussion]\n\nWhy are new data (and citations) being introduced in the Discussion? These are more appropriately included in the Results. The evidence cited in support of the statement “IL-1α and IL-1β act in the proliferation, differentiation and migration of keratinocytes, resulting in reepithelization” is inappropriate. It is not clear to this reader, what role Nrf2 and NF-κB have in angiogenesis? (Hint, they are substantial!)\n\nWhat is the evidence that “…accelerating the inflammatory phase… consequently closing the wound”?\n\nWhat is the evidence “TGF-β1 contributes to epidermis and dermis thickness”?\n\nWhat is the evidence “VEGF… increases rate of wound contraction (sic) and, consequently, early skin healing”? Is the author aware that cutaneous wound healing in mice (…most mammals!) is unlike cutaneous wound healing in humans? This statement has no relevance for cutaneous wound healing in humans?\n\nI repeat [page 7 of 12], “NF-kB” is NOT a “pro-inflammatory cytokine”!\n\nWhat is the evidence “high levels TNF-α inhibits cell migration, increases MMPs synthesis and ECM degradation”? Which cell population is inhibited by TNF-α?\n\nHow does “NF-kB also acts on inflammation, proliferation and cell migration during skin repair, through the mediation of growth factors, collagen synthesis and MMPs, promoting re-epithelialization and tissue remodeling”? What is meant by “mediation”?\n\nIt is not clear, to this reader, how “Generally, these mediators were able to induce fibroblast and keratinocytes proliferation, hypertrophy and migration, resulting in a regenerated skin with a new epidermis and dermis.” (sic) Mammalian skin includes many distinct cell populations (and phenotypically distinct sub-populations). I think that while it is convenient (for the sake of simplicity), it is disingenuous to claim that biological mediators are solely responsible for “regenerated skin”. Multiple sources of evidence indicate the ECM has an equivalent, if not dominating, role in mammalian skin repair. Elements of the ECM are also ‘molecular mediators’.\n\nFinally, as stated above, skin does NOT regenerate. Rather skin 'heals' and 'repairs'. Please look up what is the difference?\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? No\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/11-465
https://f1000research.com/articles/9-1394/v1	03 Dec 20	{ "type": "Research Article", "title": "Evaluation of possible biological control of Fusarium sp. using plant extracts and antagonistic species of microbes in vitro", "authors": [ "Mohammed Faruk Hasan", "Mohammed Asadul Islam", "Biswanath Sikdar", "Mohammed Faruk Hasan", "Mohammed Asadul Islam" ], "abstract": "Background: Fusarium species is one of the most devastating fungi responsible for fruit and vegetable crops rot worldwide. The present study was designed to find an ecofriendly control measure for pathogenic Fusarium species, using suitable bioagents. Methods: Medicinal plant extracts were evaluated or their antifungal activities against Fusarium species using the poisoned food method. Antagonistic potency of some nonpathogenic microbes was also assessed on Fusarium species using the dual culture method. Results: Highest inhibition of growth of Fusarium sp. was observed with 68.1% (0.389 mg per 90 mm Petri plate) of mycelia on Coccinia grandis plant leaf extract, in comparison to the control grown with 100.0% (1.22 mg/dish). The highest inhibition of radial growth was observed using Trichoderma viride on Fusarium sp. (46.01% inhibition). Conclusions: The findings of present study would be benevolent for antifungal drug development to control Fusarium sp. causing fruit and vegetable rot.", "keywords": [ "Fusarium sp.", "Plants extract", "Non-pathogenic microbes", "Antagonisms", "Biocontrol" ], "content": "Introduction\n\nFusarium species are a large genus of hyaline ﬁlamentous mold fungi, responsible for fruit and vegetable crop rot (Al-Najada & Gherbawy, 2015; Ziedan et al., 2018). Fusarium species are deeply invasive and can cause hematogenously disseminated infections with high mortality in neutropenic patients (Dignani & Anaissie, 2004). Numerous species of Fusarium contribute to yield loss and reduced quality to varying degrees by infection with some mycotoxins (O’Donnell et al., 2009). They also cause decay of various fruit in storage and postharvest conditions (Whiteside et al., 1988). The fruit rot caused by Fusarium incurs enormous yield losses and is often observed in fields and markets (Baria et al., 2015). The application of antagonistic agents and plant extracts in agriculture are becoming a major focus of plant protection research. Different preservatives or fungicide treatments are frequently applied to manage fruits diseases and decay, which is an alarming health concern (Munhuweyi et al., 2020). Since most chemical fungicides are highly toxic to humans and animals and they frequently cause water and soil pollution (Al-Najada & Gherbawy, 2015). Moreover, continuous and indiscriminate use is leading to the development of fungicide resistant strains of pathogens (Tafinta et al., 2013).\n\nIn Bangladesh, fruit rot is a destructive disease caused by Fusarium species on pre-harvest and postharvest fruits. To the best of our knowledge, there is no previous research on biological control of this pathogenic fungus. Therefore, the main objective of the study was to find an ecofriendly control system of Fusarium sp., to decrease fruit and vegetable rot in Bangladesh.\n\n\nMethods\n\nA pure culture of Fusarium sp. was previously isolated and identified (Accession No. MT856371) from postharvest Citrus reticulata fruit rot (Hasan et al., 2020). The culture was preserved at the Department of Genetic Engineering and Biotechnology, University of Rajshahi (Rajshahi, Bangladesh).\n\nFor antifungal activities screening, six healthy, mature medicinal plants, Allium sativum, Zingiber officinale, Coccinia grandis, Brassica juncea, Ocimum tenuiflorum and Hibiscus rosa-sinensis were collected from Mirzapur, Binodpur and Kajla village, Motihar, Rajshahi, Bangladesh.\n\nCollected plants were washed with water to remove dust from the plants’ surface and dried in room temperature. Plant extract preparation and fractionation was performed according to the method by Kader et al. (2018). Different parts of selected plants (bulb of Allium sativum; rhizome of Zingiber officinale; leaves of Coccinia grandis, Brassica juncea, Ocimum tenuiflorum; and flowers of Hibiscus rosa-sinensis plants) were cut into small species and ground by blender to form fine powder. The dried powder of the plants (100gm of each plant) were rinsed in methanol (500ml) using a conical flask, and were incubated in a shaking incubator with occasional shaking for fourteen days. The liquid contents were pressed through Markin cloth followed by filtration using Whatman no. 1 filter paper. Obtained filtered liquids were dehydrated in vacuo to leave a blackish and sticky mass. The extracts were collected in vials and preserved in a refrigerator at 4°C.\n\nThe inhibitory effect of different plant extracts was measured by following the poisoned food technique (Balamurugan, 2014). For this, 20µg of each plant extract was added to 20ml of potato dextrose agar (PDA) to fill a 90mm size Petri plate and mixed well. After solidification, seven day old 6 mm size fungal plug was placed in the center of the Petri plates. The Petri plates were incubated at 35°C for seven days in static condition.\n\nFor evaluation of antagonistic effects, six non-pathogenic pure microbe cultures, Escherichia coli, Rhizobium phaseoli, Rhizobium leguminosarum, Neofusicoccum mangifera, Trichoderma viride and Pestalotiopsis sp. were used against Fusarium species. The pure microbe cultures were kindly provided by Dr. Md. Salah Uddin, Associate Professor and Director, Microbiology Lab., Department of Genetic Engineering and Biotechnology, University of Rajshahi, as the part of a collaboration.\n\nTo assess the antagonistic effects, the dual culture technique was used, as previously described (Vethavalli & Sudha, 2012). A mycelial disc of 6 mm diameter was cut from the periphery of both antagonist cultures and the test pathogen and placed on a Petri plate with PDA media. For the control, only the test pathogen was placed in the centre of a Petri plate. The Petri plates were incubated at 35°C in darkness.\n\nThe inhibition percentage of mycelial growth= [(Gc-Gt)/Gc] × 100; Where, Gc = Mycelial growth in terms of colony diameter in control set, Gt= Mycelial growth in terms of colony diameter in treatment set. The inhibition percentages of Fusarium species growth were calculated using the following formula: Inhibition percentage (%) =100× (dc– dt)/dc; Where, dc = radial growth of pathogen in control, dt = radial growth of pathogen in dual culture. Mean values were compared through least significant different test using SAS software, version 9.4M5 (SAS Inc., Cary, NC, USA). All the experiment and test were replicated thrice.\n\n\nResults\n\nAll plant extracts showed a degree of growth inhibition of the tested fungus at the same concentrations. The highest inhibition of growth of the isolates was observed at 68.1% of mycelium on Coccinia grandis, which was followed by 64.1% on Allium sativum, in comparison to the control culture (100.0%). Hibiscus rosa-sinensis showed the lowest inhibition of mycelium with 29.6% against the fungal isolate in comparison to the control. The results are presented in Figure 1 and Figure 2.\n\nMycelia were collected after seven days of incubation on potato dextrose agar at 35°C in darkness. 90 mm Petri plates were used to culture the tested fungus.\n\n(A) Allium sativum, (B) Zingiber officinale, (C) Coccinia grandis, (D) Brassica juncea, (E) Ocimum tenuiflorum, and (F) Hibiscus rosa-sinensis. Mycelia were collected after seven days of incubation on potato dextrose agar at 35°C in darkness.\n\nThe highest percentage inhibition of radial growth was observed with Trichoderma viride (46.01%) against Fusarium, which was followed by 43.33% and 32.05% on Escherichia coli and Rhizobium phaseoli, respectively (Figure 3). The antagonistic agent Rhizobium leguminosarum did not show any inhibitory activity against the isolated fungus (Figure 3). The control group also did not show any inhibition of radial growth of Fusarium sp.\n\n(A) Escherichia coli, (B) Rhizobium phaseoli, (C) Rhizobium leguminosarum,(D) Neofusicoccum mangifera, and (E) Trichoderma viride and (F) Pestalotiopsis sp., Mycelia were collected after seven days of incubation on potato dextrose agar at 35°C in darkness.\n\n\nDiscussion\n\nFruit rot caused by Fusarium species is very common in Bangladesh. The main objective of the present study was to study biological control measures for this fungus. Plant extracts are now a superior choice to control different plant pathogens, as reported by several previous studies (Hasan et al., 2020; Kareem & Al-Araji, 2017; Parveen et al., 2014). In our study, we found that the plant extracts Allium sativum, Zingiber officinale, and Coccinia grandis have significant inhibitory effects on mycelial growth of Fusarium species. Hosen & Shamsi (2019) also found significant antifungal activity using Allium sativum (53.85%), Ocimum sanctum (48.72%) and Zingiber officinale (49.35%) against Fusarium solani and F. oxysporum. Our current results were also supported by data from Khatun et al. (2020) and Kareem & Al-Araji (2017). Confirmation of the potential of antagonists on the radial growth of the pathogen in dual culture have been previously reported by Akhtar et al. (2010). By contrast, Nasrin et al. (2018) reported 87% inhibition potency on mycelium growth of Fusarium oxysporum f. sp. lycopersici by Calotropis proceraon plant extract. In our study, Trichoderma viride, Escherichia coli, Rhizobium phaseoli and Alternaria sp. showed significant antagonistic activity against Fusarium species. Trichoderma viride showed 45.88% growth inhibition on Fusarium merismoides fungi in a study by Hosen & Shamsi (2019), which support our present findings. Nasrin et al. (2018) also reported 82% inhibition radial growth by Trichoderma sp. against Fusarium oxysporum f. sp. lycopersici. In contrast, Bashar & Chakma (2014) reported that volatile substances produced by T. viride, A. niger, A. flavus and A. fumigatus showed 29.75, 20.15, 15.78 and 12.25% growth inhibition, respectively, on F. oxysporum.\n\nIn the current investigation, there are some limitations. Although this study showed that some plant extracts and nonpathogenic microbes could control the fungal stain, the number of plant extracts and microbes was limited. In addition, we used only methanol solvent for extraction and did not use other extractions. Moreover, we performed only in vitro techniques for antifungal potency screening and did not use any in vivo techniques. Therefore, we need to perform further studies to detect ecofriendly control this devastating fungal stain in the future.\n\n\nConclusions\n\nWe evaluated different biological control measures for the devastating Fusarium fungi. Various medicinal plant extracts and non-pathogenic microbes showed promising inhibitory activities on Fusarium sp. in vitro. These identified control measures of Fusarium species show the importance of further research on Fusarium taxonomy to decline the risk of Fusarium-caused fruit rot in Bangladesh.\n\n\nData availability\n\nFigshare: Effect of plant extracts on inhibition of mycelial growth of the Fusarium species. https://doi.org/10.6084/m9.figshare.13096262 (Hasan, 2020a). This project contains the images of Petri plates for each treatment condition.\n\nFigshare: Effect of antagonistic agents on inhibition of mycelial growth of the Fusarium species. https://doi.org/10.6084/m9.figshare.13096325 (Hasan, 2020b). This project contains the images of Petri plates for each treatment condition.\n\nFigshare: Effects of different plants extract by methanol on inhibition percentages of mycelial growth of the Fusarium species, https://doi.org/10.6084/m9.figshare.13134953 (Hasan, 2020c).\n\nFigshare: Effect of antagonistic agents on inhibition of mycelial growth of the Fusarium species, https://doi.org/10.6084/m9.figshare.13134962 (Hasan, 2020d).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nA previous version of this article is available on Figshare: https://doi.org/10.6084/m9.figshare.13012517 (Hasan, 2020e).\n\n\nReferences\n\nAkhtar J, Kumar V, Tiu KR, et al.: Integrated management of banded leaf and sheath blight disease of maize. Plant Disease Research. 2010; 25(1): 35–38. 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Publisher Full Text\n\nO’Donnell K, Sutton DA, Rinaldi MG, et al.: Novel multilocus sequence typing scheme reveals high genetic diversity of human pathogenic members of the Fusarium incarnatum-F. equiseti and F. chlamydosporum species complexes within the United States. J Clin Microbiol. 2009; 47(12): 3851–3861. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParveen S, Wani AH, Ganie AA, et al.: Antifungal activity of some plant extracts on some pathogenic fungi. Archives of Phytopathology and Plant Protection. 2014; 47(3): 279–284. Publisher Full Text\n\nTafinta IY, Shehu K, Abdulganiyyu H, et al.: Isolation and Identification of Fungi Associated with the Spoilage of Sweet Orange (Citrus Sinensis) Fruits In Sokoto State. Nigerian Journal of Basic and Applied Science. 2013; 21(3): 193–6. Publisher Full Text\n\nVethavalli S, Sudha SS: In vitro and in silico studies on biocontrol agent of bacterial strains against Fusarium oxysporum f. sp. lycopersici. Research in Biotechnology. 2012; 3(2): 22–31. Reference Source\n\nWhiteside J, Bennett J, Holtzblatt K: Usability engineering: our experience and evolution. In M. Helander (Ed.), Handbook of human-computer interaction. New York, North Holland, 1988; 791–817. Publisher Full Text\n\nZiedan ESH, Khattab AA, Sahab AF: New fungi causing postharvest spoilage of cucumber fruits and their molecular characterization in Egypt. Journal of Plant Protection Research. 2018; 58(4): 362–371. Publisher Full Text" }	[ { "id": "80330", "date": "13 Apr 2021", "name": "Natarajan Amaresan", "expertise": [ "Reviewer Expertise Microbial Diversity", "Plant-Microbe interaction", "Phytoremediation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors are reporting some interesting results which will be of significance to the readership however, the manuscript lacks indepth research.\nThe effect of the bacteria and botanical extracts should be tested in the pot studies for their effectiveness. In vitro studies alone will not warrant their suitability to be used as bioinoculants for management of pathogens.\nWithout the in vivo studies the work appears as shallow research. The authors may be encouraged to do pot studies and update the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "89306", "date": "26 Jul 2021", "name": "Shaikh Jamal Uddin", "expertise": [ "Reviewer Expertise Pharmacological evaluation of ethnomedicinal plants", "natural products chemistry and drug discovery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 presented the antifungal activity of some plant extract as a eco-friendly control measure for pathogenic Fusarium species using the poisoned food method. They found some plant extracts showed promising (>65%) inhibitory activity against Fusarium sp. and suggested to use as bioagents to control fungal affected fruit and vegetable rot.\nHowever, the authors can improve the article if they consider and revise the below points:\n\nAuthors did not mention any MIC values. It will be good if they included MIC values of each extracted.\n\nIn introduction section author might include a table on the selected plant with their traditional uses and list of active constituents.\n\nIn the discussion section, there is a lacking of discussion between antifungal activity of selected plant extracts and their traditional uses and reported phytoconstituents.\n\nIs the work 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": "96967", "date": "15 Dec 2021", "name": "Mustafa M. El-Zayat", "expertise": [ "Reviewer Expertise Microbiology", "Ecology", "Nanotechnology", "Phytochemistry", "Fermentation 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 research dealt with an important topic in the safe control of the fungus Fusarium sp. that causes damage to many types of fruits without the use of chemicals that represent a danger to the environment and health.\nThe author should indicate which species of Fusarium has been used.\n\nIt is too difficult to extract 100 grams of the plant using only 500 ml methanol.\n\nIt has been mentioned that E. coli is not pathogenic while it is pathogenic strain.\n\nThe photos need to be somehow more clear because its resolution is low.\n\nIs the work 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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1394
https://f1000research.com/articles/11-464/v1	27 Apr 22	{ "type": "Review", "title": "Latest developments and scope of Health Technology Assessment in India: Tapping into the future", "authors": [ "Eti Rajwar", "Shradha S. Parsekar", "Prachi Pundir", "Helmut Brand", "Angela Brand", "Eti Rajwar", "Shradha S. Parsekar", "Prachi Pundir", "Angela Brand" ], "abstract": "Health Technology Assessment (HTA) is a multifaceted approach for informing policy by considering social, medical, economic and ethical aspects in a systematic and transparent manner. HTA is an important tool for decision-making, priority-setting and for resource allocation, leading to Universal Health Coverage (UHC). The importance of HTA becomes more pronounced in developing countries such as India. In this review, we have summarised the status and future scope of HTA in India after referring to the scientific literature available in the form of journal articles and government reports. HTA supports prioritisation of healthcare services to be included in UHC and the provision of affordable and quality healthcare services in India. India established ‘HTA in India’ (HTAIn) in January 2017 as a starting point in HTA activities and this centre conducts HTA studies of various methodologies upon receiving requests from the different central and state departments. HTA in India is a relatively new concept and the processes are not streamlined compared to countries where HTA is established. Although an HTA manual is present for guiding the HTA process in India, there is lack of clarity on the HTA information considered for translating evidence into policy or practice. Additionally, there is a dearth of individual capacity to undertake high quality HTA in the country. HTAIn initiative showcases India’s political commitment towards achieving UHC and for HTA. However, there is an imminent need to expand the role of the technical partners and regional resource hubs in capacity building in HTA by involving the private sector in HTA processes.", "keywords": [ "India", "Health Technology Assessment", "Universal Health Coverage", "narrative review" ], "content": "Introduction\n\n“Universal health coverage” (UHC) as defined by the World Health Organisation (WHO) is “ensuring that all people have access to needed health services of sufficient quality to be effective while also ensuring that the use of these services does not expose the user the financial hardship” (UHC for Sustainable Development). UHC is fundamental to achieving the “Sustainable Development Goal (SDG) three that aims at ensuring healthy lives and promoting well-being for all at all ages” (UHC for Sustainable Development). The government of India is committed towards providing UHC to its 1.3 billion population by initiating improvement in the availability, affordability and quality of health services as highlighted in the last “12th five-year plan (2012–2017)”. The challenging task of providing healthcare to all its citizens is possible with optimal utilisation of resources.1,2\n\nHealth Technology Assessment (HTA) is a multifaceted approach for informing policy by considering social, medical, economic and ethical aspects in a systematic and transparent manner.2–5 HTA ensures that technology choices are well informed by consideration of evolving technologies, scientific evidence, effectiveness regarding patient-relevant benefits, unintended harmful effects, cost-effectiveness, social values and ethical and legal implications.3–7 Therefore, HTA supports decision-making and prioritisation of limited resources in order to implement and achieve a sustainable UHC and subsequently SDG 3. For the aforesaid reasons, the global development agencies, WHO, World Bank and other agencies are persuading healthcare authorities to use HTA as it is a prerequisite for priority-setting for mobilising the healthcare resources.5 HTA has been included by the government of India as a systematic tool for priority-setting and allocation of resources so that the questions related to ‘what’ to provide to ‘which’ population or subset of population and ‘how much’ to provide can be answered. As India is a diverse country with different health problems and healthcare demands, the provision of any healthcare policy and health technology should be based on many factors that include expected benefits, harms, financial, social, cultural, ethical and legal aspects. Such a comprehensive assessment is possible with the approach of HTA.\n\nThe growing demand and interest in HTA in India can be observed through the published literature and the healthcare reforms undertaken by the Indian government e.g., investment in HTA for streamlining evidence-informed decision-making and effective healthcare spending. Previously published literature included topics such as the importance of HTA and recommendations to strengthen HTA in India,7–9 the status of HTA policymaking in India,10 the need for an HTA database,11 and an instrument to map the status of HTA in some selected countries.12 As we progress ahead, the previous literature becomes outdated, and therefore, the objective of this narrative review is to summarise the status of HTA in India based on the currently available information. This will assist HTA organisations, clinical guideline developers, finance providers, industry, research institutions, patient organisations and other stakeholders in assessing the development of HTA to strengthen prospects.\n\nA literature search was undertaken in PubMed and Google Scholar to identify articles related to HTA in India. Additionally, a search was conducted for grey literature on government websites of the “Ministry of Health and Family Welfare”, “Health Technology Assessment in India” (HTAIn), “Department of Health Research” (DHR) and the “International Decision Support Initiative” (IDSi). The search was conducted in August 2019 and updated in March 2021. Reviews or reports describing the status and process of HTA in India were included.\n\nIndia has a complex healthcare system that includes public and the dominant private sector. Information on the healthcare system and healthcare expenditure in India can be found in the extended data.13 With only 1.2% of “Gross Domestic Product” (GDP) spent on the public health infrastructure, the provision of UHC is an ambitious feat, especially at a time of rising burden of non-communicable diseases in India in addition to infectious diseases (including COVID-19) and malnourishment.14 Government health spending as GDP (%) in India is similar to Indonesia (1.1% of GDP)15 but, lower than other low- and middle-income countries such as Bhutan (2.5% of GDP)16 and contrastingly different than that of 7.3% of the GDP in the UK.17 Therefore, prioritising resources based on evidence becomes important for improving efficiency and to get the maximum value for money.18 To address problems of inequitable and unaffordable healthcare and to move towards more effective allocation of resources, the government of India has shifted its focus towards the concept of HTA. Discussions about HTA have been incorporated in the government policies, viz. the 12th five-year plan and the National Health Policy.2,19\n\nIn India, it was stated that HTA, via a viable HTA system, would help in the decision-making process for allocation and proper utilisation of resources. It will provide more transparency related to treatment options for different patients with the same disease.8 HTA provides evidence about the effectiveness and affordability of newer drugs and technologies by comparison of the risks and costs, therefore, providing information about the indications of use for a newly introduced health technology to medical practitioners.20 The other applications for HTA in India include supporting development of a pricing strategy for newer drugs and technology for the entire nation or state; thereby helping in providing value-based pricing for the drugs and technology.1,18 Additionally, it will support in preparation of clinical practice guidelines for maximum efficiency of interventions.18 HTA assists the government in priority decision-making, and during purchase of health services from the private health sector. It is beneficial for providing evidence related to equity and social justice, which are important areas to focus on while making decisions regarding priority-setting for allocation of resources.1 The users and producers of HTA evidence in India are given in Figure 1.1,21\n\nFor institutionalising HTA and to support transparent evidence informed decision-making process, the DHR, India, established HTAIn in January 2017, which was formerly known as the “Medical Technology Assessment Board” (DHR HTAIn). HTAIn conducts full HTAs (various methodologies) upon receiving requests from the different central and state departments. HTAIn aids these departments with scientific evidence for proper resource allocation and priority-setting.\n\nHTAIn consists of three core bodies, the “HTAin Secretariat”, the “HTAIn Technical Appraisal Committee” or “HTAIn Technical Advisory Committee”, and the “HTAIn Board”. “Regional Resource Hubs” (RRH) and “Technical Partners” (TPs) are academic and research institutions, usually under the government (Central or State), which have capacity, expertise and experience in HTA. RRH and TPs are explained in detail under the section HTA networks in India.13 The user departments are the central and state health ministries, or any government healthcare provider or agency that are directly or indirectly involved in the health sector in India. The structure of the HTAIn is depicted in Figure 2.21 The detailed information about the HTAIn core bodies is given in the extended data.13\n\nHTA must be rooted firmly in research and scientific method. It employs principles of benefit-harm assessment and economic evaluation to identify beneficial and safe health technologies and allows assessing their incremental cost-effectiveness ratios. The various steps of HTA that are followed by HTAIn are listed in Figure 321; viz. the research proposal must be explicit, relevant and transparent. It must incorporate appropriate methods to assess benefits, harms and costs, safety and address the issues of generalisability and transferability. Table 1 lists the key data sources for HTA in India that are used for conducting HTA analysis.22 All key stakeholder groups should be included in the HTA process. Currently, an HTA undertaken by HTAIn, RRHs or TPs typically takes six months to one year or more for completion, followed by report publication and policy brief. More information on the steps is given in the extended data.13\n\n(Source Downey et al., 201822).\n\n\n\n• Demographics\n\n• Communicable and non-communicable diseases\n\n\n\n• Health Management Information System (HMIS), National Family Health Survey (NFHS), District House Survey Data (2015-2016), Sample Registration System (SRS)\n\n• Integrated Disease surveillance Program (IDSP), National Program for the Control of Blindness, ICMR Cancer Registry Program\n\nSince 2017, HTAIn has published six policy documents. Priority topics have been researched and the reports are available in the public domain (HTAIn Policy Documents).23 Other examples of HTA conducted in India are: a) HTA conducted by the “National Health System Resource Centre” in 2017 successfully demonstrated a reduction in the average cost of drug eluting cardiac stents from INR 121,000 (Approx. USD 1,650) to INR 29,600 (Approx. USD 405).23 b) To guide policy decisions for health innovations in India, HTA was conducted for one of the health innovations from India i.e. “FnCas9 Editor Linked Uniform Detection Assay” (FELUDA), to test its addition in the COVID testing policy. It was concluded that the FELUDA test was less costly and equally effective as an RT-PCR (“Reverse Transcriptase-Polymerase Chain Reaction”) test for COVID-19 diagnosis. Future HTAs, using field level effectiveness data were recommended for the FELUDA test, also, it was recommended that the scale up of this test would be more sustainable for the health system.24\n\nThe government of India is committed to institutionalising HTA as a part of the priority-setting exercise in the “12th five-year plan”, by the “National Institution for Transforming India (NITI) Aayog”. To achieve this, a “national program for capacity building in HTA” was initiated by the government.25 This was a locally tailored capacity building approach supported by the iDSI, active from June 2015 to 2019. Important components of this capacity building program were 1) Developing a framework for capacity building; 2) Mapping and engaging relevant stakeholders; 3) Assessing need, assets and gaps for capacity building in HTA; 4) Developing a capacity development response; 5) Implementing the capacity development response which was done by environment capacity development i.e., recognition of the political economy of HTA, building HTA capacity building and resource networks in India, developing a central nodal point for HTA in the country (the HTAIn), and individual HTA capacity building training; 6) Evaluating capacity development via a Monitoring Evaluating and Learning Framework; and 7) Measuring the impact of the capacity development program.25 Furthermore, workshops were held for discussing the need of HTA and sensitisation of clinician/stakeholders on evidence-based medicine.7,8\n\nThere is an apparent gap in the HTA curriculum in India with few institutions offering HTA as a component in regular undergraduate or higher educational courses. These courses are pertaining to the fields of medical technology, biostatistics, and health economics.26 HTA as an independent curriculum is not offered by any of the institutions across India; however, two medical institutions (“School of Public Health, Postgraduate Institute of Medical Education and Research”, Chandigarh and “Amrita Institute of Medical Science”, Kerala jointly with Ruskin University, UK) offer HTA certificate courses through a virtual platform.26\n\n\nDiscussion\n\nThis review was intended to summarise the currently available information on the status of HTA in India. HTA is in the early stages of development and is not a prerequisite for reimbursement by social health insurances such as “Pradhan Mantri Jan Arogya Yojana” (PM-JAY). There is an absence of information on the consideration and use of HTA during the preparation of PM-JAY packages, costing, premium and reimbursement rates.\n\nThe complex health system of India has imposed a challenge for successful application of HTA recommendations.10 Furthermore, the healthcare market in India is diverse and unregulated, and it may influence the implementation of HTA regulations. Political ideology influences decision-making related to resource allocation and use of technology in healthcare.27 There is a high data requirement for conducting HTA analysis; absence of a robust data infrastructure might prove to be an important challenge.10,27 Limited human resource capacity for health economics, mathematical modelling and evidence synthesis is an impediment. Other challenges are quality of data and data availability (e.g., non-availability of quality of life tariff), tackling transparency and ethical aspects of the data, refusal to comply with guidelines by stakeholders.10,27 The “National Health System Cost Database” was introduced in 2016, for conducting costing studies and economic evaluations by collecting data based on standard methodology.28 A pan-India “Cost of Healthcare Services in India study” has been commissioned by the DHR to reduce the gaps related to price setting and estimation of resource requirements. The findings of aforesaid study will be added to the “National Health System Cost Database”.29 Additionally, challenges for implementation of HTA in India are: questionable health system readiness, high out-of-pocket expenditure, appropriate dissemination of information, perspective of the medical and allied health professional communities, availability of mechanisms for monitoring and evaluation.1,19 Some of the barriers to institutionalisation of HTA, which complicate priority-setting were identified as multiple insurance schemes, fragmented healthcare, and rising healthcare costs.30\n\nAs mentioned by the “International Working Group for HTA Advancement”, an important aspect of HTA is its link to decision-making.3,4,31,32 Although there is a manual on how to perform HTA prepared by the DHR, there is no clarity on whether HTA information is considered for translating evidence into policy or practice. HTA in India is a relatively new concept and the processes are not streamlined as compared to countries where HTA is established. The mechanism or checklists for quality assessment of HTA reports in India is not available in the public domain. The “International Network of Agencies for Health Technology” (INAHTA) has developed a checklist for the assessment of HTA quality and is being used during preparation and assessing credibility of HTA for policy and practice (INAHTA Briefs, Checklists & Impact Frameworks). It does not provide an overall score for HTA, but indicates the necessary components, which should be present in the HTA report. There is no evidence of a legislation on HTA in India and absence of long-term academic courses or trainings on HTA.30\n\nTo achieve UHC, the government of India has planned to purchase health services from the private sector as one of its key strategies (considering it being an important stakeholder), thereby involving the private sector in the entire process. HTA will be used for prioritisation of the health services that are needed to be purchased from the private sector. The reaction of the private sector to the entire process is yet to be seen.1\n\nThe strength of this review is that it collates latest developments of HTA in India including comprehensive information about HTAIn. The consolidated information provided, can be utilised by researchers and stakeholders working in HTA. However, this review has limitations. First, our literature search was restricted to selected databases, and to English language publications, which may lead to a substantial selection bias. Second, several important documents may not be published through journals or websites, leading to a potential publication bias. We tried to limit this risk by including grey literature in our review. We may have missed aspects that are important today or in the future. Fourth, the included studies had their own weaknesses limiting the information in our review. For example, the information within the reports and studies were heavily focused on the expert opinion and there is possibility that the experts might have been chosen through personal contacts.\n\n\nConclusions and future of HTA in India\n\nIndia needs strong political commitment to introduce and maintain HTA. To make healthcare decision-making a transparent process, patient and public involvement (PPI) in such decision-making is an encouraging step. PPI in healthcare (e.g., HTA process) can give more insights into quality of life and problems they encounter. Additionally, it helps in making people aware of the healthcare-related cost and challenges, thereby enhancing group and individual responsibility towards health.33 In addition to the demand generation by HTAIn, states can prioritise topics for HTA, as they are responsible for health financing.27 The HTAIn should publish reports on the use of HTA in the country, which will help in understanding the impact of HTA in India. Similarly, HTA can be used for appropriately calculating reimbursement packages under PM-JAY and other health insurance in India. Making HTA mandatory for health insurance, the pharmaceutical industry and the medical device industry can bring transparency in pricing of drugs and devices.23\n\nThe quality assessment checklists from countries with established HTA (such as a checklist by INHATA) can be localised to Indian settings. Countries (such as India) at the formative stages of HTA processes can learn from the experience of nations where HTA is well-established. However, the methods followed in other countries with established HTA should be adopted with caution for India because of contextual diversity.27 Further research and action should focus on the extent of coverage, utility and quality of HTA, barriers to implementing HTA in India, and comparing HTA processes between countries, especially in the south Asian region. In addition, systematic education on HTA should be increased and a curriculum including the Indian HTA process should be developed.27 Training programmes for capacity building of human resources can help overcoming the shortage of trained manpower.\n\nThe path to a robust healthcare system built on the foundation of explicit evidence, evidence-based research and cost-effectiveness, and more transparent decision processes might be lengthy, but it holds a prominent future for the healthcare of India.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: Extended data for ‘Latest developments and scope of Health Technology Assessment in India: Tapping into the future’, https://doi.org/10.6084/m9.figshare.19203245.v1.13\n\nThis project contains the following extended data:\n\n• Healthcare system in India\n\n• Healthcare expenditure in India\n\n• Administrative structure of HTAIn in India\n\n• Methods for conducting HTA in India\n\n• A figure explaining the process of selecting HTA topic by HTAIn\n\nData are available under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0)", "appendix": "Acknowledgements\n\nWe would like to acknowledge Public Health Evidence South Asia, Prasanna School of Public Health, Manipal Academy of Higher Education for the logistics and administrative support.\n\n\nReferences\n\nRao NV, Downey L, Jain N, et al.: Priority-setting, the Indian way. 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PubMed Abstract \| Publisher Full Text\n\nPrinja S, Brar S, Singh MP, et al.: Process evaluation of health system costing - experience from CHSI study in India. PloS One. 2020; 15(5): e0232873. PubMed Abstract \| Publisher Full Text\n\nMacQuilkan K, Baker P, Downey L, et al.: Strengthening health technology assessment systems in the global south: a comparative analysis of the HTA journeys of China, India and South Africa. Glob. Health Action. 2018; 11(1): 1527556. PubMed Abstract \| Publisher Full Text\n\nNeumann PJ, Drummond MF, Jönsson B, et al.: International Working Group for HTA Advancement. Are key principles for improved health technology assessment supported and used by health technology assessment organizations?. Int. J. Technol. Assess. Health Care. 2010 Jan; 26(1): 71–78. PubMed Abstract \| Publisher Full Text\n\nKristensen FB, Husereau D, Huić M, et al.: Identifying the need for good practices in health technology assessment: summary of the ISPOR HTA council working group report on good practices in HTA. Value Health. 2019; 22(1): 13–20. PubMed Abstract \| Publisher Full Text\n\nCleemput I, Christiaens W, Kohn L, et al.: Acceptability and perceived benefits and risks of public and patient involvement in health care policy: A Delphi survey in Belgian stakeholders. Value Health. 2015; 18(4): 477–483. PubMed Abstract \| Publisher Full Text" }	[ { "id": "136107", "date": "30 May 2022", "name": "Lorna Guinness", "expertise": [ "Reviewer Expertise Health economics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is a narrative review looking at the status of HTA in India. It draws on existing English language literature (published and grey) to document the importance of HTA for India, the structure of HTAIn (the government funded body responsible for HTA at the national level), the studies conducted and the challenges faced for a successful HTA programme.\n\nI suggest that the methods for the narrative review are provided in more detail and some restructuring of the paper would help strengthen it. Specific suggestions are provided below:\n\nAbstract:\nSentence 2: HTA doesn’t “….leading to Universal Health Coverage” per se. Change this to something along the lines of “… in the journey to UHC”.\n\nLine3/4 - please add something to explain why it becomes more pronounced; e.g. because the health budget is so constrained.\n\n6th sentence (line 6) – Please explain that this is what HTA in India proposes to do rather than make it a statement of fact.\n\nLast line – regional resource hubs needs to be explained for the abstract; perhaps re-phrase this as regional government funded HTA resource centres.\n\nIntroduction:\n1st para, line 6/7 – please refer to the 13th five year plan (i.e. the current one).\n\n1st para, last line – do we really know that this is true? India has one of the lowest healthcare spends in the world relative to GDP. Optimal utilisation will help but healthcare for all is not necessarily affordable within current spending patterns. I suggest re-phrasing to reflect this.\n\n3rd para line 6 – your objective is to carry out a narrative review to summarise the status of HTA in India. Can you expand on what you mean by status e.g. institutional development; capacity development; methods development; research produced; decisions made?\n\nResources for the review (should this be called Methods?):\nYou provide a very brief summary of the literature search methods. Can you explain your methods in more detail? What search terms did you use – what concepts were you looking for in this search? And what time period did you cover? Did you also rely on expert advice/prior knowledge/ reference lists of papers identified? And finally, what information/topics did you look for in your review of the literature. Did you have any exclusion criteria (eg. Non English language). It would also be useful to state what a narrative review is (as opposed to a systematic review).\n\nImportance of the Indian healthcare system:\n1st para - This section is good background but does not really tell us about the status of HTA in India. I recommend shifting this to the background.\n\n2nd para – this seems to be a summary of the reasons/ rationale/proposals behind the introduction of HTA in India and the plans for it as laid out at the start. Please explain that this is the case. In addition, do you have a source for figure 1 or was this developed by the author team; please can you also explain if these are proposed users and producers or current users and producers.\n\nAdministrative structure of HTAIn:\n1st para – Please add the sources for all these statements.\n\n2nd para, last sentence – please add a summary sentence relating to the functions of each of these bodies.\n\nMethods used for conducting HTAs:\nThis section provides an overview of the procedural steps for the conducting HTA but does not provide detail on the actual methods followed; some discussion of the HTAIn reference case for Economic evaluation would be a useful reference here and some discussion of the non-economic aspects of HTA and whether these are included in the processes.\n\nHTA analysis studies conducted:\nPlease can you give some detail on what topics have been covered in the policy documents? It is not clear why the review focuses in on the results of two studies. It would be more useful to map out the topic areas covered across the HTAIn policy documents as well as these two studies; also there are some systematic reviews of economic evaluation in India which would help make this section complete (https://pubmed.ncbi.nlm.nih.gov/26449485/; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525785/).1,2 Is there any documentary evidence of the analysis results being taken up in decision-making? Also, can you add some reflections on how topics have been chosen and why?\n\nYour discussion makes some strong points. Perhaps this section should be titled: Current challenges to HTA in India\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? No\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "180958", "date": "18 Jul 2023", "name": "Kanchan Mukherjee", "expertise": [ "Reviewer Expertise Public Health", "Health Policy", "Economic Evaluation", "Health System Analysis", "HTA", "Pharmaeconomics. Detailed research interests can be viewed at: https://tiss.edu/view/9/employee/kanchan-mukherjee/" ], "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 attempts at documenting the current status of HTA in India based on a narrative review of literature (published and grey, in English language). It also attempts at providing insights for the future based on secondary literature alone. It is a good attempt but is lacking in scientific rigour and missing several key elements of HTA in India, which has implications for the future. This article would benefit from more in-depth assessment regarding the role and practice of HTA in India. Therefore major revisions are recommended. The major revisions required are as follows:\nDetails of the methods need to be made more explicit for making it replicable. The methods for the narrative review should be provided in more detail.\n\nThe scope of a secondary literature review for understanding HTA in India is limited since there are many unpublished HTA developments. Hence, the article could have benefited immensely from Key Informant (expert) interviews of individuals from academia, Indian Administrative Service (IAS) (from the federal and state level) and the private healthcare sector, who have been involved in HTA.\n\nWhile the study has focused on the national/federal body (HTAIn) for conducting HTA in India, the role of state governments (there are 28 states), private sector, universities involved in HTA, and the use of economic evaluation (which is the core element of HTA) in India for policy decisions at the state level and for the health systems is missing. It is important to capture this because of two reasons:\nA. In India, healthcare is a state specific subject as per the Constitution, which provides autonomy to state level policymakers to engage local HTA researchers and use the HTA /economic evaluation for health policy/ programme decisions and implement them. Some of these studies have also been published1,2,3,4 and their inclusion in this review would added value. The evidence from cost-effectiveness of childbirth strategies for PMTCT of HIV among mothers receiving Nevirapine1, resulted in the first use of economic evaluation for a health policy decision in India, in the state of Tamil Nadu in 2008. References 1-4 are also examples of successful evidence to policy translation in India.\n\nB. The recent (2020) new definition of HTA explicitly links the focus of HTA with health system performance. This definition has been developed by an International task force led by HTAi and INAHTA and provides the future directions for HTA work. Hence, including this definition would have helped shaped the discussion on the future of HTA in India.\nMy specific comments are as follows:\nThe review has not included or referred to the latest definition of HTA developed by the International task force led jointly by the HTAi and INAHTA and its relevance to health systems.5,6\n\nFigure 1: The authors can consider including ‘Federal government’ and ‘State governments’ as users of HTA. Also, authors have mentioned ‘stakeholders’- public and private insurance companies and hospitals as 'producers' of HTA. However, they are 'users' of HTA. The authors can also add academic universities as producers of HTA. The word ‘research’ in producers of HTA is ambigous. Do the authors mean research organizations?\n\nIn the section on methods used for conducting HTAs in India, the authors can include the use of mixed methods to conduct HTA in India. The use of mixed methods and its contextual important for HTA in India has also been published.7\n\nIn the section on 'HTA analysis studies conducted by HTAIn' the study 'b' was not conducted by HTAIn, but at the Centre for Health Policy, Planning and Management (CHPPM) of the School of Health Systems Studies (SHSS) at the Tata Institute of Social Sciences (TISS) (Mumbai).The reference 24 referred by the authors for this study also state the above. Hence, this needs to be corrected.\n\nIn the section, 'HTA capacity building in India' , the review mentions that HTA is not offered as an independent curriculum. However, HTA is a multidisciplinary activity targeting healthcare interventions (including technologies). Hence, academic programs offering the multidisciplinary perspectives, knowledge and skills required to assess healthcare interventions, fulfill the requirement of HTA capacity building. Related to this point, the review states in the discussion that there is 'absence of long term academic courses or trainings on HTA'. This statement needs correction. Currently, there is one university in India (and the only one in South Asia)- TISS, which offers HTA integrated with MPH curriculum specific to the Indian context. It is a two year post graduate MPH (Health Policy, Economics and Finance) programme developed in collaboration with the LSE, offered by the SHSS at TISS and started in 2010. Hence, there is limited availability of long term academic courses but not 'absence' of academic courses. The lessons learnt from this academic programme is also published7 and provides insights on the future of HTA in India.\nThe review has rightly identified the complex nature of HTA in India and hence, the conclusions would have benefited from context specific implications/recommendations for the future. For example, the review mentions that HTA would lead to UHC. However, there are countries wherein, HTA was institutionalized after the country had achieved UHC. Hence, the Indian context is very different. Therefore, a discussion on what specific contribution can HTA (with its current complexities) make towards the path of UHC in India would add value.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? No\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-464
https://f1000research.com/articles/11-237/v1	25 Feb 22	{ "type": "Opinion Article", "title": "On the development of B-Raf inhibitors acting through innovative mechanisms", "authors": [ "Luca Pinzi" ], "abstract": "B-Raf is a protein kinase participating to the regulation of many biological processes in cells. Recent studies have demonstrated that this protein is frequently overactivated in human cancers, especially when it bears activating mutations. In recent years, few ATP-competitive inhibitors of B-Raf have been marketed for the treatment of melanoma and are currently under clinical evaluation on a variety of other types of cancer. Although the introduction of drugs targeting B-Raf has provided significant advances in cancer treatment, responses to such ATP-competitive inhibitors remain limited, mainly due to selectivity issues, side effects, narrow therapeutic windows, and the insurgence of drug resistance. Impressive research efforts have been made so far towards the identification of novel ATP-competitive modulators with improved efficacy against cancers driven by mutant Raf monomers and dimers, some of them showing good premises. However, several limitations could still be envisioned for these compounds, according to recent literature data. Besides, increased attentions have recently arisen around approaches based on the design of allosteric modulators, polypharmacology, PROTACs and drug repurposing for the targeting of B-Raf proteins. The design of compounds acting through such innovative mechanisms is rather challenging. However, novel valuable therapeutic opportunities can be envisioned on these drugs, as they act through innovative mechanisms in which limitations typically observed for approved ATP-competitive B-Raf inhibitors are less prone to emerge. In this article, the most recent approaches adopted for the design of non-ATP competitive inhibitors targeting B-Raf are described, discussing also on the possibilities, ligands acting through such innovative mechanisms could provide for the obtainment of more effective therapies.", "keywords": [ "B-Raf", "allosteric inhibitors", "polypharmacology", "drug repurposing", "PROTACs", "drug discovery and development", "small molecule inhibitors." ], "content": "\n\nThe Serine/Threonine protein kinase B-Raf is one among the most widely studied targets for cancer treatment.1,2 Under physiological conditions, this protein participates as key player in the Ras-Raf-MEK-ERK signaling pathway to the regulation of a number of cellular processes.3,4 In cancer cells, B-Raf is often upregulated, especially when it bears activating mutations, thus promoting oncogenic cellular processes as uncontrolled proliferation, tumor growth and metastasis.5–9 Of note, several studies have reported that B-Raf is frequently mutated in human cancers,6,10,11 with more than forty oncogenic mutations currently being described for this kinase.12 For these reasons, wild type (WT) and mutant B-Raf proteins have gained remarkable relevance for the development of anticancer drugs over the last years. Several drugs selectively targeting B-Raf proteins have also been approved for the treatment of metastatic melanoma,13,14 providing remarkable advantages in therapeutic regimens, and are currently under evaluation against colorectal cancer.15 However, the therapeutic use of the majority of these drugs is still hampered by drug resistance and side effects issues, resulting in responses that often remain temporary and rarely complete, the median time progression, e.g., for Vemurafenib against melanoma being six to seven months.16 In particular, recent findings have demonstrated that drug resistance often characterizing approved B-Raf inhibitors is mainly driven by feedback deregulation and overexpression of several other kinases.17–20 Nevertheless, clinical evidences have demonstrated that the use of selected B-Raf inhibitors in therapeutic regiments can also result in Raf paradoxical activation, which promotes cellular hyperproliferation of certain secondary skin lesions.21–26 In particular, it has been reported that when a B-Raf monomer is bound to specific inhibitors (e.g., Dabrafenib and Vemurafenib), it can dimerize promoting the aberrant activity of a second drug-free protomer, which cannot be targeted due to protein conformational rearrangements.21 This event in turn promotes abnormal proliferation in cells harboring other oncogenic mutations, through the activation of the MEK–ERK pathway.27 In this context, major research efforts have been devoted so far on the development of novel ATP-competitive kinase inhibitors binding to different αC-helix conformations of B-Raf (i.e., αC-OUT and αC-IN) (Figure 1).13,21,22,28,29 However, the obtainment of clinically safe ATP-competitive inhibitors showing high selectivity towards selected kinases is often elusive. With regards to B-Raf, low efficacy deriving by non-optimal therapeutic windows and the establishment of allosteric priming could be observed, for example, for αC-IN binders, albeit they can abrogate aberrant Raf dimerization-derived activities, through the inhibition of both the monomer and dimer of the kinase.13,30,31 Examples of recently reported compounds binding to this type of conformation of B-Raf are the pan-Raf inhibitors RAF-265, MLN-2480, TAK-632, LY3009120, CCT196969 and CCT241161,32–34 some of them having been evaluated in clinical trials (e.g., ClinicalTrials.gov identifiers: NCT02014116, NCT01425008 and NCT00304525), also in combination with MEK blockers (e.g., ClinicalTrials.gov identifier: NCT01352273). On the contrary, αC-OUT B-Raf inhibitors demonstrated to provide good selectivity profiles and wider therapeutic windows compared to αC-IN binders.13,13,28 Examples of such compounds are, among the others, Vemurafenib, Dabrafenib and Encorafenib, which potently inhibit mutant B-RafV600E monomers, but resulted to be ineffective on tumor cellular contexts driven by aberrant Raf dimerization.13,28 Moreover, compounds binding to the αC-OUT conformation of B-Raf have also been reported to promote paradoxical activation.13,35 Besides, other studies have been focused on the development of compounds as PLX7904 and PLX8394, able to escape paradoxical activation of B-Raf (i.e., the so-called “paradox breakers”) and to overcome some of known resistance mechanisms associated to previously reported Raf inhibitors,36,37 the latter ligand currently being evaluated in Phase I/IIa clinical trials (ClinicalTrials.gov identifier: NCT02428712). The development of paradox breaker compounds is expected to provide significant impact to anticancer therapy, as they allow to efficiently modulate the activity of mutant B-RafV600E, while circumventing protein dimerization.38 However, such compounds might also result in limited efficacy towards cancers with mutated Ras, according to recent literature data.38 Despite the advantages that can be observed in latest-generation ATP-competitive inhibitors of this kinase, several evidences encouraged a number of research groups to develop B-Raf modulators acting through innovative mechanisms. These include, for example, the modulation of the kinase activity of B-Raf with type III and IV highly selective allosteric binders, as well as through multi-target approaches (i.e., polypharmacology) (Figure 1). The design of allosteric kinase inhibitors of B-Raf is particularly challenging, for example, due to missing activity data or the lack of crystallographic structures suitable for the investigations, whose “activation loop”, “activation segment” and “αC-helix” are often not clearly solved. However, recent advancements in crystallographic and in silico techniques will certainly help to overcome several of the issues currently encountered in kinase allosteric ligand design.39,40 The identification of allosteric inhibitors of B-Raf holds great premises in cancer therapy. Indeed, such targeting approaches are expected to help identifying ligands with higher selectivity towards B-Raf with respect to classic ATP-competitive binders, and to help to overcome drug resistance, similarly to as postulated for other kinases.41–45 Unfortunately, neither type III, nor type IV small molecule, allosteric modulators of B-Raf have been reported so far. However, potent type III inhibitors binding to an allosteric pocket in proximity to the regulatory αC-helix have been reported for several other kinases, such as BCR-ABL, MEK, EGFR and CDK2,46–52 providing valuable structural clues for the design of innovative B-Raf modulators. These results have also been fueled by the recent crystallographic resolution of type III allosteric inhibitors of mutant EGFR and MEK (e.g., see references 45,49,53). In this regard, our research group has recently reported the design of previously unseen type III allosteric inhibitors of the CDK2 kinase showing anticancer activity.46,54 Moreover, we have also reported the identification of structurally novel allosteric modulators of WT and double mutant EGFR, exhibiting inhibitory activity towards non-small cell lung cancer (NSCLC).47 More recently, our research group has demonstrated that B-Raf possesses an allosteric pocket similar to that of EGFRT790M, adopting a DFG-IN/αC-OUT conformation potentially druggable by type III modulators.55 Remarkably, the presence of such allosteric pocket in B-Raf has also been indirectly confirmed in a recent study by Cotto-Rios et al.56 In particular, in their study the authors firstly identified Ponatinib as an inhibitor of B-Raf within a drug repositioning campaign, this compound presenting a methyl-piperazine moiety that allocated into the allosteric site in proximity to the kinase regulatory αC-helix. Then, medicinal chemistry optimizations were also performed on Ponatinib, leading to the identification of a compound (i.e., PHI1) that showed selectivity towards Raf dimers in cancer cells.56 Together with previous considerations and literature data, the results of this study suggest that the design of allosteric inhibitors of B-Raf is feasible. Moreover, these results also suggest that the design of such allosteric ligands would also open to novel strategies enabling the full arrest of the B-Raf kinase activity, potentially either via single agents or combination therapies. Nevertheless, the results prospected in this study paved the way towards the identification of innovative B-Raf inhibitors among approved drugs (i.e., drug repurposing) (Figure 1),56,57 this approach being already navigated also with natural products and clinically safe candidates, on different medicinal chemistry research areas.58–64 Similar considerations can also be argued for the design of allosteric ligands binding to the B-Raf dimer interface (i.e., type IV). The interest on such type IV allosteric ligands for the targeting of both wild type and mutant B-Raf has steadily increased over the past few years, with a number of small polypeptides able to disrupt protein dimerization and transactivation being reported.43,44,65,66 In particular, Beneker et al.43 were among the first to report the identification of a small set of polypeptides binding to the Raf dimerization interface. The results achieved in their study not only demonstrated that such a type of targeting is a feasible endeavor on B-Raf, but also that type IV allosteric ligands could provide remarkable results when used in combination with known mutant-selective ATP-competitive inhibitors promoting paradoxical activation of the ERK signaling.43 On the same line, Gunderwala et al.44 more recently reported the identification of Braftide, a small polypeptide designed through a computational strategy blocking Raf dimerization. Notably, Braftide demonstrated to efficiently inhibit Raf dimerization and to provide degradation of the MAPK complex. Moreover, Braftide has also proved to synergize with Vemurafenib and Dabrafenib,44 further supporting the potential application of type IV allosteric B-Raf inhibitors on therapeutic regiments with approved ATP-competitive drugs. The possibility to identify allosteric ligands of this kinase could also open to novel therapeutic approaches promoting simultaneous blockade of B-Raf at different sites, for example, if used in combination with approved ATP-competitive drugs. Indeed, such a complementary therapeutic approach is being under study against EGFR-mutant lung cancer,52 and it is expected to provide also valuable opportunities for B-Raf targeting. Allosteric inhibitors acting at a site different to those of the type III and type IV ligands described above have already been investigated for other kinases, in some cases with promising results.67 In the specific case of B-Raf, the identification of these types of inhibitors is still at a preliminary stage, albeit remarkable therapeutic opportunities arising on these grounds could be envisioned for the near future.\n\nNovel valuable opportunities could also arise from the identification of compounds exhibiting activity on B-Raf, in selected combinations of targets. The therapeutic advantages deriving by the simultaneous modulation of multiple targets involved in the physiopathology of a disease, either by using a combination of drugs, or with ligands endowed with tailored polypharmacology properties, have already extensively discussed in literature.68–71 Indeed, the use of approved B-Raf inhibitors is now mainly framed in combined regiments including modulators of other therapeutic relevant targets. For example, the B-RafV600E inhibitor Dabrafenib in now mainly used for the treatment of patients with unresectable or metastatic melanoma in combination with Trametinib (a MEK ATP-noncompetitive modulator).72,73 Similarly, Encorafenib (a B-RafV600E inhibitor) is used in combination with Binimetinib for the treatment of the same diseases, since their approval in 2018.72 The importance of B-Raf as a therapeutic relevant target in polytherapies is also testified by the number of combinations including Vemurafenib, Dabrafenib and Encorafenib, with the MEK inhibitors Trametenib and Binimetinib, and Cetuximab that are currently under clinical evaluation, for example, against colorectal cancer (e.g., ClinicalTrials.gov identifiers: NCT03727763, NCT03693170 and NCT04673955). Besides, several clinical studies have also been reported on the investigation of mutant selective B-Raf inhibitors, with modulators of non-kinase proteins, one among the most studied being Hsp90 (e.g., ClinicalTrials.gov identifiers: NCT01657591 and NCT02721459).68–70 The selection of the most suitable targets for combination therapy approaches is generally driven by their involvement in relevant oncogenic processes. For example, several mechanisms by which tumor cells can exert drug resistance to B-Raf inhibitors derive by deregulation or overexpression of other oncoproteins,18–20,74,75 many of them being “clients” of Hsp90.76,77 Consequently, the simultaneous targeting of Hsp90 and B-Raf have represented an attractive strategy to overcome drug resistance to B-Raf inhibitors so far. Indeed, several biological studies and clinical evidence demonstrated that the inhibition of Hsp90 helps to overcome resistance to known blockers of B-Raf, and that their combined inhibition provides synergistic effects in different cancer-related contexts.78–80 In line with the polypharmacology concept, further advantages can also be envisioned for cancer treatment on the design of ligands endowed with multi-target activity (Figure 1).\n\nIn the case of B-Raf, several studies describing the design of multi-target ligands have been reported so far.81–86 In particular, Anighoro et al.81 reported in 2017 the identification of the first two compounds endowed with activity towards B-Raf and Hsp90, demonstrating that these targets share overlapping chemical spaces. The compounds reported in this study were identified by means of an integrated in silico strategy and represent valuable starting points for the development of innovative B-Raf/Hsp90 dual inhibitors, especially considering that they showed balanced multi-target activity and low molecular weight. Of note, Hsp90 and B-Raf belong to different protein families and present distinct binding site architectures, which makes the design of dual ligands of these proteins a difficult task. The interest around Hsp90 and B-Raf as partners in polypharmacology strategies has also been further explored more recently within an effort to identify ligands endowed with Hsp90/PDHK1/B-Raf multi-target activity.82 The identification of compounds with such a tailored polypharmacology profile would enable the modulation of multiple pathways important to survival and proliferation of tumor cells, thus resulting in more effective anticancer therapies. However, the obtainment of Hsp90/PDHK1/B-Raf multi-target inhibitors is very challenging, as several, often conflicting, structural requirements should be taken into account in the ligand design process.\n\nBesides, B-Raf has also been framed into multi-target ligand design projects including other kinases, such as VEGFR-2, p38α and EGFR.83–86 In particular, several studies reported the identification of dual inhibitors of the B-Raf and VEGFR-2 kinases.85,87,88 The rationale behind the selection of B-Raf and VEGFR-2 for the development of multi-target inhibitors stands on the fact that these proteins fulfill complementary leading roles on processes related to cancer development and progression.89,90 For similar reasons, research efforts have also been performed for the design of B-Raf/p38α dual inhibitors.86,91 Of note, continuous research has also been done for the targeting B-Raf and EGFR, either via combination of selective kinase inhibitors, or with polypharmacology ligands. For example, the inhibition of these targets has already been explored on colorectal cancer by means of combination of drugs, as drug resistance that derives by overexpression and activation of EGFR could be overcome through the blockage of B-Raf, according to recent studies.20,92 The design of B-Raf/EGFR dual inhibitors has also been probed as a strategy to overcome drug resistance observed on melanoma and colorectal cancers to approved B-RafV600E drugs, providing promising results. Whereas reservations have been very recently raised on the dual inhibition of these targets as a therapeutic opportunity for NSCLC patients.93 Although particularly challenging, possibilities on the identification of innovative B-Raf/EGFR dual inhibitors could also be envisioned on type III allosteric contexts. Indeed, B-Raf and EGFR present structurally similar type III allosteric pockets,55 which make them ideal candidates for the design of multi-target ligands, for example, by means of computational structure-based approaches as docking.94,95\n\nIn recent years, increased research interests have also arisen around PROTACs (i.e., proteolysis targeting chimeras) for targeting several therapeutic targets (Figure 1).14,96,97 Such an approach generally employs bidentate molecules bearing to two covalently bounded chemical moieties, one with high affinity towards the target of interest and the other recruiting specific components of the proteasomal degradation system, to promote selective intracellular proteolysis (e.g., an E3 ligase as VHL).98,99 At present, PROTACs with high substrate specificity have been reported for targeting different protein kinases,14 including also mutant B-Raf.14,100,101 One example of such B-Raf mutant-selective PROTACs comes from a recent study by Alabi et al.,102 in which the authors designed a compound (e.g., SJF-0628) showing high selectivity towards degradation of B-RafV600E. Although being still at their infancy, approaches based on the targeting of B-Raf with PROTACs technology are expected to provided novel valuable opportunities for cancer treatment. Indeed, such approaches allow to also promote complete removal of the protein scaffold other than blocking its catalytic functions, which might represent a valuable advantage over already reported ATP-competitive inhibitors.\n\nTargeting protein kinases, such as B-Raf, has provided several therapeutic advantages in cancer treatment so far, as also testified by the number of approved drugs and clinical candidates modulating the activity of these proteins that are currently under investigation.14 B-Raf has acted as a major player in this context, with some of its ATP-competitive inhibitors (e.g., Sorafenib, Vemurafenib, Dabrafenib and Encorafenib) being approved for the treatment of patients with unresectable or metastatic melanoma in the last two decades.14 Although B-RafV600E inhibitors provided remarkable advantages in anticancer therapeutic regimens, several limitations could still be envisioned for these compounds, the most relevant being the establishment of drug resistance, paradoxical activation mediated by Raf dimerization and transactivation, and low efficacy towards Ras mutated cancers.38 Different strategies based on classic ATP-competitive single-targeting approaches are still being under study to overcome such limitations, some of them showing good premises.13 However, novel, and perhaps more valuable, opportunities can be envisioned on approaches targeting the allosteric sites of B-Raf proteins. Indeed, such approaches have already demonstrated to provide remarkable opportunities on other therapeutic-relevant kinases exhibiting high structural similarity with B-Raf.52 Moreover, the activity of allosteric kinase inhibitors is less prone to be affected by insurgence of drug resistance deriving by site point mutations with respect to ATP-competitive binders,41 this being a significant advantage in anticancer therapy. Importantly, the modulation of B-Raf by means of allosteric ligands would open to complementary approaches including also already reported ATP-competitive inhibitors to promote more efficient arrest the kinase activity, which is expected to result in improved therapeutic outcomes.43,44 In the near future, increasing research efforts will also be addressed towards the identification of multi-target inhibitors modulating B-Raf activity. Indeed, the importance of polypharmacology approaches for kinase targeting is well established,103 as also testified by the increasing number of dual inhibitors targeting B-Raf reported in the literature over the last years.81–86 The selection of suitable combinations of targets for the rational design of B-Raf polypharmacology ligands is of primary importance in this context, the identification of those providing the highest therapeutic effectiveness being very often difficult. However, recent innovations on computational approaches are expected to aid on their identification.104 Similar considerations can also be drawn for the identification of inhibitors targeting B-Raf through innovative mechanisms among already approved drugs, as recently observed for Ponatinib.56 Moreover, approaches based on PROTACs technology are also expected to bring significant chemical novelty on future B-Raf inhibitors design, as such compounds exert their activity through molecular mechanisms that are completely different with respect to those of approved drugs and compounds under investigation. The design of either allosteric, polypharmacology or PROTAC ligands targeting B-Raf proteins is challenging, especially with respect of classic kinase APT-competitive binders. However, the recent advancements on understanding cancer cells biology and the improvements on experimental techniques and in silico approaches available for the analysis of information reported in public databases, will certainly facilitate the identification of novel B-Raf inhibitors acting through such innovative mechanisms.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nAuthor contributions\n\nL.P.: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing.", "appendix": "Acknowledgments\n\nThe author is pleased to acknowledge Professor Giulio Rastelli (University of Modena and Reggio Emilia) for the helpful advices and fruitful discussion.\n\n\nReferences\n\nDienstmann R, Tabernero J: BRAF as a Target for Cancer Therapy. Anti Cancer Agents Med. Chem. 2011; 11(3): 285–295. Publisher Full Text\n\nKarasarides M, Chiloeches A, Hayward R, et al.: B-RAF is a therapeutic target in melanoma. Oncogene 2004; 23(37): 6292–6298. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "125510", "date": "04 Mar 2022", "name": "Federico Falchi", "expertise": [ "Reviewer Expertise Drug design and drug development by means of computational techniques." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nice perspective, Pinzi summarizes the current state-of-the-art in the development of B-Raf inhibitors acting through innovative mechanisms. After a brief introduction about the traditional inhibitors (Atp-competitive) with their big limitations, Pinzi describes the most challenging approaches such as the design of allosteric modulators, the polypharmacology, and the PROTACs design with an eye to the computational strategies.\nThe manuscript is well understandable, offering both a biological and medicinal chemistry point of view. The references are adequate. I do believe that work can be accepted certainly for indexing.\nI have only a very few minor remarks:\nThe sentence below is too long:\nBesides, other studies have been focused on the development of compounds as PLX7904 and PLX8394, able to escape paradoxical activation of B-Raf (i.e., the so-called “paradox breakers”) and to overcome some of known resistance mechanisms associated to previously reported Raf inhibitors,36,37 the latter ligand currently being evaluated in Phase I/IIa clinical trials (ClinicalTrials.gov identifier: NCT02428712).\nPlease change for example as:\nBesides, other studies have been focused on the development of compounds such as PLX7904 and PLX8394, able to escape paradoxical activation of B-Raf (i.e., the so-called “paradox breakers”) and to overcome some of the known resistance mechanisms associated with previously reported Raf inhibitors.\nPLX8394 is currently being evaluated in Phase I/IIa clinical trials (ClinicalTrials.gov identifier: NCT02428712).\n\nThe sentence below is too long:\nNevertheless, the results prospected in this study paved the way towards the identification of innovative B-Raf inhibitors among approved drugs (i.e., drug repurposing) (Figure 1), this approach being already navigated also with natural products and clinically safe candidates, on different medicinal chemistry research areas\nPlease change for example as:\nNevertheless, the results prospected in this study paved the way towards the identification of innovative B-Raf inhibitors among approved drugs (i.e., drug repurposing) (Figure 1). This approach is already been conducted also with natural products and clinically safe candidates in different medicinal chemistry research areas.\n\nThe abbreviation NSCLC is not straightforward, the first time an abbreviation is used it should be explained.\n\nIn the image: - a B in B-RAF is missing - the bullets points are not all the same - sometimes a period is used at the end of the sentence, other times not\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "8111", "date": "26 Apr 2022", "name": "Luca Pinzi", "role": "Author Response", "response": "I would like to thank the Reviewer for their precious comments. The manuscript was carefully revised, and long sentences split to make the text clearer to readers, in agreement to the Reviewer suggestions. Moreover, the full name of the reported abbreviations was added in the text. Figure 1 was revised to remove typos as suggested and to improve the image contents." } ] }, { "id": "125513", "date": "15 Mar 2022", "name": "Andrew Anighoro", "expertise": [ "Reviewer Expertise Drug discovery", "computational chemistry." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author gives an overview of B-Raf as a known target for cancer related diseases. Then, provides an account of design strategies that may overcome some limitations of typical ATP competitive drugs.\nThe author mostly refers to B-Raf as a target validated by \"recent\" evidence/studies, while it has been described in the literature as an oncogene at least from the 90s and Vemurafenib began clinical testing in 2008 to be approved in 2011 by FDA followed shortly by other drugs. I don't think this makes the article less interesting, but I feel that B-Raf should be referred sometimes in the text with adjectives such as \"well established\" rather than \"recent\".\n\nWould it be possible to give the reader an idea of how the αC-helix is structurally related to the ATP binding site? Maybe through a sentence in the text and/or marking it in Figure 1?\n\nWhen writing “activation loop”, “activation segment” and “αC-helix” are italics and quotes necessary? αC-helix has already been used several times in the text up until then without quotes or italics.\n\nI think that the sentence \"recent advancements in crystallographic and in silico techniques will certainly help to overcome several of the issues currently encountered in kinase allosteric ligand design\" should be followed by at least mentioning some examples of techniques that the author is certain are going to help.\n\nCan it be explained more explicitly why Ponatinib/PHI1 do not qualify as allosteric inhibitors?\n\nWould it be possible to include a comment on the challenge of rationally designing kinase multitarget inhibitors given the selectivity issues mentioned by the author?\n\nThe first time that the term PROTAC is mentioned, it should be by the full name followed by the abbreviation, proteolysis targeting chimera (PROTAC).\n\n\"(e.g., an E3 ligase as VHL)\" Probably, CRBN should be mentioned as well. Both should be mentioned by their full name first.\n\nProbably, \"e.g.\" in Figure 1 when addressing E3 ligases can be removed as it makes it look like E3 ligases can be replaced by something else in the context of PROTACs. I am not aware if that's actually possible, but if that's the case, it should be mentioned in the text.\n\nIn the abstract and in the main text the word premises is used a few times instead of promise, I believe.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "8112", "date": "26 Apr 2022", "name": "Luca Pinzi", "role": "Author Response", "response": "I would like to thank the Reviewer for the comments and the precious suggestions. The manuscript was revised to better contextualize the development of innovative B-Raf targeting approaches, with respect to established efforts (e.g., ATP-competitive inhibitors development). Accordingly, the descriptions referring to literature data were also revised under a more contemporary perspective. A brief description of the main elements characterizing the ATP-binding site of the kinases was reported in the revised version of the manuscript. This helped to delineate how the regulatory αC-helix is related to the other structural regions in the kinases ATP binding site. Besides, Figure 1 was also revised to better highlight the position of the αC-helix, with respect to the hinge region in the B-Raf active site to which ATP-competitive inhibitors bind to, and to improve the image contents. The text was also revised to provide further insights on some of the experimental techniques and in silico approaches that are (and are also expected to be even more in the future) of help for kinase allosteric drug design efforts. Moreover, additional references to relevant literature data were included in the revised version of the manuscript to better contextualize the application of some of these techniques, on both B-Raf related research and a more general perspective. An explanation of how Ponatinib and PHI1 can be classified according to their experimentally derived binding mode is now provided in the text, as suggested by the Reviewer. As revised, the text is expected to help better contextualize the example reported on Ponatinib/PHI1, with respect to the type III B-Raf allosteric pocket mentioned within this article. The main challenges that might be faced when rationally designing multi-kinase inhibitors are now discussed in the text. Moreover, the main E3 ligases that have been exploited so far for the design of PROTACs are now reported within the text. The manuscript was carefully revised, to remove typos and to make it clearer to readers. Moreover, the full names of the reported abbreviations were added within the text where needed." } ] } ]	1	https://f1000research.com/articles/11-237
https://f1000research.com/articles/11-461/v1	26 Apr 22	{ "type": "Research Article", "title": "Evaluation of lead toxicity on the retina of pregnant rats and their pups: the possible ameliorative role of pomegranate juice", "authors": [ "Abd El-Fattah B. M. El-Beltagy", "Karoline K. Abdelaziz", "Amira M. B. Saleh", "Hassan I. H. Elsayyad", "Reham A. Gahnem", "Karoline K. Abdelaziz", "Amira M. B. Saleh", "Hassan I. H. Elsayyad", "Reham A. Gahnem" ], "abstract": "Background: Lead (Pb) exposure even at a low dose can induce functional and structural impairments in both humans and experimental animals. The present study evaluated the potential ameliorative role of pomegranate juice (PJ), as a powerful antioxidant fruit against histopathological and ultrastructural changes caused by Pb in the retina of pregnant rats and their neonates. Methods: 24 pregnant female rats were selected and randomly divided into four groups (n=6): control, PJ supplemented (100 μL PJ), Pb treated (18.5 mg / kg B.Wt), and Pb co-supplemented alternatively with PJ group. After birth, the mother rats, as well as their neonates at different ages (7, 14, and 21 days old), were dissected and the eyes were removed for histological, ultrastructural, and immunohistochemical investigation of the retina. Results: The obtained results revealed deleterious histological and ultrastructural lesions in all retinal cell layers of Pb-treated female rats and their offspring. Such lesions included hypertrophied cells of retinal pigmented epithelium (RPE) with pronounced vacuolated mitochondria and fragmented Bruch's membrane. The outer and inner segments of photoreceptors appeared fragmented and detached from the RPE. Additionally, the outer nuclear layer (ONL) and inner nuclear layer (INL) appeared disorganized with vacuolated cytoplasm and pyknotic nuclei. The immunohistochemical results displayed glial fibrillary acidic protein (GFAP) weak expression and P53 strong expression in the retinal sections of the Pb-treated group of female rats and their neonates if compared with control. Conclusions: PJ successfully alleviated the deleterious histological and ultrastructural as well as immunohistochemical changes induced by lead.", "keywords": [ "Lead", "Pomegranate", "Retina", "Histopathology", "TEM" ], "content": "Introduction\n\nLead (Pb) is a prominent environmental toxic heavy metal that has adverse effects on human health. Pb and its compounds can easily accumulate in air, water, and soil1. After absorption, Pb is transported to most body organs especially the liver, kidney, brain, and finally accumulates in bone throughout life.2,3 It had been reported that there is no safe dose of Pb exposure and even very low doses have toxic effects on both humans and animals.1 Moreover, prolonged exposure to Pb can cause neurological, hematological, cardiovascular, gastrointestinal, immunological, and fertility disorders.4 During gestation, the maternal stored Pb can cross the placenta by diffusion resulting in adverse effects on the developing fetuses.5 Another study revealed that there is a positive correlation between maternal and umbilical cord blood Pb levels, indicating the transfer of Pb from mother to fetus.6 Also, Pb can transfer to neonates during lactation via the breast milk.7,8 It had been claimed that neonates are more sensitive than adults to Pb.9 The toxic response for Pb depends on several factors, like the dose, the age, the life stage of a woman, duration of exposure, and health and nutritional status of the individual.10 Accordingly, Pb constitutes a persistent public health problem.11 Previous studies claimed that the primary site of action of Pb is the central nervous system (CNS).12,13 Due to the lineal correlation between eyes and the CNS, there is no doubt that the ability of Pb to resist the development of the CNS will also affect eye development in fetuses. Pb exposure leads to reduced sensitivity of photoreceptors, blurred vision and cataracts, and optic neuritis.14\n\nCertain fruits and vegetables contain high levels of phenolic compounds that typically act as natural antioxidants.15 Pomegranate (Punica granatum) is a sweet fruit that have an ancient history and is mentioned in many Holy Scriptures such as the Torah, the Bible, and the Holy Quran.16 The pomegranate fruit has essential polyphenolic compounds in different parts of the fruit that include the peel, seeds, and arils. Other compounds such as organic acids, sugars, minerals and vitamins have been reported in pomegranates by many instigators.17,18 Moreover, the peel part of pomegranate contains bioactive phenolic compounds such as ellagitannins, flavonoids, and proanthocyanidin as well as minerals and complex polysaccharides.19,20 The arils part contains 85% H2O, 10% total sugars, especially fructose and glucose, pectin, organic acids such as malic acid, ascorbic acid, and citric acid as well as phenolic compounds and flavonoids like anthocyanins.18 The pomegranate seeds are rich with total lipids such as linoleic, stearic acid, punicic acid, palmitic acid and oleic acid.21,22 Also, the seeds are rich with protein, minerals, vitamins, pectin, crude fibers, sugars, isoflavones and polyphenols.23\n\nVarious medicinal uses have been documented for pomegranate extracts.24,25 The pomegranate is widely used in various medicinal practices such as wound-healing,26 as well as having anti-tumor properties,23,27 anti-atherosclerotic capacities,28 antihepatotoxic agents,29 and in the amelioration of cardiovascular diseases.30 Moreover, it has been documented to have anti-diabetic,25,31 anti-inflammatory,32 and antimicrobial33 activities as well as ameliorative properties for general metabolic health.34,35 Accordingly, the present study was mainly designed to evaluate the potential ameliorative role of pomegranate juice against Pb-induced retinal cell damage in pregnant rats and their neonates.\n\n\nMethods\n\n\n\n- Ethical considerations: The ARRIVE guidelines were followed. All efforts were made to ameliorate the suffering of animals. This study was approved by the Bioethics Committee of Damanhur University, no. EA 23122, 2020. All experiments inclusive of animal handling and sacrifice were conducted as per the guidelines of the Bioethics Committee of Damanhur University.\n\n- Equipment: Light microscope fitted with digital canon camera, electron microscope, flow cytometry system (FL2-H), blender, microwave rotatory vacuum.\n\n- Chemicals: Lead acetate (C4H6O4Pb3H2O) in the form of white crystals, GFAP and P53 antibodies were purchased from Sigma Chemical Company, Cairo, Egypt.\n\nFresh pomegranates fruits were washed, crushed, squeezed using a blender and then treated with pectinase to obtain PJ and by-products. The PJ was filtered, pasteurized, concentrated using microwave rotatory vacuum and atmospheric heating process respectively, and stored at -20°C. 20 mL of concentrated PJ was diluted in 500 mL of distilled water and kept in refrigerator until use. A total of 2.5 mL diluted PJ includes 100 μL PJ, which is equivalent to 2.8 μmol total polyphenols was supplemented to rats each other day.36,37\n\nFor this study, 32 Wistar albino rats (24 females and 8 males) were obtained from the Holding Company for Biological Products and Vaccines (VACSERA, Cairo, Egypt). They were 2-3 months old and had an average weight of 180 g. Animals had the same housing conditions of a clean house, regular dark-light cycle, normal ventilation and an adequate, stable, balanced diet with water ad libitum. After an acclimatization period of two weeks, the animals were mated in the special matting cages (1 male: 3 females) overnight. After 3-4 days and ensuring of pregnancy via observation of vaginal plug and using vaginal smear method, pregnant females were separated from males. The pregnant rats were randomly divided into four groups (n=6): group 1, control; group 2, pomegranate juice (2.5ml of diluted PJ, every other day); group 3, lead (oral dose, 18.5 mg/kg B. Wt, each other day);38 and group 4, Pb & PJ (Pb alternatively with PJ). Researchers were aware of the group allocations. Groups 2, 3 and 4 were exposed to the appropriate dose of treatment from the 4th day of pregnancy till the end of weaning. The offspring (six from each group) were weighed postnatal and examined at the ages of one week, two weeks and three weeks. The mothers were also examined and processed at the end of weaning period. The study took place from August 2020 – August 2021. No rats were excluded from the final analysis.\n\nExperimental procedure\n\nInvestigated parameters\n\nData were expressed as mean + standard error, (n=6 per group). Statistical analyses were one-way ANOVA followed by post hoc test, performed in Excel using the data analysis – ANOVA function. Means in the same row with different superscript () are significantly different (p>0.05), significant at p-value>0.05, significant at p-value>0.01 and significant at p-value>0.001.\n\nHistological sections by hematoxylin and eosin\n\nThe animals were euthanized using di-ethyl ether and the eyes of mothers (at the end of weaning) and their neonates (7, 14 and 21 days old, 3 offspring for each age from each group) were removed immediately, washed in normal saline and fixed in 10% neutral buffered formalin.39 After fixation, the eye specimens were dehydrated with ascending grades of ethanol, cleared with xylene, and embedded in paraffin. A 5 μm circular cut sections was made up vertical to the corneal margin and parallel with the optic nerve. The sections of the eye were stained in Mayer’s haematoxylin and eosin and processed for investigation of the retina under bright field light microscope and photographed.\n\nImmunohistochemical labeling of glial fibrillary acidic protein (GFAP) and P53\n\n5 μm thick paraffin sections were cut, mounted onto positively charged slides, de-paraffinized and rehydrated in descending series of alcohol. Endogenous peroxidase activity was inhibited using 3% H2O2 in methanol for 40 min at room temp. The retinal sections were retained at 25°C and processed for antigen retrieval by digestion in 0.05 % trypsin. After thorough washing in Tris buffered saline (TBS), pH 7.6, the sections were treated with normal swine serum and divided into two groups for further incubation with the appropriate primary antibody. For demonstration of GFAP activity, the sections were incubated for 30 minutes with polyclonal rabbit anti- GFAP antibody (1:300 dilutions, DAKO Corporation, California, and USA). After three washes in TBS the sections were incubated for 30 minutes with anti-rabbit antibody raised in swine. After thorough washing with TBS the sections were incubated with horseradish peroxidase-rabbit anti-horseradish peroxidase complex (PAP) for 30 minutes and washed for 30 min. Other sections were processed for demonstration of P53 activity by incubation for 45 min with diluted 1:10 monoclonal primary antibody (Anti-p53; clone DO-7 Dako). Slides were then rinsed in PBS and subsequently incubated in the presence of the secondary antibody for 20 min. For all sections, the complex sites were shown brown using 3, 3 diaminobenzidine tetrahydrochloride with fresh hydrogen peroxide substrate. The sections were counterstained with Mayer’s haematoxylin, mounted and photographed by phase-contrast Olympus light microscope fitted with digital canon camera. Incidences of cellular accumulations of GFAP and p53 protein were determined for each group.\n\nThe ultrastructural investigation was mainly focused on the retina of the mother rats and their neonates at 21 days old. Immediately after dissection of the selected animals, small pieces about 1 mm2 of retina were placed in freshly prepared 2.5% cold glutraldehyde as a fixative for about 5 hours. Samples were then washed in two changes of cold phosphate buffer, PH 7.3 for 1 hour. The specimens were post-fixed for 1-2 hours in buffered 1% Osmium tetraoxide, followed by washing in buffer then dehydrated in ascending series of cold ethyl alcohol, cleared in propylene oxide and mounted in epoxy resin. Semi-thin sections of 0.7μm thickness were cut with glass knives on the 6000 MT RMC ultramicrotome. The sections were mounted on glass slides and stained with 0.25% toluidine blue. For the electron microscope preparations, thin sections were cut from a preselected area and mounted in copper grids and stained with lead citrate and uranyl acetate according to.40 The sections were then examined and photographed by a JEOL 1200 EX11 transmission electron microscope in the EM Unit at Mansoura University.\n\nSlices from retinas of all studied groups were taken, and cell suspension was prepared with Tris-EDTA buffer (pH7.4) (Sigma-Aldrich Co.). Cell suspension was fixed in ice-cold 96-100% ethanol (Sigma) at 4 °C overnight, centrifuged at 1,500 rpm for 10 min, and then resuspended in PBS containing 50 μg/mL propidium iodide (PI) (Sigma-Aldrich Co.). For each sample, since analysis was based on measurement of 10000 cells. Single cell suspensions were prepared from retinas from at least six rats of each of the age of the animals, and 1.5-3×106 cells were stained for expression of the designated lineage markers. Cells were harvested by centrifugation, washed in ice-cold PBS, and fixed in 80% ethanol that had been prechilled to 20°C. They were then re-pelleted and re-suspended at a concentration of 0.1-0.3 106/ml in PBS containing 18 mg/ml propidium iodide (PI; Sigma) and 8 mg/ml RNase A (Sigma; PI solution). After incubation in the dark for at least 1 h, cell cycle profile analysis was performed on 10,000-20,000 cells on a FAC Sort flow cytometer using the Cell quest analysis program (Becton Dickinson, Sunnyvale, CA, USA).\n\n\nResults\n\nThe mean body weights of Pb-exposed mothers were significantly lower (p<0.001) than control. On the other hand, the mean body weights of mother rats of group 4 (Pb and PJ) did not appear to be significantly different (p>0.05) compared with the control group. Also, for all studied neonates maternally induced with Pb (7, 14, and 21 days old), the mean body weights were significantly lower (p<0.001) than their corresponding ages of the control. The mean body weights of 7 and 14 day-old neonates of group 4 appeared to be non-significant (p>0.05) in comparison with those of control group, however a significant (p<0.05) decrease in body weight was still recorded in 21 days neonates (Table 1).\n\n Significant at p-value≤0.05,\n\n Significant at p-value≤0.01 and\n\n* Significant at p-value≤0.001. PJ: pomegranate juice; Pb: lead.\n\n(The software used to edit the images and labels: Adobe photoshopCS8me).\n\nAbbreviations: Inner limiting membrane (ILM), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), outer limiting membrane (OLM), photoreceptors cell layer (PCL), pigment epithelium (PE) and choroid (Ch).\n\nIn control and PJ supplemented groups of mother rats, the retinal sections appeared with normal histological architecture. On the other hand, the retinal layers of Pb-treated group showed obvious deleterious histological changes especially in the pigmented epithelium (RPE), photoreceptors cell layer (PCL) and ganglion cell layer, however mild changes were recorded in outer nuclear layer (ONL) and the outer plexiform layer (OPL). The RPE cells appeared hypertrophied with bulged peripheral nuclei. The PCL appeared detached from the RPE with fragmented and vacuolated outer segments. The nuclei of the ONL were aggregated in clusters and separated from the bases of photoreceptors. The OPL appeared relatively thin and disintegrated if compared with control. Moreover, the cells of GCL appeared hypertrophied, highly vacuolated and lost its normal striation pattern.\n\nIn Pb group of mother rats co-supplemented with PJ, the retina showed marked amelioration in its histological architecture. Such ameliorations were represented by restoration of retinal detachment and normal arrangement of photoreceptors without vacuoles. The RPE appeared normal with centrally located nuclei. Also, the ONL appeared homogenous and adjacent to the bases of photoreceptors inner segments. The OPL layer appeared compact and in contact with the ONL. The retinal ganglion cells become highly organized in one row and their axons formed a continuous nerve fiber layer.\n\nIn the different ages of neonates (7, 14, 21 days old) maternally induced with Pb, the RPE illustrated pronounced wavy and hypertrophied cells, detached outer segments of PCL. On the other hand, marked histological improvement was recorded in the retina of neonates of group 4 (Pb plus PJ) where the outer nuclear layer and the RPE were tight.\n\nChanges in RPE (Figure 2)\n\n(The software used to edit the images and labels: Adobe photoshopCS8me). Abbreviations: BI: basal in folding; BM: Bruch's membrane; CC: choriocapillaris; CP: cytoplasmic processes; DBI: descending basal infolding; DBM: disintegrated Bruch's membrane; GB: Golgi bodies; L: lysosomes; M: mitochondria; N: nucleus; OS: outer segments; Ph: phagosomes; RER: rough endoplasmic reticulum; SER: Smooth endoplasmic reticulum; V: vacuoles; VM: vacuolated mitochondria.\n\nIn control and PJ groups of mother rats, the TEM investigation revealed that RPE consists of a single layer of low cuboidal cells which have numerous basal (scleral) infoldings and apical (vitreal) processes enclosing the tips of outer segments of photoreceptors. The cells of RPE appeared rich with smooth endoplasmic reticulum (SER), lysosomes (L), and rough endoplasmic reticulum (RER). In addition, the phagosomes of photoreceptor outer segment are observed.\n\nIn Pb-exposed mothers and their neonates, the cells of RPE displayed multiple ultrastructural changes like fragmentized Bruch’s membrane, cytoplasmic vacuolation, vacuolated mitochondria, disintegrated basal infoldings and apical processes as well as loss of RER. Moreover, the nucleus appeared pyknotic with irregular nuclear membranes. On the other hand, the RPE cells of Pb-exposed rats co-supplemented with PJ displayed remarkable improvement in their subcellular architecture. Such improvements were represented by rebuilding of Bruch’s membrane and extension of the cytoplasmic processes in between the photoreceptors’ outer segments. The mitochondria appeared normal with obvious regular cristae. The SER and RER appeared regularly scattered in the cytoplasm especially near the nucleus. Further observation revealed that the nucleus becomes vesicular with their intact membrane.\n\nChanges in Photoreceptors and ONL (Figures 3 and 4)\n\n(The software used to edit the images and labels: Adobe photoshopCS8me). Abbreviations: BM: Bruch's membrane; CP: cytoplasmic processes; DIS: damaged inner segment; FOS: fragmented outer segment; IS: inner segment L: lysosomes; M: mitochondria; MD: membranous disc; N: nucleus; OLM: outer limiting membrane; ONL: outer nuclear layer; OS: outer segments; V: vacuoles.\n\n(The software used to edit the images and labels: Adobe photoshopCS8me). Abbreviations: ONL: outer nuclear layer; PN: pyknotic nuclei; V: vacuoles; VM: vacuolated mitochondria.\n\nOur results by TEM revealed that the photoreceptors of control and PJ supplemented mother rats and their offspring appeared regular, and their outer segments interdigitate with the cytoplasmic processes of the RPE cells. The outer segments (OS) of photoreceptors consist of organized membranous discs, however the inner segments displayed rounded to oval nuclei. The outer limiting membrane appeared intact. In Pb treated mother rats, the OS of photoreceptors appeared fragmented and separated from the IS. Furthermore, the IS of photoreceptors were dislocated from the ONL and became aggregated in clusters with pronounced loss of their mitochondria. The neonates of Pb-exposed rats didn’t show any changes in their photoreceptors.\n\nCo-supplementation of PJ to Pb-exposed rats successfully restored the deleterious ultrastructural changes induced by Pb, whereas the OS of photoreceptors displayed a regular pattern of membranous discs and the IS appeared in direct contact with the ONL. In Pb treated mother rats and their neonates, the ONL appeared with multiple pyknotic nuclei and fragmented cell membranes with wide intercellular spaces. Furthermore, the ONL of mother rats and their neonates of group 4 appeared normal with less intercellular spaces.\n\nChanges in INL of retina (Figure 5)\n\n(The software used to edit the images and labels: Adobe photoshopCS8me). Abbreviations: AC: amacrine cells; BC: bipolar cells; HC: horizontal cells; IPL: inner plexiform layer; MC: Muller cells; RER: rough endoplasmic reticulum; V: vacuoles.\n\nGenerally, among mammals, the inner nuclear layer is made up of four categories of neuronal cells: bipolar cells (BC), horizontal cells (HC), amacrine cells (AC) and Muller cells (MC). The BCs are numerous, rounded or oval in shape and each is prolonged into an inner and an outer process. The HCs lie in the outer part of the INL and possess somewhat flattened cell bodies. Their dendrites divide into numerous branches in the OPL, while their axons run horizontally for some distance and finally ramify in the same layer. The ACs are localized in the inner part of the INL, they have not yet been shown to possess axis-cylinder processes. Their dendrites undergo extensive ramification in the inner plexiform layer. The MCs display dark, elongated nuclei. The Muller cells form an almost continuous layer separating the sclerally located BC and HC from AC somata are mostly positioned at the vitreal side of the inner nuclear layer.\n\nIn lead acetate treated mother rats and their neonates, the INL exhibited marked degeneration of horizontal cells, bipolar cells and amacrine cells. Such cellular degeneration was represented by disintegration of most cytoplasmic organelles, nuclear pyknosis and damage of cell membranes. Furthermore, the cells of the INL of Pb-exposed rats post-supplemented with PJ (group 4) appeared normal with less vacuolation still found in the cytoplasm in some cells.\n\n(The software used to edit the images and labels: Adobe photoshopCS8me).\n\nThe arrows heads indicate the degree of immunoreactivity of GFAP and P53.\n\nImmunohistochemical reaction for GFAP\n\nThe immunoreactivity of GFAP appeared strong in the retinal sections of control neonates of rats if compared with their mothers. However, the retinal sections of Pb-exposed mother rats and their neonates displayed weak to moderate expression of GFAP if compared with control. Further observations revealed that the retinal sections of group 4 (Pb plus PJ) expressed a moderate to strong immunoreactivity for GFAP that was approximately similar to that of the control. For all studied groups the immunoreactivity of GFAP appeared prominent in ONL and INL rather than the other layers (Figure 6A-C1).\n\nImmunohistochemical reaction for P53\n\nThe retinal sections of control mother rats and their offspring showed negative to weak immune expression for P53 protein. In contrast, an intense reaction for P53 was observed in all retinal cell layers of Pb-exposed mother rats and their neonates. In Pb-treated mother rats post-supplemented with PJ and their offspring, the retinal sections showed pronounced decrease in the degree of P53 immuno-reactivity (Figure 6A2-C3).\n\nNote: highly % value of apoptotic cells in Pb-exposed groups of mothers rats and their offspring (83.12%, 50.12%) in comparing with control value (48.59%, 36.53%) respectively. Pomegranate juice successfully restored the mean % values of apoptotic cells near to the normal values.\n\nThe obtained flow cytometric results revealed that the mean percentage value of cellular apoptosis in the retina of Pb-treated mothers (83.12% versus 48.59%) and their offspring (54.84% versus 36.53%) was significantly different than that of control. Post-supplementation of PJ to Pb-treated group, the mean % values of apoptotic retinal cells were significantly decreased compared to the control values (50.12% for mother rats and 38.52% for offspring).\n\n\nDiscussion\n\nLead exposure is known to disrupt most of body processes due to its toxicity to our vital organs, particularly our bones, heart, kidneys and nervous system.1,2,41 However, there has been little research into its direct effects on eye, a fundamentally cognitive process. Due to the direct correlation between the development of eyes and the central nervous system (CNS), there is no doubt that the potency of lead can prohibit the development of the CNS will inevitably cause effects on the eye. These effects are collectively termed “optic atrophy” or “blurred vision”, appearing only in cases of lead poisoning severe enough to cause brain damage.14,42 Pomegranate has a protective role as a phytotherapeutic agent due to its active ingredients like tannins,43 phenolic acids44 estrogenic flavonoids and conjugated fatty acids.45,46 Accordingly, this work was done for the first time to evaluate the possible ameliorative effects of pomegranate juice against lead acetate-induced retinal toxicity in pregnant rats and their neonates. Previous studies revealed that Pb compound can induce toxic effects on the mothers and their pups due to its transfer from the mothers to their pups during gestation and suckling periods.8\n\nThe result of the present work revealed that the mean body weights of Pb-exposed mothers and their offspring were significantly lowered than the control group. However, post supplementation of PJ to Pb-induced rats the body weight was successfully restored near to that of control. The obtained results are in agreement with previous researchers.47,48 Aprioku and Siminialayi49 reported that the decreased body weights of embryos maternally induced with Pb compound may be attributed to oxidative stress-mediated deleterious alteration in the ovarian and/or placental functions which interfere with fetal nutrition and O2 utilization. Additionally, placental disorders may also result in inhibition of transport of essential compounds to the fetuses, and consequently inhibit the normal growth rate of embryos. Another study explained that the toxic ions are one of the factors which decrease the body weight via the mechanism involving disturbance in metabolic enzymes.50 Restoration of body weight after supplementation of PJ to Pb exposed group of mother rats and their offspring may be attributed to the antioxidant constituents of PJ which allow easy assimilation of all the nutrients found in the blood stream by the body.51\n\nSeveral histological lesions were recorded in the retinal cell layers of Pb-treated mothers’ rats and their offspring. Such alterations were represented by hypertrophied RPE, detached photoreceptors with fragmented outer segments and cytoplasmic vacuolation as well as the nuclei of the ONL and INL were aggregated in irregular clusters. Additionally, the GCL was highly vacuolated and lost its normal striation pattern. Fox and Chu52 reported that adult rats’ exposure to low-level Pb results in long-term selective photoreceptor deficits. Another study revealed that lead acetate or its derivatives could induce deleterious histological changes on the embryonic development of RPE and photoreceptors resulting in retinal detachment.53 Raafat et al.38 found that lead toxicity in mother rats could induce folding of pigment epithelium and fragmentation of photoreceptors outer segments. It had been postulated that the fragmentation of photoreceptors segments as well as appearing of cytoplasmic vacuolation under lead neurotoxicity is mainly due to rupture of bi-membranous discs and mitochondria.54 Elgohary et al.55 elucidated that aggregation of nuclei is an evident sign for pyknosis. Raafat et al.38 proved that animals that received lead acetate have swollen ganglion cell layer. The histopathological signs caused by lead acetate were more pronounced in the mothers and their offspring of 21 days old in comparing with the other two ages. Such variation in the degree of effects may be attributed to long period of exposure to lead acetate.\n\nThe results of the TEM study revealed that Pb led to the creation of deleterious ultrastructural changes all-over the retinal cell layers especially in the RPE and photoreceptor cell layers. In lead acetate treated mother rats and their offspring of 21 days old, the RPE cells displayed pyknotic nuclei, swollen and vacuolated mitochondria, vacuolated cytoplasm and remarkable disassembled Bruch’s membrane. Similar observations were recorded in the RPE of rabbit56 and rat neonates.57 Also similar alterations were noticed in the RPE of monkeys that were exposed to mercury54 and in humans exposed to cadmium.58,59 Vacuolation of the RPE cells post lead acetate-treatment may represent a type of cellular defense against its toxicities as well as a source of accumulating toxic agents interfering with its biological interactions in cell metabolism.60 Schlötzer-Schrehardt et al.61 reported that retinal detachment is mainly attributed to fragmentation of Bruch’s membrane.\n\nFurther observations by the TEM study revealed that the photoreceptors of lead acetate treated mothers and their offspring at 21 days old exhibited severe deleterious changes especially in mothers. Such changes were apparently represented by fragmented outer and inner segments with obvious vacuolation among them. Similar ultrastructural findings were recorded in retina of experimental animals after exposure to heavy metals.62,63 Furthermore, the previous experimental studies revealed that the low or moderate Pb dose could induce disorganization of photoreceptors in the mothers and children.64 Sanders et al.65 suggested that degeneration of photoreceptors under the influence of Pb is mainly due to the binding of lead and calcium to the internal metal-binding site of the mitochondrial transition pore, subsequently open the transition pore, and stimulate the cytochrome C-caspase activation cascade leading to death of photoreceptors. Moreover, the ONL and INL of lead acetate treated mothers and their offspring at 21 days old showed severe pyknotic nuclei, cytoplasmic lysis and vacuolation as well as remarkable wide intracellular spaces. Similar ultrastructural alterations were recorded in the two nuclear layers of adult rats66 and mice67 after exposure to low level of lead during gestation period.\n\nGFAP is an intermediate filament protein that is expressed by numerous cell types of the central nervous system (CNS) including astrocytes68 and ependymal cells.69 GFAP is involved in many important CNS processes, including cell communication and the functioning of the blood brain barrier.70 Also, GFAP has been shown to play a role in mitosis by adjusting the filament network present in the cell.71 Our results revealed that the retinal sections from Pb treated mothers and their offspring exhibited weak expression for GFAP compared with the control group. The reactivity for such protein was more confined to the nerve cell fibers of ganglion layer, Muller cells and ONL. Similar observations were recorded in the brain of rat offspring72 and adult male rats post-treated with lead acetate.73 Chen et al.74 reported that depletion of GFAP activity plays a crucial role in activation of caspase-3 which would lead to increased apoptosis.\n\nP53 is a tumor suppressor gene that in an inactivated form tends to be associated with a high risk of certain cancers and inhibition of apoptosis.75 Moreover, most evidence suggests that the key contribution of P53 to apoptosis is primarily dependent on transcriptional activity. P53 has the ability to activate transcription of various proapoptotic genes.76 Therefore, increased P53 activity can also trigger apoptosis by repression of antiapoptotic genes, such as surviving, thus promoting caspase activation.77 In compared with control, strong immunohistochemical reactivity for P53 protein was markedly observed all-over the retinal cell layers of Pb treated mothers and their offspring. According to the previous discussion, the decreased GFAP activity and overexpression of P53 reactivity in the retina of lead acetate groups confirmed an elevated risk of cellular apoptosis and that lead is the strongest neurodegenerative agent. Further examination to confirm the apoptosis was carried out by flow cytometric analysis to detect DNA damage. The results of the present work showed a significant increase in the percentage of cellular apoptosis in Pb treated groups in relative to control and pomegranate groups. Jia et al.78 revealed that lead acetate could induce a progressive loss in human mesangial cells viability together with a significant increase in the number of apoptotic cells using the assay of flow cytometry. Another study explained that Pb could induce oxidative stress, DNA damage and alteration of P53, Bax and Bcl-2 expressions in mice.79 The main mechanism of DNA damage caused by heavy metal ions, which contributed to genotoxicity, is the excess liberation of free radicals. These free radicals attack DNA double chains and break them. If the broken DNA strands cannot be repaired timely, it will affect the function of DNA and result in genotoxicity.80\n\nIn the present work, the pomegranate juice exerts ameliorative effects against the retinal histopathology as well as the immunohistochemical alterations induced by lead acetate. The pomegranate has been regarded as a “healing food” that plays a role in regulation of metabolism.80 It was commonly used as an anti-inflammatory, and in the treatment of ulcers, diarrhea, hemorrhages, microbial infections and respiratory diseases.32,81,82 Improvement of pomegranate juice against lead acetate treated retina may explain the reduction of inflammation, re-differentiation of retina and regulation of apoptosis as well as maintain normal pattern of GFAP and P53. All of these beneficial effects could be attributed to the polyphenols inclusion of pomegranate which has antioxidant capacity.\n\nDetailed studies are required to understand the mechanism by which pomegranate juice can exert beneficial effects on the retina.\n\n\nConclusion\n\nOn the basis of our findings, the oral administration of PJ to lead acetate-treated rats during gestation and lactation could ameliorate the damage to retinal layers and restore their structure as well as enhance the antioxidative defense system due to presence of bioactive polyphenolic compounds which play a role in scavenging free radicals, and also prevent DNA damage.\n\n\nData availability\n\nfigshare: Raw data for body weight. https://doi.org/10.6084/m9.figshare.19276061.v283\n\nThis project contains the raw data on rats’ body weighs.\n\nfigshare: Data of Flow cytometry. https://doi.org/10.6084/m9.figshare.19294352.v284\n\nThis project contains the flow cytometry data.\n\nfigshare: Underlying data.pdf. https://doi.org/10.6084/m9.figshare.19237473.v485\n\nThis project contains the images used in the manuscript.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe underlying image data for this study are too large to share openly. The image files are predominately JPG files, and approximately 200 MB (210,739,647 bytes) in size. Considering the large size and multiple images (n=280), the image files will be shared on request to readers. Please contact the corresponding author (beltagyaaa@yahoo.com) if you would like to request access to the image files. Representative images are shown in the figures and can be found in the Figshare repository.\n\n\nReporting guidelines\n\nfigshare: ARRIVE checklist for ‘Evaluation of lead toxicity on the retina of pregnant rats and their pups: the possible ameliorative role of pomegranate juice’. https://doi.org/10.6084/m9.figshare.19237293.v3", "appendix": "Acknowledgments\n\nThe authors would like to thank the Light and Electron microscope unit of Mansoura University, for providing facilities to bring to this work.\n\n\nReferences\n\nWani A, Ara A, Usmani JA: Lead toxicity; A review. Interdiscip. Toxicol. 2015; 8(2): 55–64. 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PubMed Abstract \| Publisher Full Text\n\nElbeltagy A: Raw data for body weight. figshare. Dataset. 2022. Publisher Full Text\n\nElbeltagy A: Data of Flow cytometry. figshare. Dataset. 2022. Publisher Full Text\n\nElbeltagy A: Undelying data.pdf. figshare. Figure. 2022. Publisher Full Text" }	[ { "id": "146540", "date": "30 Aug 2022", "name": "Matt Rutar", "expertise": [ "Reviewer Expertise I am a retinal cell biologist with particular interest in retinal degenerations and neuroimmunology. Techniques include cellular (histology", "cytometry) and molecular (RNA profiling) methods for interrogating changes in retinal pathophysiology." ], "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 by El-beltagy and colleagues sought to determine a retinal neuroprotective effect of pomegranate juice in rats exposed to lead toxicity. The authors investigated this by treating lead-affected mice with pomegranate juice and then performed comparative histological analysis on retinal tissue sections, as well as rudimentary cytometry and immunostaining. Though the overall premise of the study is reasonable, I have major concerns with methodology and analysis of the data throughout the manuscript. In general, there is a severe lack of (1) adequate quantification and presentation of the data, (2) appropriate description in the methods, and (3) mechanistic insight. The most glaring of these are detailed below:\nThe figures documenting histological changes in retinal structure are entirely qualitative, making the authors assertion of a protective effect of PJ questionable. Moreover, the representative retinal tissue sections presented are not clear and are of variable quality (Figure 1 in particular). At a minimum, the cell layer thickness, hypertrophy, or other parameters must be measured and quantified with respect to each group, and with appropriate statistical analysis included. Ideally, ERG recordings should also be included to determine whether PJ confers a protective effort with respect to vision function.\n\nThe GFAP and P53 staining figures are inadequate to draw a meaningful conclusion (Figure 6). The staining presented is difficult to resolve clearly, as is any change with regard to PJ treatment, and there is also no quantification of the data. GFAP staining should be apparent within GCL astrocytes and muller cell processes, though the data presented appears to show staining throughout the retina (particularly in Figure 6 C). The authors should also include in any negative or positive controls in their analysis (either as part of the figure or a supplementary).\n\nThe cytometry data are confusing (Figure 7). It is not abundantly clear (1) what they are actually analysing, (2) what the overall data show, and (2) how this was undertaken (methods for this are threadbare at best). I assume that it would be apoptotic cells, though it is not described how the data presented in Figure 7 relate to this. The figure appears to show merely the raw data taken from the cytometer, with no indication of what retinal cell type they assessing, and no apparent statistical correlation between groups. The figure also does not mesh with the text description – the data show ‘counts’, though the text refers to ‘percentages’.\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": [] }, { "id": "148791", "date": "05 Sep 2022", "name": "Hidehiro Oku", "expertise": [ "Reviewer Expertise Neuroprotection." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reviewer has some concerns about histological findings using light microscope.\n\n1. Figure 1. It seems very hard to segment each retinal layer in rats 7 days after birth. In addition, some vacuolar changes exist in the INL even in the control rats of 14 and 21 days after birth.\n2. Muellar cells are primary glial cells in the retina which run transversely throughout whole retinal layers. Please correct the following description: “the inner nuclear layer is made up of four categories of neuronal cells: bipolar cells (BC), horizontal cells (HC), amacrine cells (AC) and Muller cells (MC)”.\n3. In addition, please discuss less immunoreactivities to GFAP in rats treated with Pb exposure. Retinal injuries almost always accompany activation of Muellar cells and increase immunoreactivities to GFAP.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-461
https://f1000research.com/articles/11-460/v1	26 Apr 22	{ "type": "Study Protocol", "title": "Effectiveness of comprehensive cognitive and balance training strategies (CCBTS) on gait, balance, quality of life, and fall-related self-efficacy among institutionalized elderly in Udupi District: A mixed-method study protocol", "authors": [ "Anil Raj Assariparambil", "Judith Angelitta Noronha", "Anice George", "Anil Raj Assariparambil", "Judith Angelitta Noronha" ], "abstract": "Background: The geriatric population is at high risk for gait and balance-related issues as they grow older. The gait and balance-related issues then causes them to be more vulnerable to a fall. Following a fall, the psychological impact could result in fear of falling and decreased quality of life (QoL) in later life. This mixed-method study protocol was developed to evaluate the effectiveness of cognitive and balance training on gait, balance, fall-related self-efficacy, and QoL among institutionalized older adults. Methods: A sequential explanatory study has been designed with phase I as quantitative (QUAN) and phase II as qualitative (qual). A randomized controlled trial will be conducted to determine the effectiveness of combined cognitive and balance training among older adults. An in-depth interview will be conducted in phase II with thematic analysis to determine the phase II objectives. A nested sampling technique will be used, wherein the phase II participants will be selected from phase I. In phase I, the intervention arm will receive both cognitive and balance training whereas the control arm will receive only balance training. The phase II participants will be selected from the institutionalized older adults who show improved and not improved fall-related self-efficacy and QoL after comprehensive cognitive and balance training strategies (CCBTS) training. The outcome variables included in the phase I study are gait, balance, fall-related self-efficacy, and QoL. Conclusions: Cognitive training and balance training as a stand-alone intervention has proved their impact among older people. The gait and balance issue could impact from the consequences of both cognitive decline and physical decline; hence the proposed research would highlight the need for combined intervention to enhance overall well-being among the geriatric population. Registration: This study is registered with The Clinical Trials Registry - India (CTRI); CTRI reference ID: CTRI/2016/11/007449; registered on 08/11/2016.", "keywords": [ "Circuit-based exercise", "Gait", "Postural balance", "Quality of life", "Aged", "Fear", "Randomized Controlled Trial", "Qualitative research" ], "content": "Introduction\n\nOver the past two decades the age structure of the world’s population has taken a momentous shift due to declining fertility and mortality (Private, Health, Conference, & Insurance, 2014). Globally, the portion of older population (aged 60 years and above) has increased from 9% in 1994 to 12% in 2013, which is expected to reach 21% by 2050 (UN, 2013).\n\nThe annual growth rate of the geriatric population will be almost triple the growth rate of the total global population. In absolute terms, the percentage of the geriatric population has almost doubled between 1994 and 2014. From 1994 to 2014, there has been a major increase in the number of older adults in Asian countries (225 million), accounting for almost two thirds (64%) of global growth (UN, 2014).\n\nIn India, an individual aged 60 years and older is known as a ‘geriatric population’ (Giri, 2011). The demographic summary portrays that, in the years 2000-2050, the total population in India will rise by 55%, while the population of senior citizens will increase by 36%. Census of India 2001 data showed that older adults made up 7.7% of the whole population, which increased to 8.14% in the 2011 census. The predictions for the older population in the succeeding four censuses are: 133.32 million (2021), 178.59 (2031), 236.01 million (2041) and 300.96 million (2051) (MOHFW, 2011). The geriatric population in India has gone up from 6.0% in 1991 to 8.0% in 2011, whereas in Karnataka the total percentage of older adults constitute 8.4% (Census of India, 2011).\n\nDue to advancing age, older adults usually complain about forgetting names, dates and appointments. Another major effect of age-related change is cognitive decline and its impact on gait and balance (Doumas, Rapp, & Krampe, 2009; Segev-Jacubovski et al., 2011). Impairment in any of the executive functions like visuo-spatial perception, self-awareness, response inhibition, attention or dual tasking may affect one’s ability to walk efficiently and safely (Salthouse et al., 2003; Salthouse, 2005).\n\nAmong the Indian geriatric population, fall prevalence was about 14-53%. The factors contributing to the occurrence of fall are multifactorial and age is one of the factors (Krishnaswamy & Usha, 2006). World Health Organization (WHO) declared fall among older adults as a public health burden because of the highest number of injuries and death related to fall (Todd & Skelton, 2004).\n\nThe geriatric population usually limit their daily activities due to psychological consequences of fall, i.e., fear of falling. The incidence of fall among the geriatric population does not normally occur during their normal walk, whereas when they are doing a secondary task (talking while walking, concentrating on other things, changing an object, walking across a busy road etc.) along with walking, they are more prone to a fall incident (Silsupadol, 2008).\n\nFear of falling among older adults may also lead to reduced activity, enhanced decline in physical functioning, and general diminishment of quality of life (QoL). Furthermore, restricting mobility because of fear of falling may itself be a risk factor for falls. The strong and consistent association of fear of falling with reduced QoL is an important public health issue (Kato et al., 2008).\n\nA systematic review done in the USA (Kueider, Parisi, Gross, & Rebok, 2012) on computerized cognitive training with older adults, shows that compared to traditional cognitive training programmes, computer assisted cognitive training programmes have numerous benefits like customized training based on individual ability and can be used as a recreational modality among institutionalized older adults. There is an usual misconception about older adults that they won’t enjoy learning how to use new technology. Regardless of many older individuals reporting anxieties about using new technology at the commencement of training, after training most described great levels of understanding (Lee, Chen, & Hewitt, 2011).\n\nA study conducted (Smith-Ray et al., 2015) in Chicago, with an objective to evaluate the effectiveness of a cognitive enhancement training intervention on gait and balance among cognitively intact older adults revealed that there was a significant improvement in Berg Balance Scale (BBS) score (F (1, 31) = 4.709, p = .038), gait speed (F (1, 29) = 6.57, p = .016) and mean distracted gait speed (I; μ = –0.86, C; μ = –0.39) in the intervention arm compared to the control arm.\n\nA study was done by Verghese, Mahoney, Ambrose, Wang, and Holtzer (2010) in New York to evaluate the effectiveness of cognitive training on gait among sedentary older adults. It concluded that compared to baseline, gait velocity during usual walking (68.2 ± 20.0 vs 76.5 ± 17.9 cm/s, p = .05) and talking while walking (36.7 ± 13.5 vs 56.7 ± 20.4 cm/s, p = .002) improved a lot after the cognitive remediation.\n\nAn investigation was conducted by Theill, Schumacher, Adelsberger, Martin, and Jäncke (2013) in Switzerland to find the combined effect of physical and cognitive training among older people. The study results show that the concurrent training of cognitive and physical capabilities facilitated to progress cognitive and motor-cognitive dual task performance. Between the groups there was a significant difference in gait velocity reduction (F (1.58) = 3.165, p = .05, R2 = .098) but not in increase of step-to-step variability, offering better potential on activities of daily living among older adults.\n\nA systematic review was conducted at Manipal, Karnataka (Dsouza, Rajashekar, Dsouza, & Kumar, 2014) to identify fall prevention strategies for older adults. They reviewed a total of 18 research articles. They report that the common strategies adopted to prevent fall among the geriatric population are nutritional interventions, visual and depth perception, balance training, balance and mobility training, ankle exercises, balance training under dual task conditions with graded sensory context, yoga, and Ayurveda.\n\nMany research studies were done to improve motor strength as well as gait and balance among older adults. At the same time new research is being done to improve the gait and balance through cognitive training as well. But the geriatric population are an age group where both physical and cognitive decline occurs and we need to address it together, not as stand-alone issues. So, this research is interested in evaluating the combined efficacy of cognitive and physical training in improving gait, balance, fall-related self-efficacy and QoL among institutionalized older adults, as no such studies have been done so far among the Indian geriatric population.\n\nThe literature search and the researcher’s experience in dealing with older adults revealed that, older individuals are prone to have gait and balance disturbance due to aging. As age increases there will be physical as well as cognitive decline, which results in impairment in gait and balance, thus increasing the risk of fall among them. Fear of falling is the major hindering factor contributing to the lack of confidence in doing daily activities among older adults, which in turn leads to poor QoL.\n\nTherefore, the researchers felt the need to conduct a study to evaluate the effect of comprehensive balance and mobility training strategies among institutionalized older adults to improve gait, balance, fall-related self-efficacy and QoL. The proposed research study will help older adults to build confidence to manage their activities of daily living.\n\nThe objectives of the study are to:\n\n• determine the effectiveness of comprehensive cognitive and balance training strategies (CCBTS) in terms of improvement in:\n\n▪ gait\n\n▪ balance\n\n▪ fall-related self-efficacy\n\n▪ QoL\n\n• describe the experiences of institutionalized older adults undergoing CCBTS with regard to change in (improved and not improved) QoL and fall-related self-efficacy.\n\n\nMethods\n\nBased on the nature of the problem being studied, and the purpose and objectives of the study, a mixed method approach was found to be feasible to evaluate the effectiveness of CCBTS in improving gait, balance, fall-related self-efficacy and QoL among institutionalized older adults. This study protocol is reported in line with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines (Assariparambil et al., 2022b).\n\nFrom the different research designs under the mixed method paradigm, explanatory sequential design (Creswell & Clark, 2018) quantitative (QUAN) → qualitative (qual), is found suitable for the present study as the researcher was interested to find reason or in-depth information (in the qual second phase) from the positive-performing exemplars and outlier results of the quantitative (randomized controlled trial (RCT)) first phase.\n\nPhase I: QUAN phase\n\nFor the first phase of the mixed methods explanatory sequential study, an RCT is found to be appropriate, as the study focuses on evaluating the effectiveness of CCBTS in terms of improvement in gait, balance, fall-related self-efficacy and QoL among institutionalized older adults. Phase I of the study has two arms, i.e., intervention and control arm. Subjects in the intervention arm will receive both cognitive and physical training as the treatment along with routine care and the control arm subjects will receive physical training alone along with routine care (Machin & Fayers, 2010) (Jadad, 1998). Participants will be informed that trial enrolment is purely on a voluntary basis, and they can quit or discontinue the trial at any time. Once participants are allocated to the study arms, they will not be allowed to change the arm. If any participants would like to withdraw from the study, they can do so by informing the trial coordinator/researcher. As the intervention is done under the supervision of the researcher, it would enhance the intervention.\n\nType of randomization: To reduce the chance of encountering selection bias and to ensure balance in participants’ allocation within the subgroups, a restricted randomization scheme, Stratified Block Randomization will be adopted.\n\nStratification: Age wise strata is considered for the study. A total of three strata will be included based on the participant’s age; 60-65 years: Strata I, 66-70 years: Strata II, and 71-75 years: Strata III.\n\nSequence generation: The random allocation sequence will be generated adopting the principles of probability by using an online randomizer software, Research Randomizer (Research Randomizer, RRID:SCR_008563), and with the help of a statistician.\n\nAllocation concealment: Allocation of the participants to the groups will be concealed until the end by employing Sequentially Numbered, Opaque, Sealed Envelopes (SNOSEs). SNOSEs will be prepared by an external person, who is not directly involved in this project. The prepared SNOSEs will be kept with the research supervisor for the central allocation. Upon completion of the screening assessment, the research supervisor will be contacted to get the allocation status of those who meet the eligibility criteria and provide consent based on the strata in which the participant belongs to.\n\nBlinding: The outcome assessors will be unaware of the allocation status, and they will do the post-test assessment at the 5th and 10th week of intervention. Inter-rater reliability of the outcome assessors needs to be established before the outcome assessment.\n\nManipulation/intervention\n\nThe manipulation/intervention in this study i.e., CCBTS refers to a combination of cognitive and balance training program given to the institutionalized older adults with a purpose of enhancing their gait, balance, fall-related self-efficacy and QoL. It includes:\n\nCognitive training: In this study three computer based cognitive training games will be used to target the cognitive domains like visuospatial working memory, speed of processing, inhibition and attention. It is a researcher supervised training, it includes Double Decision, divided attention and target tracker developed by Posit Science, Brain HQ.\n\nBalance training: In this study balance training refers to researcher-supervised exercises intended to improve muscle strength and balance among institutionalized older adults. This training is adopted from the National Health Service (NHS) training for older adults, it includes exercises like sit to stand, mini squat, calf raise, sideways walking, and one leg stands.\n\nThe experimental arm will receive a combination of both cognitive and balance training along with routine care as the researcher intends to determine the combined effect of both cognitive and balance training in improving the functional status of institutionalized older adults. Cognitive exercises will be practiced by the participants along with the researcher using laptops in a face-to-face, one-to-one interaction. The balance training will be done after the cognitive exercises.\n\nPhase II: qual phase\n\nTo accomplish the objective of the second phase of this mixed method study, a qualitative approach will be adopted. The aim of the qual strand is to understand in depth experiences of institutionalized older adults in undergoing CCBTS intervention program in view of its impact over fall-related self-efficacy and QoL. In-depth interviews will be conducted among institutionalized older adults to determine who shows improvement and who does not show any improvement in fall-related self-efficacy and QoL in the intervention group. A thematic analysis (TA) method of the qualitative design will be adopted for the present study. TA (Braun et al., 2019; Braun & Clarke, 2006, 2019; Clarke & Braun, 2017) is a qualitative method aimed at understanding the pattern or themes within qualitative data collected from the participants. Table 1 represents the schematic representation of the study design.\n\nResearch setting\n\nInstitutions for older adults in Udupi District. As per the ministry of social justice and empowerment, Government of India directives, all care homes across the country have to be registered with their respective district administration. The list of care homes will be obtained from district senior citizen welfare department under the district administration.\n\nPopulation\n\nInstitutionalized older adults of Udupi District will be the target population.\n\nSample and sampling technique\n\nThe sampling technique will be based on the advanced typology of mixed methods sampling design by Onwuegbuzie (2007), a nested sampling technique will be adopted for this study.\n\nPhase I: QUAN: A purposive sampling technique will be used to recruit potential trial participants initially. Institutionalized older adults who meet the sample inclusion criteria will be recruited. Later those participants will be randomly assigned in to experimental or control arm.\n\nPhase II: qual: A criterion sampling under the purposeful sampling technique (Patton, 2005) is found to be most appropriate for the qual strand of this mixed method study. The criteria used in this study is from the phase I intervention arm, institutionalized older adults who show improved and not improved fall-related self-efficacy and QoL after CCBTS training.\n\nPhase I: QUAN\n\nInclusion criteria\n\n➢ Aged 60-75 years\n\n➢ Those who are able to read or write English or Kannada\n\n➢ Score of ≥ 26 on the Montreal Cognitive Assessment (MoCA)\n\n➢ Score of ≤ 8 on the Short-form Geriatric Depression Scale\n\n➢ Muscle strength of ≥ 4 for the lower limb as per the Medical Research Council London\n\nExclusion criteria\n\n➢ Presence of balance or walking impairment\n\n➢ Those who completed a balance training program within the previous year\n\n➢ Visual acuity of < 20/80\n\n➢ On psychotropic medications\n\n➢ Score ≤ 74 on Modified Barthel Index (MBI)\n\n➢ Diagnosed with chronic health conditions (symptomatic cardiac condition, post stroke, parkinsonism, cancer patients on active treatment, diagnosed vestibular disorders)\n\nPhase II: qual\n\nInclusion criteria\n\nInstitutionalized older adults from the phase I intervention arm, those who show improved (reduction in Falls Efficacy Scale (FES) score ≥ 20 and increase in QoL score ≥ 15 from baseline) and not improved (reduction in FES score ≤ 10 and increase in QoL score ≤ 10 from baseline) fall-related self-efficacy and QoL after CCBTS training.\n\nSample size: (QUAN)\n\nSample size is calculated by using the following formula\n\nZ1-α/2 –1.96 at α 0.05 level of significance\n\nZ1-β – 0.84 for power of 80%\n\nσ-Standard deviation of the observation = 1.3 based on previous study (Nitz & Choy, 2004)\n\nd – Clinically significant difference =0.8 for TUG Scale\n\nAttrition rate = 30%\n\nCalculated sample size is 60 in each group (three strata, block size: eight, total blocks: five)\n\nFor the qualitative approach, data will be collected after the 10-week intervention, from the intervention arm, each stratum (i.e., from those who improved and did not improve QoL and fall-related self-efficacy) until data saturation occurs.\n\nThe trial participants are the residents of the care homes and they are performing the intervention under the direct supervision of the researcher. Repeated contact and frequent follow-ups will be made, hence drop outs can be minimized. To promote the retention of participants, frequent reinforcements will be given, and they will be encouraged to continue with the exercise and cognitive training.\n\nData collection instruments\n\nInstruments used for screening the participants:\n\nPhase I: QUAN\n\nTool 1: Sociodemographic proforma\n\nThis tool (Assariparambil et al., 2022a) is developed by the researcher to collect sociodemographic data from the institutionalized older adults. The proforma consists of 20 items such as age, sex, religion, educational status, marital status, previous occupation, number of children, and residential area of family.\n\nTool 2: Basic health check-up\n\nThe basic health check-up tool (Assariparambil et al., 2022a) comprises of 13 items to determine general well-being of the institutionalized older adults. The items included are height (in cm), weight (in kg), body mass index (BMI) (kg/m2), Heart rate (HR), Respiratory rate (RR), and Blood pressure (BP).\n\nTool 3: Modified Barthel Index (MBI)\n\nMBI is a tool derived from Barthel Index (Mahoney & Barthel, 1965) instrument that aims to measure an individual’s ability to perform activities of daily living and mobility. A score ≤ 74 in MBI is an exclusion criterion of this trial.\n\nTool 4: Short-form geriatric depression scale\n\nThe short-form geriatric depression scale (Sheikh & Yesavage, 1986) is a self-rating scale that consists of 15 dichotomous items used to assess symptoms of depression among the older population. Individuals with a score ≥ 8 will not be included in this trial.\n\nTool 5: Montreal Cognitive Assessment (MoCA)\n\nThe MoCA is an instrument used to screen cognitive abilities of an individual to determine mild cognitive impairment. The total score that can be obtained is 30 and scores ≥ 26 are considered as normal thus institutionalized older adults with scores ≥ 26 will be included in this study.\n\nTool 6: Muscle strength examination\n\nMuscle strength examination of the participants will be assessed based on the Manual Muscle Test (MMT), a standardized test that is used to assess muscle strength and function (Jellinger, 2001). As per the MRC muscle strength assessment, any institutionalized older adults with ≥ 4 score will be enrolled in the trial.\n\nInstruments used for outcome assessment\n\ni. Balance\n\nTool 1: The Berg Balance Scale (BBS)\n\nBBS (Berg et al., 1989) is a 14 item instrument used to assess the balance and fall risk among individuals.\n\nTool 2: Timed Up and Go Test (TUG)\n\nThe TUG (Podsiadlo & Richardson, 1991) is a standardized test used to determine the mobility, balance, walking ability and risk for fall among older adults.\n\nTool 3: Functional Reach Test (FRT)\n\nFRT (Berg et al., 1992) is used to determine the patient’s stability in balance and functional mobility.\n\nTool 4: Lateral Reach Test (LRT)\n\nLRT (Brauer et al., 1999) is used to determine the medio-lateral postural stability of older adults to identify balance ability as a predictor of risk for fall.\n\nii. Gait\n\nTool 1: 10-meter walk test (10 mWT): Gait Speed\n\nTo identify the functional mobility and gait of an older individual, a 10 mWT (Bohannon et al., 1996) is found to be effective and will be adopted for the study.\n\nGait speed and distracted gait speeds: during walking 10 meter at a usual or comfortable pace as well as engage in a secondary visuospatial task while they walk at a usual or comfortable pace.\n\niii. Fall-related self-efficacy\n\nTool 1: Tinetti FES\n\nTinetti FES (Tinetti et al., 1990), is a standardized instrument used to determine the level of fear about fall among older adults.\n\niv. QoL\n\nTool 1: WHOQOL-OLD tool\n\nA standardized instrument developed by WHO to assess the QoL among older adults (Power et al., 2005).\n\nPhase II: Interview guide\n\nThe in-depth interview (IDI) guide for the qual strand of the study will be prepared by the researcher and finalized after validation by experts. The main lead question is to describe the experience of older adults in undergoing CCBTS in improving or not improving fall-related self-efficacy and QoL.\n\nEthical considerations\n\nAs the present study involves human participants, the researcher ensures that it will follow all the ethical principles laid down by the World Medical Association (WMA) as the Declaration of Helsinki (World Medical Association, 2013). Hence, the researcher will ensure that there is no breach of rights of participants during the conduct of the study.\n\nApproval: The study was approved by Kasturba Medical College and Kasturba Hospital, Manipal ethics committee on March 2016 (IEC 130/2016).\n\nAdministrative permissions: Permissions to conduct the study will be obtained from the Head of the institutions, and the administrators of the care home who will permit to conduct the study.\n\nTrial registration: This study is registered with The Clinical Trials Registry - India (CTRI);CTRI reference ID: CTRI/2016/11/007449; registered on 08/11/2016.Complete study information will be given to the participants through the ‘Participants Information Sheet’ and written informed consent will be obtained from the participants who are willing to participate in the study. The data monitoring committee from the IEC will monitor the adverse events, protocol deviations, and trial conduct.\n\nPlan for data analysis\n\nPhase I QUAN data will be analysed using SPSS version 15 (SPSS, RRID:SCR_002865) after entering the coded data into the software. Only the research team will have access to the stored data in the password protected personal computer of the researcher. Descriptive statistics will be used for analysing sample characteristics. Inferential statistics like Chi Square, and repeated measures of ANOVA will be used to measure the outcome variables within and between the groups. Intention to treat analysis and per protocol analysis will be computed as per the data availability and missing data. Phase II qual data will be analysed using TA with NVivo 12 software (NVivo, RRID:SCR_014802) using the institutional subscription.\n\nDissemination\n\nThe research team has planned to disseminate the findings of the study through various national and international geriatric domain specific scientific conference presentations. Team has also planned to publish two scientific papers from the study findings in peer-reviewed, indexed, international journals and institutional repository. The significant study findings will also be discussed with various stakeholders, NGOs and institutional administrators to enhance QoL at care homes.\n\nStudy status\n\nOngoing study is closed for participant recruitment and follow-up is going on with the study participants (both experimental and control arm). Second phase qualitative strand data collection is ongoing.\n\n\nDiscussion\n\nPopulation aging is a demographic phenomenon faced by many countries across the globe at a different pace. India is one of the fastest aging regions both in low- and middle-income countries (LMICs) and in the world. However, the impact of an aging population is more or less the same across all regions (Rechel et al., 2009). The health trend among the older population is very unique, as in, some regions there is a declining trend in severe disabilities whereas in some other regions there is an increasing trend (Oliver et al., 2014). The pace with which age-related changes appear among the older population is also not the same among older people. While dealing with older adults it is essential to understand the common age-related normal changes among them. Out of all age-related changes among the older population, cognitive and physical decline have a major impact among older individuals (Harada et al., 2013). During the process of normal aging, cognitive abilities such as processing speed, attention, visuospatial abilities, executive functioning, conceptual reasoning, and memory decline gradually over time. However, the changes are not homogenous among older adults (Deary et al., 2009). This normal cognitive aging or age-related decline in cognitive abilities will eventually make older adults at high risk for impaired dual tasking and subsequently putting them at risk for fall and fall-related injuries (Hedden & Gabrieli, 2004). Thus, every attempt was made to enhance the cognitive abilities of older adults by engaging them in cognitive retraining so as to build their cognitive reserve. One way this will help them to be cognitively active and the other way it should aim at preventing the consequences of cognitive decline, balance, and gait issues among them (Salthouse, 2012; Salthouse, 2010). The present study protocol is also addressing the major concern of the geriatric population, the cognitive decline. In this study the researcher intends to identify the effect of cognitive training in gait speed, balance, fall-related self-efficacy and to enhance QoL among older adults.\n\nAge-related decline in physical activity is another important contributing factor towards the issue pertaining to mobility and balance among the aging population. Due to a decrease in muscle mass and strength, the geriatric population experiences nearly 40-60% decline in their physical activity (Gregory et al., 2013). Hence, there is an increased likelihood of issues related to impaired physical activity such as balance issues, gait abnormalities, and fall-related injuries among older adults (Gopinath et al., 2018; Notthoff et al., 2017; Taylor, 2014; Virlando Suryadinata et al., 2020). Studies have even proved that if anyone is physically active, their momentum of cognitive decline would be very slow when compared with ones who are physically inactive. It is also proven that if a person is physically and cognitively active it will have a positive impact on their physical, emotional, and social well-being, hence aiding to a better QoL (Gill et al., 2013). With the present study, the researcher is addressing another important aspect of older adults, the physical decline by employing the muscle strength and balance training through various exercises for the institutionalized older adults.\n\nAccordingly, the main focus of research studies in the field of geriatric medicine and gerontological nursing is to overcome the undesirable impacts of age-related changes and to enhance the overall wellbeing among the geriatric population (Gardette et al., 2007). A systematic review was conducted by Cadore et al., (2013) to determine the impact of various exercises on fall risk, gait, and balance issues among older adults. The review includes various exercise regimens like multi-component exercise program, resistance, endurance and balance training exercises. Results of the review summarised that, out of 10 trials seven found less fall-incidence, out of 11 trails six of them revealed a better gait stability, and seven out of 10 trials showed an enhancement in balance among older adults. The review concluded that the physical training by means of multicomponent exercise regimen is found to be effective in improving physical abilities among the older population. A systematic review of RCTs conducted by de Labra et al., (2015) on exercise interventions among older adults recommended that for improving the executive functionality of older people, physical training with targeted cognitive training would be better as it has got greater impact than physical and cognitive training alone. Out of multiple studies carried out among older adults to improve gait and balance as well as to prevent fall, the majority of them are either with physical exercise interventions executed separately and it often is not combined with cognitive training. Rather than an isolated implementation of cognitive or physical training, a combination of both is assumed be significantly beneficial in improving the walking and cognitive functionality among older people. Therefore, the researchers are interested in evaluating the combined effect of cognitive and balance training among older people in terms of gait, balance, and fall-related self-efficacy, which are all very common age-related issues among the geriatric population, and eventually to enhance their QoL.\n\n\nConclusions\n\nBeing the second most populous region in the world, ‘population aging’ the demographic phenomenon is more prominent in India and the issues and concerns related to the older population are also increasing over time. Amongst all the normal age-related changes among older people, cognitive decline and physical dependence are the most common and significant concern in the field of geriatrics. However, it would be better to treat both cognitive decline and physical strength as one component rather than treating it separately, as they complement each other and then there would be a better positive result. If the combination of physical and cognitive training is found to be effective in improving the gait, balance, and QoL among older individuals, it could be proposed to be implemented in all care homes by sensitizing various stake holders for effective implementation of such training program among older adults.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: PERSONAL PROFILE.docx (tool to collect sample characteristics and demographic profile). https://doi.org/10.6084/m9.figshare.19494506 (Assariparambil et al., 2022a).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: SPIRIT checklist for ‘Effectiveness of comprehensive cognitive and balance training strategies (CCBTS) on gait, balance, quality of life, and fall-related self-efficacy among institutionalized elderly in Udupi District: A mixed-method study protocol’. https://doi.org/10.6084/m9.figshare.19497503 (Assariparambil et al., 2022b).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.", "appendix": "Acknowledgments\n\nWe would like to acknowledge the contributions of all doctoral advisory committee members for their guidance and suggestion in conceptualizing the study.\n\n\nReferences\n\nAssariparambil AR, George A, Noronha J: PERSONAL PROFILE.docx. figshare. 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Dual-Task Training on Balance Performance Under Dual-Task Conditions in Older Adults With Balance Impairment: a Randomized, Controlled Trial. A Dissertation. 2008. (September).\n\nSmith-Ray RL, Hughes SL, Prohaska TR, et al.: Impact of Cognitive Training on Balance and Gait in Older Adults. J. Gerontol. Ser. B Psychol. Sci. Soc. Sci. 2015; 70(3): 357–366. PubMed Abstract \| Publisher Full Text\n\nTaylor D: Physical activity is medicine for older adults. Postgrad. Med. J. 2014; 90: 26–32. PubMed Abstract \| Publisher Full Text\n\nTheill N, Schumacher V, Adelsberger R, et al.: Effects of simultaneously performed cognitive and physical training in older adults. BMC Neurosci. 2013; 14: 103. PubMed Abstract \| Publisher Full Text\n\nTinetti ME, Richman D, Powell L: Falls efficacy as a measure of fear of falling. J. Gerontol. 1990; 45(6): P239–P243. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "161518", "date": "20 Feb 2023", "name": "Bart Visser", "expertise": [ "Reviewer Expertise Human Movement Sciences" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper describes a protocol for a mixed-method study. The quantitative part focus on the additional value of cognitive training to balance training and is set-up as randomised clinical trial (RCT). The qualitative part has a focus on the experiences of the participants were a distinction will be made between those who have improved and those who didn't with respect to quality of life and fall-related self-efficacy.\nThe Phase 1:QUAN RCT is well described in general. I have only a minor comment:\nFrom the description it is not completely clear if participants of the experimental group receive their balance training separate or together with the control group.\nThe Phase II: qual need some clarification:\nHow is improvement (no improvement) defined?\nAre there cut-off points defined in advance for each outcome measure?\nOr are quartiles / deciles used\n\nAre subjects included who (not)improve on both QoL and Fall-related Self efficacy or on either of the outcomes.\n\nIf QUAN shows no effectiveness, what will be the rationale of including only subjects from the intervention arm?\n\nWhy not include subjects from the control group anyway, so you can get insight of the added value of the cognitive training.\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": [] }, { "id": "179174", "date": "17 Jul 2023", "name": "Ragab Elnaggar", "expertise": [ "Reviewer Expertise Physical Therapy and Health Rehabilitation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a mixed-method study protocol that was developed to evaluate the effectiveness of cognitive and balance training on gait, balance, fall-related self-efficacy, and quality of life among institutionalized older adults. Although the study protocol plans for an interesting topic, I have some major/minor concerns that the authors need to address before resubmission. Please find them below.\nThe introduction section is poorly written. It doesn’t properly provide background information or context on the topic.\n\nI can see too much space devoted to the definition of the “elderly population” and current and expected future epidemiology. This can be revised for brevity and conciseness.\n\nThe conceptual basis for the combined intervention (cognitive and balance training) is not clear. Authors need to provide a synopsis of the potential implications of the combined intervention in the “Introduction” and elaborate on this point in the “Discussion”.\n\nA better rationale would lead to the statement of the study hypothesis, which is currently lacking.\n\nWhy the study objectives are put down using “bullet” points? Unless this is a journal requirement, it should be revised and re-written as plain text.\n\nMixed methods research, which combines both quantitative and qualitative research methods, has gained popularity in recent years due to its ability to provide a more comprehensive understanding of complex research questions. However, there are some disadvantages and limitations. In terms of generalizability, mixed-methods research may not be as generalizable as single-method research. The qualitative data collected may not be representative of the larger population, and the quantitative data may not capture the nuances and complexities of the research question. Also, researcher bias can be a concern in mixed methods research, particularly in the qualitative phase. Researchers may be more likely to focus on qualitative data that supports their hypotheses, leading to a confirmation bias. I would like to see a comment from the authors in this respect.\n\nThe authors will adopt a stratified randomization approach and create three age-based strata (60-65, 66-70, and 71-75). I am curious to know what drove them to consider this stratification. Should there be a difference between the elderly population in these age groups that might affect the results?\n\nAs I understood, the authors planned for a two-arm study (intervention arm: combined training and control arm: balance training). This would not adequately offer a conclusive analysis of the intervention effect. I would have liked it if the authors considered three arms (balance training vs cognitive training vs combined training).\n\nAlthough this is a protocol for a study that is ongoing or about to be undertaken, the interventions and assessment procedures should be described more clearly so that they can be easily understood and replicated.\n\nQualitative research involves a range of data collection techniques, including interviews, which are used to gather rich and detailed insights into people's experiences, attitudes, and perspectives. There are several types of interviews that can be used in qualitative research, each with its own strengths and limitations. Which type of interviews (structured, unstructured, semi-structured, or group interview) will be carried out?\n\nThe discussion section of the study protocol needs extensive revision to provide complete insights to the readers. Please, comment on the main points of the study in light of prior research, emphasizing the significance of the proposed study and its potential contribution to the field.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/11-460
https://f1000research.com/articles/11-312/v1	14 Mar 22	{ "type": "Research Article", "title": "Predictors of social response to COVID-19 among health care workers caring for individuals with confirmed COVID-19 in Jordan", "authors": [ "Heyam Dalky", "Adam Khraisat", "Anas H. Khalifeh", "Sawsan Abuhammad", "Ayman Hamdan-Mansour", "Adam Khraisat", "Anas H. Khalifeh", "Sawsan Abuhammad", "Ayman Hamdan-Mansour" ], "abstract": "Background: The outbreak of COVID-19 forced public health authorities around the world to call for national emergency plans. Public responses, in the form of social discrimination and stigmatizing behaviors, are increasingly being observed against individuals with confirmed COVID-19 and healthcare workers (HCWs) caring for those individuals. Therefore, this study aimed to investigate the perception of social discrimination and coping strategies, and explore predictors of social discrimination and coping with COVID-19 among HCWs and individuals with confirmed COVID-19. Methods: This study used a cross-sectional descriptive-comparative design to collect data, using a convenience sample of 105 individuals with confirmed COVID-19 and 109 HCWs using a web-based survey format. Results: In this study, individuals confirmed with COVID-19 reported a high level of social discrimination compared with HCWs (t = 2.62, p < 0.01), while HCWs reported a high level of coping with COVID-19 compared to individuals with COVID-19 (t = -3.91, p < 0.001). Educational level, age, monthly income, and taking over-the-counter medication were predictors of social discrimination and coping with COVID-19 among HCWs and individuals with confirmed COVID-19. Conclusions: The findings showed that individuals with confirmed COVID-19 were more likely to face social discrimination, and HCWs cope with COVID-19 better than ordinary individuals with confirmed COVID-19.", "keywords": [ "Social Response", "COVID-19", "Health Care Workers", "Coping", "Jordan." ], "content": "Introduction\n\nOver the past two years, the COVID-19 pandemic has been threatening human lives around the world, and health-care professionals are among those who are at higher risk due to immediate exposure to infection and infected people (Gan et al., 2020). Waves of COVID-19 outbreak are continuingly taking different forms and series adding further long-term burden on healthcare professionals. A recent study showed that the general population, due to COVID-19, were suffering stress, anxiety, depression, and psychological disturbances (Hamaideh et al., 2021). Nevertheless, health-care workers (HCWs) have demonstrated exceptional courage and commitment despite their fears of being at higher risk of infection and assumed to infect others (Liu et al., 2020). Approximately 14% of WHO-reported COVID-19 cases are HCWs (Organization, 2020a). In addition, HCWs are also considered a source of virus transmission according to public perception (Dalky et al., 2020).\n\nThe increasing reports of spread of COVID-19 has provoked fear and concern among people globally (Tang et al., 2020). Important concerns include a stigma associated with COVID-19 and the issues related to unknown pathophysiology, treatment, and effectiveness of vaccination (Dalky et al., 2020; Organization, 2020b). This situation has created a new form of stigma and discrimination against individuals with COVID-19 (Tang et al., 2020). There has been a harsh attitude toward those who have been infected and HCWs caring for individuals confirmed with COVID-19 (Bhanot et al., 2020). The stigmatizing behaviors demonstrated the dread of the unknown and the unexplained negative attitudes toward infected people or suspected of being infected, as well as, those suspected of spreading the virus such as HCWs (Bhanot et al., 2020). Societies have blamed people infected with COVID-19 for being ignorant and indifferent (Morin, 2021). This had indicated the need for more emphasis on research to understand the public responses and forms of understanding such dilemmas.\n\nThe pandemic has put an extraordinary psychological burden on HCWs due to work in high-demanding environments forcing them to isolate themselves for fear of transmitting the infection and further accusations of spreading the infection (Rodríguez and Sánchez, 2020). In addition, it has been noticed that the use of social media to negatively reporting and addressing the COVID-19 pandemic has led to social discrimination against those confirmed with COVID-19 and HCWs, as well (Singh and Subedi, 2020). Regrettably, HCWs are labeled, faced loss of status and discrimination due to the stigma associated with COVID-19 (Singh and Subedi, 2020). Public responses in the form of social discrimination and stigmatizing behaviors are increasingly being observed against individuals with confirmed COVID-19 and HCWs caring for individuals with COVID-19 (Abuhammad et al., 2021; Bhanot et al., 2020). Discriminatory behaviors are observed in the form of denial of infection, concealment of being infected, or refusal of the COVID-19 test. Thus, the need to emphasize such an issue would enable understanding reasons and contributing factors related to burnout and psychological distress among HCWs.\n\nThe ongoing transmission, the increasing number of COVID-19 cases, and the growing need to study the effects of community response, discrimination, and stigmatization against HCWs and individuals with confirmed COVID-19 infections are important areas that needs further exploration (Dalky et al., 2020). This makes addressing social discrimination practiced against individuals with COVID-19 and HCWs a significant issue (Dalky et al., 2020). Because of their long and intense exposure to various stressors, it is important to address and understand coping strategies used by HCWs and individuals with COVID-19 to manage stigma practiced against them. This will provide information guidance on what interventions could be implemented to maintain the best care for individuals infected with COVID-19 and maintaining the mental wellness of HCWs, as well. Although studies are increasingly published in the context of psychological consequences of COVID-19 worldwide, there is still a need to address public responses and how it is affecting HCWs’ coping abilities; in particular, in the Arab culture. This study investigated the perception of social discrimination and coping strategies, and predictors of social discrimination and coping toward COVID-19 among HCWs and individuals with COVID-19.\n\nThe aims of this study were:\n\n• To compare coping and social discrimination to COVID-19 among HCWs and individuals with COVID-19.\n\n• To assess sociodemographic or personal factors that could predict coping and social discrimination toward COVID-19 among HCWs and individuals with COVID-19.\n\n\nMethods\n\nThis study utilized a cross-sectional design using a descriptive-comparative approach of research. Data collected in relation to social discrimination and coping from individuals infected with COVID-19 and their health-care workers caring form them was collected using a self-reported online survey format.\n\nThe sample included individuals who had been infected with COVID-19 and HCWs caring of individuals with COVID-19. A convenience sampling technique was used to recruit the participants of this study. Inclusion criteria for HCWs were: 1) providing direct care to those infected with COVID-19. HCW were excluded if he/she reported being infected. For public, to be eligible, required 1) being aged 18 years or above, 2) have access to software to fill out the survey, and 3) be able to read and write in Arabic. Those with physical, mental or cognitive disabilities were excluded as it may interfere with their ability to understand the questions and make their responses.\n\nThe Arabic versions of the tools were used in this study. WHO guidelines for translation and adaption were used to translate the survey. The tools used were as follows:\n\nSocial discrimination was measured using the Social Discrimination Scale (Dinos et al., 2004). The Arabic version of the social discrimination subscale was developed and used before by Dalky et al. (2020) and thus used in this study. This subscale is composed of 12 items and individuals are requested to choose their response on a Likert scale formed of five possible responses ranging from 1 (strongly disagree) to 5 (strongly agree). Those who had a higher score on the scale were more likely to experience a higher level of social discrimination. Previous studies showed good internal consistency with Cronbach's alpha of 0.80 (Dalky et al., 2020).\n\nLifestyle management and psychosocial adaptation with COVID-19 were measured using the FANTASTIC survey (Wilson et al., 1984). This scale focuses on physical and health-related fitness of individuals. The scale is formed of 17 items measuring nine domains with each domain name represented by the first letter of the word “FANTASTIC”: F for Family and Friends, A for Activity, N for Nutrition, T for Tobacco and Toxics, A for Alcohol Intake, S for Sleep, Seatbelts, Stress, and Safe sex, T for Type of behavior, I for Insight, and C for Career. The scale investigates individuals' perception during the previous month. The total score indicates the category in which the individual falls, ranging from needing improvement (0-35) to excellent (85-100). The scale has good reliability with Cronbach's alpha of r = 0.88.\n\nSociodemographic variables\n\nAn author-developed profile was developed to collect information in relation to age, gender, marital status, and other sociodemographic information from both patients and HCWs. Specific information was collected from HCWs regarding their work placement and experiences.\n\nThe software package IBM-SPSS v.25 was used to analyze the collected data. Central tendency measures and dispersion measures were used to describe the variables of the study. Pearson‘s r coefficient was used to assess correlation magnitude and direction. A t-test for two independent samples and ANOVA were used to test differences and compare the HCWs’ and individuals with COVID-19 responses, respectively. A multiple logistic regression test was used to examine predictors of social discrimination. The alpha significance level was set to 0.05.\n\nThe study was granted ethical approval (reference number 113/132/2020), as suggested by both academic authors’ institutions and hospitals administration systems relevantly. The authors received written informed consent from HCWs and individuals with confirmed COVID-19 participants. The consent form included detailed information about the study aims, and the need to conduct such studies, as well as their approval for the publication of this manuscript for research purposes to increase knowledge in this area of COVID-19 impact.\n\n\nResults\n\nIn this study, the total number of individuals with confirmed COVID-19 who completed the survey was 105. Their age ranged from 18-60 years with a mean (M) of 34 (SD=10.4) years (see Table 1). The total number of HCWs who participated was 109. The age of HCWs ranged from 23-65 years with a mean of 33.7 (SD = 7.6) years (see Table 2).\n\nJD = Jordan Dinar.\n\nThe results of the t-test showed that there was a significant difference between mean score of HCWs and individuals with confirmed COVID-19 in relation to social discrimination (t = 2.62, p < 0.01) (see Table 3). The total mean item score of individuals with confirmed COVID-19 was higher (M = 2.64, SD = 0.867) than the total mean item score of HCWs (M = 2.07, SD = 0.19). The item-to-item comparison showed that individuals with confirmed COVID-19 mean item scores were significantly higher than HCWs in all items. The highest mean scores for individuals with confirmed COVID-19 were observed for the items “People insulted me for being diagnosed with the Coronavirus” and “I have not had any problems due to Coronavirus diagnosis” (M = 3.87, SD = 0.856; M = 3.66, SD = 1.192; respectively). The lowest mean items for individuals with confirmed COVID-19 were observed for “I was discriminated against because of my diagnosis of the Coronavirus” and “After I suffered due to a diagnosis of Coronavirus, I feel that life is unfair” (M = 1.99, SD = .098; M = 2.74, SD = 1.010; respectively).\n\nThe results of the t-test showed that there was a significant difference between HCWs and individuals with confirmed COVID-19 in coping with COVID-19 (t = -3.91, p < .001) (see Table 4). The total mean item score of HCWs was higher (M= 51.8, SD = 0.86) than the total mean coping item score of individuals with confirmed COVID-19 (M = 34.3, SD = 1.19). The item-to-item comparison showed that, in general, individuals with COVID-19 had significantly lower mean item scores than HCWs for all items. The highest mean items for HCWs were observed for “I am satisfied with my job or role” and “I give and receive affection” (M = 4.28, SD = 0.859; M = 4.14, SD = 0.976), respectively. The lowest mean item scores for HCWs were observed for “I am vigorously active for at least 30 minutes per day e.g., running, cycling, etc.” and “I eat a balanced diet (see explanation)” (M = 1.56, SD = 1.013; M = 2.44, SD = 1.287), respectively.\n\nRegarding predictors of coping with COVID-19 in relation to demographic variables of the whole group, a multiple regression test was conducted. The results showed that the model was significant (F = 14.88, p = 0.001). Educational level was a significant predictor (B = 0.541, p < 0.001) indicating that those with higher educational levels had higher scores in coping with COVID-19. Age was also a significant predictor (B = 0.187, p =0.007) indicating that older people have higher coping score. The third significant predictor was monthly income (B= -0.338, p < 0.001) that has negative association imposing income as risk factor. In addition, taking over-counter medication was a significant predictor (B = 0.54, p < 0.001) indicating that those who use over-counter medication are more likely to have higher coping scores (see Table 5).\n\n* p < 0.01.\n\n p < 0.001.\n\nTo investigate discrimination perception associated with COVID-19 among both groups, a multiple regression test was conducted. The results showed that the model was significant (F =14.21, p = .001). The predictors were educational level (B = -0.447, p < 0.001) which indicated that as the level of education increased, individuals experienced less discrimination associated with COVID-19; age (B = -0.162, p = 0.02): as age increased the discrimination against COVID-19 was decreased; and monthly income (B = 452, p < 0.001), which means with a higher income experienced more discrimination associated with COVID-19. The other predictor was taking medication over the counter (B = 0.447, p < 0.001) which means people taking medication over the counter experienced more discrimination associated with COVID-19 (see Table 6).\n\n p < 0.001.\n\n\nDiscussion\n\nResponses to COVID-19 might influence the process of treatment and willingness to collaborate. Therefore, differences in perception of social responses in the form of social discrimination between HCWs and individuals with confirmed COVID-19 is a core component in coping with the disease. The results of this study showed that individuals with confirmed COVID-19 had a higher perception of social discrimination and a lower level of coping with COVID-19 compared to HCWs. The results indicate that the general and ordinary individuals are more likely to be exposed to discrimination and possess a lower level of ability to cope with the disease and related factors than the HCWs. Moreover, it is possible that HCWs might have a higher level of knowledge and competency to manage discrimination phrases or cues or have lower sensitivity for such expressions that contributed to their feelings of being discriminated against. It is expected due to their education and training that HCWs are more capable of coping with the disease and manage discrimination than the general population (Chew et al., 2020). Such major differences in perception of being discriminated against may cause conflicts in understanding, communicating, or commitment to a treatment plan that interferes with the achievement of health-care outcomes. The results do support previous reports in which social discrimination and fear of communicable diseases hampered the response of the public (Brooks et al., 2020; Liebrenz et al., 2020). It has also been noted that social discrimination has forced people to negate their positive results of infection to avoid discrimination leading them to avoid seeking healthcare services and lacking protective health measures that endangered others' health conditions and lives (Brooks et al., 2020; Liebrenz et al., 2020).\n\nWhile COVID-19, due to its pandemic nature and global influence pattern, is considered a stress-inducing illness, coping strategies are still required to manage the disease and its consequences (Hamaideh et al., 2021). In this study, HCWs used coping strategies more effectively than individuals with COVID-19. In previous reports, HCWs were challenged in managing their responsibilities due to stigma and discrimination (Dalky et al., 2020). Nevertheless, they were able to better use the coping strategies than ordinary people. It is worth saying that social isolation and job burden are factors that have contributed to increased job stress among HCWs (Bani-Hani and Hamdan-Mansour, 2021; Singh and Subedi, 2020). Thus, HCWs might have depended largely on their learned adaptation skills to manage job stress to be able to handle discrimination and cope better COVID-19. On the other hand, ordinary people probably lack the skills and knowledge to manage discrimination due to fear of losing their social and occupational privileges (Chew et al., 2020). There were differences in using coping strategies between HCWs and individuals with confirmed COVID-19. Such difference might negatively affect HCWs, due to the fact that they are more vulnerable to COVID-19 infection compared to the general population. It has been confirmed that such differences might influence and explain the level of care provided by the HCWs and the low level of compliance and collaboration of individuals with confirmed COVID-19 (Kar et al., 2021). The degree to which a person fears COVID-19 is an element that could be important in understanding the coping process. Fear is an emotional state that stimulates self-defense behaviors; thus, fear of infection would influence coping strategies leading to poor prognosis (Kim et al., 2020).\n\nBesides, many factors could be related to social discrimination against individuals with confirmed COVID-19 or HCWs caring for those people. Such factors might be related to the collectivist culture that puts high social pressure on people and affects their decisions to seek healthcare services (Al Ali et al., 2017; Aldalaykeh et al., 2019). For instance, fear and anxiety from the spread of COVID-19 may lead to social discrimination in people having disease, places that are considered sources of the infection such as hospitals, and even people who were in quarantine (CDC, 2020). Furthermore, social discrimination and stigmatized behaviors are extensively noted in mental health research; however, these variables in the context of COVID-19 seem to lead to the same negative effect on health-care outcomes (Brooks et al., 2020). The notion that social discrimination is a multifaceted factor infers that a reciprocal relationship exists between the bio-psycho-social and cultural components of human wellbeing (Dalky et al., 2020). In other words, the social discrimination might take various forms depending on the cultural definition of discrimination. People with COVID-19 who complied with the quarantine have reported higher levels of psychological disturbances such as stress and anger (Brooks et al., 2020). Such a critical health situation in addition to poor coping and discrimination, might be a threat to successful endorsement and implementation of public healthcare plans against COVID-19 leading to poor healthcare outcomes (Fu et al., 2021).\n\nFurthermore, the study found that HCWs and individuals with confirmed COVID-19 who have a high level of education and use over-the-counter medication showed greater coping with COVID-19. The results support previous equivalent studies that indicated using emotional-based coping among low educated people compared to using problem-based coping among those with a high level of education (Mohammadzadeh et al., 2020; Shamsi et al., 2021). This reflects the differences in the behavioral responses related to individual knowledge and experience. In addition, the results showed that HCWs who earned a high income showed greater coping with COVID-19. These findings are in line with previous studies that found financial constraints were linked to and predicted higher levels of stress and lower levels of effective coping (Atchison et al., 2020; Barbara et al., 2020; Cluver et al., 2020). Similarly, age and literacy play a positive role in predicting effective coping and positive responses (Darraj et al., 2016; Magsamen-Conrad et al., 2019; Moukaddam and Shah, 2020; Volk et al., 2021), older people and more literate ones were found to use more effective coping strategies with COVID-19 than younger and illiterate people. Such findings were consistent with previous studies that reported a higher level of coping connected to higher levels of resiliency (Pearman et al., 2020).\n\nThere were two limitations of our study. First, data was collected using cross sectional sampling utilizing an online survey format which limits ability to derive causal relationships. Secondly, using a self-reporting format might not allowed to draw objective data affected by recall bias.\n\n\nConclusions\n\nThis study focused on exploring predictors of coping and responses to social discrimination in the form of stigma among HCWs and individuals with confirmed COVID-19. The findings showed that individuals with COVID-19 were more likely to face social discrimination than HCWs. Yet, in dealing with COVID-19, HCWs used more effective coping strategies with COVID-19 than non-medical infected individuals. The main conclusion of this study is that predictors of social discrimination and coping were educational level, age, monthly income, and taking over-the-counter medication. Although both social discrimination and coping are complicated and may be influenced by a variety of factors, we must reconsider and find ways to reinforce them in light of the probable recurrence of COVID-19 and other future global pandemic risks. Innovative strategies are to be granted to clinical practice and the public sectors to best tackle the challenges associated with the COVID-19 pandemic. In addition, public media also has to be targeted to combat discrimination and support individuals confirmed with COVID-19 and HCWs caring for them.\n\nThis study has implications relevant to clinical practice, as well as policymakers and public health officers. The results emphasize the need to enhance mutual understanding of the effect of discrimination on both ordinary people and HCWs. Training and enhancement of psychological skills is needed for both ordinary people and HCWs and has to be included in treatment protocols. Furthermore, HCWs are in need of peer and organization support to enable them to manage job burden and discrimination. Management and administrative personnel can guide and support the HCWs by reforming or modifying the current clinical practice to best accommodate and cope with extra or unexpected demands added to the HCWs shoulders, as those seen during the COVID-19 pandemic.\n\n\nData availability\n\nZenodo: Data repository of Predictors of Social Response to COVID-19 among Health Care Workers Caring for Individuals with Confirmed COVID-19 in Jordan, https://doi.org/10.5281/zenodo.6044084 (Dalky et al., 2022).\n\nThis project contains the following underlying data:\n\n- Project1 (2) (1).xlsx (raw survey data)\n\nZenodo: Data repository of Predictors of Social Response to COVID-19 among Health Care Workers Caring for Individuals with Confirmed COVID-19 in Jordan, https://doi.org/10.5281/zenodo.6044084 (Dalky et al., 2022)\n\nThis project contains the following extended data:\n\n- COVID-19 in Jordan.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAbuhammad S, Alzoubi KH, Khabour O: Fear of COVID-19 and stigmatization towards infected people among Jordanian people. Int J Clin Pract. 2021; 75(4): e13899. PubMed Abstract \| Publisher Full Text\n\nAl Ali NM, Alqurneh MK, Dalky H, et al.: Factors affecting help-seeking attitudes regarding mental health services among attendance of primary health care centers in Jordan. Int J Ment Health. 2017; 46(1): 38–51. Publisher Full Text\n\nAldalaykeh M, Al-Hammouri MM, Rababah J: Predictors of mental health services help-seeking behavior among university students. Cogent Psychol. 2019; 6(1): 1660520. Publisher Full Text\n\nAtchison C, Bowman L, Vrinten C, et al.: Perceptions and behavioural responses of the general public during the COVID-19 pandemic: A cross-sectional survey of UK Adults. medRxiv. 2020.\n\nBani-Hani MA, Hamdan-Mansour AM: The moderation effect of locus of control on the relationship between job demand and job satisfaction among nurses. Int J Nurs Pract. 2021; 27(1): e12876. PubMed Abstract \| Publisher Full Text\n\nBarbara G, Facchin F, Micci L, et al.: COVID-19, lockdown, and intimate partner violence: some data from an Italian service and suggestions for future approaches. J Womens Health. 2020; 29(10): 1239–1242. PubMed Abstract \| Publisher Full Text\n\nBhanot D, Singh T, Verma SK, et al.: Stigma and Discrimination During COVID-19 Pandemic. Front Public Health. 2020; 8: 577018. PubMed Abstract \| Publisher Full Text\n\nBrooks SK, Webster RK, Smith LE, et al.: The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020; 395(10227): 912–920. PubMed Abstract \| Publisher Full Text\n\nCDC: 2020. Reduscing stigma. Retrieved November Reference Source\n\nChew QH, Chia FLA, Ng WK, et al.: Perceived stress, stigma, traumatic stress levels and coping responses amongst residents in training across multiple specialties during COVID-19 pandemic—A longitudinal study. International Journal of Environmental Research and Public Health. 2020; 17(18): 6572. PubMed Abstract \| Publisher Full Text\n\nCluver L, Lachman JM, Sherr L, et al.: Parenting in a time of COVID-19. Lancet. 2020; 395(10231): e64. PubMed Abstract \| Publisher Full Text\n\nDalky HF: Arabic translation and cultural adaptation of the stigma-devaluation scale in Jordan. J Ment Health. 2012; 21(1): 72–82. PubMed Abstract \| Publisher Full Text\n\nDalky HF, Hamdan-Mansour AM, Amarneh BH, et al.: Social discrimination perception of health-care workers and ordinary people toward individuals with COVID-19. Social Influence. 2020; 15(2-4): 65–79. Publisher Full Text\n\nDalky H, Khraisat A, Khalifeh A, et al.: Data repository of Predictors of Social Response to COVID-19 among Health Care Workers Caring for Individuals with Confirmed COVID-19 in Jordan [Data set]. Zenodo. 2022. Publisher Full Text\n\nDarraj HA, Mahfouz MS, Al Sanosi RM, et al.: Arabic Translation and Psychometric Evaluation of the Depression Literacy Questionnaire among Adolescents. Psychiatry J. 2016; 2016: 8045262–8045267. PubMed Abstract \| Publisher Full Text\n\nDinos S, Stevens S, Serfaty M, et al.: Stigma: the feelings and experiences of 46 people with mental illness. Qualitative study. Br J Psychiatry. 2004; 184: 176–181. PubMed Abstract \| Publisher Full Text\n\nFu L, Wang B, Chan PSF, et al.: Associations between COVID-19 related stigma and sleep quality among COVID-19 survivors six months after hospital discharge. Sleep Med. 2021. PubMed Abstract \| Publisher Full Text\n\nGan WH, Lim JW, Koh D: Preventing intra-hospital infection and transmission of coronavirus disease 2019 in health-care workers. Saf Health Work. 2020; 11(2): 241–243. PubMed Abstract \| Publisher Full Text\n\nHamaideh SH, Al-Modallal Ha, Tanash M: Depression, anxiety and stress among undergraduate students during COVID-19 outbreak and “home-quarantine”. Nursing Open. 2021; 9: 1423–1431. PubMed Abstract \| Publisher Full Text\n\nKar N, Kar B, Kar S: Stress and coping during COVID-19 pandemic: Result of an online survey. Psychiatry Res. 2021; 295: 113598. PubMed Abstract \| Publisher Full Text\n\nKim R, Nachman S, Fernandes R, et al.: Comparison of COVID-19 infections among healthcare workers and non-healthcare workers. PLoS One. 2020; 15(12): e0241956. PubMed Abstract \| Publisher Full Text\n\nKing M, Dinos S, Shaw J, et al.: The Stigma Scale: development of a standardised measure of the stigma of mental illness. Br J Psychiatry 2007; 190(3): 248–254. PubMed Abstract \| Publisher Full Text\n\nLiebrenz M, Bhugra D, Buadze A, et al.: Caring for persons in detention suffering with mental illness during the Covid-19 outbreak. Forensic Science International. Mind and law. 2020; 1: 100013. PubMed Abstract \| Publisher Full Text\n\nLiu Q, Luo D, Haase JE, et al.: The experiences of health-care providers during the COVID-19 crisis in China: a qualitative study. Lancet Glob Health. 2020; 8(6): e790–e798. PubMed Abstract \| Publisher Full Text\n\nMagsamen-Conrad K, Dillon JM, Billotte Verhoff C, et al.: Online health-information seeking among older populations: Family influences and the role of the medical professional. Health Commun. 2019; 34(8): 859–871. PubMed Abstract \| Publisher Full Text\n\nMohammadzadeh F, Delshad Noghabi A, Khosravan S, et al.: Anxiety Severity Levels and Coping Strategies during the COVID-19 Pandemic among People Aged 15 Years and Above in Gonabad, Iran. Arch Iran Med. 2020; 23(9): 633–638. PubMed Abstract \| Publisher Full Text\n\nMorin A: Why We're Blaming Victims During COVID-19. 2021. Retrieved November Reference Source\n\nMoukaddam N, Shah A: Psychiatrists beware! The impact of COVID-19 and pandemics on mental health. Psychiatric Times. 2020; 37(3).\n\nOrganization, W. H: Multilingualism and translation tools. 2018. Retrieved November Reference Source\n\nOrganization, W. H: Keep health workers safe to keep patients safe: WHO. 2020a. Retrieved NovemberReference Source\n\nOrganization, W. H: Social Stigma associated with COVID-19. 2020b. Retrieved November. Reference Source\n\nPearman A, Hughes ML, Smith EL, et al.: Mental health challenges of United States healthcare professionals during COVID-19. Front Psychol. 2020; 11: 2065. PubMed Abstract \| Publisher Full Text\n\nRodríguez BO, Sánchez TL: The Psychosocial Impact of COVID-19 on health care workers. Int Braz J Urol. 2020; 46(suppl.1): 195–200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShamsi T, Mohammad Al-Din ZH, Sarfraz M: Coping Strategies Used by Tax-paying Male Bankers During COVID-19 Induced Lockdown-A Bangladeshi and Pakistani Perspective. Indian Journal of Public Health Research and Development. 2021; 12(2).\n\nSingh R, Subedi M: COVID-19 and stigma: Social discrimination towards frontline healthcare providers and COVID-19 recovered patients in Nepal. Asian J Psychiatr. 2020; 53: 102222. PubMed Abstract \| Publisher Full Text\n\nTang B, Wang X, Li Q, et al.: Estimation of the Transmission Risk of the 2019-nCoV and Its Implication for Public Health Interventions. J Clin Med. 2020; 9(2): 462. PubMed Abstract \| Publisher Full Text Reference Source\n\nVolk AA, Brazil KJ, Franklin-Luther P, et al.: The influence of demographics and personality on COVID-19 coping in young adults. Personality and Individual Differences. 2021; 168: 110398. PubMed Abstract \| Publisher Full Text\n\nWilson DMC, Nielsen E, Ciliska D: Lifestyle Assessment: Testing the FANTASTIC Instrument. Canadian Family Physician. 1984; 30: 1863–1866. Reference Source" }	[ { "id": "127956", "date": "12 Apr 2022", "name": "Sergul Duygulu", "expertise": [ "Reviewer Expertise Nursing management", "community health nursing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for giving me the opportunity to review this manuscript. The subject covered in the study is a current and important issue. In particular, the research has addressed an important issue in terms of addressing the problems experienced by healthcare professionals and individuals diagnosed with COVID-19 at the beginning of the pandemic.\nThis study aimed to explore perceptions of social discrimination and coping strategies, and to explore predictors of social discrimination and coping with COVID-19 among healthcare workers and individuals with confirmed COVID-19. The theoretical basis of the study is well explained and it is clearly stated that the results of the study will meet which need in the literature. However, it can be put forward more clearly why the determinants of social discrimination and coping with COVID-19, which are discussed in this study, should be determined. Another suggestion is that in this study, there is a need to include the answers to the questions of why individuals and healthcare professionals with a history of being diagnosed with COVID-19 and healthcare professionals at the same time and why a comparative study is needed, albeit briefly, in the introduction. The aim of the study is stated. However, it would be appropriate to include research questions/hypotheses.\nThe design of the study is appropriate. Sampling inclusion and exclusion criteria are specified. There is no statement that the adequacy of the sample size is evaluated. It would be appropriate to write. The study's sample “individuals with confirmed with COVID 19 and healthcare workers” is considered to be the limitation of this study. However, this limitation is acceptable as the results of the study address an important issue. However, as a limitation, it would be appropriate to refer to the sample size in the limitation session of manuscript.\nAlthough it has been stated that data collection is carried out online, the data collection procedure can be explained in more detail. How did the process work?\nThe findings, discussion and conclusion of the study were reported in accordance with the aims of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8138", "date": "28 Apr 2022", "name": "Heyam Dalky", "role": "Author Response", "response": "Thank you very much for giving me the opportunity to review this manuscript. The subject covered in the study is a current and important issue. In particular, the research has addressed an important issue in terms of addressing the problems experienced by healthcare professionals and individuals diagnosed with COVID-19 at the beginning of the pandemic. This study aimed to explore perceptions of social discrimination and coping strategies and to explore predictors of social discrimination and coping with COVID-19 among healthcare workers and individuals with confirmed COVID-19. The theoretical basis of the study is well explained and it is clearly stated that the results of the study will meet which needs in the literature. However, it can be put forward more clearly why the determinants of social discrimination and coping with COVID-19, which are discussed in this study, should be determined. Response: Social discrimination has been reported widely in the literature during the pandemic and people reacted differently to the diagnosis. We thought HCP also might have this and decided to study it and also the predictors to better plan for future occurrences of the unexpected pandemics. Another suggestion is that in this study, there is a need to include the answers to the questions of why individuals and healthcare professionals with a history of being diagnosed with COVID-19 and healthcare professionals at the same time and why a comparative study is needed, albeit briefly, in the introduction. Response: It is known in the literature that experience or perception of social discrimination and stigma are personal in nature and varies among individuals or groups. This perception could lead to various negative consequences that require further investigations to study and plan the suitable interventions to tackle and treat them. Yet, a comparative study or approach usually presents differences among both groups to highlight the interventions needed accordingly. The aim of the study is stated. However, it would be appropriate to include research questions/hypotheses. Response: This comment is fully considered. The requested information is added in red-colored text after the study aims in the introduction section. Research Questions: What is the level of social discrimination against COVID-19 among HCWs and individuals with COVID-19? What is the level of coping with COVID-19 among HCWs and individuals with COVID-19? Which are the study factors that could predict coping and social discrimination toward COVID-19 among HCWs and individuals with COVID-19? The design of the study is appropriate. The sampling inclusion and exclusion criteria are specified. There is no statement that the adequacy of the sample size is evaluated. It would be appropriate to write. Response: This information is added as suggested. See red-colored text in the methods section; sample. A minimum number of 100 per each group was determined as necessary to achieve the study's desired power of 80 % at the level of significance of α =0.05. The study's sample of “individuals with confirmed with COVID 19 and healthcare workers” is considered to be the limitation of this study. However, this limitation is acceptable as the results of the study address an important issue. However, as a limitation, it would be appropriate to refer to the sample size in the limitation session of manuscript. Response: These comments are fully considered. The sample size in this study was convenient and appropriate to drown the conclusions derived. Although it has been stated that data collection is carried out online, the data collection procedure can be explained in more detail. How did the process work? Response: These comments are fully considered, the information is added for more clarifications. See methods added red-text. In this study, data was collected via an online survey. Participants were contacted online via social media available for HCWs and individuals with COVID-19. Upon agreeing to participate in the study, signed the consent participants were encouraged to complete the online survey. Authors' information was available shall they have questions or queries and assistance was available accordingly. The findings, discussion and conclusion of the study were reported in accordance with the aims of the study. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Yes" } ] } ]	1	https://f1000research.com/articles/11-312
https://f1000research.com/articles/10-922/v1	15 Sep 21	{ "type": "Research Article", "title": "Reproductive characteristics of the giant gurami sago strain (Osphronemus goramy Lacepède, 1801): basic knowledge for a future hatchery development strategy", "authors": [ "Azrita Azrita", "Hafrijal Syandri", "Netti Aryani", "Azrita Azrita", "Netti Aryani" ], "abstract": "Background: The giant gourami sago strain (Osphronemus goramy Lacepède) has been approved in 2018 as a candidate for freshwater aquaculture in Indonesia. However, information on the species’ reproduction is minimal. This study analyzed the reproductive characteristics of the gourami sago strain broodfish to provide basic knowledge for a future hatchery development strategy. Methods: A total of 10 female and 10 male mature gourami sago strain broodfish were measured for body weight and length, and were evaluated for their reproductive characteristics. Breeding fish were spawned naturally in a 2×1×0.6 m concrete pond with a male-female sex ratio of 1:1. Egg weight and diameter were measured in 25 eggs per female using, respectively, ACIS AD- 600i scales with 0.01 g accuracy, and a microscope (Labo model L-711) using Canon Digital Camera Software 3 . Semen was collected using plastic syringes in 3 mL aliquots, then placed in an insulated ice-cooled container, and analyzed within two hours of collection. Results: Average weights of female and male broodfish before spawning were 2180±159.78 g and 3060±134.99 g, respectively. The relative fecundity and egg diameter were 1029±36 eggs kg-1 and 2.42±0.05 mm, respectively. The hatching rate and embryo survival to an eyed-egg stage were respectively 76.40±2.27% and 94.76±0.42%. Sperm characteristics showed that volume was 0.60±0.12 ml kg-1 and percentage of motile sperm was 70.04±2.27%. Female broodfish weight after spawning was strongly correlated with the weight before spawned (r2 = 0.999) and absolute fecundity was also strongly correlated with female broodfish weight before spawning (r2= 0.921). Sperm concentration was moderately correlated with sperm motility (r2 = 0.556) and duration of sperm motility (r2 = 0.502). Conclusions: The gourami sago strain broodfish has suitable reproductive characteristics for the development of hatcheries. Successful natural spawning should be followed by larval weaning and feeding technology to increase growth and survival.", "keywords": [ "Aquaculture", "giant gourami", "broodfish", "egg", "sperm", "hatchery performance." ], "content": "Introduction\n\nFreshwater aquaculture practiced in inland waters such as lakes, rivers, reservoirs, floodplains, oxbow lakes, and freshwater ponds have expanded during the last decades in Indonesia.1-5 Approximately 77.57% of fish produced in freshwater aquaculture in Indonesia are sourced from freshwater ponds and inland waters.6 However, their development depends upon many factors, such as fish species, aquaculture systems, water depletion, fish diseases, farmers’ knowledge, and aquaculture practices.4,7-10\n\nFreshwater aquaculture is one of the fastest-growing aquacultures in Indonesia, with more than 3,378,298.92 metric tons produced in 2018.6,11,12 Nile tilapia (Oreochromis niloticus) contributed 37.93% of the total aquaculture production, African catfish (Clarias gariepinus) 33.35%, Pangasius catfish (Pangasius hypophthalmus) 12.38%, common carp (Cyprinus carpio) 9.28%, and giant gourami (Osphronemus goramy) 6.96%.13-15\n\nIndonesian giant gourami strains include the local \"tambago”, “palapa”, “soang”, “galunggung” and “blusafir\" strains, which have been grown semi-intensively in small-scale farms for decades.8,13,14,16 However, they have not been able to contribute majorly to freshwater aquaculture production in Indonesia. Although the gourami sago strain has been approved as a candidate for aquaculture production, its distribution is limited in the West Sumatra Province of Indonesia.17,18 Therefore the development of its hatchery is necessary to be able to contribute to the production of freshwater aquaculture. The gourami sago strain is considered to support food security along with many other freshwater fish species in Indonesia.\n\nThe gourami sago strain was approved as a candidate for freshwater aquaculture in 2018 (Decree of the Ministry of Marine and Fisheries, Republic of Indonesia No.56/KEPMEN-KP/2018).19 However, data on its reproductive characteristics are still limited. The evaluation of reproductive performance in other fish species has had beneficial impacts in the development of freshwater aquaculture in Asia.20-24 In contrast, there are still gaps in knowledge of giant gourami sago strain broodfish regarding size at oocyte maturity, age of sexual maturity, sperm characteristics, egg hatchability, survival after eyed-egg stage, larva weaning, and growth rate. These factors were identified as key challenges for successful giant gourami sago strain hatchery performance in the future. Therefore, the present study was conducted to evaluate the reproductive characteristics in the giant gourami sago strain to provide basic knowledge for a hatchery development in the future.\n\n\nMethods\n\nThere are no required permits from the government of the Republic of Indonesia to evaluate reproductive characteristics in gourami sago strain broodfish (Osphronemus goramy) as a candidate for future aquaculture. The study was funded by the Research and Community Service, Universitas Bung Hatta under a competitive grants scheme called the research of Professor in 2021 (Contract number: 06.02.1.46.03.2021). This grant included ethical approval and permits to collect fish specimens, rear and spawn giant gourami sago strains in the Aquaculture Laboratory Faculty of Fisheries and Marine Science, Universitas Bung Hatta facilities. There was no animal suffering involved in this study and gourami sago strain broodfish were still in good condition when returned to the pond. Ethical approval was granted by the Ethics Commission for Research and Community service at Universitas Bung Hatta (023/LPPM/Hatta/I-2021).\n\nJuvenile fishes were selected about six years ago from a local hatchery in Luhak District, Lima Puluh Kota Regency, West Sumatra Province. The juvenile fish were kept in tanks and transported by truck to the Aquaculture Laboratory, Faculty of Fisheries, and the Marine Science University of Bung Hatta. A total of 200 individuals giant gourami sago strain juvenile fishes were reared for five years until sexual maturity, in an 8.4 m3 (6 × 2 × 0.7 m) concrete freshwater pond. During the rearing of the fishes to sexual maturity, the juvenile fish were fed with commercial fish pellets (781-2 with 30% crude protein content and 4% crude fat; PT Japfa Comfeed Indonesia Tbk).\n\nAfter the giant gourami sago fish were reared for five years, 20 mature individual broodfish were separated according to sex and reared in two 18 m3 (6 × 2 × 1.5 m) concrete freshwater ponds for 60 days. The broodfish were fed twice daily (09:00 AM and 16:00), with extruded feed pellets containing 39.50% crude protein and 12.21% fat with a predetermined quantity of 3% of fish weight per day. Besides that, the fish were given sente leaves (Alocasia macrorriza L.) at amounts of 1% of fish wet weight per day. The leaves contained 2.85% protein and 0.47% crude fat (of wet weight). Each concrete freshwater pond had a 50 mm drain in the middle, which was covered with a net of 0.5 cm mesh size to prevent the fish from escaping and predators from entering. Water was pumped from borehole wells at a rate of 5 liters per minute.\n\nA total of 20 broodfish with mature oocytes were selected, consisting of 10 females and 10 males. Before spawning, female and male broodfish were weighed using scales (OHAUS model CT 6000-USA with 0.1 g accuracy), and body length was measured using a meter ruler with 1mm accuracy. The average weight and length of the 10 female broodfish were 2140 ± 159 g and 39.70 ± 1.77 cm, while those of the male broodfish were 3060 ± 135 g and 43.1 ± 1.79 cm.\n\nReproductive parameters in gourami sago strain broodfish were analyzed using the following formulae:\n\n• Condition factor (CF) = wet weight in gram/length3 × 100\n\n• Ovulated egg weight (g) = fish weight before spawning (g) – fish weight after spawning (g)\n\n• Ova somatic index (%) = egg weight ovulation (g)/fish weight before spawning (g) × 100\n\nAbsolute fecundity was the total number of eggs estimated per nest, and relative fecundity was the total number of eggs per kg body weight.\n\nStarting in August 2020 onwards, the broodfish were checked monthly for eggs and semen production. The broodfish were captured with a hand net and anesthetized by oral ingestion of Tricaine methanesulfonate (MS-222, ethyl 4-aminobenzoate methanesulfonate 98%, Sigma Aldrich Co, USA, MO; 50 mg L−1), based on the dosage used for Hemibagrus wyckii.21 Oocyte maturation was assessed for each individual. The oocyte maturity in giant gourami females was assessed from oocytes sampled by intraovarian biopsies using a flexible polyethylene catheter.21 Egg diameter was measured using Labo microscope model L-711 and Canon Digital Camera Software 3.\n\nNatural spawning of broodfish was carried out in 1.2 m3 (2 × 1 × 0.6 m) concrete freshwater ponds with a male-female sex ratio of 1:1. Before the broodfish spawned, the ponds were drained, cleaned, and all other species removed. Then, palm fibers were placed on top of a bamboo raft in the pond. The pond was then filled with water, and the female and male broodfish were released into the spawning pond. The male broodfish made a nest for five to seven days, after which spawning took place and the female broodfish laid eggs. Spawning occurred in the afternoon (between 3.00 to 5.00 PM). Due to the presence of a very large oil globule, giant gourami eggs float.8 After the broodfish had finished laying eggs, the eggs were kept by the female broodfish in the nest. After the eggs had been kept by the broodfish for four hours in the nest, the eggs were collected and transferred to an incubation tray, which was placed in a ventricular hatching system. A total of 100 eggs for each broodfish were incubated in incubation trays. Meanwhile, the broodfish were returned to their pond once spawned, and no mortality occurred.\n\nEgg weight and diameter were measured for 25 eggs per female using SHIMADZU-model AY 220 scales with 0.1 mg accuracy and a microscope (Labo model L-711) using Canon Digital Camera Software 3. A total of 25 eggs were randomly sampled 16 hours after spawning to determine the fertility rate (FR). The hatching rate (HR) was determined by counting all hatched fry 48 hours after spawning. Then, the endogenous feeding period of the fish larvae was counted until the egg yolks ran out (in days), and embryo survival rate (%) to the eyed-egg stage was measured.\n\nTo stimulate the spermiation process in male broodfish, an LHRH-preparation (Ovaprim, manufactured for Syndel Laboratories Ltd, 2595 Mccullough Rd Nanaimo B. C 9VS 4m9 Canada) was injected into male fish, with a dosage of 0.5 ml per kg of the brooder. Semen samples were obtained from 10 gourami sago broodfish randomly selected from the farm. The male broodfish were first anesthetized with 50 mg Lˉ1 of MS-222,25 then fish weights (MaW) and total lengths (MaL) were measured. Special care was taken to avoid any contamination of semen with urine, feces, mucus, and water. Semen samples were collected using plastic syringes in 3 mL aliquots and then placed in an insulated ice-cooled container, transported to the laboratory, and analyzed within two hours.\n\nThe sperm assessment included gross (visual) and microscopic examination as reviewed by Rurangwa et al.26 and Cabrita et al.27 The gross examination was based on visual and physical observation of parameters like semen volume, semen pH, sperm concentration, motility, and duration motility. Semen volume was determined by collecting the semen in a graduated cylinder. Semen pH was determined with a hand pH meter (HI8424 Hanna Instruments, USA). Microscopic examination was carried out using the Olympus model CX40, with × 10 and × 25 magnification, to determine parameters such as motility (MO) percentage and duration, by observing water-activated semen placed on a glass slide. Motile sperm were observed and expressed as a percent of non-moving sperm. Motility duration (DMO) was determined as the period between movements of the sperm to a cessation of any progress using Neubauer’s hemocytometer and calculated as the number of sperm ml−1.28 Semen pH was determined with a hand pH meter (HI8424 Hanna Instruments, USA).\n\nWater samples were collected in the spawning pond and incubation trays to determine alkalinity, hardness, and pH. The protocol for determining alkalinity by standard methodology is presented by Rice et al., 2012.29 pH values were determined with a pH meter (digital mini pH meter, 14pH, IQ Scientific, Chemo-science Thailand Co., Ltd, Thailand). An oxygen meter (YSI model 52, Yellow Spring Instrument Co., Yellow Springs, OH, USA) was used in situ, and water temperature in the spawning pond and incubation trays were measured with a thermometer (Celsius scale).\n\nResults were given as the mean values (± SD). Simple linear regression analyses were performed using SPSS software (version 16.0 for Windows; SPSS Inc., Chicago, IL). The standard deviation of each parameter was determined. For linear regression analyses, correlations were considered significant at p < 0.05, and trends or tendencies were considered significant at p < 0.05.\n\n\nResults\n\nThe reproductive characteristics of female broodfish in sago giant gourami are summarized in Table 1. The total number of eggs per nest (absolute fecundity) varied from 2000 to 2650, while relative fecundity (total number of eggs per kg female brooder) varied between 977 and 1071. The fertility rate ranged from 76 to 84%, and the hatching success rate ranged from 72 to 80%. Endogenous feeding period ranged from 10 to 12 days, and embryo survival rate to eyed-egg stage varied between 94.73 and 95%.\n\nReproductive characteristics for male broodfish and sperm samples are presented in Table 2. The average live weight of the males was 3340 ± 275.68 g. Sago giant gourami male broodfish were found to be slightly bigger than female broodfish. Gonad weight ranged from 25 to 30 g, whereas gonad somatic index ranged from 0.83 to 0.93%.\n\nThe linear correlation (r2) between variables of reproduction characterization parameters in sago strain of giant gourami females broodfish results are shown in Table 3. In this study, the reproductive parameters that showed strong correlations with the absolute fecundity were female fish weight before spawning (r2 = 0.921) and female fish weight after spawning (r2 = 0.864). Similarly, results revealed significant correlations between egg diameter and hardened egg diameter (r2 = 0.833), and between egg diameter and percentage of the hardened diameter (r2 = 0.699). Meanwhile, egg diameter and fertility rate were moderately correlated (r2 = 0.568). In contrast, the egg diameter was not strongly related to absolute fecundity (r2 = 0.169) and relative fecundity (r2 = 0.096). On the other hand, the survival rate of larvae (10 days) also had strong correlations with the hatching rate (r2 = 0.998) and endogenous feeding period (r2 = 0.757).\n\nLinear correlation analysis results (r2) between variables of reproduction characterization parameters in sago giant gourami male broodfish are shown in Table 4. The reproductive parameters that had a strong correlation with gonad weight were somatic index of gonads (r2 = 0.836), while semen volume (r2 = 0.521), semen pH (r2 = 0.521) and sperm concentration (r2 = 0.506) were moderately correlated with gonad weight. In contrast, the gonadal weight negatively correlated with sperm motility (r2 = 0.017) and duration of motility (r2 = 0.275). In addition, the sperm concentration was also moderately correlated with the sperm motility (r2= 0.556) and duration of motility (r2= 0.502).\n\nThe physico-chemical water quality parameters in the spawning ponds and incubation trays for embryo development were as follows: water alkalinity ranged from 50.5 mg L−1 to 52.5 mg L−1 HCO3−, hardness varied from 65.5 mg L−1 to 67.5 mg L−1 CaCO3, pH ranged from 6.4 to 6.6, oxygen ranged from 6.1 mg L−1 to 6.7 mg L−1, and temperature varied from 28°C to 30°C.\n\n\nDiscussion\n\nIn our study, body weight in female sago giant gourami broodfish before spawning ranged from 1958 to 2500 g per fish, and ova somatic index ranged from 1.43 to 1.65%. Body weight in female sago strain gourami broodfish was smaller than that of giant gourami belonging to the galunggung strain, which ranged from 2500 to 3500 g.8 Conversely, the ova somatic index of galunggung strain is found to be slightly bigger than that of sago giant gourami, which ranged from 3.7 to 4.6%.8 The differences in reproductive characteristics in broodfish can be explained by strains, brood size, age of broodfish, previous spawning history, and the production setting.23 Absolute fecundity in sago giant gourami ranged from 2000 to 2650 eggs fish−1 and relative fecundity (RF) ranged from 977 to 1071 eggs kg−1. Egg produced in kg fish−1 (RF) is thought to be more informative than absolute fecundity. RF in sago strain of giant gourami was smaller compared to those in galunggung strain, palapah strain, and blusafir strain.8,16,31 On the other hand, the difference in relative fecundity can also be related to differences in broodfish size and age used.23 Environmental factors such as rainfall also influenced the number of eggs per spawning in giant gourami broodfish, while the water temperature negatively related to the number of eggs per spawning.15 Furthermore, egg diameter in sago strain of giant gourami was found to be almost the same as other strains of giant gourami (Table 5). In this study, average egg diameter was 2.42 ± 0.05 mm, consistent with those reported by other researchers; 2.18 ± 0.19 mm for the giant gourami,30 2.40 ± 0.05 mm for blusafir strain,16 and 2.5 ± 0.05 mm for galunggung strain.8 The differences in the RF, ova somatic index, egg diameter, and hatching rate of giant gourami can be influenced by differences in the strains. Furthermore, egg diameter has been influenced by dietary protein level,32-35 age of broodfish,36 and spawning season.37,38 In our study, egg diameter was shown to be positively correlated with egg weight, hardened egg weight, and egg weight increase. The egg weight of rainbow trout also increased after the hardening process and was positively correlated with the viability of eggs.39 Other egg quality metrics, such as hatching rate, and survival to first feeding, have been correlated with good egg quality.21\n\nIn this study, the hatching rate of the embryo in the sago strain of giant gourami was smaller than those of other strains of giant gourami.8,16,31 This condition might be affected by the egg and sperm quality in giant gourami sago strain broodfish. In the present study, whether eggs and sperm quality of sago giant gourami breeders are affected by feed type was poorly understood. Broodfish sex ratio did not influence egg quality.8 The reproductive parameters that were strongly correlated with the hatching rate were fertility rate (r2 = 0.703) and survival rate (10 days) (r2 = 0.998). According to Sink et al.,40 the biochemical composition of broodfish eggs is strongly correlated with egg quality. In this study, we did not evaluate the biochemical composition of an egg, because the relationship between egg quality and biochemical composition is difficult to interpret.41\n\nThe keys regulator hormones of fish reproduction are gonadotropins, follicle-stimulating hormone, luteinizing hormone, and sex steroids.42,43 In addition, oocyte development and maturation are also regulated by locally acting paracrine and autocrine signaling.44,45 However, there is no information about the effects of such factors on oocyte development in giant gourami sago. Still, the extrusion feed enriched with vitamin E (d-α-tocopherol) at concentrations of 137.8, 238.05, 338.72 and 439.39 mg per kg feed affected markers of reproductive functions of giant gourami broodfish, such as the sexual maturity cycles, ovum somatic index, relative fecundity, and egg diameter.31\n\nVarious efforts have been made by scientists to increase the reproductive performance of female broodfish, such as increasing dietary protein levels for Xiphophorus helleri,33 Channa marulius,46 and Ictalurus punctatus.40 Additionally, implantation of 17ß-estradiol has also improved the reproductive performance in Hemibagrus nemurus.47 Currently, whether the increase in the protein level of feed and use of hormones can increase the reproductive potential in sago giant gourami is poorly understood. Therefore, we recommend the use of proteins in feed (at levels of 25%, 30%, or 35%) and the addition of 17β-estradiol (for example 200, 400, or 600 μg/kg body weight) to increase the reproductive potential of giant gourami sago in the future.\n\nAverage semen volume in giant gourami sago was lower (0.4 to 0.6 ml) than those of Hemibagrus wyckii (0.60 to 1.20 ml),21 but higher than those of Pterygoplichthys gibbiceps.48 It appears that the semen volume depends on fish species.21,49,50 Many factors influenced sperm quality and quantity such as genetic, physiological, spawning season, and environmental factors.26,49,51,52 On the other hand, improvements in feed nutrition of broodfish can increase gamete quality and semen volume.46 Commercial honey combined with 10% Dimethyl Sulfoxide (DMSO) was also shown to increase sperm motility.53 Synthetic hormones such as gonadotropin-releasing hormone analogs (GnRHa), with or without dopamine antagonist, and domperidone (Dom) effectively improve sperm quality.54,55 Nevertheless, the duration of motility of sperm was strongly correlated with the water quality in ponds. In this study, the water quality parameters in spawning ponds included alkalinity of 50.5 to 52.5 mg/L HCO3− and hardness of 65.5 to 67.5 mg/L CaCO3, a pH between 6.4 and 6.6, and a water temperature between 28 and 30 oC. These water quality parameters were able to support the ability of the sperm to fertilize the egg.21\n\nThe sperm motility in sago giant gourami ranged from 68 to 75%, and the duration of motility ranged from 43 to 61 sec. These results are consistent with Genypterus blacodes and Esox lucius.50,56 Sperm motility includes the percentage of motile sperm, straight-line velocity, curvilinear velocity, average path velocity, and linearity.50 In this study, we did not investigate those parameters. In addition, the percentage of motile sperm is influenced by the addition of extenders and cryoprotectants.57-59 However, sperm motility from fresh semen was slightly greater compared to cryopreserved semen from Esox lucius.50 The fertility rate of eggs ranged from 76 and 84%; however, no significant correlation was detected between fertility rate and sperm parameters, such as semen volume, semen pH, motility, and duration of motility. Conversely, the sperm concentration was moderately correlated with sperm motility and duration of motility. The parameters commonly measured to assess sperm quality in brood were volume, density, and motility (such as the percentage of motile sperm, straight-line velocity curvilinear velocity, average path velocity, linearity, and amplitude of lateral head displacement), including fertilizing capacity.49,50,52,60 In this study, we did not investigate the ionic composition of the semen, but this phenomenon could be related to the ionic composition of semen which might have a significant influence on sperm motility and duration of motility.\n\n\nConclusions\n\nThis research analyzed the reproductive characteristics of giant gourami sago strain broodfish reared in concrete freshwater ponds, in the Aquaculture Laboratory Faculty of Fisheries and Marine Science, Universitas Bung Hatta. Relative fecundities of the giant gourami sago strain broodfish ranged from 977 to 1071 eggs, and egg diameter ranged from 2.32 to 2.46 mm. Semen volume ranged from 0.4 to 0.7 ml per kg body weight and sperm motility was comprised between 68 and 75%. A strong linear relationship was observed between absolute fecundity and female fish weight before and after spawning. Similarly, a strong, positive correlation was observed between survival rate (10 days) and hatching rate. The sperm concentration was also moderately positively correlated with the motility and duration of sperm motility. Keys to increasing the reproduction performance in gourami sago strain fish depend on broodfish weight, relative fecundity, and hatching rates. Although data on the reproductive characteristics of gourami sago strain broodfish have been obtained, there are still knowledge gaps in feeding technologies and larval weaning during rearing. Therefore, for successful practices in hatcheries, further research is recommended to determine a proper feed formulation and the development of appropriate aquaculture systems.\n\n\nData availability\n\nFigshare: Reproduction characterization of the gurami sago (Osphronemus goramy Lacepède, 1801): basic knowledge for a hatchery development strategy for the future.\n\nhttps://doi.org/10.6084/m9.figshare.14661189.v3.61\n\nThis project contains the following underlying data:\n\n- Table 1. Raw data of fish length, weight, absolute fecundity, and relative fecundity of gurami sago broodfish\n\n- Table 2. Raw data of egg diameter (mm) in sago strain of giant gourami broodfish\n\n- Table 3. Raw data of hardened egg diameter (mm) in sago strain of giant gourami broodfish\n\n- Table 4. Raw data of egg diameter increase (%) in sago strain of giant gourami broodfish\n\n- Table 5. Raw data of egg weight (mg) in sago strain of giant gourami broodfish\n\n- Table 6. Raw data of hardened egg weight (mg) in sago strain of giant gourami broodfish\n\n- Table 7. The data of egg weight increase (%) in sago strain of giant gourami broodfish\n\n- Table 8. The data of fertilization rate (%) in sago strain of giant gourami broodfish\n\n- Table 9. The data of hatching rate (%), endogenous feeding period (day), and embryo survival rate to the eyed-egg stage (%) and in sago strain gourami\n\n- Table 10. Male size, gonadal weight, and semen in sago strain of giant gourami broodfish\n\n- Table 11. Sperm concentration (109/mL) in sago strain of giant gourami broodfish\n\n- Table 12. Sperm Motility (%) in sago strain of giant gourami broodfish\n\n- Table 13. Duration motility (sec) in sago strain of giant gourami broodfish\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe authors thank Professor. Dr. Tafdil Husni the Rector of Universitas Bung Hatta for supporting this study through the competitive grant’s schema called Research Professor, 2021. The appreciation goes to all of the students (Puji Kurniawan and Ranji Rinaldi and Muhammad Vajri Djauhari) who helped the author during data collection in the field.\n\n\nReferences\n\nSyandri H, Azrita MA: Nitrogen and phosphorus waste production from different fish species cultured at floating net cages in Lake Maninjau, Indonesia. Asian J. Sci. Res. 2018; 11(2): 287–294. Publisher Full Text\n\nAryani N, Suharman I, Azrita, et al.: Diversity and distribution of fish fauna of upstream and downstream areas at Koto Panjang Reservoir, Riau Province, Indonesia. F1000Res. 2020; 8: 1435. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMungkung R, Aubin J, Prihadi TH, et al.: Life Cycle Assessment for environmentally sustainable aquaculture management: a case study of combined aquaculture systems for carp and tilapia. J. Clean. 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PubMed Abstract \| Publisher Full Text\n\nZadmajid V: Comparative effects of human chorionic gonadotropin (hCG) and Ovaprim™ (sGnRHa+domperidone) on the reproductive characteristics of wild-caught male Longspine scraper, Capoeta trutta (Heckel, 1843). Aquaculture. 2016; 463: 7–15. Publisher Full Text\n\nDumorné K, Valdebenito I, Contreras P, et al.: Effect of pH, osmolality and temperature on sperm motility of pink cusk-eel (Genypterus blacodes, (Forster, 1801)). Aquac. Rep. 2018; 11: 42–46. Publisher Full Text\n\nGolshahi K, Aramli MS, Nazari RM, et al.: Disaccharide supplementation of extenders is an effective means of improving the cryopreservation of semen in sturgeon. Aquaculture. 2018; 486: 261–265. Publisher Full Text\n\nYusoff M, Hassan BN, Ikhwanuddin M, et al.: Successful sperm cryopreservation of the brown-marbled grouper, Epinephelus fuscoguttatus using propylene glycol as cryoprotectant. Cryobiology. 2018; 81: 168–173. PubMed Abstract \| Publisher Full Text\n\nKommisrud E, Myromslien FD, Stenseth EB, et al.: Viability, motility, ATP content and fertilizing potential of sperm from Atlantic salmon (Salmo salar L.) in milt stored before cryopreservation. Theriogenology. 2020; 151: 58–65. Publisher Full Text\n\nCejko BI, Żarski D, Palińska-Żarska K, et al.: Artificial seminal plasma improves motility and fertilization capacity of common carp Cyprinus carpio L. sperm during one hour of storage. Aquaculture. 2019; 506: 224–228. Publisher Full Text\n\nSyandri H, Azrita A, Aryani N: Untitled ItemReproduction characterization of the gurami sago (Osphronemus goramy Lacepède, 1801): basic knowledge for a hatchery development strategy for the future. figshare. Journal Contribution. 2021. Publisher Full Text" }	[ { "id": "129704", "date": "06 Apr 2022", "name": "Fatimah Hashim", "expertise": [ "Reviewer Expertise Fish amoeba", "protozoology", "cell biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have evaluated this article and found the description of the approach taken for this study to be very appropriate, and each description is very detailed. Comparisons between previous studies are also fully informed in this article. I strongly support the following step of indexing of this article for the sharing of information on the reproductive characteristics of the giant gurami sago. I only found that a few corrections to the abstract need to be made, wherein the method section and the brand name need to be deleted from this section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "129705", "date": "19 Apr 2022", "name": "Mochamad Syaifudin", "expertise": [ "Reviewer Expertise Fish genetics and 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\nIn general, the article is quite good explaining the reproductive performances of gourami sago. However, the introduction needs to state the distribution of gourami strain sago in Indonesia, whether it is endemic or introduced in the region. There is less information on the general characteristics of sago compared to other strains. Table 5 should explain some specific conditions of breeding technology used in other strains.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-922
https://f1000research.com/articles/11-334/v1	18 Mar 22	{ "type": "Research Article", "title": "COVID-19 vaccination hesitancy among Iraqi general population between beliefs and barriers: An observational study", "authors": [ "Laith G. Shareef", "Ali Fawzi Al-Hussainy", "Sajid Majeed Hameed", "Ali Fawzi Al-Hussainy", "Sajid Majeed Hameed" ], "abstract": "Background: Vaccine apprehension poses a serious threat to global health. While there has been a tremendous global effort to create a vaccine against coronavirus disease 2019 (COVID-19), little is known about its reception in Iraq. Therefore, we sought to examine COVID-19 vaccine acceptance, hesitation, and related elements in the Iraqi population. Methods: Between the 19th of May and the 22nd of September 2021, a descriptive, cross-sectional web-based survey was conducted employing a quantitative approach. Respondents from both sexes aged 18 years and above who live in Iraq and agreed to participate were included. An anonymized online structured questionnaire was designed based on data from prior research on vaccine hesitation in general, and COVID-19 vaccination reluctance specifically. Results: A total of 1221 eligible participants from various regions in Iraq actively participated in the short web-based questionnaire. The overall acceptance rate of the COVID-19 vaccine was 56.2%, with a greater acceptance rate among younger male participants (p<0.05). Marital status had no significant association (p=0.834). Urbanization influenced the acceptance rate significantly (p=0.002). The barriers to receiving the COVID-19 vaccine were exemplified by the vaccine not being evaluated for a sufficient period in 51.4% of the responses, as well as concerns about future side effects in 76.6% of the responses and a lack of efficacy in 55.7% of the responses. The Pfizer-BioNTech vaccine received 39.6% preference and participants confidence, followed by the Oxford/AstraZeneca vaccine at 18.1% and the Sinopharm vaccine at 14.6%. Conclusions: COVID-19 vaccination apprehension was discovered in almost half of the study population. Lack of understanding about vaccination eligibility, anxiety about adverse events and vaccine efficacy, and distrust in the government were independently predictive of vaccine hesitation.", "keywords": [ "Perception", "attitudes", "Iraq", "COVID-19", "coronavirus", "vaccine acceptance", "SARS‑CoV‑2", "vaccine hesitancy" ], "content": "Introduction\n\nThe coronavirus disease 2019 (COVID-19) pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is still causing turmoil around the world. This pandemic seems to have had a negative influence on living standards and placed a strain on health care organizations.1 Diverse chemotherapeutic and biological treatments have already been tested in COVID-19 cases, but none have been shown to provide a definitive therapeutic efficacy.2 With little effectiveness, many preventive public health interventions such as quarantining, practicing hand hygiene and cough etiquette, as well as physical separation are all measures that were instated.3\n\nWhen vaccines were introduced during a previous influenza pandemic, the acceptancy level ranged between 8% and 67% as stated in a systematic review by Larson et al., in 2018.4 According to a meta-analysis by Xiao and Wong, the acceptance rate in the United States in 2020 was 64%.5 Also, in the United Kingdom, about half of survey respondents said they were willing to try the H1N1 influenza vaccine according to a report by Chan et al., in 2015.6 During the 2014 Avian influenza A(H7N9) outbreak in China, 50.5%of research participants were vaccinated with the A/H7N9 vaccine as soon as it was available.7 In Beijing, 59.5% of research respondents aware of H7N9 expressed that they would be willing to receive a future influenza A (H7N9) vaccination.8 Vaccine acceptability and need are multifaceted and context-dependent, altering over time periods, geography, and the beliefs influencing the scope of the community.9 According to a survey performed in Ireland, medical personnel ignored seasonal flu immunization mainly because of misapprehension regarding the efficiency and dependability of the vaccine.10 In China, demographic characteristics and public perceptions are major factors of vaccine uptake. The primary determinants of vaccine acceptance in Hong Kong are anxiety levels and vaccine experience.6 In the United States, the most important determinants of influenza vaccination adoption were anticipated vaccine efficiency, cultural influence, and insurance coverage.7 Higher introversion was related to poorer vaccination uptake in studies conducted in the United States, while greater confidence was linked to greater vaccination uptake.11 A study on parental opinions of pediatric age group immunizations in the UAE discovered that just 12% of respondents were against pediatric immunizations, according to the report, vaccination safety (17%), adverse reactions (35%), and too frequent injections (28%) were all critical factors in vaccine reluctance.12 Participant willingness to get the COVID-19 vaccine was higher among those who had previously received a seasonal flu vaccine.13\n\nThus far, several vaccines have been created, with some licensed and others continuing in clinical studies. Notably, the Pfizer-BioNTech, Moderna, and the Oxford/AstraZeneca vaccinations have all been licensed for urgent application and are now in use in a number of countries, including Iraq.14,15 Despite significant progress in vaccine research, public acceptability of the COVID-19 vaccine remains a significant barrier.16 As per the World Health Organization (WHO), vaccination reluctance is among the 10 leading threats to global health, but it is being aggravated mostly by rising conspiracy theories surrounding COVID-19 and vaccines.17 As a result, we sought to examine the acceptance, hesitation and related elements around the COVID-19 vaccine in the Iraqi population.\n\n\nMethods\n\nThis study was approved by the Al-Esraa University College Ethics Committee (approval no. 506). The Al-Esraa University College Ethics Committee waived the need for consent for the acquisition, interpretation, and publication of the prospectively collected anonymous data for this non-interventional research. Before participating in the survey, individuals were allowed to decline participation in the questionnaire. Participation in this study was entirely voluntary, free of coercion, and uncompensated. Participants provided informed, written consent as part of the questionnaire. Throughout the study, anonymity and confidentiality were maintained. All data were saved in a secure file, with only the researchers in charge of the questionnaire accessing the information. The study was carried out according to the World Medical Association’s Declaration of Helsinki.18\n\nThis was a descriptive, cross-sectional web-based survey performed between the 19th of May and the 22nd of September 2021, using a quantitative approach.\n\nRespondents from both sexes aged 18 years and above who live in Iraq and agreed to participate were included. Simple random sampling was the method of selecting participants in which the author chose a subset of participants randomly from a population. Each individual in the population had an equal probability of being considered.\n\nThe survey was disseminated through a variety of electronic means, including social media and e-mail. Therefore, any concerns regarding sample bias and generalizability may be ameliorated since the form could have been filled out by anybody with an internet connection. Furthermore, the author asserts that there are no instances of reporting bias or performance bias in their findings.\n\nThe sample size calculation was based on prior reports of COVID-19 vaccination hesitancy or refusal, ranging from 6% in Egypt to 13.9% in Italy, 23% in the United States, 30.5% in Malta, and 51.4% in Iraqi Kurdistan.19,20 This resulted in a sample size of 962 people, equating to the lowest prevalence, relative precision of 25%, and alpha error of 5%. The sample size was determined using the equation N = Zα2P (1 − P)/d2, where = 0.05 and Z = 1.96, and the permissible margin of error for proportion d is calculated to be 0.1. When we received 1221 completed surveys, we decided to end the survey.\n\nUsing data from prior research on vaccination reluctance in general, and specifically the COVID-19 vaccine,21–26 an anonymous online structured questionnaire was developed.43 The set of questions was divided into three sections: the first contained basic demographic information as well as an evaluation of understanding and source of knowledge concerning COVID-19 vaccine, the second contained an assessment of attitudes toward the vaccine, and the third contained details regarding vaccination barriers. This questionnaire was made available online using Google Forms.\n\nThe sociodemographic variables of sex, age, marital status, educational level, permanent resident, and sources of knowledge about coronavirus and COVID-19 vaccinations were included as independent key predictors, while the acceptance, hesitation, trust, and perceptions concerning COVID-19 vaccination were dependent variables.\n\nFor cleaning and coding, submitted questionnaire responses were exported from Google Forms to Microsoft Excel 2019 (Microsoft Excel, RRID:SCR_016137). The processed data were transferred to IBM SPSS software for Windows, version 25 (IBM SPSS Statistics, RRID:SCR_016479) for analysis. Means (standard deviations) and percentages were used to summarize numerical data. For parametric and non-parametric data, the median (interquartile range) was used. Categorical data were collected. Frequency and proportions were used to summarize the data. Relationships between independent factors and dependent variables were determined using chi-square test or Fisher’s exact test, and logistic regression analysis was used to evaluate variables. p<0.05 was considered statistically significant. When dealing with missing data, we relied on two primary methods for eliminating data: list-wise and deleting variables. Listwise method: This strategy deletes all data for an observation that includes one or more missing values. Only observations with a full set of data are subjected to the analysis. Dropping variables: If data are missing for over 60% of the samples, it may be best to remove if the variable is insignificant.\n\n\nResults\n\nA total of 1221 eligible participants (aged 18 and over) from various country regions actively participated in the short web-based questionnaire. The study population was mainly concentrated in urban areas (71.2% live in urban areas). Furthermore, a larger portion of responders within the age range of 18–29 years represented 40% of the study participants, and the ratio of male to female participants was 0.94:1, where the female percentage was 51.5%. A total of 719 (58.9%) were married. Most study participants held an academic degree (677, 55.4%). The overall acceptance rate was 56.2%. This research showed a higher acceptance rate of COVID-19 vaccination in younger, male participants (p<0.05). No significant association was demonstrated regarding marital status (p=0.834). Geographic area significantly impacted the acceptance rate where most of the respondents lived in urban areas (p=0.002). The level of education seemed to have no noticeable effect on vaccination acceptance rate (p=0.238) (Table 1).42\n\nMost of the participants did not have an underlying medical condition (798, 65%), 574 (47.0%) participants had been positive for COVID-19 in the past, while 815 (66.7%) reported the occurrence of COVID-19 infection in family members, from the total number of respondents 880 (72.1%) had children. The association of the above parameters with the vaccination acceptance rate showed that healthy people without a chronic illness had a higher acceptance rate of vaccination (p=0.002), patients with a past medical history of COVID-19 infection were accepting the vaccine at a significantly higher rate than patients without a history of infection (p<0.001), also there was a positive relationship between having family members who had been infected with COVID-19 with vaccination acceptance rate (p=0.001), and respondents who had children (<18 years) seemed to show a statistically higher level of vaccination acceptance (p=0.035) (Table 2).\n\nThe percentage of perceived risk of getting COVID-19 was very low; the majority of participants (47%) believed their risk ranged from 0 to 20% (Figure 1).\n\nCOVID-19, coronavirus disease 2019.\n\nTable 3 depicts the study population’s understanding of COVID-19 vaccinations; most respondents thought that the priority of vaccination should be focused on health care providers.\n\nThe barriers to receiving the COVID-19 vaccine were exemplified by the vaccine not being evaluated for a sufficient period in 51.4% of the responses, as well as concerns about future side effects in 76.6% of the responses, and a lack of efficacy in 55.7% of the respondents (Figure 2).\n\nCOVID-19, coronavirus disease 2019.\n\nThe Pfizer-BioNTech vaccine received 39.6% preference and participants confidence followed by Oxford/AstraZeneca vaccine at 18.1% and the Sinopharm vaccine at 14.6%, as shown in Figure 3.\n\nBesides the governmental platforms, social media have become a significant source for COVID-19 information; according to the findings of this study, 41% of respondents obtained medical instruction from social media, as illustrated in Figure 4.\n\nCOVID-19, coronavirus disease 2019.\n\n\nDiscussion\n\nThe vaccine is recognized as an important public health innovation of the 21st century. Its acceptability, however, varies with geography, time, socioeconomic class, ethnicity, and human-environmental behavior.27 Despite the fact that there have been few studies to investigate the intention to use the COVID-19 vaccination during the current crisis in Iraq, our findings contradict those of studies conducted in China and the United States.28,29 According to a study in China, 72.5% of members of the public intended to get the COVID-19 vaccine.29 In addition, studies conducted in the United States indicated that the COVID-19 vaccine was accepted by 80% of the eligible population.28 Throughout our research, 56.2% of participants indicated a desire to accept the COVID-19 vaccination. This study discovered there was a significant disparity in vaccination hesitancy frequency between men and women; however, vaccine hesitancy was high in both sexes.\n\nFurthermore, the individuals who were reluctant to get the COVID-19 vaccine were older. According to Robertson et al., in the United Kingdom, 21% of women and 14% of men were apprehensive about getting the COVID-19 vaccine.30 In contrast to this research, the younger respondents were less likely to refuse the COVID-19 vaccine.\n\nVaccine reluctance could be caused by a variety of scenarios, including disinformation and conspiracy beliefs.31,32 Furthermore, health inequities, economic constraints, racial profiling, and access hurdles are seen as underlying reasons for poor trust and poor uptake.33 According to Paul et al., in the United Kingdom, 16% of the study participants showed high levels of skepticism about the COVID-19 vaccines.34 Minority groups, individuals with lower levels of education, lower yearly income, and a lack of knowledge of COVID-19, and those who do not follow government COVID-19 recommendations have higher levels of distrust. They reported that 14% of respondents had chronic diseases and 23% of this chronically ill population were unsure about receiving the COVID-19 vaccine. Our participants in this study had a greater prevalence of chronic illnesses (34%) and a higher rate of vaccination unwillingness. Meier et al., indicated that people who perceived themselves to be more susceptible to COVID-19 were more likely to have a COVID-19 vaccine in the United States, which is consistent with the results in our research.35 The majority of study participants said that COVID-19 vaccination was necessary, especially for health care workers, and that it should be made mandatory once it was widely available. Corresponding findings were published by Lucia et al.36 The Centers for Disease Control and Prevention (CDC) advises that medical practitioners receive the first doses of COVID-19 vaccines because of the higher risk of exposure.37\n\nThe public’s confidence in the COVID-19 vaccine may fluctuate depending on the vaccine type. Research, for example, discovered the greatest degree of confidence in mRNA technology. Compared to AZD1222, both BNT162b and mRNA1273 had a better level of acceptability.38 Furthermore, cases of bleeding, thrombosis and platelets dysfunction following COVID-19 vaccinations in individuals with preexisting hemodynamic abnormalities or those taking particular drugs have sparked widespread alarm on social media. As a result, the use of the Oxford/AstraZeneca vaccine has been temporarily suspended in various European nations.39 Some officials were hesitant to receive the Oxford/AstraZeneca vaccine (ChAdOx1 nCov19) in May 2021 in Iraq. These officials awaited and received the Pfizer-BioNTech vaccine, which is based on mRNA. This unresponsive conduct may have contributed to apprehension about receiving the COVID-19 vaccine in Iraq.\n\nThe evidence shows that it is crucial to concentrate on building confidence in COVID-19 vaccines. This includes negotiating the COVID-19 evidence framework with credible communicators and fostering vaccination confidence via transparency and anticipatory monitoring. Societies must be included early on to respond to complaints, respond to questions, and dispel misconceptions.40 Because public trust in vaccination is low, COVID-19 immunization programs can only succeed if there is a widespread conviction that the vaccinations offered are safe and effective.41 Lucia et al., emphasized the importance of transparency to address concerns regarding the efficiency and safety of vaccine development.36 It is critical to support COVID-19 immunization through public statements and news releases, as well as to monitor and combat false news.36\n\n\nConclusions\n\nCOVID-19 vaccination apprehension was discovered in almost half of the study population. Lack of understanding about vaccination eligibility, anxiety about adverse events and vaccine efficacy, and mistrust in the government were all independent predictors of vaccination hesitancy. A heightened risk perception of COVID-19 reduced vaccine hesitancy. Concerns were raised about a lack of vaccination-related information and the vaccine launch before the release of evidence on safety and effectiveness. While vaccination indecision has decreased over time, health education programs designed to raise vaccine awareness and build trust in government authorities would be beneficial.\n\nOur study was limited by the fact that it was conducted after COVID-19 vaccination had been initiated. As a consequence, it is probable that it underestimated the initial vaccine rejection among individuals who were vaccinated and then moved to the vaccination accepting group.\n\n\nData availability\n\nZenodo: ‘Questionnaire responses for 1221 participants’. https://doi.org/10.5281/zenodo.6345333.42\n\nThis project contains the following underlying data:\n\ndata zenodo.xls (Questionnaire responses for 1221 participants)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nZenodo: ‘Research questionnaire’. https://doi.org/10.5281/zenodo.6345361.43\n\nThis project contains the following extended data:\n\n- Questionnaire.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBonyan FA, Shareef LG, Al-waily A, et al.: COVID-19 clinical characteristics and outcomes in 60 hospitalized Iraqi patients-Case series. Med. 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PubMed Abstract \| Publisher Full Text\n\nBangalee A, Bangalee V: Fake news and fallacies: Exploring vaccine hesitancy in South Africa. S. Afr. Fam. Pract. 2021; 63(1): e1–e3. PubMed Abstract \| Publisher Full Text\n\nIslam MS, Kamal AH, Kabir A, et al.: COVID-19 vaccine rumors and conspiracy theories: The need for cognitive inoculation against misinformation to improve vaccine adherence. PloS One. 2021 May 12; 16(5): e0251605. PubMed Abstract \| Publisher Full Text\n\nRazai MS, Kankam HK, Majeed A, et al.: Mitigating ethnic disparities in covid-19 and beyond. BMJ. 2021 Jan 15; 372. Publisher Full Text\n\nPaul E, Steptoe A, Fancourt D: Attitudes towards vaccines and intention to vaccinate against COVID-19: Implications for public health communications. The Lancet Regional Health-Europe. 2021 Feb 1; 1: 100012. PubMed Abstract \| Publisher Full Text\n\nMeier BP, Dillard AJ, Lappas CM: Predictors of the intention to receive a SARS-CoV-2 vaccine. J. Public Health (Oxf.). 2021 Mar 3; PubMed Abstract \| Publisher Full Text\n\nLucia VC, Kelekar A, Afonso NM: COVID-19 vaccine hesitancy among medical students. J. Public Health. 2021 Sep; 43(3): 445–449. PubMed Abstract \| Publisher Full Text\n\nMbaeyi S, Oliver SE, Collins JP, et al.: The Advisory Committee on Immunization Practices’ Interim Recommendations for Additional Primary and Booster Doses of COVID-19 Vaccines—United States, 2021. Morb. Mortal. Wkly Rep. 2021 Nov 5; 70(44): 1545–1552. PubMed Abstract \| Publisher Full Text\n\nRzymski P, Zeyland J, Poniedziałek B, et al.: The perception and attitudes toward COVID-19 vaccines: a cross-sectional study in Poland. Vaccines. 2021 Apr; 9(4): 382. PubMed Abstract \| Publisher Full Text\n\nWise J: Covid-19: European countries suspend use of Oxford-AstraZeneca vaccine after reports of blood clots. BMJ. 2021; 372: n699. Publisher Full Text\n\nCarson SL, Casillas A, Castellon-Lopez Y, et al.: COVID-19 vaccine decision-making factors in racial and ethnic minority communities in Los Angeles, California. JAMA Netw. Open. 2021 Sep 1; 4(9): e2127582. PubMed Abstract \| Publisher Full Text\n\nWouters OJ, Shadlen KC, Salcher-Konrad M, et al.: Challenges in ensuring global access to COVID-19 vaccines: production, affordability, allocation, and deployment. Lancet. 2021 Mar 13; 397(10278): 1023–1034. PubMed Abstract \| Publisher Full Text\n\nAlkinani LG: Questionnair responses for 1221 participants [Data set]. Zenodo. 2022. Publisher Full Text\n\nAlkinani LG: Research questionnaire [Data set]. Zenodo. 2022. Publisher Full Text" }	[ { "id": "128221", "date": "01 Apr 2022", "name": "Reem Hamdy A. Mohammed", "expertise": [ "Reviewer Expertise Autoimmune diseases", "clinical immunology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe given study is a descriptive observational research study that aimed to investigate the public perception of COVID-19 vaccination in the Iraqi population and it's relation to differing participation, as well as vaccine related variables that might affect perception, acceptance and decision to vaccinate.\nThe authors used a google form constructed survey divided into three sections, each concerned with specific variables that relate to the domains of concern. In building their research questionnaire and defining the sample size authors used data from the literature discussing the question of research going through detailed revision of similarly published international data as an evidence based reference. The analysis done was informative, focusing on the variables to be addressed and providing thorough descriptions of the relationships intended to be explored, including comments on the perception and acceptance for the different forms of vaccinations available.\nAuthors concluded from their questionnaire that lack of sufficient data about the potential long-term benefit versus risk of the vaccination constitutes the cornerstone behind hesitancy to vaccinate. The study is interesting and adds to the body of knowledge regarding the impact of the pandemic and the preventive strategies designed.\n\nIs the work 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": "128019", "date": "19 Apr 2022", "name": "Manal Mohammed Younus", "expertise": [ "Reviewer Expertise Pharmacovigilance and pharmacoepidemiology", "drug safety", "clinical pharmacy and pharmacy practice." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst, I would like to thank you for the opportunity to review this article. The subject is important, and there is still a need to have such studies covering it. The manuscript describes the responses of online respondents to the survey items tackling different factors that reflect the understanding of the respondents to the COVID-19 vaccination and barriers to receiving the vaccines. The study also included the COVID-19 vaccines preferences and the primary source of information related to the vaccines. The study showed that about half of the participants had hesitancy in accepting the vaccines. The barriers were related to the vaccine's eligibility, effectiveness, and safety.\nBelow are my notes:\n1. Introduction:\n\nPlease rewrite the section with references (4-13) and tell in a more clear way how the problem is not only H1NI vaccine-related.\n\nThere is no mention of COVID-19 vaccines in this paragraph, and one would assume that all that is written is related to H1N1 vaccine hesitancy. Please clarify.\n2- Methods:\nRegarding the questionnaire, please mention if internal or external validation was required and if it was adopted from other languages.\n\nAlso, why do you use only two options yes or no, and agree or disagree, and not give a third alternative option like I don't know?\n\nRegarding the statistical analysis; could you elaborate on why using Fisher’s exact test when your sample size is big enough to be analyzed using chi-square.\n\nIn the last two lines of the data analysis part; the manuscript said \"If data are missing for over 60% of the samples, it may be best to remove if the variable is insignificant.\" Is this a statement or it is the methodology that was used by the researcher. Please clarify.\n\n3- Results;\nCould you please mention what social media platform you distributed the questionnaire to and how many responses you received throughout the email!\n\n4- Discussion;\n\nPlease insert a reference to the paragraph; \"Some officials were hesitant to receive the Oxford/AstraZeneca vaccine (ChAdOx1 nCov19) in May 2021 in Iraq. These officials awaited and received the Pfizer-BioNTech vaccine, which is based on mRNA. This unresponsive conduct may have contributed to apprehension about receiving the COVID-19 vaccine in Iraq.\"\n\nIs the work 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": "8136", "date": "25 Apr 2022", "name": "Laith G. Shareef", "role": "Author Response", "response": "I appreciate the time and effort the reviewers have dedicated to providing valuable input on my manuscript. I have been able to incorporate changes to reflect most of the suggestions provided by the reviewer, and I have highlighted the changes within the manuscript. Introduction: I’m sorry for being misunderstood, I rewrote the paragraph with references (4-13) as you recommended, and please let me clarify my point of view for this section to be clearer for you and the readers: This section summarized the acceptance rate of previously developed vaccines not only in H1N1 in USA and UK (Ref. 5 & 6); but also, in AH7N9 in China (Ref. 8), seasonal flu immunization in Ireland among medical staff (Ref. 10 & 11), refusal of pediatric age group vaccination in the Arab region (UAE) (Ref. 12), and the association of flu vaccine acceptance with the COVID-19 vaccine acceptance (Ref. 13). Thank you for your recommendation regarding adding more about COVID-19 acceptance, I reinserted more about this topic in reference 17. Methods: Thank you for your comments regarding validations and other languages usage, the required details have been mentioned in the revised version. Thank you for your comments regarding the type of question. The questions in this study were adopted from the book \"Questionnaire Design: Asking Questions with a Purpose,\" https://vulms.vu.edu.pk/courses/HRM619/Downloads/How_to_ask_questions_through_questionaire.pdf and we discovered that one of the permissible kinds of close-ended questions is as follows: \"there is a variety of ways to write close-ended questions. Some required answers that fall along an implied continuum (as in a rating scale); others supply answers in no particular order (lists): ( check all that apply); others provide relevant answers but allow respondents to add others not on the list. the following section gives examples of two-options responses: No-Yes, Disagree-Agree, False-True, and Oppose-Favor.\" Thank you for your comments regarding the tests used for data analysis. I reviewed more than statistical references comparing the two tests listed above: - 1) As the name indicates, Fisher's exact test provides an exact right result regardless of sample size. https://www.graphpad.com/guides/prism/latest/statistics/stat_chi-square_or_fishers_test.htm 2) When the sample size is high enough, the chi-square p-value approaches that of a Fisher's exact. Fisher's exact also has the advantage of being applicable to large sample sizes. https://www.saem.org/about-saem/academies-interest-groups-affiliates2/cdem/for-students/cdem-voice/educational-research-column/educational-research-column-choosing-wisely-chi-square-vs.-fisher-s-exact Thank you for your comments regarding the manuscript's last two lines in the data analysis section. Actually, this was how to handle missing data. Results: Thank you for your comments regarding questionnaire distribution. As you recommended, we clarified the revised version of the manuscript, the questionnaire has been distributed throughout the most widely used social media in Iraq, like Facebook, Twitter, and Instagram. We did not get the responses by email we only distribute the questionnaire through it to relatives or co-workers who did not have been in touch over the social media. Discussion Thank you for your comment regarding inserting the reference for the aforementioned text. This was a piece of trending news over the newspapers, tv news since no reliable reference could be cited, I removed the paragraph from the revised version." } ] } ]	1	https://f1000research.com/articles/11-334
https://f1000research.com/articles/11-454/v1	25 Apr 22	{ "type": "Research Article", "title": "Preventable contributors to the neonatal healthcare-associated infections: a uni-center analytical study from South India.", "authors": [ "Usha Rani", "Leslie E. Lewis", "Kiran Chawla", "Anup Naha", "Usha Rani", "Kiran Chawla", "Anup Naha" ], "abstract": "Background: Globally, neonatal healthcare-associated infections (HAIs) are known to cause high mortality. HAIs is a preventable condition related to the healthcare environment. The current study explored the contributors to neonatal HAIs in one of the largest tertiary care referral hospitals in South India. Methods: Neonates from December 2016 to June 2018 were observed for the occurrence of healthcare-associated infections and compared with the matched control group. Various observations on neonatal demography, maternal contributors, and medical procedures were made and recorded to explore and analyse the contributors to neonatal HAIs. Univariate and multivariate analysis was carried out to find the contributors. The Odds ratio with 95% CI was also computed and reported. Results: Bloodstream infection (83%) was prevalent among neonates; the maternal contributor was only preterm labor (Odds ratio of 11.93; 95% CI; 6.47-21.98; p<.05) to acquire HAIs. On univariate analysis, mechanical ventilation for > 3days duration, NIV for > five days, and PICC line insertion procedure were significant (p<0.05) contributors to neonatal HAIs. IV cannulation for more than three times in four consecutive days was found in 100(85%) neonates considered being associated with neonatal HAIs. On multivariate analysis, NIV, PICC line, preterm labor, and low birth weight were significant (p<0.05) contributors to neonatal HAIs. Conclusion: The increased duration of invasive and non-invasive therapeutic devices and catheters contributes to neonatal HAIs. Neonates are acquiring bloodstream infections; low birth weight (LBW) neonates are more susceptible to acquiring HAIs.", "keywords": [ "Cross infection", "neonate", "healthcare", "prevention" ], "content": "Introduction\n\nReports from UNICEF and the World Bank 2018 showed a reduction in Neonatal Mortality Rate (NMR) among all the participating countries. India’s NMR is 23 as per 2017 report.1,2 Each year nearly 0.748 million newborn deaths occur in India, contributing to 26% or 1/3rd of the world’s neonatal death. As per WHO, in developing countries like South East Asia, HAIs are responsible for nearly 50% of mortality in Neonatal Intensive Care Unit (NICU).3 The rate of healthcare-associated infections (HAIs) reported by the World Health Organization (WHO) in South Asian countries ranges from 11.3-23.6%.4\n\nAccording to Liu et al., dominating causes of neonatal deaths are preterm (35%), birth asphyxia (20%), pneumonia (16%), sepsis (15%), and other causes (<10% each group), these are also the primary reasons for ICU admission and development of HAIs.5 Indian studies have reported bloodstream HAIs, Ocular infection (Healthcare-associated conjunctivitis), urinary tract infection, and skin infection among neonates.6–11\n\nNewborns needing critical care and support get hospitalized in Neonatal Intensive care units (NICUs) are at increased risk of acquiring Healthcare-associated infections (HAIs).12 HAIs have a detrimental effect on the recovery, length of stay in NICUs, and immune system acting as a vicious cycle.13\n\nNeonatal HAIs is one of the preventable leading cause for neonatal mortality. Developed countries are putting efforts to identify the risk factors to neonatal HAIs and its preventable measures.14,15 The contributing factors to neonatal HAIs in India are unexplored among neonates.16 The rate of HAIs and their related mortality and morbidity is also not explored in Indian literature. To contain the spread of microorganism, domestic preventable contributors to neonatal HAIs needs to be explored. The objective of the study was to identify the contributors to neonatal HAIs in one study centre.\n\n\nMethods\n\nThe study collated and analysed the contributing factors from December 2016 - June 2018. The study was conducted in one of the largest tertiary care teaching referral hospital in south India. Manipal Academy of Higher Education (MAHE) ethics committee provided Institutional Ethics Committee (IEC) approval was taken, approval ID: MUEC/014/2016-17. The study setting is equipped with a 30-bedded level III Neonatal Intensive Care Unit (NICU) with mechanical ventilation, indwelling catheters, intravenous fluid, phototherapy, and an advanced monitoring facility.\n\nICU hospitalized neonates were followed to development of neonatal HAIs. A case-control study design was adopted with controls matching the gestational age to identify maternal and neonatal direct contributors. The neonates were defined from the day of birth ‘0’ days to ‘28’ days; however, each day of life was counted from 24 hours of birth, not at midnight.\n\nIn order to compute the sample size, the probability of HAIs in current settings was obtained from a pilot study data of the same study setting and found to be 8 per 100 admissions >48 hrs. We assumed the probability of acquiring neonatal HAIs (p) as 0.08 and the probability of not acquiring neonatal HAIs (q) as 1-p = 0.92. With a 95% confidence interval using estimation of single proportion sample size calculation method d (accepted error) = 0.05. Sample size computation was made as below: s=Z2pqd2 = 113 neonates with HAIs for infinite population and 106 for the finite population. We assumed 10% missed events; hence another 12 cases were added in the sample pool leading to 118 samples of neonates acquiring HAIs with similar numbers in the control group.\n\nThe case detection and confirmation were carried out for any newborn admission to NICU >48 hrs using a combination of CDC and WHO recommended clinical findings and diagnostic findings, where the presence of at least any two variables of each category confirms the case label as HAIs.17,18 The following clinical and diagnostic criteria were used to determine the neonate acquiring HAIs. The presence of any two clinical and diagnostic tests was considered as the case of positive neonatal HAIs (Table 1). The Case Record Form (CRF) was prepared keeping objectivity as priority to clearly discriminate between neonate acquiring HAIs versus other sick babies. The CRF was pilot tested for construct, content and criterion validity before the commencement of the study.\n\nNeonates admitted from other hospitals with sepsis and maternal infections like UTI/Chorioamnionitis/Pneumonia were excluded from the study. Each morning of a working day the duty clinician as well as the nurse in charge was enquired for any probable case of HAIs by the researcher. If it was a holiday or weekend the next working day the enquiry was raised on suspected case of neonatal HAIs who was in NICU since last 48 hours. Only infections originating at the study site after 48 hours of admission were identified and included in the study as per the clinician ascertainment and the diagnostic criteria determining HAIs. The data on these neonates were obtained from medical records.\n\nBed occupancy days, admission and discharge in total per month, and further inborn and outborn categorization is reported in numbers per month. The rate of HAIs was calculated per 100 neonatal admissions for >48 hours.\n\nRate of HAIs per 100 neonates admitted for >48 hours = (# of new HAIs/# of all admissions in respective month >48 hours) × 100\n\nBacteraemia rate was calculated by dividing the number of new cases by the total number of bed occupancy days and multiplying it by 1000.\n\nBacteraemia rate per 1000 days = (#of HAIs per month/Total number of Bed Occupancy days in the respective month) × 1000\n\nA Chi-square test was performed on the birthing place and HAIs to find any statistical significance among two variables where the level of significance was fixed at <0.05.\n\nThe variables were entered in Microsoft Excel sheet within 48 hours of data collection and the neonate was followed till its outcome as improved/succumbed/discharge against medical advice. If the any variable input was missed the clinician and nurse care giver was asked to provide the relevant information on missed data. Missed data was manually filled post completion of the enquiry. The descriptive variables were reported using mean and standard deviation when normally distributed. Median with interquartile range was reported for skewed data. The frequency and percentage table were used for the nominal and categorical variables. Boxplot was used for skewed data to identify and report the outliers, however, further analysis with or without outliers was not performed.\n\nMaternal risk factors like mode of delivery, premature rupture of membrane (PROM), maternal peripartum infection, preterm labor, foul vaginal discharge, maternal urinary tract infection, intrapartum fever >38°C, and uterine tenderness was recorded and analyzed for their role in HAIs. A case record form was used, and details on maternal history were obtained from the neonatal record file at the time of admission.\n\nNeonatal healthcare-associated infections have been related to many variables, the patients’ data related to demography like gestational age, birth weight, mode of delivery and congenital deformity, gender, date of birth, date of admission, date of discharge/death/DAMA (Discharge Against Medical Advice) etc. were captured and analyzed.\n\nMedical invasive interventions, number of intravascular lines, duration of invasive and mechanical ventilation, central line, a peripherally inserted central catheter (PICC line), medication delivery, and any other invasive procedure record were captured. Medical non-invasive interventions like duration of non-invasive mechanical ventilation, phototherapy, and routine care were recorded.\n\nThe score for neonate acute physiology (SNAP II) score for each neonate in their first 24 hours of admission was calculated and reported. Clinical diagnosis and patients’ vitals were recorded to identify cases of HAIs.\n\nClinicians’ ordered diagnostic biochemistry laboratory investigations, and the result of microbiological test reports in the proforma were recorded, which helped identify the cases with HAIs.\n\nA record on the number of catheters, cannula/tubing attached, and the procedures carried on to the neonate was captured. Later retrospectively, the records were screened to find any invasive or non-invasive procedure, the medical treatment, the feeding schedule and the type of feeding given, the number of times the catheter site was touched, the number of invasive catheters, and the duration of each catheter in the body and any sign or symptom of infection was recorded. This proforma was filled within 48 hours of diagnosing HAIs in neonates. The controls of 1:1 were taken from the medical record of the neonates matching the gestational age. The recorded data was captured, and observations on the control could not be carried out.\n\nThe descriptive variables were reported using mean and standard deviation when normally distributed. Median with interquartile range was reported for skewed data. The frequency and percentage table were used for the nominal and categorical variables. A cluster bar diagram was used to summarise the categorical variables. Chi-square test was used with a level of significance at <0.05 for analyzing maternal and neonatal contributors for HAIs. The Odds ratio for demographic variables and the use of devices to ascertain HAIs was determined. Analysis of the odds of vascular catheter insertion for more than three times to HAIs was not ascertained due to insufficient controls data.\n\nUnivariate logistic regression followed by Backward Wald to identify the independent coefficient of each covariate was carried out using IBM SPSS Statistics for Windows, version 26 (IBM Corp., Armonk, N.Y., USA). Multivariate analysis of the variables found to be significant in univariate analysis was carried out to find the adjusted contributors to HAIs.\n\nClinical Trial Registry India (CTRI) registration was done before starting the project, and the confirmation ID was: CTRI/2017/08/009538.\n\n\nResults\n\nThe total number of admissions to NICU was 2278 neonates, with 1223 neonates hospitalized for >48 hours in nineteen months duration. Neonates hospitalized for less than 48 hours with outcome or with infection were excluded (Table 2). The rate of HAIs was 9.6±4.1 per 100 admissions, and the bacteremia rate was 5.2±1.6 per 1000 days (Table 2).\n\n* Gestational age was taken for matching.\n\nThe median length of stay for all the admissions to NICU was 10 days (IQR=8.9-11), and for cases with HAIs, it was 30 days (IQR=16-45), we did not find any correlation of length of stay with any specific microorganism as found in other studies.19 The length of stay for cases with HAIs ranged from four days to 147 days. Neonatal stay in NICU for >48 hours was found in 45% of all admissions to NICU.\n\nThe SNAP II score ranged from 0 to 94, where 79% of neonates had a score of ‘0’; 5% had a score of 5, and there were 11% neonates with a score of >5 within 24 hours of admission. Among the 5% of neonate in case group and all the patients in control group; the SNAP II score could not be calculated due to the unavailability of the required data. The SNAP II score was computed to describe the severity of sickness among the neonate on admission to NICU and was not considered as contributor to HAIs.\n\nThere were four neonates with congenital issues where two had intrauterine growth retardation (IUGR), one with hypoxic-ischemic encephalopathy, and one had hypoglycemia and seizures. Since the data was captured from medical records and were also verified by physician there was no variable considered to be contributing to HAIs had missing data.\n\nMothers of the neonate in the very preterm category had experienced more premature rupture of the membrane (PROM) (57%) as compared to others. These neonates had a better outcome (72%) and were improved with treatment even though they acquired HAIs. However, any neonate born to a mother with a history of PROM >18 hours was excluded from the study.\n\nMothers of moderate to late preterm neonates acquiring HAIs experienced preterm labor (44%) more compared to any other group and was found to be statistically significant at p=0.000. There was 24 (27%) mortality among neonates with HAIs born to mothers with preterm labor, whereas 63 (69%) neonates had recovered. There was no statistically significant association (p=0.2504) between maternal contributors and the neonate’s outcome (mortality vs. improved).\n\nPreterm labour was noted among ninety-one mothers of neonate acquiring HAIs but the first culture sample among all these neonates did not grow any microorganisms. No mother was identified with intrapartum fever >38°C or uterine tenderness or Maternal leukocytosis >15000/mm3. We found preterm labor with an Odds ratio of 11.93 (95% CI; 6.47-21.98; p<0.0005) contributed to the development of HAIs in univariate analysis,\n\nEighty-three (70%) neonates acquiring HAIs were delivered through cesarean section (C-Section), similar to studies reported from India.19 Most of the neonates were improved and discharged (71%) from NICU; however, mortality (25%) and discharge against medical advice (4%) outcome was recorded, in control group there was 19 (16%) neonates died and 99 (84%) neonates improved (Table 2). The overall mortality during the study period in the entire NICU was 5% among all neonatal admissions, whereas there was 25% mortality among the neonates acquiring HAIs, contributing to nearly 1/4th mortality (24%) among all the admissions to NICU. The Odds of mortality were found to be insignificant (Odds ratio=1.777; 95% CI=0.93-3.39; p=0.0814) among cases with HAIs compared to non-HAIs cases.\n\nThe male gender (59%) acquired HAIs more than the female gender (41%). Moderate to late preterm neonates (38%) acquired HAIs more compared to the rest of the categories. Mean gestational age was 32±4.3; the gestational age showed near-normal distribution among neonates with HAIs.\n\nOn univariate analysis, we did not find any birth weight category as a contributor to neonatal HAIs both the Odds ratio and chi-square test were insignificant to HAIs; ELBW (p=0.339), VLBW (p=0.7833), LBW (p=0.6353), NBW (p=0.09).\n\nMost of the neonates acquiring HAIs were born through C-section (71%), were VLBW with a median birth weight of 1370 (IQR=530, 3860) grams. The majority of the neonate acquiring HAIs were male gender (59.3%) and were moderate to late preterm (39%) with VLBW (35%). Further analysis between gender and birthweight showed that the male gender with VLBW (19%) was more prone to HAIs than females in a similar birth weight category (16%) (Table 2).\n\nOverall extreme preterm neonates (38%) had detrimental outcome (mortality) as compared to the rest of the gestational age group. Neonate with moderate to late preterm with LBW acquiring HAIs had higher mortality than any other gestational age or birth weight. Neonates had birth weight ranging from 520 g to 3850 g showed marked improvement in health conditions over time and were discharged (Figure 1).\n\nInvasive and non-invasive types of assisted ventilation were provided to 82% of the neonates who acquired HAIs, and 56% of neonates with HAIs were provided invasive mechanical ventilation for <5 days. Maximum neonates acquiring HAIs had <3 days of invasive mechanical ventilation and <6 days of non-invasive ventilation (Table 3).\n\nMechanical ventilation > 3days on the chi-square test was significant at p<0.05 to neonatal HAIs. The Odds of neonates on mechanical ventilation were at 2.1 times higher risk (Odds ration=2.11; 95% CI=1.24-3.59; p=0.0056) of acquiring HAIs compared to those who were not mechanically ventilated (Table 3).\n\nBefore diagnosing HAIs, apnoea and bradycardia were observed in 55 (47%) and 75 (64%) neonates. Both apnoea and bradycardia were observed in 41 (35%) neonates and an Irregular heart rate with apnoea was noted among 26 (22%) neonates that required immediate respiratory support, and later in two to three days’ time, neonates were identified as acquiring HAIs.\n\nThe odds of acquiring HAIs due to utilization of NIV was three times higher (Odds ratio=3.07; 95% CI=1.80-5.23; p<0.0001) compared to non-utilization of NIV. In 69 (58%) neonates, Feed intolerance was noted before acquiring HAIs.\n\nThere were 100 (85%) neonates who received >3 times IV cannulation procedure and developed HAIs within 72-96 hrs of the procedure. There were 35 (30%) neonates having either peripherally inserted central catheter or umbilical catheter who acquired HAIs when these lines were in situ. Such neonates’ blood samples were sent within 72-96 hrs to the microbiology lab for culture and sepsis screen. The presence of a PICC line on univariate analysis was a significant (p<0.0003) contributor to neonatal HAIs. PICC line presence carries an Odds of 5.5 times higher risk (Odds ratio=5.538; 95% CI=2.192-13.99; p≤0.001) to pose HAIs. In contrast, the presence of an umbilical catheter did not pose threat to cause HAIs (Table 4). The Chi-square (chi-square statistic is 15.5361) test was also significant for the PICC line (p=0.000081) but not for umbilical line insertion for HAIs (Table 4).\n\n* Chi-square test was performed with 95% CI and level of significance at p<0.05.\n\nChange of IV cannula for more than three times in less than four days was observed in 100 (85%) neonates who acquired HAIs later on. Those neonates who had confirmed BSI 98 (83%) among them 85 (87%) neonates had IV cannulation changed for > three times in the last four days before the occurrence of BSI. There were 33 (28%) neonates who had both PICC lines and had a change of IV cannulation > three times before the development of HAIs. We could not carry out further analysis due to the lack of recorded data on IV line insertion in the control group.\n\nMultivariate analysis showed NIV (p=0.000; 95%CI), PICC line (p=0.005; 95%CI), preterm labour (p=0.000; 95%CI) and LBW (p<0.05; 95%CI) as contributors to neonatal HAIs. Odds of neonate on NIV posed a 2.1 times higher risk (Odds ratio 2.133; 95% CI=1.097-4.149) to the development of HAIs. The presence of a PICC line carries 6.5 times higher risk (Odds ratio 6.595; 95% CI=2.104-20.665) to HAIs. Preterm labour (Odds ratio 14.911; 95% CI=6.514-34.134) and very low birth weight (VLBW) (Odds ratio 3.371; 95% CI=1.169-9.717) pose 14.9 and 3.4 times higher risk respectively to the occurrence of neonatal HAIs. However, mechanical ventilation, umbilical catheter, PROM, ELBW, LBW, and normal birth weight (NBW) do not pose a statistically significant risk to acquiring HAIs found on multivariate analysis.\n\n\nDiscussion\n\nAs per a systematic review burden of HAIs ranged from 3.6 to 11.6 per 100 neonatal admissions, whereas in low-middle income countries, it ranges from five to 19 per 100 neonatal admissions.4 The incidence density of HAIs in the USA and Europe ranges from 13.0 to 20.3 incidence per 1000 hospital bed days. A multicentre study from Canada and another one from Germany reported the rate of HAIs as 23.5% and 12.3% among 100 neonatal admissions.20,21\n\nIn Brazil rate of HAIs ranged from 12.3 % in NBW neonates and up to 51.9% in very low to extreme low birth weight neonates (ELBW). The overall incidence density bacteremia rate was 24.9 per 1,000 hospital bed days.22,23 Another large study from Germany has reported BSI incidence as 6.5 per 1000 hospital bed days.24 In South Asia, the incidence density of neonatal HAIs reportedly is 9.8 per 1000 live births.25\n\nThe rate of HAIs and the bacteremia rate in the current study was similar to a study conducted in Italy where the rate of HAIs was 9%, but incidence density was lower 3.5 per 1000 hospital bed days compared to the current study.26\n\nNeonates acquiring HAIs and delivered to mother with PROM had no different outcome, however, is considered as a risk for HAIs27,28 and were born to very preterm and moderate to late gestational age. Maternal peripartum infection,29 UTI and leaking per vaginal had no different outcomes among neonates acquiring HAIs. However, the number of cases with HAIs were very few to analyse.\n\nMothers with preterm labor observed high neonatal mortality (35%), India witnesses the highest preterm deliveries in the globe and 50% of neonatal death occurs in preterm (<37 weeks gestation).30 We observed high mortality among moderate to late preterm neonates (34%) born to mothers with preterm labor, probably due to the high number of neonates in this category acquiring HAIs.\n\nNeonates acquiring HAIs and delivered to mother with PROM had no different outcome, however, is considered as a risk for HAIs27,28 and were born to very preterm and moderate to late gestational age. Maternal peripartum infection,29 UTI and leaking per vaginal had no different outcomes among neonates acquiring HAIs. However, the number of cases with HAIs were very few to analyse.\n\nMothers with preterm labor observed high neonatal mortality (35%), India witnesses the highest preterm deliveries in the globe and 50% of neonatal death occurs in preterm (<37 weeks gestation).30 We observed high mortality among moderate to late preterm neonates (34%) born to mothers with preterm labor, probably due to the high number of neonates in this category acquiring HAIs.\n\nAnother known contributor to neonatal HAIs is mothers unpasteurized breast milk that was contaminated with MRSA leading to neonatal HAIs in one of the studies, however, we did not find any such contributors during our study.31\n\nPreterm labour, that was significantly associated with neonatal HAIs alone may not be able to contribute to HAIs, it can lead to lower gestational age and LBW delivery of the neonate that might contribute to a more significant extent on acquiring HAIs.\n\nBasu et al.32 found no significant association between the occurrence of HAIs and mode of delivery but contradictory to another study published from North-eastern India where they found a significant association between vaginal delivery (p=0.002) and occurrence of HAIs,33 we did not find any association with mode of delivery.\n\nThe mortality of near 25% was similar to the study by Bammigatt et al.34 where they also reported 24% mortality with no statistically significant (p>0.05) among cases with HAIs.\n\nHAIs are always known to cause higher related mortality and cause of concern among all the risk factors for mortality.12,35 Neonatal mortality among developing nations due to HAIs ranges from 4% to 56% of all causes of mortality during the neonatal period,36 and our study found 24% of mortality was due to HAIs.\n\nWe found a predominance of male (59.3%) gender in neonatal HAIs in similar studies from the same region where they found a male predominance of 62.3%, and in another study, it was found to be 1.3:1 for the male to female ratio in acquiring HAIs in India.8,28 A systematic review on neonatal sepsis found that the male gender (OR: 1.3, 95% CI: 1.02, 1.68) is a risk factor.37\n\nThose born preterms had a 92% increase in the risk of getting HAIs compared to other gestational ages, as reported in studies from developed and developing nations where preterm birth is a risk of acquiring HAIs.25,38,39 The number of moderate to late preterm neonates who acquired HAIs was more than other gestational age groups, and the reason could be the higher number of admission for 48 hours and beyond to NICU of moderate to late preterm neonates compared to other gestational age.40,41 The majority of neonates in this category was admitted for respiratory distress syndrome.\n\nThe neonatal HAIs among neonates with birth weight >1500 grams (62%) was reported as significant from north India.42 Another large study from Germany has reported a high prevalence of HAIs among VLBW neonates.24\n\nLBW neonates are more susceptible to neonatal HAIs than NBW categories,26 but our findings were in contrast. In our study, most of the neonates were in the VLBW category (35%); there was an equal proportion of birth weight distribution among ELBW (23%) and LBW (23%); however, the NBW neonates (19%) were very few in numbers.\n\nVLBW and prematurity were identified as factors contributing to neonatal HAIs in a study from western India.43 VLBW with very preterm neonates and moderate to low birth weight with moderate to late preterm were found susceptible to HAIs in our study, similar to a study from Northern India that reported LBW as a risk factor to neonatal HAIs.44\n\nHowever, in this study, we found only three VAP cases, and the majority were bloodstream infections still, the presence of mechanical ventilation was identified as a contributor to HAIs. Mechanical ventilation with intubation was found to be a risk to neonates causing HAIs.45,46 The neonates exposed to non-invasive ventilation had an insignificant risk of acquiring HAIs, similar to other studies showing that the increased duration of NIV utilization poses a higher risk of acquiring HAIs.24,47\n\nIn this study, 97 (82%) of the neonates were kept nil per oral (NPO) before developing HAIs that could have been a reason for lower immunity and infection as reported in the literature.48,49\n\nThe placement of the umbilical catheter line did not show significant risk to neonates for HAIs in contrast to other studies where the author found an association in umbilical catheter insertion to HAIs.40,50 We did not find any study evaluating association with the cannulation procedure in less than four days to greater than one day to the occurrence of HAIs.\n\nWe found a frequent change of IV cannula (for > 3 times) leading to multiple pricks in the skin and use of mechanical ventilation (< 3 days) was found frequent among neonates acquiring HAIs. However, further analysis could not be carried out. A study by M. Takrouri et al. suggests changing the IV cannula every 48 hours to prevent colonization and further infection to the patient,51 but our study finds a frequent change in IV cannula poses a higher risk to neonatal HAIs. Although umbilical catheters had no significant association with HAIs, changing the central venous catheter/umbilical catheter every 10 days or less to prevent HAIs may have a better outcome.51 Different therapeutic procedures, both invasive and non-invasive, significantly contribute to neonatal HAIs as per the large multicentre study of the German Neonatal Network.52 To curb the development of HAIs, it is necessary to reduce the number of procedures, duration of invasive and non-invasive procedures, especially to neonates born due to premature labor and very low birth, as they pose a higher risk of acquiring HAIs.\n\nThis study had a few limitations as data collection was carried out in one study centre. In order to identify potential case of HAIs the researcher was dependent on the discretion of the duty physician and nurse. Physician initiated studies and in closed ICU would have provided further insight to the outcome. It would have been better to have cohort study rather case-control study design keeping track of all the patients irrespective of the underlying disease or HAIs. The record on number of IV site pricks and reason to remove existing IV line for all the neonate was not available and hence further analysis on this potential factor could not be carried out. Maximum data was captured from medical records of the neonate rather prospectively collected. There could be miss cases of HAIs due to selection bias created by the study data collection method that could affect the results of the study. There was no practice on collection of swab samples from various parts of ICU except on two occasions, where there was suspected outbreak of two microorganism during the study. During these two occasions identified source was from the cradle of the neonate and washbasin of NICU. Probability of missing source of infections cannot be ignored while interpreting the findings. There are many other cofactors that could be contributor to neonatal HAIs like hand hygiene of healthcare workers,53 this was analysed and reported elsewhere but its establishment with cases of HAIs was not ascertained. There are other factors that were identified as contributor to neonatal HAIs elsewhere like ELBW, MV, umbilical catheter, were not found as contributor in this study setting.\n\nAs this was a uni-centre study, the results cannot be generalized to the whole population. The explored factors are similar to the published literature but some new factors that were identified and highlighted need further exploration to determine as the contributor to neonatal HAIs. These contributors could be related to only this study setting considering the policy and practices of healthcare workers. Furhter multicente cohort studies considering capturing the data on this list of factors could help to bring out associated preventable contributors. There are variabilities on neonatal care practices that could be a potential contributor requiring further analytical studies like decision on removal of a central line/umbilical catheter. There is a dearth of published literature from India requiring further research and reporting on factors contributing to neonatal HAIs.\n\n\nConclusion\n\nBloodstream infection (83%) was prevalent, causing neonatal HAIs. Mechanical ventilation, NIV, and PICC line on univariate analysis contributed to neonatal HAIs. Although on univariate analysis, mechanical ventilation for > three days duration, NIV for > five days, and PICC line insertion procedure were contributors to neonatal HAIs but on multivariate analysis, NIV, PICC line, preterm labor, and LBW were found as contributors to neonatal HAIs. IV cannulation more than three times in four consecutive days was associated with neonatal HAIs; this needs further studies to find any correlation as a contributor to the neonatal HAIs.\n\nHowever, mechanical ventilation, umbilical catheter, PROM, ELBW, LBW, and NBW did not pose a statistically significant risk to acquiring HAIs. A larger multicentric study from India will be required to establish further evidence.\n\n\nData availability\n\nFigshare: Dataset on neonatal Healthcare Associated Infections of a uni-center analytical study from South India. DOI: https://doi.org/10.6084/m9.figshare.1940362454\n\nThis project contains the following underlying data:\n\n- The data set is on the contributors to neonatal HAIs from an Indian NICU of a tertiary care teaching hospital.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthors’ contributors\n\nAll the authors conceptualized the research idea and contributed to the manuscript. Usha Rani conceptualized, designed, analyzed, and reported the data. Leslie E. Lewis carried out technical guidance, data analysis verification, and data reporting. Usha Rani and Kiran Chawla developed a data extraction form, and later interpreted the analyzed data. Usha Rani, and Anup Naha, analyzed and reported the data. Usha Rani prepared the discussion based on the results. All the authors have proofread and has provided their intellectual contributions in the manuscript. All authors have approved the final results of the manuscript for publication.", "appendix": "References\n\nUN Inter-agency Group for Child Mortality Estimation: Mortality rate, neonatal (per 1,000 live births)\|Data.2019. Accessed 4 Jan 2020. Reference Source\n\nUNICEF: The urgent need to end newborn deaths: Every Child Alive.2018.\n\nWHO, World Health Organisation: Newborns: reducing mortality.2018. Accessed 26 Oct 2018. Reference Source\n\nWHO: Report on the Burden of Endemic Health Care-Associated Infection Worldwide. WHO Libr Cat Data. 2011. Reference Source\n\nLiu L, Johnson HL, Cousens S, et al.: Global, regional, and national causes of child mortality: An updated systematic analysis for 2010 with time trends since 2000. Lancet. 2012; 379: 2151–2161. PubMed Abstract \| Publisher Full Text\n\nAfjeh SA, Sabzehei MK, Karimi A, et al.: Surveillance of ventilator-associated pneumonia in a neonatal intensive care unit: characteristics, risk factors, and outcome. Arch. Iran. 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Publisher Full Text\n\nAbramczyk ML, Carvalho WB, Carvalho ES, et al.: Nosocomial infection in a pediatric intensive care unit in a developing country. Braz. J. Infect. Dis. 2003; 7: 375–380. PubMed Abstract\n\nGeffers C, Baerwolff S, Schwab F, et al.: Incidence of healthcare-associated infections in high-risk neonates: results from the German surveillance system for very-low-birthweight infants. J. Hosp. Infect. 2008; 68: 214–221. PubMed Abstract \| Publisher Full Text\n\nChaurasia S, Sivanandan S, Agarwal R, et al.: Neonatal sepsis in South Asia: Huge burden and spiralling antimicrobial resistance. BMJ. 2019; 364: k5314. PubMed Abstract \| Publisher Full Text\n\nCrivaro V, Bogdanović L, Bagattini M, et al.: Surveillance of healthcare-associated infections in a neonatal intensive care unit in Italy during 2006–2010. BMC Infect. Dis. 2015; 15: 152. PubMed Abstract \| Publisher Full Text\n\nPathak A, Singh P, Jain S, et al.: Incidence and determinants of health care associated blood stream infections at a neonatal intensive care unit in Ujjain, India: Results of a prospective cohort study. Int. J. Infect. Dis. 2014; 21: 48. Publisher Full Text\n\nGaurav J, Sugandha A, Rajni G, et al.: Sepsis with extended-spectrum β-lactamase producing Gram-negative bacteria in neonates - Risk factors, clinical profile and outcome. J. Pediatr. Infect. Dis. 2011; 6: 195–200.\n\nSiddique M, Memon KN, Sulehri MA: Neonatal sepsis - Its determinants & outcome among newborns admitted in Neonatal Intensive Care Unit (NICU), Liaquat University Hospital Hyderabad. Med. Forum. Mon. 2013: 1–7.\n\nBlencowe H, Cousens S, Chou D, et al.: Born Too Soon: The global epidemiology of 15 million preterm births. Reprod. Health. 2013; 10: S2. Publisher Full Text\n\nKato H, Ide K, Fukase F, et al.: Polymerase chain reaction-based open reading frame typing (POT) method analysis for a methicillin-resistant Staphylococcus aureus (MRSA) outbreak through breast-feeding in the neonatal intensive care unit. IDCases. 2018; 12: 1–3. Publisher Full Text\n\nBasu S, Kumar R, Tilak R, et al.: Candida blood stream infection in neonates: Experience from a tertiary care teaching hospital of Central India. Indian Pediatr. 2017; 54: 556–559. PubMed Abstract \| Publisher Full Text\n\nVijayakanthi N, Bahl D, Kaur N, et al.: Frequency and Characteristics of Infections Caused by Extended-Spectrum Beta-Lactamase-Producing Organisms in Neonates: A Prospective Cohort Study. Biomed. Res. Int. 2013; 2013: 1–8. PubMed Abstract \| Publisher Full Text\n\nBammigatti C, Doradla S, Belgode HN, et al.: Healthcare associated infections in a resource limited setting. J. Clin. Diagnostic Res. 2017; 11: OC01–OC04.\n\nPayne V, Hall M, Prieto J, et al.: Care bundles to reduce central line-associated bloodstream infections in the neonatal unit: A systematic review and meta-analysis. Arch. Dis. Child Fetal Neonatal Ed. 2018; 103: F422–F429. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: The burden of healthcare-associated infection worldwide: A Summary. World Health Organization; 2011; 3.\n\nMurthy S, Godinho MA, Guddattu V, et al.: Risk factors of neonatal sepsis in India: A systematic review and meta-analysis. PLoS One. 2019; 14: e0215683. PubMed Abstract \| Publisher Full Text\n\nHarrison W, Goodman D: Epidemiologic Trends in Neonatal Intensive Care, 2007-2012. JAMA Pediatr. 2015; 169: 855–862. PubMed Abstract \| Publisher Full Text\n\nNeelagund YF, Vinodkumar CS: Acinetoacbacter septicaemia in neonates. Indian J. Med. Microbiol. 2004; 22: 71.\n\nPicone S, Aufieri R, Paolillo P: Infection in late preterm infants. Early Hum. Dev. 2014; 33: 871–882.\n\nStoll BJ, Hansen N, Fanaroff AA, et al.: Late-onset sepsis in very low birth weight neonates: The experience of the NICHD Neonatal Research Network. Pediatrics. 2002; 110: 285–291. Publisher Full Text\n\nJajoo M, Manchanda V, Chaurasia S, et al.: Alarming rates of antimicrobial resistance and fungal sepsis in outborn neonates in North India. PLoS One. 2018; 13: e0180705. PubMed Abstract \| Publisher Full Text\n\nWadile R, Bhate V: Study of clinical spectrum and risk factors of neonatal candidemia. Indian J. Pathol. Microbiol. 2015; 58: 472–474. PubMed Abstract \| Publisher Full Text\n\nDatta S, Roy S, Chatterjee S, et al.: A five-year experience of carbapenem resistance in enterobacteriaceae causing neonatal septicaemia: Predominance of NDM-1. PLoS One. 2014; 9: e112101. PubMed Abstract \| Publisher Full Text\n\nYalaz M, Altun-Köroğlu Ö, Ulusoy B, et al.: Evaluation of device-associated infections in a neonatal intensive care unit. Turk. J. Pediatr. 2012; 54: 128–135.\n\nBhargava A, Gur R, Kaur H, et al.: Risk factors and outcome analysis of gram-positive and gram-negative neonatal sepsis: A case-control study. Can. J. Infect. Control. 2017; 32: 98–103.\n\nWójkowska-Mach J, Merritt TA, Borszewska-Kornacka M, et al.: Device-associated pneumonia of very low birth weight infants in Polish Neonatal Intensive Care Units. Adv. Med. Sci. 2016; 61: 90–95. PubMed Abstract \| Publisher Full Text\n\nCernada M, Brugada M, Golombek S, et al.: Ventilator-associated pneumonia in neonatal patients: An update. Neonatology. 2014; 105: 98–107. Publisher Full Text\n\nHalder D, Haque ME, Zabidi MH, et al.: Nosocomial bacterial sepsis in babies weighing 1000-1499 g in Kelantan. Med. J. Malaysia. 1999; 54: 52–57. PubMed Abstract\n\nWang W, Zhao CL, Ji QL, et al.: Prevention of peripherally inserted central line-associated blood stream infections in very low-birth-weight infants by using a central line bundle guideline with a standard checklist: a case control study. BMC Pediatr. 2015; 15: 1–6. Publisher Full Text\n\nChawla G, Diwakar KK: Comparison of umbilical cord cleansing using sterile water and povidine iodine-spirit during early neonatal period: A double blind randomized control trial. J. Clin. Diagnostic Res. 2015; 9: 6–8. Publisher Full Text\n\nTröger B, Göpel W, Faust K, et al.: Risk for Late-onset Blood-culture Proven Sepsis in Very-low-birth Weight Infants Born Small for Gestational Age. Pediatr. Infect. Dis. J. 2014; 33: 238–243. PubMed Abstract \| Publisher Full Text\n\nRani U, Chawla K, Lewis LE, et al.: Hand Hygiene Practices and Training Gap in a Neonatal Intensive Care Unit at Coastal Karnataka India. Indian J. Public Heal. Res. Dev. 2020; 11: 59. Publisher Full Text\n\nRani U: Dataset on neonatal Healthcare Associated Infections of a uni-center analytical study from South India. Figshare. 2022. Accessed 25 Mar 2022. Reference Source" }	[ { "id": "137206", "date": "16 May 2022", "name": "Nitesh Kumar", "expertise": [ "Reviewer Expertise I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Editorial Team,\nI am very much pleased to add my review report on the title 'Preventable contributors to the neonatal healthcare-associated infections: a uni-center analytical study from South India.' submitted by Usha Rani, Leslie E. Lewis, Kiran Chawla and Anup Naha.\nI commend the writers for selecting a topic that will benefit neonatal research in India. The paper's sequencing is well-planned. The research gap reviews are also self-evident, and the approach and instruments employed in this study offer greater relevance to the current environment. The paper's analytical section seems promising, identifying the cases of healthcare-associated infections through diagnostic and clinical evidence and later identifying the contributors in a systematic way is very challenging that is addressed in the current paper. Highlighting the contributors like frequent changes in IV cannula would guide further research in this field. Univariate and multivariate analysis of various contributors is carefully done and explained in a meaningful manner.\nThey explain clearly their limitations and provide further direction and scope for research.\nThe entire paper was written flawlessly in terms of language and was also educational to the entire research community.\n\nThe study's implications are also explicitly discussed.\nI am glad to read this paper adding value to the research community and opening doors for further research, hence, I place my recommendation for indexing the article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "143494", "date": "13 Jul 2022", "name": "Vijay Ivaturi", "expertise": [ "Reviewer Expertise Predictive healthcare analytics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI am pleased to review this paper titled: Preventable contributors to neonatal healthcare-associated infections: a uni-center analytical study from South India. The study focuses well on the target population, geographical location, and the preventable contributors to neonatal healthcare-associated infections. The study topic and epidemiological reporting have reported essential findings among the neonatal population considering India as one of the top countries witnessing the world's highest neonatal mortality.\nReporting of introduction and importance of the study is adequate. The study meets the standard reporting guidelines. The methodological justification satisfies the criteria and is adequate for the inquiry. The study used easy-to-repeat, specific criteria to evaluate whether a neonate had a healthcare-associated infection. This article helps to identify the specific cases of healthcare-associated infections using diagnostic and clinical information, which is challenging. The analytical component of the article appears to have merit. The statistical tools and methods are appropriately selected and reported as per the reporting standards.\nThe category of results is well-defined and meticulously planned. A novel contributor to research that was not reported earlier, like several pricks while inserting an IV cannula, was reported in this study.\nDetailed comparative discussion with global and national peer publications is adding more insight into the contributors to neonatal HAIs. Discussion is exhaustive, ensuring comparability with similar studies.\nLimitations of the study are meticulously reported and justified, which would help researchers plan future studies by overcoming these limitations.\nThis study investigated the potential causes of healthcare-associated infections in newborns. Even though the study was single-center and might not be generalized to the broader population or healthcare facilities, the paper contained substantial information about preventable contributors.\nI believe that the study's outcomes will offer different perspectives to individuals who contribute to neonatal healthcare-associated illnesses. I thus suggest that this study can be considered for publication.\n\nIs the work 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/11-454
https://f1000research.com/articles/11-452/v1	22 Apr 22	{ "type": "Research Article", "title": "Written corrective feedback: A comparative study of the preferences and beliefs of EFL teachers and learners in Saudi Arabia", "authors": [ "Hani Hamad M Albelihi" ], "abstract": "Background: Corrective feedback plays the role of enabling both teachers and learners to gauge their performance and reflect on their development. It can vary in nature, amount, and focus; nevertheless, its centrality to the classroom cannot be ignored. At the same time, what makes it effective is the way it is communicated. The role of Written Corrective Feedback (WCF) is vital as both a corrective measure and durability for future reference. Foreign language classrooms are an active foreground for feedback practices, given that the bulk of correction is multifaceted and multimodal. However, teachers are left to their devices to formulate best practices in the absence of defined classroom feedback mechanisms. The purposes of this study are (i) to evaluate Saudi English as a foreign language (EFL) instructors’ real practices in supporting their students with corrective feedback; and (ii) to check the students’ beliefs about the feedback they receive from their instructors. Methods: Using the writing output of 92 EFL learners from Qassim University, Saudi Arabia, and the nature of WCF provided to them by three university instructors who were free to choose their feedback strategies in two phases of writing and correction, followed by learners’ cumulative response to the two, the study concludes that a number of difficulties surrounding the scope of feedback need to be researched. Results: The study found that the most used type of WCF is direct in regard to grammatical errors, vocabulary, syntax, and content evaluation. However, the Saudi EFL learners prefer direct corrective feedback for grammatical errors but indirect coded feedback for content correcting their writing assignments. Conclusions: The study encourages EFL teachers to focus on the different types of WCF when reverting to their learners. Furthermore, students’ preference for feedback should be the cornerstone teachers begin with while giving the WCF.", "keywords": [ "Feedback Efficacy", "Learners' Beliefs", "Preference", "Types of Written Corrective Feedback", "WCF in EFL" ], "content": "Introduction\n\nAssessment is an institutional and an individual need in learning situations such as English as a foreign language (EFL) for learners to get their language production assessed and corrected while progressing toward fluency in the target language. Making mistakes is a natural phenomenon in language learning, especially when it comes to a complex skill: writing. Feedback may minimize the frequency and seriousness of errors in the learner’s journey towards fluency and accuracy. Nevertheless, some factors need to be taken into consideration, such as the types of written corrective feedback (WCF), the time given to supporting learners with WCF, and even the psychology of the learners when considering whether to accept the feedback or even their motivation in applying the feedback in further language tasks or activities. Feedback as a pedagogical strategy for teaching English as a second language (ESL) writing has received growing attention in the academic literature.1–3 Some previous studies focus on how learners respond to feedback.3–5 WCF has also engaged educationists and researchers for a long time, so much that a reasonable amount of literature exists on the topic. The success of feedback can only be traced to the development of students’ written production. It has been proposed that both teachers and students cooperate to form the desired dimensions of WCF.6 Previous researchers are divided on studying the influence of WCF on EFL learners, the alignment between teachers’ beliefs on WCF and their actual practices in the classroom6–8; while some others focus on the types of feedback9,10; and some considered the learners’ feelings.4,11 However, none of the investigations of WCF have examined the learners’ beliefs about the teachers’ WCF and the types of feedback they preferred in one setting; therefore, there is a need for a genuine inquiry into such perceptions.\n\nFurthermore, the great majority of previous research has been teacher-centred. Thus, it is still necessary to focus on learner-centeredness, gather accurate comments on students’ writing, and check the types of feedback the students themselves desire to be given. Accordingly, the study aims to bridge the gaps in the existing research by reviewing the learners’ perceptions of feedback, analysing instructors’ actual practices in supplying feedback to their students, and finally, shedding light on the preference of the type of WCF of the Saudi EFL learners.\n\nEnglish language teachers may supply learners with ineffective content or content that does not meet individual preferences, despite the variety of ways to deliver it. As a result, it is vital to consider EFL learners’ preferences in the teaching and learning process as much as in teachers’ feedback. Accordingly, the main objective of this study is to discover the Saudi EFL learners’ preferences for the type of Corrective Feedback (CF) they would like to receive from their teacher on a written project. Teachers provide different types of CF on students’ compositions (e.g., direct, indirect). This study explores types of CF that Saudi EFL students prefer to receive from their teachers. It also aims to investigate the students’ attitudes and perceptions towards their teachers’ CF in general.\n\nThe study answers the following questions:\n\n1. What are the types of feedback provided by Saudi EFL instructors?\n\n2. What is the Saudi EFL learners’ perception about the role of WCF in enhancing the quality of their writing output?\n\n3. What are the Saudi EFL learners’ preferences in WCF in EFL?\n\n\nLiterature review\n\nCorrective feedback is a term that refers to critical error correction (CER).12 CER is the process of identifying mistakes in students’ written work and fixing them by commenting on the types of errors and providing suggestions for corrections. It is also perceived as the type of feedback teachers/instructors provide to their students.6 According to previous research, there is a strong correlation between WCF and learners’ success and the potential for English to be taught successfully in various EFL learning/teaching contexts, including China and Saudi Arabia. The clearer and more effective CF that the teachers support their learners with, the more enhancement in students’ writing can be seen. Nevertheless, there is a disparity between teachers’ beliefs and actual practices in the context of WCF.6,8,13 These issues will be dealt with in the following sections.\n\nStudies focused on EFL instructors’ perceptions and practices on WCF are many.6–8,14 The majority of these studies showed that misalignments usually exist between beliefs and practices. Amrhein and Nassaji14 explored the perceptions of both EFL teachers and learners regarding the kinds and quantity of WCF and found that many contradictions appeared in WCF practices. Mao and Crosthwaite6 investigated Chinese teachers’ beliefs and actual practices regarding WCF in students’ essays and found some agreements, and at least three discrepancies: teachers believed they provided indirect feedback to students when they directly gave their feedback. Secondly, teachers said they wrote down their comments on students’ work in the margin; in reality, they did not. Finally, teachers were found to give more importance to local issues while focusing more on global issues. In the Omani context, Trabelsi8 explored the alignment and misalignment between instructors’ beliefs and practices and students’ congruent preferences with their instructors’ WCF. The study revealed more misalignment and a few alignments. The mismatches stemmed from explicitness, focus, and redrafting, whereas alignment was reached to correct and identify the mistakes. Furthermore, the study found that the number of alignments between students’ preferences and teachers’ beliefs are more significant than the misalignments. The agreement was reached on the source, amount, and explicitness of feedback. The incongruence was found in the comments and focus of the feedback.\n\nLearner participants in this study took part in writing compositions and submitted them to their teachers while investigating their feelings regarding the corrective feedback. Learner’s engagement with CF is characterized by Ellis15 based on how learners respond to that feedback, which includes cognition, behavior, and emotion. The subsequent research has expanded to include how learners process and respond to WCF.3–5 Chen et al.4 explored Chinese learners’ perceptions and preferences in WCF. The study showed that learners showed neutral opinions regarding explicit grammar instruction; they revealed a positive tendency towards the comments in the margins.\n\nIn addition to variations between people, learners’ engagement with WCF is concurrently influenced by learners’ variables and context.15–17 Similarly, Guerrettaz and Johnston12 considered learners’ participation in WCF as part of classroom life, reflected in the vast range of learners’ features and contextual factors. A degree of complication occurs due to the interplay of so many elements to enhance learning, which causes negative pressures among learners.\n\nThe topic of corrective feedback is saturated in the Saudi context.7,18,19 Written corrective feedback in an EFL context has been investigated from different dimensions in much previous research.13,20–24 The investigations include teachers’ beliefs and actual practices and the learners’ preferences. Al-Shahrani and Storch13 studied the form of WCF that Saudi instructors provided their learners following the guidelines of institutions, instructors’ beliefs on the most effective feedback, and learners’ preferences. The findings showed that teachers’ beliefs were not consistently enacted in their practices. Their indirect feedback focused on grammar and ignored learners who preferred direct feedback. Similarly, Albogami18 studied L2 instructors’ and learners’ perspectives on the relevance of written feedback and the components of successfully written feedback at King Saud University. The results showed awareness in both learners and instructors on the importance of written feedback in boosting learning, strengthening confidence, improving autonomous learning, and increasing interaction between learners and instructors in the English writing classroom. Furthermore, Alkhatib7 studied teachers’ beliefs and practices and learners’ preferences in the Saudi context. The findings showed that teachers’ beliefs and practices were congruent in the type and quality of feedback. They diverged regarding the source of feedback, the directness and indirectness, and implicitness and explicitness of feedback.\n\nHalim et al. explored EFL learners’ perceptions of remedial feedback.22 They found that learners viewed teachers’ feedback positively as a vital learning tool for their learning path. Alzahrani’s20 study revealed that Saudi instructors ascertain that using unfocused corrective feedback probably develops learners’ abilities to correct their writing errors. Learners felt that receiving coded, unfocused feedback encouraged them to write better, and it was found that this type of feedback was best suited to higher-levels of learning where better standards of improvement are expected.\n\nRajab23 found that both instructors and learners showed a high interest in WCF. Learners believed that getting well-structured WCF helped them familiarize themselves with how language works and thus accelerated their language acquisition. Findings showed that congruence was poor between learners’ and instructors’ beliefs: learners were interested in unfocused WCF while instructors preferred coded feedback.\n\nNomenclature in WCF has been a subject of much debate and consequent confusion.25 WCF strategies may be classified as direct feedback, coded feedback, uncoded feedback and, marginal feedback. Furthermore, another classification was supplemented by Tedick and Gortari,10 who identified six types of corrective feedback strategies: Explicit correction, recast, clarification request, metalinguistic cues, elicitation, and repetition. Still, a third classification given by Ellis9 mentioned there are three types of feedback strategies: Direct, Indirect, and Metalinguistic. Direct feedback is when the error is marked along with the correction. In Indirect feedback, the error is marked, but correction is not given. Metalinguistic feedback is an explicit type of corrective feedback that poses questions or provides comments or information that enable the learner to spot the error and correct it.\n\n\nMethods\n\nThe current study aimed to examine the types of EFL teachers’ WCF on the written output of learners in EFL classrooms and learners’ beliefs and attitudes towards WCF. The study was qualitatively conducted based on the content analysis of teachers’ comments on students’ writing. Data have been collected in two forms: One, the types of the WCF provided by three EFL teachers in a Saudi university. These data were coded according to the four types of WCF (see Table 1); Two, responses to a questionnaire that examined learners’ preferences and beliefs towards the different types of WCF as tools in language improvement. Therefore, the written output generated for the study was not evaluated for its quality, and focus was diligently kept on the WCF.\n\nThe study was conducted in the Department of English and Translation at Qassim University, Saudi Arabia. The intake class, consisting of 184 students, were informed about the purpose of the research and invited to participate in the study. They enrolled in the first semester and studied Writing 1 Course for 2021-2022. The participants had the same cultural background and had studied English for the same number of years. Their median age was 21 and their native language was Arabic. The researcher told them to provide accurate answers and informed them that all their information would remain anonymous and would only be used for research purposes. They were informed that their data would be published publicly, but anonymously in line with their agreement.\n\nFrom the population of 184 students (the total intake for the session), the researcher identified a convenience sample of 92 first-year EFL learners across three levels/sections in the department of English. It may be noted that EFL learners have some exposure to English literature in their senior school years and this component is integrated into their language curriculum. The Greek drama Antigone is taught to EFL learners both in senior high school and in the first year of English education in the university. Using this background, the researcher organized a viewing of the dramatized version of the play, freely available on YouTube (https://www.youtube.com/watch?v=DPPE5Wq90MQ). This version carries English subtitles and is slightly more than 150 minutes long. Based on their viewing, the learners were informed beforehand that they would be requested to compose a short character sketch of the central character Creon. The screening was completed in one sitting by requesting adjustments from other teachers. No writing exercise was conducted on the same day. On the following day, writing output samples were collected as data on the teachers’ use of WCF. These samples were collected in one phase to ensure authentic output. Assigning writing tasks to EFL learners is part of the language enrichment activities prescribed in the syllabus. This made the researcher’s task more manageable as the participants did not need to take extra time to complete the writing samples. In all, the study comprised 92 writing samples which were coded anonymously using numbers instead of the students’ names, to ensure teachers’ unbiased feedback. Copies were made of all the 92 samples, and three EFL teachers in two language departments at the university were given a set each to complete WCF in two phases. For this, prior written permission was secured from the Rector, and teachers were assured that the exercise was for research purposes only and would not be used to evaluate them. Corrected copies were requested to be returned at the end of a week to compile any WCF patterns in each phase. The aim behind employing two phases was to ensure that all possible feedback choices formed the dataset.\n\nAt the end of phase two, a five-point Likert Scale-based questionnaire on all possible strategy combinations used by the teachers was administered to the participants to identify their preferences in WCF in EFL. The questionnaire aimed to measure students’ perceptions of the WCF and their opinions. The questionnaire comprised 11 close-ended items, as presented in Table 2. These items reflected students’ perceptions of the types of the WCF pertained by their instructors and the focus of their instructors on the elements of language such as vocabulary, grammar, syntax, and discourse. A copy of the questionnaire can be found in the Extended data.28\n\nThe researcher produced a rubric of all possible feedback combinations to classify the WCF generated in the two phases of correction. The rubric consisted of five strategies in a column, i.e., direct, metalinguistic, clarification, elicitation, and repetition, and rows consisting of grammar, syntax, vocabulary, and discourse. The researcher checked each of the teachers’ WCF samples and classified them according to the strategy types and language systems for each student’s marked papers. The researcher then carefully parsed each corrected script to identify every instance of WCF and recorded it in the rubric. Finally, all the classified WCF test papers were calculated to get the frequency for each type.\n\nInstrument validity and reliability were established by a panel of five subject experts whose suggestions were incorporated into the questionnaire before pilot testing it with a group of thirty EFL learners not included in the final survey. The questionnaire items were coded as 1 (totally disagree) to 5 (totally agree). SPSS 22 version was used to calculate the findings. Descriptive analysis was conducted including the presenting of percentage.\n\nEthical approval was obtained from the Committee of Research Ethics in the College of Language and Translation, Qassim University (approval number: 30-Eng-2021). Informed verbal consent from all the students who participated in the study was obtained. The researcher explicitly told them that the study aimed to explore the EFL Saudi instructors’ use of WCF while they revise their compositions. The researcher informed them that their anonymized data would be published publicly.\n\n\nResults\n\nTable 1 below reflects the data collected. which includes the instructors’ WCF comments on students’ compositions. It may be noted that the frequency of the occurrence of a strategy as applied by the teachers is the total across the two phases since teachers would use any of the five strategies suitable with the type of errors made by students. Furthermore, the analysis of the collected WCF showed that teachers who applied WCF seemed to be constant and accurate. Based on the previously reviewed literature, five strategies could be identified and were used in the rubric.\n\nFor ease of use, the data were graphically represented in Figure 1 below.\n\nFigure 1 clearly shows that teachers show a preference for direct strategies in WCF across the written samples in all the four parameters. This preference was identified by the many direct comments they made on students’ compositions that followed the strategies, viz., grammar, syntax, vocabulary, and content. This finding answers the first research question, which concerns the preferred strategy applied by the EFL teachers. This finding diverged from Al-Shahrani and Storch’s13 who reported that Saudi teachers preferred to use indirect feedback. This could be because the level of the learners in our study was so elementary that they could not understand the indirect feedback or deduce what the indirect feedback suggested in a different context. It may also be noted that the instructors belonged to two different universities, which may mean that the WCF patterns identified could be generalized for the larger EFL teacher community in Saudi universities. Similar results were reported by Alsolami and Elyas,26 who explored several forms of CF. This study showed that metalinguistic feedback, elicitation, repetition, and explicit correction, were far more successful in eliciting improvement but were rarely used in EFL courses.\n\nTo answer the remaining two research questions, a five-point Likert Scale based questionnaire (with 1 = totally disagree, 2 = disagree, 3 = neither agree nor disagree, 4 = agree, 5 = totally agree) seeking learners’ opinions on the role of WCF in enhancing the quality of their writing output and their preference for specific strategies for language components was used. Table 2 below summarizes the findings from the questionnaire responses of the final survey participants. The full dataset can be found in the Underlying data.28\n\nThe study aimed to identify the nature of the WCF applied by EFL instructors at Saudi universities and, to this end, three subject teachers evaluated 184 writing samples in a phased manner. The other two research questions that the study set out to answer were the Saudi EFL learners’ perception about the role of WCF in enhancing the quality of their writing output; and their preferences with respect to WCF in writing output. Table 2 summarizes learners’ perceptions of available WCF practices at two Saudi universities. All four correction parameters were considered in the study, viz., grammar, vocabulary, syntax, and content. Data presented in Table 1 showed that direct feedback is the most preferred strategy by the teachers. However, learners’ preferences in Table 2 appear unfulfilled, although they show agreement that WCF is necessary for their learning as reflected in statement 2, to which 92.4% of the respondents disagreed to on the item “I prefer to get no written feedback in the English class”. This finding is in line with many previous studies’ findings.4,8 Trabelsi8 reported that teachers’ and students’ perceptions of WCF was identifying and correcting errors. Furthermore, Chen et al.4 reported that Chinese learners positively welcomed their teachers’ written feedback on their writing. Regarding getting direct feedback on grammar, 83.6% of the respondents feel that the direct feedback strategy is most suitable, and it helps them improve on this count in future assignments (80.4% agree with statement 3). Learners in item 4 reported their unlikeliness to discover the mistakes in grammar themselves while the teachers pointed them out. This finding is confirmed by Alzahrani’s (2016) study, which reported that direct feedback is suitable at a basic level.20 Similarly, direct feedback is appreciated in regard to vocabulary (66.6% disagreed when asked in statement 5 if the teacher needs to correct all erroneous vocabulary) and in syntactic errors, with 71.7% of the respondents voting for it in statement 9. However, direct feedback in writing output was rejected by 67.3% of the respondents (statement 11) who believe that it does not help them improve their output (60.8% do not want complete corrections or direct feedback in cohesion and coherence issues) and feel that it takes away the liberty of expression from them (73.9% of the responses to statement 6).\n\nGreater focus on WCF literature could produce interesting findings that account for a more holistic understanding of the phenomena. Information on WCF in more contexts is needed as the majority of studies focus on one context. As mentioned in the literature, WCF may be changed and affected by learning settings; therefore, further research should be undertaken to compare learning environments and the similarities and differences in beliefs and practices and learners’ preferences. Also, more research is required to determine the level of content analysis in teachers’ feedback to make use of them along with grammar and vocabulary alike in the development of learners’ writing abilities.\n\nA number of limitations need to be noted regarding the current study. First, this study is restrictive in nature as it sought learners’ attitudes on 11 items without allowing the freedom to express their opinions on types of WCF and their needs. Second, it confirms and digresses from previous findings without setting new dimensions for the process of WCF management or moving forward to set a framework to guide writing teachers to focus not only on the linguistic elements of the learners’ writings but also the discursive elements. Finally, caution must be applied with a small sample size, as the results might not be transferable to similar contexts.\n\n\nConclusions\n\nThis study set out to holistically review some of the queries on WCF in the Saudi context. One of the most significant findings to emerge from this study is that Saudi EFL instructors had a preference for applying direct strategies to correct their learners’ written assignments. In contrast to earlier findings, however, this finding diverged from Al-Shahrani and Storch’s13 who reported that Saudi teachers preferred to use indirect feedback. This finding is consistent with Alharbi27 who examined the relative efficacy of three distinct forms of written corrective feedback and feedback vs. no feedback in the context of learners’ writing quality. Furthermore, the study revealed that Saudi EFL learners believed in the importance of getting WCF on their writings. They believed that such feedback accelerated their proficiency in writing skills, and both students and instructors were aware of the importance of direct feedback, which is confirmed by Alzahrani.20 The results indicated that, of the three forms of feedback, direct written corrective feedback was the most successful in enhancing learners’ writing quality, and the instructors preferred it most. In the current study, too, direct feedback is preferred by EFL learners in regard to grammar, vocabulary, and syntax but not in content. This pushes and renews our knowledge in WCF scholarship.\n\n\nData availability\n\nFigshare: Dr. Hani Hamad - Questionnaire.docx. https://doi.org/10.6084/m9.figshare.19322408.v1.28\n\nThis project contains the following underlying data:\n\n- Dr.Hani Hamad - Raw Data.xlsx\n\n- Hani’s Writing Samples.docx\n\nThis project contains the following extended data:\n\n- Dr.Hani Hamad - Questionnaire.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.", "appendix": "Acknowledgements\n\nI am eternally grateful to the participants for their time and efforts.\n\n\nReferences\n\nTseng S-S, Yeh H-C: The impact of video and written feedback on student preferences of English speaking practice. Lang. Learn. Technol. 2019; 23(2): 145–158. Publisher Full Text\n\nWilson J, Czik A: Automated essay evaluation software in English Language Arts classrooms: Effects on teacher feedback, student motivation, and writing quality. Comput. Educ. 2016; 100: 94–109. Publisher Full Text\n\nZheng Y, Yu S: Student engagement with teacher written corrective feedback in EFL writing: A case study of Chinese lower-proficiency students. Assess. Writ. 2018; 37: 13–24. Publisher Full Text\n\nChen S, Nassaji H, Liu Q: EFL learners’ perceptions and preferences of written corrective feedback: a case study of university students from Mainland China. Asian. J. Second. Foreign. Lang. Educ. 2016; 1(1): 1–17. Publisher Full Text\n\nRoothooft H, Breeze R: A comparison of EFL teachers and students’ attitudes to oral corrective feedback. Lang. Aware. 2016; 25(4): 318–335. Publisher Full Text\n\nMao SS, Crosthwaite P: Investigating written corrective feedback: (Mis) alignment of teachers’ beliefs and practice. J. Second. Lang. Writ. 2019; 45: 46–60. Publisher Full Text\n\nAlkhatib N: Written corrective feedback at a Saudi University: English language teachers’ beliefs, students’ preferences, and teachers’ practices. (Doctoral dissertation), University of Essex.2015. Reference Source\n\nTrabelsi S: (Mis) alignment in Relation to Written Corrective Feedback: the Teachers’ Beliefs and Practices vs the Students’ Preferences in an EFL Context. Int. J. Lang. Linguist. 2021; 9(1): 6–16. Publisher Full Text\n\nEllis R: A typology of written corrective feedback types. ELT J. 2008; 63: 97–107. Publisher Full Text\n\nTedick DJ, De Gortari B: Research on error correction and implications for classroom teaching. ACIE Newsletter. 1998; 1(3): 1–6.\n\nGuerrettaz AM, Johnston B: Materials in the classroom ecology. Mod. Lang. J. 2013; 97: 779–796. Publisher Full Text\n\nEllis R: Corrective feedback and teacher development. L2 Journal. 2009; 1(1) Publisher Full Text\n\nAl-Shahrani A, Storch N: Investigating teachers’ written corrective feedback practices in a Saudi EFL context: How do they align with their beliefs, institutional guidelines, and students’ preferences?. Aust. Rev. Appl. Linguist. 2014; 37(2): 101–122. Publisher Full Text\n\nAmrhein HR, Nassaji H: Written Corrective Feedback: What do Students and Teachers Think is Right and Why?. Can. J. Appl. Linguist. 2010; 13(2): 95–127. Reference Source\n\nEllis R: Epilogue: A framework for investigating oral and written corrective feedback. Stud. Second. Lang. Acquis. 2010; 32(2): 335–349. Publisher Full Text\n\nHan Y: Written corrective feedback from an ecological perspective: The interaction between the context and individual learners. System. 2019; 80: 288–303. Publisher Full Text\n\nKartchava E: Learners’ beliefs about corrective feedback in the language classroom: Perspectives from two international contexts. TESL Can. J. 2016; 33(2): 19–45. Publisher Full Text\n\nAlbogami M: An inquiry into effective written feedback from EFL teachers’ and students’ perspectives at a Saudi University. (Doctoral dissertation). University of Exeter.2020. Reference Source\n\nAl-Shahrani AA: Investigation of written corrective feedback in an EFL context: beliefs of teachers, their real practices and students’ preferences. (Masters by Coursework & Shorter thesis), School of Languages and Linguistics, The University of Melbourne.2013.\n\nAlzahrani HF: Teachers’ stated beliefs on coded unfocused corrective feedback in EFL writing at a Saudi university. TESOL Int. J. 2016; 11(1): 52–64.\n\nGamlo NH: EFL learners’ preferences of corrective feedback in speaking activities. World J. Engl. Lang. 2019; 9(2): 28–45. Publisher Full Text\n\nHalim T, Wahid R, Halim S: EFL students’ attitudes toward corrective feedback: a study conducted at undergraduate level. Saudi Journal of Language Studies. 2021; 1(1): 40–49. Publisher Full Text\n\nRajab H: EFL teachers and learners’ perceptions, beliefs and practices on written corrective feedback in the Saudi higher education context. University of Exeter; 2018.\n\nAl-Shahrani A: Investigation of written corrective feedback in an EFL context: beliefs of teachers their real practices and students’ preferences.Noor Publishing; 2017.\n\nRobb T, Ross S, Shortreed I: Salience of feedback on error and its effect on EFL writing quality. TESOL Q. 1986; 20: 83–93. Publisher Full Text\n\nAlsolami EH, Elyas T: Investigating teachers’ corrective feedback and learners’ uptake in the EFL classrooms. Int. J. Educ. Investig. 2016; 3(1): 115–132.\n\nAlharbi SH: Efficacy of different types of written corrective feedback on EFL university students’ writing quality. Int. J. Engl. Linguist. 2020; 10(4): 217. Publisher Full Text\n\nHamad H: Dr. Hani Hamad - Questionnaire.docx. figshare. Dataset. 2022. Publisher Full Text" }	[ { "id": "142560", "date": "04 Jul 2022", "name": "Mahdi Aben Ahmed", "expertise": [ "Reviewer Expertise Culture and Intercultural Communication", "Professional and workplace communication", "English for Specific Purposes", "Business English", "CEA Accreditation", "Second Language Teaching" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA review on Written corrective feedback: A comparative study of the preferences and beliefs of EFL teachers and learners in Saudi Arabia\nCorrective feedback is thought to be a good way that helps learners to master their writing ability.\nThe abstract includes all the required elements, i.e., background, purpose, methods, findings, and conclusion. They are expressive and meaningful. However, the lengthy background initiated the abstract.\nIntroduction\nA good argument is shown in the introduction. The author shifted from one idea into another smoothly. The reader can follow him. Furthermore, showing the importance of written corrective feedback was assigned from the first step. Another good point is the ability of the author to show the gap in previous research and set the shift from a teacher focus to students in a fundamental and logical reason per see to conduct the research.\nThe author used the phrase \"complex skill: writing\", which seemed to be fragmented. It would be more natural to rewrite it as a \" complex skill like writing.\"\nLiterature review\nThe author begins his review by defining the term (written corrective feedback), its type, and its use. Then some studies which discussed the practice of EFL teachers to WCF were summarized in a readable way. Besides, the research focused in the next section on the learners' beliefs on WCF. Likely, studies on WCF in the Saudi context were summarized. The author mentioned the various conducted in the Saudi context; he showed the gaps in them and made his research a necessary and prevalent to bridge such gaps. In the last section, the authors reviewed the types of WCF. Interestingly, the organizational way of the literature review helped the author to answer his research questions in the results.\nMethods\nThe methods section covered (research design, participants, instrument, data analysis, and ethical approval/consent). The author answered all the comments that usually criticized the methods because of their complexity and how to make them duplicated to any researcher. I congratulate him on his great job.\nResults\nThe results are demonstrated in the table and figures. Sufficient explanations were also shown. The results are designed to answer the three research questions. However, there are a few problems:\n\nThe first line seemed run-on: \"Table 1 below reflects the data collected. which includes the instructors’ WCF\". The full stop between the words \"collected\" and \"which\" should be omitted.\nPage 6. \"This finding diverged from Al-Shahrani and which \" two mistakes are spotted. First, the reference number is missed, and the author's use of the word \"and\" seemed ungrammatical.\nIn table 2, it would be more valid if the author added average mean scores at the end of the table, which would help him conclude the students' answers. Furthermore, Table 1 is not clear. What do the numbers in front of the WCF types mean? The author should add the word frequency to the first raw of the table.\nAs the results and discussion are done under the title \"results\" so, the word \"discussion\" should be added to the results section.\nThe researcher listed some recommendations for future research using content analysis to check teachers' feedback in other disciplines. Moreover, some limitations were identified regarding the close-ended questionnaire with just 11 items. Both recommendations and limitations are strongly synthesized from the research findings.\nFinally, a brief and comprehensible conclusion decorated the research. It showed the purpose and main findings of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "142482", "date": "07 Jul 2022", "name": "Ruiying Niu", "expertise": [ "Reviewer Expertise Second language acquisition and second language writing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 deals with the types of written corrective feedback (WCF) that teachers provide and students’ perceptions of teacher WCF in the Saudi Arabian EFL context. WCF is an important topic in both second language acquisition and second language writing research. As it is controversial whether teacher WCF benefits students’ language learning and written accuracy and which type of WCF is more beneficial, plenty of empirical studies have been conducted to examine these issues. Against this background, it is important to understand teachers’ WCF performances and students’ perceptions. Thus, such a relevant study would definitely contribute to the field.\nRegarding the study under review, there are several issues that need to be noted.\nFirst, in the literature review, types of WCF had better be reviewed earlier than the empirical studies. It is also notable that the study focuses on WCF, so do not confuse WCF and oral corrective feedback. They are classified in different ways. Such corrective feedback strategies as explicit correction, recast, clarification request, metalinguistic cues, elicitation, and repetition, as mentioned on P. 4 (of the pdf), belong to oral corrective feedback rather than WCF. In Table 1, the types of strategies listed are also confusing.\nSecond, still in the literature review, the studies conducted in the Saudi Arabian context do not have to be reviewed in a separate subsection. Instead, they should be integrated together with other related studies.\nThird, regarding methods, “study population” is not the proper term; the proper one is “participants”. According to the findings, the study did not use SPSS actually. The study is not a qualitative one but a descriptive quantitative study. In fact, for studies on students’ perceptions, the qualitative method is often adopted. Particularly, interviews can be used for data collection. In the present study, a questionnaire is used to understand students’ perceptions. It is acceptable to use a questionnaire, but the items should be carefully designed. In the present study, however, it is not clear how the items in the questionnaire are connected with the teachers’ feedback strategies.\nFourth, the findings and the discussion had better be written in different sections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-452
https://f1000research.com/articles/11-451/v1	22 Apr 22	{ "type": "Systematic Review", "title": "Effectiveness of pedometer-based exercise program in phase 1 and phase 2 cardiac rehabilitation: A Systematic Review", "authors": [ "Vanamala Lakshmi Vasavi", "Janhavi Khandekar", "Vijay Pratap Singh", "Stephen Rajan Samuel", "Molly Cynthia D’souza", "Vanamala Lakshmi Vasavi", "Janhavi Khandekar", "Stephen Rajan Samuel", "Molly Cynthia D’souza" ], "abstract": "Background: Physical Inactivity (PI) and sedentary behaviour are the most important and modifiable risk factors to prevent cardiovascular diseases (CVD). Cardiac rehabilitation (CR) is a multidisciplinary program for patients who have undergone cardiac surgeries. Through CR, monitoring physical activity (PA) is possible and may reduce the rate of rehospitalization. The pedometer is a device that is useful to track the step count of the person day to day. It helps in self-monitoring of PA. The purpose of this review was to summarise the evidence about the effect of pedometer-based exercise program in phase 1 and phase 2 CR. Methods: Databases such as MEDLINE, Cochrane, Scopus, Embase, and Web of Science were searched. This search is limited to randomized controlled trials (RCTs), human trials, a 10-year period, and English language journals. Based on inclusion criteria of pedometer-based exercise programs in phase 1 (inpatient phase) and phase 2 (outpatient phase) of CR and exclusion criteria of studies using pedometer in maintenance phase CR. The primary outcome of the study is step count, while secondary is PA (in terms of time), heart rate, sedentary behaviour, and quality of life. This study quality was assessed by the Downs and Black’s checklist. Results: The study includes 6 RCTs based on the inclusion and exclusion criteria. Most studies show a considerable increase in step count associated with increased patient PA. Few studies have explained a significant increase of PA in the study group and maintained for longer terms. More research is needed to determine the impact of cardiovascular risk factors.", "keywords": [ "Cardiac rehabilitation", "pedometer", "physical activity", "step count", "fitness tracker", "inpatient phase cardiac rehabilitation", "outpatient phase cardiac rehabilitation" ], "content": "Introduction\n\nCardiovascular diseases (CVD) are a group of diseases that involve the heart and the blood vessels. Coronary heart diseases, peripheral heart diseases, congenital heart diseases come under this group of CVDs. For the past 20 years, heart disease has been the leading cause of death worldwide. The number of deaths from heart disease climbed from approximately 2 million in 2000 to almost 9 million in 2019 (World Health Organisation [WHO] reveals leading causes of death and disability worldwide). Various risk factors which cause CVD are smoking, blood pressure, lipid level, diet, diabetes mellitus, physical activity (PA), and psychosocial characteristics (Balady et al. 2007).\n\nSedentary behaviour and Physical inactivity (PI) are two of the most modifiable risk factors for CVD worldwide (Young et al. 2016). PI has been linked to systemic pathophysiology, linked to the development of metabolic and cardiovascular disorders, osteoporosis, and some malignancies. Beginning a physically active lifestyle decreases the probability of developing these conditions and fatality (Fletcher et al. 2018).\n\nPA is defined as any bodily movement produced by skeletal muscles that result in energy expenditure (Caspersen et al. 1985). A systematic review and meta-analysis (Dibben et al. 2018) focusing on PA and cardiac rehabilitation (CR) has concluded there is an increase of PA level through CR in patients with coronary heart disease. CR is an effective treatment for a wide range of heart disease patients. Cardiopulmonary fitness, psychological variables, and quality of life are improved, and morbidity and death are reduced (McMahon et al. 2017).\n\nCVD is a leading cause of morbidity and mortality worldwide (Roth et al. 2017). Secondary prevention, such as provided through CR, is essential to recovery since cardiac patients are at high risk of recurring episodes and have a decreased quality of life (Mehra et al. 2020). CR is a multidisciplinary and multiphasic approach.\n\nCR plays a significant role in preventing CVDs and helps recover the patients who have undergone cardiac surgeries. It consists of essential elements such as risk factor modification, exercise training, dietary suggestions, psychosocial counselling, and patient education. A customizable follow-up approach and simple access to a specialised team are also included in CR (European Association of Cardiovascular Prevention and Rehabilitation Committee for Science Guidelines 2010). The European Society of Cardiology (ESC) (Van de Werf et al. 2008), American Heart Association (AHA), and American College of Cardiology (ACC) (Antman et al. 2008) all recommend CR programmes in the management of patients with coronary artery disease (CAD) and chronic heart failure (CHF).\n\nThe patients' weekly amounts and intensities of physical activity frequently fell short of the prescribed limits, owing to a lack of physical activity on non-CR days (Ayabe et al. 2004). A systematic review has provided evidence of improved PA significantly associated with pedometer among out-patient adults (Bravata et al. 2007).\n\nAccording to the AHA/ACC guidelines for secondary prevention of CVDs, a recommended PA ranges from minimum of thirty minutes for five days a week up to a maximum limit of sixty minutes for seven days per week. It includes moderate intensity workouts such as aerobic exercises; while resistance training is encouraged. Patients who come under high risk are suggested to perform these under medical supervision (Smith et al. 2006).\n\nA pedometer is a form of activity tracker that helps people keep track of how many steps they take each day. It motivates the user by giving them instantaneous feedback on how many steps they have taken regularly. The self-monitoring of the PA via pedometers over an extended period impacts the outcome by increasing the PA levels (Ayabe et al. 2010). A pedometer-based intervention was linked to increased PA, improved psychological health, and improved cardiorespiratory fitness (Butler et al. 2009).\n\nPeople can use a pedometer to assist them in boosting their PA level (Kaminsky et al. 2013). The use of pedometer determines step count for PA in CR. The PA results were significantly influenced by adherence to the step goal (Schneider et al. 2006).\n\nMany researchers have previously conducted various systematic reviews, such as pedometer adherence to PA in the CHD population (Bravata et al. 2007) and also a few on maintenance phase of CR (Hannan et al. 2019). Furthermore, no systematic review has been conducted on studying pedometer use in inpatient and outpatient phases of CR.\n\nIn this systematic review, we aim to study the randomised control trails (RCTs) that are done phase 1 and phase 2 CR using a pedometer-based exercise program. The primary outcome measure of this study is pedometer-based step count. While the secondary outcomes are PA, sedentary behaviour, heart rate, and quality of life.\n\n\nMethods\n\nThis systematic review includes full-text articles limited to RCTs, human trials and published in English language journals. Studies that use a pedometer as part of the intervention in either the inpatient or outpatient phase of CR were included.\n\nSingle group studies, non- RCTs, quasi-experimental studies, case series and studies, cross-sectional studies, and qualitative studies are also excluded. The studies which use pedometer-based mobile applications are excluded because according to the analyses, when compared to the pedometer, all the mobile applications had an unsatisfactory error percentage (Orr et al. 2015).\n\nThe protocol of this systematic review was registered in Open Science Framework (OSF) with the registration DOI: 10.17605/OSF.IO/73WX4. MEDLINE (RRID:SCR_002185; URL: http://www.nlm.nih.gov/bsd/pmresources.html), Cochrane (RRID:SCR_013000; URL: http://www.cochrane.org/reviews/clibintro.html), Scopus (Elsevier, RRID:SCR_013811; URL: http://www.elsevier.com), Embase (RRID:SCR_001650; URL: http://www.elsevier.com/online-tools/embase), and Web of Science (Clarivate Analytics, RRID:SCR_017657; URL: https://clarivate.com) are the databases used to screen the articles. A search strategy was established for each database using keywords along with the MeSH terms and Boolean operators such as “AND” & “OR”. A search was performed to identify the articles using various keywords such as, for CR – cardiovascular rehabilitation, heart rehabilitation, coronary rehabilitation; for pedometer – activity tracker, fitness tracker, accelerometer, PA tracker, personal tracker; step count; for PA – aerobic exercises, aerobic training and physical exercise. This search was narrowed to the years 2011–2021 to include more relevant and recent research. This review aimed at performing a meta-analysis for synthesizing high-quality evidence.\n\nThe searches were performed by using the search strategy mentioned above and it retrieved the total number of 989 articles. With the help of a reference managing tool Zotero (version: 5.0.96.2; RRID:SCR_013784; URL: https://www.zotero.org), the duplicates of 292 articles were removed. The articles which did not fit the inclusion criteria are excluded during the title and abstract screening. Two authors screened the full-text articles, and a total of 6 articles were finalised for the review. The PRISMA flow chart of the review is presented in Figure 1.\n\nThe process of assessing the study quality of the included studies was done by two authors independently using the Downs and black’s checklist (Downs and Black, 1998) (https://www.researchgate.net/figure/Downs-and-Black-Checklist-for-Quality-Assessment_fig2_269766057). This checklist provides a 27-list questionnaire that determines the quality of the studies. It assesses the studies by reporting characteristics, external validity, internal validity-bias, internal validity-confounding, and power. The studies had been rated according to the questionnaire values and divided into excellent (26–28), good (20–25), fair (15–19), and poor (less than or equal to14) upon a total of 28.\n\nFinalised articles were reviewed, and the primary author extracted relevant data. Data such as title, author, year of publication, total sample size, division of samples into intervention group and control group, and primary and secondary outcome measures were taken. The data tables of each article were screened for mean-standard deviation, confidence interval, and median – interquartile range.\n\nWe extracted secondary outcome data such as PA, heart rate, sedentary behavior, and quality of life. This data was from pre-intervention (baseline) and post-intervention time points. The percentage of completed samples, inclusion criteria, given intervention, frequency of sessions, dropout, and adverse events were extracted from all finalised articles. A second author checked this data while a third author resolved discrepancies. For additional data of the included studies, two authors were contacted and provided with the information (Midence et al. 2016; Thorup Msn et al. 2016).\n\n\nResults\n\nAll the included studies were RCTs. All the articles were eligible for the methodological quality assessment using the Downs and Black’s checklist. One study fell into the category of poor, given the insufficient information regarding blinding, allocation concealment, and follow-up details of patients (Thorup Msn et al. 2016). All the other studies were considered of good quality scoring in a range of 20–25 (Houle et al. 2011; Sangster et al. 2015; Midence et al. 2016; ter Hoeve et al. 2018; Gama Lordello et al. 2020). Randomisation was done in all the studies, where most of the studies mentioned the type of randomisation, blinding and allocation concealment. The adverse events were mentioned only in one study (Gama Lordello et al. 2020). The checklist of above study quality is presented in Table 1.\n\nOut of the 6 RCTs, three studies were three arm trails. Three articles mentioned primary outcome data which was step count in the format of Mean and standard deviation (Houle et al. 2011; ter Hoeve et al. 2018; Midence et al. 2016) (n=3, 50%), while one article had mentioned the data in the format of the median and interquartile range (Gama Lordello et al. 2020) (n=1, 16.67%). This step count data is presented in Table 2. Some articles did not mention step count values (Sangster et al. 2015), and few did not take the step count of the control group in account (Thorup Msn et al. 2016). Among all the studies, three studies performed pedometer-based cardiac telerehabilitation (n=3, 50%). Two studies considered cardiovascular risk factors (n=2, 33.33%). There was only one study which was of inpatient phase CR. The details of the interventions and outcome measures are summarised in Table 3.\n\nOwing to the variability among the included studies in terms of methodology and outcome measures presented, the results of the individual studies have been summarised and provided narratively since it was not possible to pool the data in a meta-analysis approach.\n\nImprovement in step count: Significant increase in step count has been shown in three studies, while one study could not show a statistical significance in the step count. ter Hoeve et al. (2018) reported an overall significant increase of step count in the intervention group (p=0.010) with a pedometer and face-to-face counselling sessions compared to the tele-rehabilitation group and control group (p=0.307). In the Gama Lordello et al. (2020) study, the total number of steps taken in the study group were 2183 (1729–2772) and in the control group were 2006 (1517–2657). However, there was an increase in steps taken in the study group, although the difference was not statistically significant (p=0.167).\n\nImprovement in PA: Out of six studies, four studies observed the patients' PA levels in adherence to the pedometer. Though there was an increase in both the study and control group, only a few studies noted a significant increase of PA in the study group that was maintained for longer terms (Sangster et al. 2015; ter Hoeve et al. 2018). Sangster et al. (2015), saw an improvement of PA from p=0.23 to p=0.15, while ter Hoeve et al. (2018), observed the difference between the PA levels by p=0.165 (CR+F [cardiac rehabilitation along with face-to-face counselling) vs. CR] and p = 0.331 [CR+T (cardiac rehabilitation along with telerehabilitation) vs. CR].\n\nImprovement in cardiovascular risk factors: Houle et al. (2011), and Gama Lordello et al. (2020), considered cardiorespiratory fitness and observed the pre- and post-intervention differences in both the study and control groups. Based on heart rate, both the studies showed a significant statistical effect. The study by Houle et al. (2011) described the lipid profile, fasting blood glucose, waist circumference, blood pressure, and smoking. Of which, waist circumference was shown to have a statistically significant effect, while smoking was seen to have a less significant role. Midence et al. (2016) also observed that the factors such as heart rate and smoking had no significant difference.\n\nImprovement in sedentary behaviour and psycho-social factors: One study observed the sedentary behaviour in their participants but no changes were seen in the results from baseline to the post-intervention sessions (ter Hoeve et al. 2018). Psycho-social factors are explained in three out of six studies in the form of either social support, quality of life, self-reported reasons for decreased walking, and self-efficacy expectation. Increased quality of life (QoL) has been seen in the study of Midence et al. (2016) while social support showed no significant changes in the same study. Reasons reported by the patients for decreased walking were noted in the study by Gama Lordello et al. (2020).\n\n\nDiscussion\n\nThe significant finding of this review is a substantial increase in PA noted as step count on pedometer adherence in patients undergoing phase 1 and 2 CR. The possible reasons that cause an increase in the step count results stated by the studies were- the instantaneous input on step activity leading to conscious awareness of walking action (Thorup Msn et al. 2016); objective feedback by the pedometer itself along with the follow-up counselling sessions (ter Hoeve et al. 2018).\n\nA pedometer is a helpful device that helps track the person’s PA while performing the exercise. It enhances adherence by giving visual feedback of the achieved PA target and acts as an incentive for the upcoming goals (Bravata et al. 2007). Patients were motivated to meet their step objectives because they expected health advantages and had become aware of walking as a beneficial activity (Thorup et al. 2016). This may be considered as extrinsic motivation from an self-determination theory (SDT) viewpoint since the incentive is not the act itself (walking), but the expected outcomes of the act (health benefits), however the motivation appears to be integrated (Deci and Ryan 2000).\n\nIn this review, a study that have used pedometer-based interventions suggested exercises like gymnastic exercises, running/brisk walking, sports activities, and relaxation exercises (ter Hoeve et al. 2018). According to the Borg scale, the researchers have set an exercise goal of increasing patients’ walking activity by 3000 steps daily of moderate-intensity (Houle et al. 2011). Along with this pedometer, the patients maintained a step count diary to record their regular PA or to note the daily step count achieved.\n\nOnly one study has performed an inpatient phase CR. In this study, the intervention group was given a cycle ergometer for arm and leg exercises. In contrast, the control group were recommended regular exercises such as activity for lower and upper limbs, open kinematic chain, flexion-extension of the shoulder, diagonal movements - cross body, straight leg raises, hip and knee flexion-extension, ankle pumps in the intensive care unit (ICU). As the patients were shifted out of ICU, additional exercises such as breathing exercises, stretching, progressive PA according to individual capacity followed by ICU exercises were given in both the groups (Gama Lordello et al. 2020).\n\nFew studies followed up via telephonic sessions for the aftercare program (Houle et al. 2011; Sangster et al. 2015; Thorup Msn et al. 2016; ter Hoeve et al. 2018). While lifestyle management counselling was also given by two out of six studies (Sangster et al. 2015; ter Hoeve et al. 2018). The telephonic sessions may have proved to be encouraging in terms of constant monitoring on the patient’s PA and their results.\n\nThis systematic review comprises the information of various forms of pedometer-based interventions. Unfortunately, a meta-analysis could not be done as the data obtained was heterogeneous. In terms of research design and statistical analysis, the four studies chosen for the meta-analysis were diverse in their approach. Two of the four studies were three-group studies, with one having two intervention groups and one control group (ter Hoeve et al. 2018), and the other having three separate intervention groups (Midence et al. 2016). The outcome variables in the other two trials were mean-standard deviation and median-interquartile range, respectively (Houle et al. 2011; Gama Lordello et al. 2020). This review included all RCTs and tried to provide high-level evidence to support objective of the study. Adding on, five out of six studies included in this study have a low risk of bias and one with a moderate risk of bias.\n\nThis systematic review has included all possible evidence obtained on the pedometer and CR. The exclusion of certain mobile application-based pedometers may have created a window of bias in this review. Furthermore, a meta-analysis was not possible as less evidence was available in the RCT study design. Limited reporting of statistical analysis and relevant data tables could have reduced the chances of a meta-analysis (Sangster et al. 2015; Thorup Msn et al. 2016; Su and Yu 2019).\n\nMore research should be done to compare the effects of using a pedometer in both control and intervention groups. As the heart rate was not examined in many trials, potentially widening the information gap, this analysis indicates that future RCTs might be developed using heart rate as one of the key outcome measures. According to one study, step rates of 130, 140, and 150 steps per minute are equivalent to the exercise intensity required to improve cardiorespiratory fitness levels (65%–75%) heart rate maximal (Lubans et al. 2009).\n\n\nConclusion\n\nAccording to the findings of this systematic review, using a pedometer in the early phases of CR may encourage PA and help patients adhere to their exercise regimen. This may aid in preventing CVD and the re-hospitalization of patients.\n\n\nData availability\n\nOpen Science Framework: “Effectiveness of pedometer-based exercise program in phase 1 and phase 2 cardiac rehabilitation.” https://doi.org/10.17605/OSF.IO/8TPJ5\n\nThis project contains the following underlying data:\n\nData file 1- Table 1: Quality assessment using Downs and Blacks checklist\n\nData file 2- Table 2: Step count data\n\nData file 3- Table 3: Intervention description and the outcome measures\n\nData file 4- Figure 1 – PRISMA flow chart\n\nData file 5- PRISMA checklist\n\nData file 6 – Systematic review protocol\n\nOpen Science Framework: “Effectiveness of pedometer-based exercise program in phase 1 and phase 2 cardiac rehabilitation.” https://doi.org/10.17605/OSF.IO/8TPJ5\n\nThis project contains the following extended data:\n\nData file 1- Supplementary table 1\n\nData file 2- Supplementary table 2\n\nOpen Science Framework: PRISMA Checklist for article “Effectiveness of pedometer-based exercise program in phase 1 and phase 2 cardiac rehabilitation”, https://doi.org/10.17605/OSF.IO/8TPJ5.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAuthor contributions\n\nVanamala Lakshmi Vasavi- Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nJanhavi Khandekar- Methodology, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nDr. Vijay Pratap Singh- Conceptualization, Methodology, Writing – Original Draft Preparation.\n\nDr. Stephen Rajan Samuel- Conceptualization, Writing – Review & Editing,\n\nMolly Cynthia D’souza- Supervision, Writing – Review & Editing.\n\nSupplementary table 1- Keywords used: Strategy builder\n\nSupplementary table 2- Synonyms and keywords", "appendix": "References\n\nAntman EM, Hand M, Armstrong PW, et al.: 2007 Focused Update of the ACC/AHA 2004 Guidelines for the Management of Patients With ST-Elevation Myocardial Infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines: developed in collaboration With the Canadian Cardiovascular Society endorsed by the American Academy of Family Physicians: 2007 Writing Group to Review New Evidence and Update the ACC/AHA 2004 Guidelines for the Management of Patients With ST-Elevation Myocardial Infarction, Writing on Behalf of the 2004 Writing Committee. 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PubMed Abstract \| Publisher Full Text\n\nWHO reveals leading causes of death and disability worldwide: 2000-2019: [accessed 2021 Jun 7]. Reference Source\n\nYoung DR, Hivert M-F, Alhassan S, et al.: Sedentary Behavior and Cardiovascular Morbidity and Mortality: A Science Advisory From the American Heart Association. Circulation. 2016; 134(13): e262–e279. PubMed Abstract \| Publisher Full Text" }	[ { "id": "182739", "date": "16 Aug 2023", "name": "Eiichi Watanabe", "expertise": [ "Reviewer Expertise Arrhthmia" ], "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\nLakshmi Vasavi and colleagues conducted a systematic review of randomized controlled studies to examine the effects of a pedometer-based exercise program. Six studies were included based on predetermined exclusion criteria.\nMajor comment: While the methodology is clearly stated, the paper's structure could be improved in terms of presenting results. Despite the inclusion of heterogeneous populations and study methods, a summary figure analyzing the effects on quality of life, physical activity, dietary habits, social activity, BMI, and changes in serial steps during the study (though partly shown in Table 2) would be helpful. Description of the 6 studies is hard understand the overview of the pedometer-based studies.\nAbstract: Results was not well written. Only subjective descriptions and there are no objective findings and has no Conclusions.\nMinor comments: Introduction is too long and redundant.\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": [] }, { "id": "139602", "date": "16 Nov 2023", "name": "Shweta Gore", "expertise": [ "Reviewer Expertise physical activity", "COPD", "Heart failure", "multimorbidity" ], "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 premise of this study adds little value to the scientific body of evidence. Pedometers have been previously studied in systematic reviews and have shown to significantly underestimate activity. The validity of pedometers has been questioned in several studies.\n\nThe introduction is choppy and the paragraphs need to connect better. For example, introducing CVD is the leading cause of morbidity in paragraph 4 is redundant as this was already introduced in paragraph 1. Also the operational definition of CVD does not fit in the introduction but in other section.\nThere are grammatical errors and typos throughout the manuscript that need special editing. Example: trial is spelt 'trail'; 'that are done phase 1'.. instead of 'in phase 1'.\nFurthermore, the purpose of the review is not mentioned. Need to phrase the research question instead of talking about the approach in this section.\n\nMethods:\nInclusion criteria: use of both past and present tense in this section.\nExclusion criteria: again, sentence structure needs attention. Studies 'were' excluded since this has already been done.\n\nLiterature search: What was the inter-rater reliability of the two independent reviewers. How was conflict resolution attained. What were the qualifications of the reviewers. Did the same two reviewers also complete quality appraisal? Was inter-rater reliability established?\nStudy quality:\n\nWhy was a standardized quality appraisal tool such as the Cochrane risk of bias tool not used for quality appraisal? Downs and Black is an old checklist.\nTable 2: Why does it not have information on 2 other included studies (Sangster and Theroupe)? This table only shows information from 4 studies.\n\nResults:\nStep count: reporting p values is insufficient. What was the confidence interval?\n\nConclusion:\nConcluding that pedometer is effective is an erroneous conclusion when only 6 studies were included with 1 study of poor methodological quality, and only 3 studies showing improvement . Additionally, improvement in sedentary behavior and QOL was only sporadically noted.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] } ]	1	https://f1000research.com/articles/11-451
https://f1000research.com/articles/11-450/v1	22 Apr 22	{ "type": "Genome Note", "title": "The complete mitochondrial genome sequencing of the Asian lesser white-toothed shrew (Crocidura shantungensis Miller, 1901; Eulipotyphla: Soricidae) from Jeju of Korea", "authors": [ "Seo-Jin Lee", "Dong-Hee Kim", "Hang Lee", "Seo-Jin Lee", "Dong-Hee Kim" ], "abstract": "We report the complete mitochondrial genome of Crocidura shantungensis (Asian Lesser White-toothed Shrew). The complete mitochondrial genome is 17,160bp in length and contains 13 protein coding genes, one replication origin region, 22 transfer RNA, two ribosomal RNA, and one D-loop (control region). The mitogenome is A+T rich, with a composition of A: 32.5%, T: 32.6%, C: 21.7%, and G: 13.1%. In the phylogenetic tree, the Crocidura genus species formed a solid monophyletic group in the family Soricidae, C. shantungensis was well grouped with sister group to C. sibicica within the Genus Crocidura clade, and each species noted against each other well.", "keywords": [ "mitogenome", "Phylogenetic analysis", "Crocidura shantungensis", "Asian Lesser white-toothed shrew" ], "content": "Introduction\n\nThe Crocidura shantungensis (Asian lesser white-toothed shrew) Miller (1901) is classified under order Eulipotyphla, family Soricidae and genus Crocidura. The taxa that was considered C. suaveolens in East Asia was phylogenetically separated from C. suaveolens inhabiting Europe and is now treated as a separate species, C. shantungensis (Dubey et al. 2006, Jiang and Hoffmann 2001, Motokawa et al. 2003, Ohdachi et al. 2004). C. shantungensis is listed as 'Least Concern' on the IUCN (International Union for Conservation of Nature) Red List and the China Red List (Hutterer 2005, Jiang et al. 2016, Temple 2016). The Asian lesser white-toothed shrew is endemic to East Asia including Korea, Taiwan, China, Russia, Tsushima Island of Japan, and Mongolia (Hutterer 2005, Jiang and Hoffmann 2001, Motokawa et al. 2005). It is the smallest species of white-toothed shrew in South Korea and is widely distributed on the South Korean mainland as well as in its southern peripheral islands and Jeju Island (Yoon et al. 2004).\n\n\nMethods\n\nThe voucher specimen was from Seogwipo-si (33°26'21.4\"N 126°49'24.2\"E) in Jeju Special Self-Governing Province. Voucher specimen was deposited at the National Science Museum (2021, Daejeon, South Korea. Korean Natural History Research Information System [NARIS, https://www.naris.go.kr/main.do], Hyunjong Kim and ttliu@nsmk.kr) under the voucher number NSMK-OB-MM-210300881. Our research falls under the study using a part of dead organism or animal tissue, so it does not need to undergo deliberation and approval from the committee [IACUC Standard Operating Guidelines (Revised on Dec. 2020)].\n\nThe total genomic DNA was extracted from tissues of C. shantungensis using the QIAGEN blood and tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction. The amplified mitochondrial DNA was prepared to sequencing library using QIAseq FX single cell DNA library kit (QIAGEN), and DNA library was sequenced by HiseqX platform (Illumina, San Diego, CA, USA) in GnC Bio Co. (Daejeon, South Korea). The sequence assembly and gene arrangement were performed by Geneious Prime® 2022.0.2 (Biomatters, Auckland, New Zealand).\n\nThe nucleotide dataset for phylogenetic analysis included 13 protein coding genes (PCGs) of 33 Soricidae species. Two Muridae species (Apodemus agrarius: JN629047, A. peninsulae: HQ660074) was used as outgroup. The maximum-likelihood analysis with 1000 replication of rapid bootstrapping options was performed by PhyML version 3.1 with GTR+I+G model (Guindon et al. 2010).\n\n\nResults\n\nThe mitochondrial complete genome sequences of C. shantungensis (NCBI. No. OM038325) was 17,160 bp in length and contains consists of 13 protein coding genes (PCGs), 22 transfer RNA, two ribosomal RNA, one replication origin region (OL), and one control region (D- loop). Total length of the 13 protein coding genes is 11,344 bp long, all of which are encoded on the same strand except for ND6 in the light strand. The gene arrangement was exactly in accordance with other Soricidae species. The 9 PCGs (ND1, COX1, COX2, ATP8, ATP6, COX3, ND4L, ND4, ND6, CYTB) were started with initiation codon ‘ATG’, and the rest of three PCGs (ND2, ND3, ND5) were ‘ATT’. The three PCGs (COX3, ND3, and ND4) have incomplete stop codon, ‘T—’. The complete stop codon ‘TAA’ were showed in eight PCGs (ND1, COX1, COX2, ATP8, ATP6, ND4L, ND5, ND6). Also, ND2 and CYTB were terminated ‘TAG’ and ‘AGA’, respectively. The base nucleotide composition of the mitochondrial genome of C. shantungensis from Jeju in Korea is A: 32.5%, T: 32.6%, C: 21.7%, and G: 13.1% with a strong bias toward A+T (65.1%).\n\nIn the phylogenetic analysis result, the Crocidura species formed a solid monophyletic group in the family Soricidae. The result showed that C. shantungensis was well grouped with sister group to C. sibicica within the Genus Crocidura clade (Figure 1). Our result is consistent with the previous study by Jiang et al. (2019) and Li et al. (2021). The C. shantungensis from Jeju in Korea used in this study differed from C. shantungensis from other specimen from Korea, from GenBank accession number JX968507 (location is Odaesan National Park in South Korea, Kim et al. 2013) by 91 SNPs, from GenBank accession number KR560064 (exact location is unknown, expect Korea; unpublished) by 18 SNPs and three indels (two 1 bp and one 2 bp in the length). There are many reports of morphological and genetic differences between Jeju Island and the inland in various biota of Korea, and the biota of Jeju Island is sometimes classified into subspecies or species (Kim et al. 2018, Komiya 1985, Lee and Oh 2021, Oh et al. 2012). The C. shantungensis is distributed throughout the Korean Peninsula, and we presented the complete nucleotide sequence of the mitochondrial genome for the C. shantungensis, which is distributed on Jeju in Korea. We reported genetic differences from JX968507 (location is Odaesan National Park in South Korea, Kim et al. 2013). These findings are useful for inferring biogeographic and phylogenetic studies within the Soricidae family, and especially for the crocidura genus. The genomic resources of Jeju in Korea obtained through this study can be used as reference data for genetic research as well as the phylogenetic relationship and evolutionary history of Crocidura species.\n\nNumbers on branches represent bootstrap values.\n\n\nData availability\n\nGenBank: Crocidura shantungensis mitochondrion, complete genome, Accession number OM038325: https://www.ncbi.nlm.nih.gov/nuccore/OM038325\n\nBioProject: Crocidura shantungensis (Asian lesser white-toothed shrew), Accession number PRJNA791823: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA791823\n\nSRA: crocidura shantungensis, Accession number SRR17417922: https://www.ncbi.nlm.nih.gov/sra/?term=SRR17417922\n\nBioSample: animal sample from Crocidura shantungensis, Accession number SAMN24371516: https://www.ncbi.nlm.nih.gov/biosample/?term=SAMN24371516\n\nMendeley Data: Complete mitochondrial genome of Crocidura shantungensis, http://dx.doi.org/10.17632/k9y6mkfh5z.2 (Lee 2022).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nDubey S, Zaitsev M, Cosson J-F, et al.: Pliocene and Pleistocene diversiication and multiple refugia in a Eurasian shrew (Croci-dura suaveolens group). Mol. Phylogenet. Evol. 2006; 38: 635–647. PubMed Abstract \| Publisher Full Text\n\nGuindon S, Dufayard JF, Lefort V, et al.: New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 2010; 59(3): 307–321. PubMed Abstract \| Publisher Full Text\n\nHutterer R: Order Soricomorpha. Wilson DE, Reeder DM, editors. Mammal species of the world. Baltimore, MD: Johns Hopkins University Press; 2005; 220–311.\n\nJiang H, Li F, Wang X, et al.: The complete mitochondrial genome of the Siberian shrew, Crocidura sibirica (Mammalia, Soricidae). Mitochondrial DNA. 2019; 4(1): 916–917.\n\nJiang X, Hofmann RS: A revision of the white-toothed shrews (Crocidura) of Southern China. J. Mammal. 2001; 82: 1059–1079. Publisher Full Text\n\nJiang Z, Jiang J, Wang Y, et al.: Red List of China’s Vertebrates [J]. Biodiv. Sci. 2016; 24(5): 500–551. Publisher Full Text\n\nKim H-N, Kim H-U, Jo Y-S, et al.: Development of 17 polymorphic microsatellite loci from Jeju striped field mouse, Apodemus agrarius chejuensis (Rodentia: Muridae), by 454 pyrosequencing. Hereditas. 2018; 155: 30. PubMed Abstract \| Publisher Full Text\n\nKim H-R, Park J-K, Cho JY, et al.: Complete mitochondrial genome of an Asian Lesser White-toothed Shrew, Crocidura shantungensis (Soricidae). Mitochondrial DNA. 2013; 24(3): 202–204.\n\nKomiya Y: Studies on the Trichochrysea-species of Japan, Ryukyu Archipelago, Taiwan and Korea (Coleoptera: Chrysomelidae: Eumolpinae). Elytra. 1985; 12(2): 11–25.\n\nLee S-J: Complete mitochondrial genome of Crocidura shantungensis. Mendeley Data. 2022; V2. Publisher Full Text\n\nLee J-W, Oh H-S: External and Cranial Characteristics of Mustela sibirica quelpartis on Jeju Island. Anim. Syst. Evol. Divers. 2021; 37(3): 205–211.\n\nLi H, Zhang G, Liu G, et al.: Characterization of the complete mitochondrial genome of Dongyangjiang White-toothed Shrew, Crocidura dongyangjiangensis (Eulipotyphla: Soricidae) and its phylogenetic analysis. Mitochondrial DNA. 2021; 6(3): 1004–1006. PubMed Abstract \| Publisher Full Text\n\nMiller SG: Descriptions of three new Asiatic shrews. Biol. Soc. Wash. 1901; 14: 157–159.\n\nMotokawa M, Lin L-K, Harada M, et al.: Morphometric geographic variation in the Asian lesser white-toothed shrew Crocidura shantungensis (Mammalia, Insectivora) in East Asia. Zool. Sci. 2003; 20: 789–795. PubMed Abstract \| Publisher Full Text\n\nMotokawa M, Yu H-T, Harada M: Diversification of the white-toothed shrews of the genus Crocidura (Insectivora: Soricidae) in East and Southeast Asia. Mammal Study. 2005; 30: S53–S64. Publisher Full Text\n\nOh D-J, Kim T-W, Chang M-H, et al.: Migration route estimation of the Jeju striped field mouse Apodemus agrarius chejuensis (Rodentia, Muridae). Mitochondrial DNA. 2012; 24(2): 137–144. PubMed Abstract \| Publisher Full Text\n\nOhdachi SD, Iwasa MA, Nesterenko VA, et al.: Molecular phylogenetics of crocidura shrews (insectivora) in East and Central Asia. J. Mammal. 2004; 85: 396–403. Publisher Full Text\n\nTemple H: Crocidura shantungensis (errata version published in 2017). The IUCN Red List of Threatened Species 2016: e.T5617A115077696.2016.\n\nYoon M-H, Han S-H, Oh H-S, et al.: The mammals of Korea. Seoul: Dongbangmedia; 2004." }	[ { "id": "179809", "date": "18 Jul 2023", "name": "Rogelio Rosas-Valdez", "expertise": [ "Reviewer Expertise Molecular systematics and Biogeography" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMethods. Para. 2: Please provide information of the tissues used for extraction of DNA.\nNeed justification:\nWhy PCGs where chosen for phylogenetic analysis?\n\nWhy were Muridae species were used as outgroup, based in previous analyses?\n\nGenes can show different patterns in the nucleotide composition, Why used the GTR+I+G model for 13 genes? How it was obtained?\nQuestion: Have you tried Bayesian inference?\n\nResults, authors claim: \"The 9 PCGs (ND1, COX1, COX2, ATP8, ATP6, COX3, ND4L, ND4, ND6, CYTB)\"; but they are ten, please fix.\nThe phrase: \"The result showed that C. shantungensis was well grouped with sister group to C. sibicica within the Genus Crocidur clade (Figure 1).\" is not appropriate, a correct option should be: C. sibirica is the sister group of C. shantungensis. Please fix.\nFigure 1. The ML trees always present length of branches, these help to understand relationships and differences among groups. Please show the length of branches.\nDifferences among C. shantungensis (SNPs) generate changes in amino acids translation? Are they synonymous mutations?\nPlease clarify, where you found the indels?\nPlease check the correct spelling of the scientific names throughout the document, and also the grammar.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] }, { "id": "188520", "date": "05 Oct 2023", "name": "Sadık Demirtaş", "expertise": [ "Reviewer Expertise Mammalian systematics and phylogeography" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 well in generally, despite some parts that need to be changed and improved.\nComments to Author:\nAbstract\n“Asian Lesser White-toothed Shrew” change to “Asian lesser white-toothed shrew”\nIntroduction\nThe “taxa” are a plural noun, not a singular noun and its singular is taxon. Please correct the verb. Namely, the taxa that “were”…..East Asia were…..\nMethods\nThe parentheses after the National Science Museum are incomprehensible. Namely, (2021, Daejeon, South Korea. Korean Natural History Research Information System [NARIS, https://www.naris.go.kr/main.do], Hyunjong Kim and ttliu@nsmk.kr)\n\n33 Soricidae species must have been misspelled because the ingroup contains 32 species (see Figure 1). Also, Mogera spp is not Soricidae. Please rearrange it.\n\nI and G values must be specified in the selected GTR+I+G model.\nResults\n“(NCBI. No. OM038325)” Please change it to (NCBI accession OM038325)\n\nThe 9 PCGs (ND1, COX1, COX2, ATP8, ATP6, COX3, ND4L, ND4, ND6, CYTB) were... Please change it to The 10 PCGs.\n\naccession number JX968507 change to NC_021398, because it given a reference genome for the species now.\n\nI don't see in Kim et al. 2023 this location (Odaesan National Park in South Korea). Please specify if personal communications as “pers. comm.”\n\nPlease review and rearrange to the SNP numbers and specify if there are non-synonymous mutations from them.\n\nPlease remove the parentheses. it has already been mentioned above “We reported genetic differences from JX968507 (location is Odaesan National Park in South Korea, Kim et al. 2013)”.\n\nmiss-spelling of the word ‘crocidura’ to Crocidura.\nData availability\nSRA: miss-spelling of the word “crocidura shantungensis”, to Crocidura shantungensis.\nI think that the following issues should be justified.\nThe evolutionary relationship with C. suaveolens was mentioned in the introduction, but not in the result part and phylogenetic tree.\n\nWhy are Muridae species used as outgroups, Talpidae members more appropriate?\n\nPlease show the length of branches in ML tree because genetic differences between groups/species are not clearly understood.\n\nSome groups in the ML tree were low statistically supported by bootstrapping analysis (Figure 1). I strongly recommend that you display the results of posterior probability values in Bayesian analysis on an ML tree.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-450
https://f1000research.com/articles/11-449/v1	21 Apr 22	{ "type": "Research Article", "title": "Aesthetic development of children", "authors": [ "MASATOSHI HAMADA" ], "abstract": "Background: The dynamics between conceptualizing art-making and responding to aesthetic phenomena in relation to aesthetic development of children (ADC) are unclear. This study aimed to investigate what facilitates the transition between conceptualizing art-making and responding to aesthetic phenomena in terms of metaphors of life and nature in ADC. Methods: We adopted an ethical methodology prioritizing movement and respecting children’s autonomy toward positive emotions. The participants were eight Japanese children (age range: 7−15 years; 6 girls, 2 boys). They were in the same painting class to ensure nearly identical aesthetic conditions. Results: Four states according to children’s ages were observed in their perceptions of metaphors of life and nature in conscious and unconscious ways in the iterative dynamics between conceptualizing art-making and responding to aesthetic phenomena. Conclusion: Two important findings from systems-oriented perspectives are that emotional communication in art can be theorized, and that the four states of the aesthetic development of children seem to be related to children's age-specific tension-flow rhythms. By making the process of art therapy for ADC, we can expect art therapy and art-based research to be more developmentally appropriate for children.", "keywords": [ "aesthetic development", "children", "metaphors of life and nature", "emotional communication in art", "tension-flow rhythms", "conceptualizing art- making", "responding to aesthetic phenomena", "consciousness and unconsciousness" ], "content": "Introduction\n\nStudies on the aesthetic development of children (ADC) have been crucial both in our understanding of child development in education (Burrill, 2010) and of children’s cultural contexts (Manifold, 2012). The implementation of ADC in art therapy has recently been thought to be essential because the current context, such as the coronavirus disease 2019 (COVID-19) pandemic, can increase the years of therapy needed for children, and children’s aesthetic senses could vary during this turbulent period.\n\nHarrison (1990) suggested that children’s classification abilities require their ability to recognize similarities. Furthermore, a child’s ability to identify emotions between visual stimuli results from their classification abilities (Winston, Kenyon, Stewardson, & Lepine, 1995). Therefore, it follows that ADC could also relate to children’s classification abilities.\n\nJessen and Grossmann (2016) suggested that conscious perception and unconscious perception are connected to emotions. Similarly, Winston et al. (1995) suggested that ADC includes not only parameters of perception but also those of emotions. Almeida-Rocha et al. (2020) studied ADC when responding to aesthetic phenomena and validated Parsons’s (1989) theory that the parameters of ADC varied according to the children’s ages. Therefore, it is understood that ADC, when responding to aesthetic phenomena, could relate to emotions stemming from both the conscious and unconscious mind and vary according to children’s ages.\n\nChildren’s aesthetic sense and artistry are nurtured by receiving alternating feedback from external and internal factors. (Manifold, 2012). Thus, conceptualizing art-making and responding to aesthetic phenomena is necessary for ADC. The study of conceptualizing art-making related to children’s emotions, however, has only just begun to be investigated (Chilton et al., 2015). This could be an explanation as to why the dynamics of ADC between conceptualizing art-making and responding to aesthetic phenomena remain unclear.\n\nThe genesis of emotions might stem from metaphors of life and nature in the conscious and unconscious mind (Chilton et al., 2015); therefore, conceptualizing and responding to metaphors of life and nature could be necessary for ADC. Thus, the following question is addressed in this study:\n\nQ1: What facilitates the transition between conceptualizing art-making and responding to aesthetic phenomena in terms of metaphors of life and nature in ADC?\n\n\nMethods\n\nA third-party non-governmental organization (NGO) in Japan, Nijiiro Creyon, collected the data used in this study. Nijiiro Creyon has official registration as an NGO working with children and communities in the Ishinomaki region of Japan. Nijiiro Creyon’s Articles of Incorporation states, as part of its code of ethics, that they can participate in non-profit activity to promote academic, cultural, and artistic activities for children; therefore, no approval from an external ethics committee was required as the study methods were part of normal activities for the NGO. The author received the data from Nijiiro Creyon and conducted the analysis; the author did not have any contact with the children or parents.\n\nOn November 14, 2020, the author sent the materials and presentation describing the method of the study to the NGO. The NGO consented to take part in the study and agreed to collect the data. Before conducting the survey, the NGO obtained verbal consent from the children and their parents after explaining the purpose of the study; the Japanese Ministry of Health, Labour and Welfare does not require written consent if no intervention is applied or samples are obtained from the human body, thus verbal consent was appropriate for this study. At the NGO, the staff use a notebook to record the discussions they have after each activity is complete. This notebook was used to record the names of the children and parents who consented to be part of the study. The notebook is kept securely, and third parties are not able to access it. The study began on February 1, 2021. After analyzing the data collected, the author explained the results to the NGO on February 12, 2021. Consent for publishing these results was obtained from the NGO on January 28, 2022.\n\nDue to the uncertainty regarding how long the COVID-19 pandemic will last, we chose to conduct a cross-sectional study. Specifically, we chose a method similar to Almeida-Rocha et al. (2020), which has been used in studies with children of various ages under the same conditions. Further, because it was difficult to find several children with the same aesthetic environment, we chose to include as many children as possible from the same aesthetic environment as our participants, even if it was only a small number. Accordingly, we decided to compare our results with those of previous cross-sectional and longitudinal studies to determine if our findings could be supported.\n\nThe study used a comparative design to explore the key developmental phenomena in ADC. We compared up to four states, as children’s senses are either conscious or unconscious of the metaphors of life and nature in both conceptualizing art-making and responding to aesthetic phenomena. Thus, two experiments were conducted: the first was to explore how children conceptualize art-making, and the second was to explore how children respond to aesthetic phenomena. We then studied the dynamics between the two experiments regarding the metaphors of life and nature.\n\nWe adopted an ethical methodology for the following reasons: The sense of metaphors of life and nature is a fundamental factor of positive emotions (Chilton et al., 2015). Therefore, ethical considerations respect children’s autonomy in increasing their self-healing power, which results in an ethical approach being recommended to allow children to participate in a natural, fun, and playful way regarding positive emotions. For this reason, the methodology prioritized movement, such as playing with art cards without any limitations.\n\nThe participants were Japanese children aged between 7 and 15 years (N = 8; 6 girls and 2 boys) who attended painting classes run by the NGO. The eligibility criterion was that all children attended the same painting class, to ensure similar aesthetic conditions. The NGO looked for participants across a wide age range. The participants and their parents were given information about the survey at the NGO’s painting classes. The NGO conducted the survey in February 2021 during the COVID-19 pandemic after all the participants and their parents provided their consent in-person. All participants wished to participate and no parent withdraw their children from the study.\n\nChildren’s classification abilities stem from recognizing similarities (Harrison, 1990) and identifying emotions (Winston et al., 1995); thus, we investigated ADC based on the participants’ classification abilities. Regarding the materials used to facilitate classification, D’Onofrio and Nodine (1981) suggested that a wide stylistic range of paintings is necessary for exploring ADC; therefore, painting cards (Boyer, 2016), which consisted of 33 paintings from the Renaissance art period and abstract art were selected for inclusion in the study. Two experiments were conducted using these cards.\n\nThe cards of people and landscape still lifes were regarded as “metaphors of life and nature,” similar to Dietrich and Hunnicutt (1948); the cards of shape, illustration, and design were regarded as “skill,” as seen in Kuscevic, Kardum, and Brajcic (2014).\n\nThe ethical methodology could help bring out children’s innate senses and unconscious mind. Burrill (2010) suggested that, through free movement and expression, children construct concrete concepts and internal images that are consistent with the environment around them. These images include emotions, a sense of life, memories, aesthetic sensations, and intellect. Further, Švachová (2016) studied Suzanne Osten, who is the founder and long-time artistic director of the Swedish research theater Unga Klara, which has focused on children and youth since 1975. The study suggested that each child’s unconscious needs and desires could be the motivation for the accompanying artistic activities. Thus, we proceeded this study with a focus on on the children’s conscious and unconscious mind.\n\nThe ethical considerations followed a positive approach, as data were collected during the COVID-19 pandemic, and participants might be experiencing extraordinary stress due to this context. The methodological approach and ethical considerations resulted in this study emphasizing that the children should participate in a natural, fun, and playful way. Thus, this research used a co-constructivist approach, similar to Richards (2014), in which the children set the pace and direction of the data generation related to their perspectives and voices in the experiments.\n\nWe conducted text mining based on Trevors et al. (2017) to avoid analysist’s subjective elements. In this article, we used the text mining tool for Japanese language (Yasuda, 2011) because the participants were Japanese children.\n\nThe following software was used for text mining.\n\nName: TinyTweetCrawler (TTM)\n\nVersion: version 0.16\n\nWebsite: https://mtmr.jp/ttc/\n\nThe following procedure was used for data analysis:\n\n1) A table was created on an Excel sheet with a tag (A to H) of the participants.\n\n2) The data collected from the interviews was entered into the table on the Excel sheet (CSV format).\n\n3) Text mining was run by the TTM software. A cross-tabulation of words and terms (number of occurrences) was output as a result.\n\n4) Non-zero number of occurrences were regarded as related words and merged words were generated.\n\n5) An Excel table of participant tags and merged words was created by inputting the number ‘1’ if a participant corresponded to the merged word, and ‘0’ otherwise.\n\n6) A correlation analysis was run in Excel and output the correlation coefficients between the merged words. Generally, an absolute value of correlation coefficient from 0.4 to 0.7 is treated as a moderate correlation and from 0.7 to 1 is treated as a strong correlation. Thus, the absolute value of correlation coefficient equal to or greater than 0.4 was adopted as a major parameter relationship.\n\nExperiment 1 took 40 minutes per participant; data was collected by interviewers who wrote down the participants’ responses. First, each child classified the 33 cards into three categories: similar, normal, and dissimilar by comparing them to their own paintings. The children were then asked why they decided to classify certain cards as similar, and how they created their own paintings. The children's responses were written down by the interviewer during the interview, and text mining was conducted to exclude the subjective factors of the questioner. Because the sense of metaphors of life and nature could be essential to art avtivities in children (Chilton et al., 2015), we assumed that children will always have this sense, whether it is conscious or unconscious. If the children answered with a word related to the metaphors of life and nature, we considered it to be conscious; if they did not, we considered it unconscious.\n\nParticipants were classified into three categories: “similar,” “normal,” and “dissimilar.” However, because the only data used in the analysis was the reason for selecting a similar picture, the children were asked to select the one picture they felt was most similar to the picture they selected as “similar,” and then asked why they selected that picture, with the intention of highlighting the reason.\n\nExperiment 2 took 40 minutes per participant and data was collected by interviewers who wrote down the participants’ responses. The children were then asked to sort the 33 cards freely, without limiting the number of groups or the number of cards in each group. We then inquired about the child’s reasons for their classifications. The children's responses were written down by the interviewer during the interview and text mining conducted.\n\nThe interviews for this study were conducted with a teacher and a student of a painting class who had already established a trusting relationship with each other. The interviews were conducted at the location of the painting class. Additionally, the analysis was done specifically on metaphors of life and nature related to positive emotions. However, since negative emotions are also necessary to magnify positive emotions and children express positive, negative, and mixed emotions when they create pictures (Chilton et al., 2015), the ratio of positive, negative, and mixed emotions in the emotions of individual children is different. Therefore, the difference in emotions between the researcher and the subjects could be a deviation.\n\n\nResults\n\nFigure 1 depicts an example of the classification of the cards in Experiment 2 with unlimited numbers of groups and cards in groups. As seen in this example, children recognize paintings irrespective of the style of the painting and regardless of the historical period. These results are shown below in relation to the children’s own painting skills, the sensations they acquired visually, and their emotions.\n\nTables 1 and 2 show the results of each experiment depicting the children from youngest (A) to oldest (H). The tables indicate how the children answered each question regarding the factors in conceptualizing art-making and responding to aesthetic phenomena. At a glance, the tables show that the ADC is so patchy that it is difficult to tell whether there are relationships between conceptualizing art-making and responding to aesthetic phenomena. However, by organizing the tables, we can visually decipher that the ADC changes with growth in the relationships. The correlation seems to be in the metaphors of life and nature of people.\n\n# Answers given by children regarding their classification of similarity.\n\n# Answers given by children regarding their free classification.\n\nFigure 2 illustrates the results shown in Tables 1 and 2 and outlines the four states found from the youngest to the oldest children regarding the metaphors of life and nature in the combination of conceptualizing art-making and responding to aesthetic phenomena:\n\nState 1: unconscious and unconscious\n\nState 2: conscious and unconscious\n\nState 3: conscious and conscious\n\nState 4: unconscious and conscious\n\nWe employed the study design to explore the developmental phenomena of children by explicitly comparing the four states. Accordingly, we found that the factors that make up the investigated transition in the research question depend on the four states of ADC, as described below.\n\nFigures 3–6 illustrate the relationships of the major parameters in each state. As shown in Figure 3, by feeling the metaphors of life and nature unconsciously, the children of State 1 conceptualize art by feeling and imaging color, and respond to aesthetic phenomena by feeling and imaging intriguing or strange images and describing color.\n\nAs shown in Figure 4, the children of State 2 started conceptualizing art-making by feeling and seeing metaphors consciously associated with color and skill, but continued to feel and see metaphors unconsciously in response to aesthetic phenomena.\n\nAs shown in Figure 5, the children of State 3 started generating metaphors in response to aesthetic phenomena related to emotions and associated with skill. As Figure 6 shows, the children of State 4 completed the transition of the metaphors from conceptualizing art-making to responding to aesthetic phenomena and feeling and seeing the metaphor unconsciously again when conceptualizing art-making. By completing the transition of metaphors in emotions, the children developed an aesthetic sense that alternates between conceptualizing art-making and responding to aesthetic phenomena.\n\n\nDiscussion\n\nIn a systems-oriented perspective, individuals are thought to develop through three combined levels of action: environmental, intrapersonal and interpersonal, and sociocultural (Demick & Wapner, 1988; Wapner & Demick, 1998). It is perhaps not surprising that our experimental results encompass the previous studies when viewed from a systems-oriented perspective. It may also simply be that good research conditions resulted in similarities with the previous studies.\n\nThe findings of this study partly support Winston et al. (1995), as ADC of responding to aesthetic phenomena was found to be associated with that of conceptualizing art-making. However, the findings suggest that ADC is more dynamic, with iterations between conceptualizing art-making and responding to aesthetic phenomena. Therefore, the findings similarly support Manifold (2012) as a child's aesthetic sense and artistry are nurtured by alternating feedback from external and internal factors. In addition, the findings suggest that aesthetic sense in relation to conscious and unconscious ways of expressing metaphors of life and nature transition to emotions through iterative dynamics. This finding supports the suggestions of Boyakova, Savenkova, and Torshilova (2020), based on more than half a century of long-term experimental research, such as the importance of valuing human life and human existence, fostering a dialectic of the mind, and encouraging openness to the external world as well as the novelty of the self. This research emphasizes that the external environment surrounding the child plays an important role in the universality of the child’s aesthetic elements and the process of art and culture that permeates the child’s entire maturation process.\n\nThe findings of this study suggest that the four states of ADC follow children’s ages when the aesthetic environment is similar. This means that ADC could be combined with the natural ability of children and the aesthetic environment by which children are surrounded, as suggested by Boyakova et al. (2020). The findings also indicate that ADC could enhance and expand children’s sense of metaphors of life and nature. The findings partly support Chilton et al. (2015), in that the senses could be a fundamental factor of positive emotions in the natural art ability of children. However, as posited by Boyakova et al. (2020), it may be the aesthetic environment instead that could enhance and expand the senses.\n\nThere are two important findings in our results: emotional communication in art may be theorizable, and the four states of ADC seem to be related to the age-specific rhythms of children.\n\nIn an environment where children are uninhibited, they could express a wide range of thoughts and emotions in creative art. When children view art, they may be smiling, calm, indifferent, or experience a variety of emotions. Between the art and the children's emotions, a non-verbal relationship is established. In art, this is called emotional communication.\n\nArt, such as painting, may not always be aware of how the viewer perceives it (Langer, 1953), but even if the emotions of the children and the viewer, or the artist and the children, are different, some kind of emotional communication in art will be established between the children's emotions and the art. Even though the effect of emotions in art have been studied since the early 20th century (Stanislavski, 1937), to our knowledge, emotional communication in art has not yet been theorized. This may be why the research on ADC has focused only on responding to aesthetic phenomena.\n\nLooking at ADC from the perspective of nonverbal communication, the four states would indicate that the two-way nonverbal communication between children and art is tied to the age of the children. The formation of this two-way nonverbal communication requires both conceptualizing art and responding to aesthetic phenomena based on the metaphors of life and nature. If research is carried out from this perspective, emotional communication in art would be theorized, and it may lead to an education method that respects children and their relationship with nature and life like Burrill (2010).\n\nInfants aged 0-5 years have age-appropriate unconscious tension-flow rhythms, and these age-specific rhythms are almost identical when infants perform dance and art (Burrill, 2011). In our discussion below, we conclude that the tension-flow rhythms may also occur at later ages under several assumptions.\n\nWe assume that the same tension-flow rhythms are present in dance and art at later ages. Our experiment was conducted while the children were playing in a free-flowing, unrestricted environment. Therefore, we believe the children had almost the same positive emotions, or at least did not have negative motivations. In this environment, the ADC showed four states. If the tension-flow rhythms were the same for dance and art, it means that there could be at least four age-specific tension-flow rhythms between ages 7 to 15. Indeed, in the context of embodiment research, there is growing interest in the phenomenon of interpersonal resonance between infants and their mothers in nonverbal communication occurring at later ages, showing almost identical tension-flow rhythms under the same emotional environment even at ages 18 to 29 (Koch & Rautner, 2017). Because tension-flow rhythms are unconscious, metaphors of life and nature in the conscious and unconscious mind would be related to children's age-specific tension-flow rhythms.\n\nInterestingly, the emotional communication and tension-flow rhythms seem to have some relationship through ADC. This relationship, derived from the fact that tension-flow rhythms change with emotion and awareness even at ages 18 to 29 (Koch & Rautner, 2017), and from the findings in our experiment, leads to the following hypotheses.\n\nState 1: Children to art and art to children are two-way unconscious emotional communication in art. Emitting tension-flow rhythms from early childhood up to the age of 5, children unconsciously perceive art with the rhythms.\n\nState 2: Children to art becomes unidirectional conscious emotional communication in art. The tension-flow rhythms emitted by the children begin to change after infancy and are updated by the children's consciousness and emotions. Meanwhile, art to children remains unidirectional unconscious emotional communication in art, and is influenced by art with tension-flow rhythms from infancy.\n\nState 3: Children to art and art to children become two-way conscious emotional communication in art. The tension-flow rhythms are updated by children's consciousness and emotions; children emit art with their own rhythms and are influenced by art with the rhythms. For this reason, the fundamental new tension-flow rhythms in various consciousnesses and emotions toward youths is formed during this period.\n\nState 4: Children to art becomes unidirectional unconscious emotional communication in art again. The tension-flow rhythms emitted by the children begin to be specific to their consciousness and emotions. Art to children remains unidirectional conscious emotional communication in art, renewing new tension-flow rhythms in various consciousnesses and emotions.\n\nThe hypotheses have a very important premise: children should enjoy doing art in a free-spirited and playful way, as we have displayed in our ethical approach. Under this condition, children feel metaphors of life and nature naturally in their mind. In the positive environment, movement becomes primary in art activities like Burrill (2010). Thus, we can expect that movement and art have similar tension-flow rhythms.\n\nWe performed this study under near-ideal conditions in which the children’s aesthetic environments were as close to identical as possible. However, implementing a longitudinal study with many participants from the same aesthetic environment was difficult during the COVID-19 pandemic. Our findings from this cross-sectional study with a small number of participants were compared with previous results; however, as this could not be fully controlled, it presents a limitation in this study, mainly because the children's aesthetic environments varied. Nevertheless, in addition to surfaces that were visible to the children such as color and skill, this study explored the underlying conscious and unconscious metaphors of life and nature from a systems-oriented perspective.\n\nPsychological interventions for children are necessary, not only for those affected by the COVID-19 pandemic (Northwest Mental Health Technology Transfer Center, n.d.), but also for those who have lived through disasters such as tsunamis (Takahashi & Setou, 2019). ADC should be introduced in art therapy while capturing the changing aesthetic sensibilities of children. This suggestion is important for research and therapy. McNiff (2019) suggests that to address the idea of how art heals is to acknowledge that dimensions of art that are shared across cultures and infinite differences in people, communities, and artistic expression exist simultaneously. Further, McNiff (2011) notes that arts-based research is especially important for arts therapies that use creative expression as a way of knowing, a way of communicating, and a way of promoting personal and social development. If we can make this process of art therapy and art research ADC-appropriate, we can expect therapy and research to be more developmentally responsive to children.\n\nIt is vital to consider the implication of this research in the context of the COVID-19 pandemic. Children may experience significant stress due to the COVID-19 pandemic (Northwest Mental Health Technology Transfer Center, n.d.); thus, during this period, children’s aesthetic sense may display variations, as seen in this study’s findings. Therefore, a discipline implementing ADC in art education and art therapy during the COVID-19 pandemic may be an ethical and appropriate approach.\n\nThe variation of the children's aesthetic environments may have affected the children’s conscious or unconscious senses of metaphors of life and nature; therefore, further studies regarding how, why, and when children feel metaphors of life and nature in their conscious or unconscious mind may be a worthwhile challenge.\n\nFurther research into emotional communication in art and children's age-specific tension-flow rhythms should be conducted from a systems-oriented perspective in a variety of settings. The findings result in the hypothesis derived from ADC, the emotional communication in art and tension-flow rhythms. We would like to test the hypothesis, but as it is far beyond our resources, so we invite other interested researchers to continue to investigate these possibilities.\n\n\nConclusion\n\nOur findings show that the ADC is more advanced in both conceptualizing art-making and responding to aesthetic phenomena. Recently, museum art materials have become a new modality for art-based therapy (Monsuez et al., 2019; Montreal Museum of Fine Arts, n.d.), and these findings support art activities in museums, and specifically, the implementation of movement with art-making and art appreciation. We found that methodology should prioritize movement and respect children’s autonomy in relation to positive emotions. The present findings are consistent with Burrill (2010) and show that movement could be crucial in ADC, especially in the context of the COVID-19 pandemic.\n\nIn conclusion, based on the present findings, four states were identified in which children perceived metaphors of life and nature in conscious and unconscious ways, which could lead to positive emotions in art activities. The factors of ADC were according to the states that followed the children’s ages if their aesthetic environments were similar.\n\nAdopting an ethical methodology which prioritizes movement and respects children’s autonomy in relation to positive emotions could be crucial in ADC, especially within the context of the COVID-19 pandemic. Therefore, our study concludes that, in seeking to acknowledge and support wider ADC, insights into metaphors of life and nature between conceptualizing art-making and responding to aesthetic phenomena are pedagogically important for the process of art therapy and art research.\n\n\nData availability\n\nZenodo: Data of Aesthetic development of children. https://doi.org/10.5281/zenodo.6353721 (Hamada, 2022)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThanks to Nijiiro Creyon, an NGO for children in the areas affected by the Great East Japan Earthquake, for the interviews with children during COVID-19 pandemic. The contribution of the NGO is a cooperation offered to the UNESCO MILX research (Grizzle & Hamada, 2019).\n\n\nReferences\n\nAlmeida-Rocha T, Peixoto F, Jesus SN: Aesthetic development in children, adolescents and young adults. Analise Psicologica. 2020; 38: XXXVIII: 1–13. Publisher Full Text\n\nBoyakova EV, Savenkova LG, Torshilova EM: The quality of aesthetic development in the context of an art festival as a condition for the overall development of children and adolescents. Propósitos y Representaciones. 2020; 8((SPE2), (SPE2)). Publisher Full Text\n\nBoyer A: Mémo tableaux. Larousse; 2016.\n\nBurrill R: The primacy of movement in art making. Teach. Artist J. 2010; 8(4): 216–228. Publisher Full Text\n\nBurrill R: Movement, art, and child development through the lens of an innovative use of the Kestenberg movement profile. Am. J. Dance Ther. 2011; 33(2): 111–130. Publisher Full Text\n\nChilton G, Gerber N, Bechtel A, et al.: The art of positive emotions: Expressing positive emotions within the intersubjective art making process (L’art des émotions positives: Exprimer des émotions positives à travers le processus artistique intersubjectif). Canadian Art Therapy Association Journal. 2015; 28(1–2): 12–25. 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Art Ther. 2019; 36(3): 162–165. Publisher Full Text\n\nMonsuez JJ, François V, Ratiney R, et al.: Museum moving to inpatients: Le Louvre à l’hôpital. Int. J. Environ. Res. Public Health. 2019; 16(2): 206. PubMed Abstract \| Publisher Full Text\n\nMontreal Museum of Fine Arts: Art therapy activities in the museum. Montreal Museum of Fine Arts; n.d.Reference Source\n\nNorthwest Mental Health Technology Transfer Center: Stress by COVID-19 pandemic. Northwest Mental Health Technology Transfer Center; n.d.Reference Source\n\nParsons MJ: How we understand art: A cognitive development account of aesthetic experience. reprint ed.Cambridge: Cambridge University Press; 1989.\n\nRichards R: The private and public worlds of children’s spontaneous art. Stud. Art Educ. 2014; 55(2): 143–156. Publisher Full Text\n\nRocha TA, Peixoto F, Jesus SN: Aesthetic development in children, adolescents and young adults. Análise Psicológica. 38(1): 1–13. Publisher Full Text\n\nStanislavski C: An actor prepares. Hapgood ER, Trans. Geoffrey Bles; 1937.\n\nŠvachová R: The childish Unga Klara: Contemporary Swedish children’s theatre and its experimental aesthetics. Brünner Beiträge zur Germanistik und Nordistik. 2016; 30(1): 51–63. Publisher Full Text\n\nTakahashi S, Setou N: Kodomo no Aimaina Soushitsu [Chapter 3 Ambiguous loss of a child]. Kurokawa M, Ishii C, Nakajima S, editors. Aimaina soushitsu to kazoku no rejiriensu [Ambiguous loss and family resilience]. Seishinshobo; 2019; (pp. 63–84).\n\nTrevors GJ, Muis KR, Pekrun R, et al.: Exploring the Relations between Epistemic Beliefs, Emotions, and Learning from Texts. Contemporary Educational Psychology January. 2017; 48: 116–132. Publisher Full Text\n\nWapner S, Demick J: Developmental analysis: A holistic, developmental, systems-oriented perspective. Damon W, Lerner RM, editors. Handbook of child psychology, 1. Theoretical models of human development. 5th ed.Hoboken: New York, Wiley; 1998 (Vol. Ed.); pp. 761–805).\n\nWinston AW, Kenyon B, Stewardson J, et al.: Children’s sensitivity to expression of emotion in drawings. Vis. Arts Res. 1995; 21(1): 1–14.\n\nYasuda M: An Investigation and Clustering of the Keywords for Color Response: Using Text Mining. The 20th International Congress of Rorschach and Projective Methods, Tokyo, Japan.2011." }	[ { "id": "140968", "date": "25 Jul 2022", "name": "Renata Martinec", "expertise": [ "Reviewer Expertise Expressive arts-therapies", "dance movement therapy", "complementary therapy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the author tried to consider the dynamics between conceptualizing art-making and responding to aesthetic phenomena in relation to the aesthetic development of children (ADC) by including terms of metaphors of life and nature in ADC. Results show that four states according to children’s ages were observed in their perceptions of metaphors of life and nature in conscious and unconscious ways in the frame of the dynamics between conceptualizing art-making and responding to aesthetic phenomena. According to obtained data, the author pointed out that emotional communication in the art can be theorized, and that the four states of the aesthetic development of children seem to be related to children's age-specific tension-flow rhythms.\nThis article is interesting and has significant potential, but the methodology is rather unclear and should be described in more detail. For example, why words related to the metaphors of life and nature were to be considered as conscious and, if they did not, considered as unconscious?\nGiven that a smaller number of respondents were included in the research, it might have been more transparent if the paper had shown examples of pairs of children's works, similar cards along with associated words.\nAlso, it should be described how the categories in table 1. and table 2. were defined (such as metaphors, skill, color, emotions, feeling, and image).\nIt is interesting premise that children's age-specific tension-flow rhythms are connected to aesthetic development and metaphors of life and nature. But are there some other variables that could influence ADC?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "180045", "date": "03 Jul 2023", "name": "Andri Savva", "expertise": [ "Reviewer Expertise Children's responses to art", "art play in early years. Museum and art education", "Citizenship and art education" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author poses questions in relation to ACD of children and their responses to aesthetic phenomena in terms of life and nature. However the research question is unclear. The author needs to explain and define the meaning of those aesthetic phenomena in relation to art. The study examines children's classifications abilities - I cannot see how this is related to emotions. I find the use of the terms \"conscious and unconscious\" problematic, and it definitely is not possible to define them in relation to children's classification or responses to art.\n\nGardner1 and Housen2 research on children's aesthetic development could be used to support theoretical positions. It is vital to clarify that this study is about aesthetic responses of children to reproduction of art paintings.\n\nThe manuscript needs proof reading. Eg. instead of material the author could use the term \"research tools\".\nI cannot understand how movement is related to the \"card play\".\n\"The children were then asked why they decided to classify certain cards as similar, and how they created their own paintings\" - Please provide clarity. Perhaps an English proofreading would help make the description clear.\nAlthough the researcher refers to the interview process - tools - questions used, are not provided. Further the author needs to refer children's verbal comments. How does the author - researcher - study the children's tension follow rhythms?\n\nPlease consider the following in relation to the section - Limitations: Though is important to include different studies or genres of art, 33 cards is a vast amount of information. The author should consider the inclusion of local or national artists and not only those that are considered to be Europeans or Great Artists in the Western World. Results can not be generalised due to small number of participants.\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? No", "responses": [] }, { "id": "180049", "date": "03 Jul 2023", "name": "Ayomi Irugalbandara", "expertise": [ "Reviewer Expertise Aesthetic Education" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, the paper provides a good and generalized background on the importance of aesthetic development in children. However, several suggestions need to be addressed:\nIn the introduction section, the author highlights the basic arguments for the study, but these arguments are not explored in depth in the discussion section. It is important to ensure that the arguments presented in the introduction are discussed and expanded upon in the subsequent sections.\n\nThe data collected for the study is from the COVID period, but this is not specifically highlighted in the results or discussion sections. It is important to mention the impact of the pandemic on the data collection process and any implications it may have on the findings.\n\nWhile the paper follows the standard structure of a research paper, it fails to convincingly establish the rationale for the study. It is essential to clearly articulate the purpose and significance of the research to provide a strong foundation for the study.\n\nThe selected group in the study sample consists of children ranging from 7 to 15 years old, which includes different age groups. It is unclear how the aesthetic sense of a 7-year-old child can be compared or deemed compatible with that of a 15-year-old. The justification for the selection of the sample is not clear. Additionally, gender may also influence their sense of creativity, so it is important to consider and discuss the potential impact of gender in the study.\n\nThe findings of the study cannot be generalized. It is important to acknowledge the limitations and scope of the study, and clearly state that the findings may not be applicable to a broader population.\nThese suggestions should be addressed to improve the clarity and coherence of the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-449
https://f1000research.com/articles/10-1069/v1	21 Oct 21	{ "type": "Research Article", "title": "Prevalence study of intermittent hormonal therapy of Prostate Cancer patients in Spain", "authors": [ "Xavier Bonfill-Cosp", "Ariadna Auladell-Rispau", "Ignasi Gich", "Javier Zamora", "Luis Carlos Saiz", "Jose Ignacio Pijoan", "Iratxe Urreta", "José Antonio Cordero", "Xavier Bonfill-Cosp", "Ariadna Auladell-Rispau", "Ignasi Gich", "Javier Zamora", "Luis Carlos Saiz", "Jose Ignacio Pijoan", "Iratxe Urreta" ], "abstract": "Background: Although intermittent androgen deprivation therapy was introduced many years ago to improve patients’ quality of life with the same carcinologic efficiency as continuous hormonal therapy, recent data suggest that those patients could be overtreated. This study aims to estimate the prevalence of prostate cancer patients receiving intermittent androgen deprivation therapy in Spain. Methods: A retrospective, longitudinal study was conducted using electronic drug dispensation data from four Spanish autonomous communities, which encompass 17.23 million inhabitants (36.22% of the total population in Spain). We estimated intermittent androgen therapy use (%IAD) and the prevalence of patients under intermittent androgen therapy (PIAD) overall and stratified by region. Other outcome variables included the pharmaceutical forms dispensed and the total direct annual expenditure on androgen deprivation therapy‐associated medications. Results: A total of 863,005 dispensations corresponding to a total of 65,752 men were identified, treated with either luteinizing hormone-releasing hormone (LHRH) analogues (353,162) administered alone or in combination with anti‐androgens (509,843). Overall, the mean (±SD) age of the patients was 76.9 (±10.4) years. Results revealed that the mean annual PIAD along the study was 6.6% in the total population studied, and the overall %IAD during the five‐year study period was 5.6%. The mean cost of hormonal therapy per year was 25 million euros for LHRH analogues and 6.3 million euros for anti-androgens. Conclusions: An important proportion of prostate cancer patients in Spain could benefit from intermittent androgen therapy during the study period while avoiding overtreatment harms associated with continuous hormonal therapy.", "keywords": [ "intermittent androgen deprivation therapy (IAD)", "LHRH analogues", "prostate cancer", "appropriateness" ], "content": "Abbreviations\n\nADT: Androgen Deprivation Therapy\n\nATC: Anatomic Therapeutic Code\n\nCAD: Continuous Androgen Deprivation therapy\n\nELD: Effect duration of the Last Dose\n\nGPC: Clinical Practice Guideline\n\nIAD: Intermittent Androgen Deprivation therapy\n\n%IAD: Percentage of time off treatment\n\nIntT: Interval Time\n\nLHRH analogue: Luteinizing Hormone-Releasing Hormone analogue\n\nPCa: Prostate Cancer\n\nPIAD: Prevalence of Intermittent Androgen Deprivation therapy\n\nPIIT: Potentially Intermittent Interval Time\n\nPSA: Prostate Specific Antigen\n\n\nIntroduction\n\nAndrogen deprivation therapy (ADT) is the most used therapeutic approach to treat patients with Prostate Cancer (PCa) who experience a biochemical relapse after radical prostatectomy. Reduction of circulating levels of androgen hormones can be achieved either with drugs, such as luteinizing hormone-releasing hormone analogues (LHRH) or with orchiectomy, which is irreversible. Nevertheless, ADT is associated with a wide array of adverse effects1 ranging from the well-known hot flushes, loss of libido, gynecomastia, or erectile dysfunction up to midterm consequences such as bone fractures, depression, diabetes, and hematologic2 and cardiovascular events.3,4 Importantly, all of these undesirable effects increase with the duration of ADT.5\n\nIn the 1990s, as a response to minimise these negative outcomes, intermittent androgen deprivation therapy (IAD) emerged as an alternative to continuous androgen deprivation therapy (CAD) but not without controversy regarding its feasibility.6 Over the years, it has been shown that IAD can improve patients’ quality of life by reducing toxicity,7 reduce costs8 and potentially delay the onset of resistance to chemical castration.9 Recent strong evidence from randomized clinical trials10–14 and systematic reviews,15,16 as well as favourable recommendations included in recently updated clinical guidelines,17–19 highlight the potential benefits of IAD, supporting its use as an alternative treatment.\n\nDespite the potential advantages of IAD, in a previous study we detected a low use of this approach in Catalonia (Spain)20 compared to recent evidence of IAD utilization in Canada.21 We conducted the current study to assess the prevalence of IAD with real-world data from four Spanish autonomous communities.\n\n\nMethods\n\nAn observational, longitudinal study was conducted using electronic drug dispensation data from four Spanish autonomous communities: Catalonia, Madrid, Basque Country and Navarra (population: 16.05%, 14.17%, 4.63%, and 1.37% of the total population in Spain respectively, representing overall 36.22% of the total population in Spain, which was about 47 million inhabitants in 2020).\n\nData used for this study were obtained from the health government agencies from each of the four territories in Spain: CatSalut in Catalonia, Servicio Madrileño de Salud in Madrid, Osakidetza in the Basque Country and Servicio Navarro de Salud-Osasunbidea in Navarra. We received all relevant dispensation data from the four respective agencies for the period between 1 January 2011 and 31 December 2016. Databases contained anonymised patient codes, date of birth, dispensing date, prescriber code (anonymised), drug name and anatomic therapeutic code (ATC), total dose per pharmaceutical form, number of dispensed drug units and the corresponding cost. The dispensation of each of the following LHRH analogues was identified: leuprorelin (L02AE02), triptorelin (L02AE04), goserelin (L02AE03), buserelin (L02AE0), histrelin (H01CA0), nafarelin (H01CA02), and the following antiandrogen drugs: bicalutamide (L02BB03) and flutamide (L02BB0), in any pharmaceutical form or dose registered in Spain. We selected ≥ 18-year-old male patients with at least one dispensation of LHRH analogues. We excluded individuals under age 18 based on the high likelihood that LHRH dispensations in this subgroup would be associated with precocious puberty, which was not within our interest. Given the specificity of ADT, we assumed that all other men ≥ age 18 were prescribed these drugs as PCa treatment.\n\nThe requested data were provided under the requirements of the Spanish data protection laws and no personal data was available to the investigators.\n\nTo distinguish between the CAD and IAD regimens, we identified all the periods during which patients received no LHRH analogue or Potentially Intermittent Interval Times (PIIT). We first identified all interval times (IntT), defined as the period between any two consecutive dispensations for each patient, together with the effect of the last dose (ELD), identified as the time period during which the patient was covered by the medication, as shown in Figure 1. Further details about the methods followed can be consulted in our previous publication.20 Table 1 summarizes the type of LHRH analogue used, the total dose of the commercially available formulation, and the corresponding total duration of action of each dose. Although an intermittency gap usually starts after a minimum of six months of continuous hormonal treatment to reconfirm patient castration (PSA < 4 ng/mL), in our study, we considered three months as the minimum potential gap in order to have a conservative approach in IAD estimation.\n\n* Exposition measure: d = day/s, m = month/s.\n\nOne of the two key study variables created to estimate the use of intermittence was the percentage of time off treatment (%IAD). This percentage was calculated by dividing the sum of all the off periods (in patients with ≥1 intermittent interval time) by the sum of all interval times on any LHRH analogue regimen (i.e., the sum of periods between the first and the last dispensation dates for each patient in the study). The %IAD was calculated overall and for each region along the study period. The other key variable to estimate the number of patients under intermittency was the prevalence of IAD (PIAD), calculated as the number of patients with ≥1 PIIT per year, divided by the total number of patients treated with LHRH analogues in the same year. Nevertheless, it should be noted that the PIAD for the first year of data (2011) was underestimated due to the prior three-month intermittency requirement (e.g., patients in the intermittent period in January 2011 could not be identified as such until the next off period). Because of that, the data from 2011 was not included in the specific analysis of intermittency. We present the costs of hormonal treatment, including LHRH analogues and anti-androgenic therapeutic groups, per drug, year, and autonomous community. We excluded the Basque Country for the calculation of the annual expenditure because of its lack of complete 2011 data. To compare the cost between communities, we normalised the data as euros per dispensation. We expressed baseline characteristics, as mean ± standard deviation for continuous variables and frequency (%) for categorical variables with the corresponding 95% confidence interval (95%CI). Calculations were performed using SPSS version 26.0 software (IBM, Armonk, NY).\n\n\nResults\n\nFor the full study period (2011-2016), a total of 863,005 dispensations corresponding to a total of 65,752 men, including either LHRH analogues administered alone (353,162) or in combination with anti-androgens (509,843), were made in Catalonia, Madrid, Basque Country and Navarra. Previously, 309 males under the age 18 were excluded together with 7,731 subjects that were dispensed only with antiandrogens, because complete castration was not guaranteed. Additionally, 2012 data from the Basque Country were considered inconsistent (likely incomplete electronic system introduction in this area) and were also excluded from the analysis. Finally, a total of 44,264 PCa patients were included in the study, as summarized in Figure 2. Overall, the mean (±SD) age of the patients was 76.9 (±10.4) years, although 81% were older than 70 years and 47% older than 80 years.\n\nOf the total men (44,264) finally included in the study and taking LHRH analogues alone, 36,775 (83%) were under CAD therapy and 7,489 (17%) under IAD therapy, considering the full five-year period of the study. However, this value of global prevalence represents an overestimation of the actual annual prevalence of IAD, as can be observed in Figure 3. For example, among the ten patient profiles simulated, only five profiles (6 to 10) fulfilled IAD requirements at least once (5/10 cases where “Potential IAD = Yes”), when considering the six years globally. Then, the global IAD prevalence was of 50%. However, when considering each year separately, the annual prevalence varied from 0 to 50%, with a mean annual prevalence of 24%. The age distribution of the patients according to the treatment regimen (IAD vs CAD) is shown in Figure 4. Patients on the IAD regimen were somewhat older compared to the total patients under continuous hormonal therapy. The proportion of octogenarians and older patients under IAD therapy (56.7%) was higher compared to those under CAD (45.1%).\n\nThe five-year prevalence (PIAD) of patients under an IAD regimen was 6.6% in the total population studied. The global prevalence of intermittency slightly increased from 8.2% in 2012 to 8.4% in 2013, but later decreased to 3.6% in 2016. We also calculated the %IAD during the five-year study period with a total %IAD value of 5.6%, which is rather consistent with the PIAD figure reported earlier.\n\nAmong the four Spanish regions, the PIAD shows that in 2012 Madrid had the highest prevalence of IAD therapy (10.7%), followed by Navarra (8.2%) and Catalonia (5.8%). Peak values obtained were up to 14.2% [2014] in Madrid, 8.5% [2013] in Navarra, 8.4% [2013] in Catalonia, and 3.8% [2015] in the Basque Country; thereafter, the use of intermittent treatment decreased to 4.3% by 2016 in Madrid, 4.0% in Catalonia, 3.4% in Navarra and 2.5% in the Basque Country. Table 2 shows the total PIAD distributed per community per year, as well as %IAD for each community.\n\n# In reference to male inhabitants older than 20 years of each Autonomous Community in 2016.\n\nNote: IAD prevalence (CI 95%) based on annual PIAD and %IAD.\n\nAbbreviations: IAD Intermittent Androgen Deprivation therapy; PIAD Prevalence of Intermittent Androgen Deprivation therapy; %IAD percentage of time off treatment.\n\nWe found that the most representative non-treatment period lasted for a median of six months (ranging from 3 to 58 months) considering all four areas. In Madrid and Catalonia, the median of the PIIT intervals was 6.2 months with an IQR of 7.1 and 9.2 months respectively, while in the Navarra and Basque Country the PIIT intervals were shorter and less variable: 5.1 months (IQR 4 months) and 4.0 months (IQR 3.1 months), respectively. In the four areas the global tendency was to increase the duration of the “off” period from four (2012) up to six months (2016).\n\nThe most prescribed drug in all four communities during the five-year study period was leuprorelin, followed by triptorelin and goserelin (representing globally a 51.72%, 34.72%, and 12.33%, respectively) of the total LHRH analogues dispensed.\n\nThe global mean cost of hormonal therapy per year was 25 million euros for LHRH analogues and 6.3 million euros for anti-androgens for the four regions. Along the study period, the total expenditure in Catalonia, Madrid and Navarra decreased from 34.7 million euros [2011] down to 25.3 million euros [2016]. Figure 5 plots the changes in total cost for each specific LHRH analogue per year. The global mean (±SD) cost per dispensation was 214.6 ± 33.5 euros in 2011, then decreased to 201.7 ± 31.9 euros in 2014, and increased again up to 213.9 ± 26.3 euros in 2016.\n\n\nDiscussion\n\nWe conducted a study that estimates the prevalence of IAD in four Spanish autonomous communities (17 million people), based on electronic dispensation information from 2011 to 2016. Our results show that, taking either the mean PIAD (6.6%) or the %IAD (5.6%) as reference, IAD use was in the range of 6%. These results are in agreement with our previous estimations from Catalonia (2008-2012): PIAD = 4.2% and %IAD = 1.7%20 and confirm a low global utilisation of IAD in Spain. Even worse, the IAD utilisation rate has evolved in a progressively decreasing manner along the current study period, ranging from 8.4% (2013) to 3.6% (2016). Among the four Spanish regions, the %IAD showed that Madrid had the highest prevalence of intermittency (11.4%), followed by Catalonia (5.2%), Navarra (4.3%) and the Basque Country (1.7%). The PIAD showed a decreasing trend along the study period until 2016 in Madrid (4.3%), Catalonia (4.0%), Navarra (3.4%) and the Basque Country (2.5%). Based on the obtained data, we interpret that the well-known role of intermittent treatment in hormonal therapy, based on sound evidence from randomised controlled trails10–14 and systematic reviews,15,16 does not appear to be influencing actual clinical practice in a significant proportion of cases. It seems that the necessary trade-off between benefits and adverse effects could be much improved in those situations where IAD is as effective as CAD, since adverse effects of IAD should be lower for a broad range of morbidities.4,13,16,22–24 An international survey conducted in 19 countries asking 441 physicians currently treating PCa patients, showed that 23% of non-metastatic patients treated with gonadotropin-releasing hormone analogues were using IAD. Additionally, the same authors agree with our results that the CAD and IAD use in Spain among men with non-metastatic prostate cancer, was of 35.7% and 6.9%, respectively.25 Reference countries like France, Italy or Germany showed higher IAD use than Spain (9.1, 13.9 and 14.3%, respectively), still based on physicians opinions. Further evidence suggesting that the use of IAD is low in Spain, comes from data collected in Manitoba (Canada), where 74% (447/601 nonmetastatic hormone-sensitive PCa patients) were using IAD for the management of their relapse.21\n\nWhen analysing the data related to the duration of the “off” periods, or halted medication periods, we observed that the median duration varied between 4 and 6,2 months in all four regions (ranging from 3 to 13 months). These off-treatment periods are concordant with the corresponding values reported by Crook et al., that ranged from approximately 20 months in the first non-treatment interval, down to three months in the 6th interval.13 Globally, there was also a tendency to increase the median duration of the pause period between 2012 (three to four months) and 2016 (five to six months). This may suggest that prescribers using IAD are progressively more confident with the risk/benefit balance of this therapeutic strategy. This is in agreement with the fact that patient-reported outcomes were significantly better in IAD versus CAD at 20 and 38 months of treatment, based on validated quality of life questionnaires in Japan.26\n\nWe calculated that the total cost of hormonal therapy in the four autonomous communities was 31.3 million euros per year (25 million corresponding to LHRH analogues and 6.3 to anti-androgens), which represents a mean of 214 euros per dispensation. The fact that the mean cost per dispensation declined from 214.6 euros in 2011 to 201.7 euros in 2014 could be related to the fact that in 2012, the Spanish health authorities introduced restrictive measures for the sustainability of the National Health System (Royal Decree 16/2012)27 and all kind medication were reduced in the whole country by as much as 12 million per month.28 However, thereafter the cost increased again up to 213.9 euros per dispensation in 2016. Obviously, the efforts to enhance the use of IAD would produce important cost savings due to the reduction in expensive drug consumption, which some authors estimate in the range of one-third of CAD.11 In the population studied, we would be talking of around half a million savings per million inhabitants per year.\n\nIf PCa patients are to be actively involved in their decision process related to hormonal therapy medications, as current clinical guidelines suggest, they should know the existing sound evidence that shows similar overall survival of both CAD and IAD users, but the broader span of morbidities associated to CAD. From the physicians’ point of view, the PSA levels (54%), patient request (48%), desire to maintain sexual function (40%), patient age and comorbidities (38%) were reported as the most frequent reasons for IAD use.25\n\nThe main strength of our study is the capacity to analyse all public health systems electronic dispensations data from the selected Spanish regions, which eliminates any potential selection bias. They constitute a very large sample size, which favours the representativeness of the whole Spanish population. Also, the reliability of the database reduces information bias and missing data problems as previously observed by other authors.29\n\nDue to the administrative nature of our data sources, our study is not able to estimate whether patients identified under IAD are clinically appropriate or not. However, taking into account the conservative approach that we used and compared to the real-world utilization in Canada21 and 19 other countries,25 we could say that the use of IAD in Spain could be significantly increased, leading to the consequent improvement in patients’ quality of life and significant savings for the National Health Service. We plan to conduct an audit in a sample of the electronic records of PCa patients who were potential IAD candidates, to assess if they fulfilled the clinical criteria for having eventually stopped the hormonal therapy.\n\n\nConclusions\n\nWe conclude that the IAD use in Spain was relatively low during the study period, although enough time has passed since the related international recommendations were published. As a consequence, an important proportion of hormone-sensitive prostate cancer patients could be currently overtreated with CAD, which would produce a significant number of avoidable adverse effects on their quality of life, apart from the important repercussion that this expense has on the health budget in times of crisis. The population-based method to estimate the IAD prevalence seems to be a quite consistent approach and opens the door to comparable international data to set up reference standards on adequate IAD utilisation.\n\n\nData availability\n\nProject Datasphere: Intermittent hormonal therapy of prostate cancer patients in Spain: a prevalence study, https://doi.org/10.34949/fzzk-ye57. This project contains the following underlying data:\n\n- CEIC_PROTOCOLO_Ca prostat.pdf (Study protocol)\n\n- IAD_CRF_v1.docx\n\n- DataDescription_v1.xlsx\n\n- BBDD_Spain_IAD_v4.xlsx (Dispensation data)\n\nData are available under the terms of the PDS Data Sharing Agreement. Any individuals who wish to access the data will need to register for an account on Project Datasphere.", "appendix": "Acknowledgments\n\nWe are grateful to those who facilitated the data for this study: Public Data Analysis Program for Research and Innovation in Healthcare in Catalonia held by the Health Quality and Assessment Agency (AQuAS, Catalonia), Carmen Marina Meseguer Barros (Pharmaceutical Information Systems Area, Madrid Health Service), Javier Gorricho Mendívil (Planning, Evaluation and Management Service, Pamplona) and Begoña San Jose Ruiz (Hospital Pharmacy Department, Hospital de Cruces, Bilbao).\n\n\nReferences\n\nBourke L, Kirkbride P, Hooper R, et al.: Endocrine therapy in prostate cancer: time for reappraisal of risks, benefits and cost-effectiveness? Br. J. Cancer. 2013; 108(1): 9–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrossmann M, Zajac JD: Hematological changes during androgen deprivation therapy. Asian J. Androl. 2012; 14(2): 187–192. 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PubMed Abstract \| Publisher Full Text\n\nCherrier MM, Higano CS: Impact of androgen deprivation therapy on mood, cognition, and risk for AD. Urol. Oncol. 2020; 38(2): 53–61. PubMed Abstract \| Publisher Full Text\n\nTsai HT, Pfeiffer RM, Philips GK, et al.: Risks of Serious Toxicities from Intermittent versus Continuous Androgen Deprivation Therapy for Advanced Prostate Cancer: A Population Based Study. J. Urol. 2017; 197(5): 1251–1257. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHigano CS: New developments in the treatment of castration-resistant prostate cancer. J. Natl. Compr. Cancer Netw. 2014; 12(5 Suppl): 773–776. Publisher Full Text\n\nLiede A, Hallett DC, Hope K, et al.: Editorial on an international survey of androgen deprivation therapy (ADT) for non-metastatic prostate cancer in 19 countries. Ann Oncol. 2016 [cited 2021 Jan 31]; 1: e000040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYokomizo A, Koga H, Ito K, et al.: Patient-reported outcomes following neoadjuvant endocrine therapy, external beam radiation, and adjuvant continuous/intermittent endocrine therapy for locally advanced prostate cancer: A randomized phase III trial. Cancer Med. 2021 May [cited 2021 May 3]; 10: 3240–3248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReal Decreto-ley 16/2012, de 20 de abril, de medidas urgentes para garantizar la sostenibilidad del Sistema Nacional de Salud y mejorar la calidad y seguridad de sus prestaciones: Boletín Oficial del Estado (BOE) 2012. Accessed November 11, 2020.Reference Source\n\nFarmaindustria: Boletín de Coyuntura del Mercado del Medicamento en España. No 113. Accessed September 2, 2020.Reference Source\n\nAcezat OJ: «Impacta el uso de las TICS en el gasto farmacéutico? Incorporación de la receta electrónica» [Impact of the use of ICT on pharmacy costs. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "97868", "date": "06 Dec 2021", "name": "Helena Colom Codina", "expertise": [ "Reviewer Expertise My expertise area is biopharmaceutics pharmacokynetic-pharmacodynamic data analysis and 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\nThis is a very interesting study to estimate the prevalence of IAD in four Spanish autonomous communities based on electronic dispensation information from 2011 to 2016. The paper is well written; clear and appropriate in it's methods and results. A very high number of data have been recorded so that very consistent results have been obtained.\n\nTo complete the results I would suggest to perform statistical comparisons of the prevalence values between communities and between the years of follow-up. The study has fulfilled the aims, i.e. to estimate the prevalence values of IAD in different Spanish communities from 2011 to 2016; however, I would have appreciated some discussion about the reasons why the prevalence of IAD in Spain is low.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8092", "date": "28 Apr 2022", "name": "José Antonio Cordero Rigol", "role": "Author Response", "response": "Thanks Dr. Colom for your comments and support. As for your suggestion \"To complete the results I would suggest to perform statistical comparisons of the prevalence values between communities and between the years of follow-up.\" Agree. The statistical comparison will be added to the version 2 but very significant differences (p<0.001) among communities and years of follow-up will be expected due to the big size of the sample analysed. We therefore concentrated on comparing the magnitude of the differences summarised in table 2. With respect to your comment that \"I would have appreciated some discussion about the reasons why the prevalence of IAD in Spain is low\". Agree. This result is quite surprising and we only have conjectures to explain that e.g., Liede at al. reported important differences in IAD use among European countries due to \"clinicians devise individualised treatment courses of optimal length based on patient characteristics while accounting for associated risks and benefits of ADT. This individualised clinical approach is represented in the variation of survey responses as it can become difficult to compartmentalise patients when deciding on treatment strategies.\" The fact that in Spain we have 4 physicians/1000 inhabitants may explain some extra healthcare pressure than in France, Italy or UK with >4.5 physicians/1000 inhabitants. In any case, the data are fairly consistent when it comes to demonstrating this weak use of IAD therapy in Spain." } ] }, { "id": "100495", "date": "13 Jan 2022", "name": "Philipp Dahm", "expertise": [ "Reviewer Expertise evidence-based medicine", "clinical practice guidelines", "prostate cancer", "urology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract, Background: I don't think the first sentence makes logical sense (\"although IADT was introduced...recent data suggest that those patients could be overtreated\"). I think what you are trying to say is that it is being underutilized.\n\nAbstract, Methods: Please clarify the denominator. Here I have the impression it is the entire (male?) population but I'd assume it to be patients with advanced/metastatic PCA.\n\nIn alignment with the study results I'd say that few patients use IADT; secondarily you can state that this likely represents underuse.\n\nIntroduction: I would state that metastatic disease is the main indication (not biochemical recurrence after radical prostatectomy which is also unnecessarily narrow).\n\nReference 20 seems to represent a similar study. You should discuss what this study will add/how it differs?\n\nThe first part of the discussion appears repetitive. If you present the results clearly, you do not need to repeat them here. Please focus on the big picture take-away points.\n\nThe RCTs and SRs you cite are likely overlapping bodies of evidence. Citing one recent high quality SR would suffice. Citing relevant guidelines would be even more compelling.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8093", "date": "28 Apr 2022", "name": "José Antonio Cordero Rigol", "role": "Author Response", "response": "Thanks Dr. Dahm for your comments and support. Let me answer step by step: \"Abstract, Background: I don't think the first sentence makes logical sense (\"although IADT was introduced...recent data suggest that those patients could be overtreated\"). I think what you are trying to say is that it is being underutilized\". Agree. Will be corrected in next version. \"Abstract, Methods: Please clarify the denominator. Here I have the impression it is the entire (male?) population but I'd assume it to be patients with advanced/metastatic PCA.\" It is correct. The prevalence estimation was referred (denominator) to the total number of patients under hormonal treatment. \"In alignment with the study results I'd say that few patients use IADT; secondarily you can state that this likely represents underuse.\" Agree. Will be corrected in next version. \"Introduction: I would state that metastatic disease is the main indication (not biochemical recurrence after radical prostatectomy which is also unnecessarily narrow).\" Agree. Will be corrected in next version. \"Reference 20 seems to represent a similar study. You should discuss what this study will add/how it differs?\" Agree. Basically, extension of the same method of prevalence estimation to other 3 Spanish autonomous communities confirming the same tendency previously observed in the first region (Catalonia). Will be added in next version. \"The first part of the discussion appears repetitive. If you present the results clearly, you do not need to repeat them here. Please focus on the big picture take-away points.\" Agree. The first sentence in the discussion will be removed. \"The RCTs and SRs you cite are likely overlapping bodies of evidence. Citing one recent high quality SR would suffice. Citing relevant guidelines would be even more compelling.\" Agree. The older SRs will be deleted." } ] } ]	1	https://f1000research.com/articles/10-1069
https://f1000research.com/articles/10-283/v1	12 Apr 21	{ "type": "Brief Report", "title": "Temporal development of research publications on SARS-CoV-2 and COVID-19", "authors": [ "Jonghoon Kang", "Erin Kang", "Matthew L. Cowan", "Manuel Orozco", "Erin Kang", "Matthew L. Cowan", "Manuel Orozco" ], "abstract": "The coronavirus disease 2019 (COVID-19) pandemic has affected daily life throughout the world. The scientific community has globally responded to the pandemic with research on an unprecedented scale to help prevent disease spread and terminate the pandemic, resulting in a proliferation of scientific publications. In this article, the temporal trend of research on COVID-19 is analyzed to describe its development and inform a prediction of its future. Four other viruses are included in the analysis as negative or positive controls to illustrate that the concerns of the general public and/or the interest of the scientific community are major driving forces in the development of research. Our analysis predicts that COVID-19 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be major topics of research until at least 2025. We discuss the implications of our analysis for three sectors of community: researchers, epidemiologists, and young students.", "keywords": [ "COVID-19", "SARS-CoV-2", "HCV", "HIV", "Ebola", "Zika", "PubMed", "Scientometrics" ], "content": "Introduction\n\nThe recent outbreak of coronavirus disease 2019 (COVID-19) has imposed an unprecedented and devastating burden on the world,1 including a serious encumbrance to health care systems.2 Collectively the scientific community has responded to the pandemic by researching the spread of the disease and its causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in order to understand and terminate the pandemic. These efforts have resulted in a vast amount of publications. We believe it would be worthwhile to analyze the trend of the publications in order to predict the future of research in this area.\n\nWe have previously demonstrated that the number of publications may be a reliable quantitative measure of the magnitude of research activity of a biological or biomedical science.3 In conjunction with regression analysis, the method of assessing research activity of a biological or biomedical discipline based on the number of publications in the field has been found to be effective in the prognostication of the future of biomedical fields by extrapolation of the best fit equation.4 The method has successfully been applied to various fields such as food sciences,5 epigenetics,6 metabolomics,7 and environmental sciences.8\n\nIn this paper, we apply the method mentioned above to COVID-19 research to quantitatively describe the temporal development of the research and predict its future. We also include four other viruses in the study; hepatitis C virus (HCV) and HIV as negative controls without any apparent outbreaks in the period from January to November, 2020, and Ebola virus disease (EVD) and Zika virus (ZIKV) as positive controls of epidemiological outbreak during the period of examination from January 2014 to November 2020.\n\n\nMethods\n\nTo quantitatively investigate the trend of research related to the five viruses (SARS-CoV-2, HCV, HIV, EVD, and ZIKV), we searched the PubMed database on December 23, 2020. Our search strategy was as follows for the different viruses: (The superscripts a and b in the search phrases represent month and year, respectively.)\n\nSARS-CoV-2: (((COVID [Title/Abstract]) OR (COVID-19[Title/Abstract])) OR (SARS-CoV-2[Title/Abstract])) AND ((“2020/Ma”[Date - Publication]: “2020/Ma”[Date - Publication]))\n\nHCV: (((HCV [Title/Abstract]) OR (“hepatitis C virus”[Title/Abstract])) AND (virus [Text Word])) AND ((“2020/Ma”[Date - Publication]: “2020/Ma”[Date - Publication]))\n\nHIV: (((HIV [Title/Abstract]) OR (“human immunodeficiency virus”[Title/Abstract])) AND (virus [Text Word])) AND ((“2020/Ma”[Date - Publication]: “2020/Ma”[Date - Publication]))\n\nEbola: ((Ebola [Title/Abstract]) AND (virus [Text Word])) AND ((“Yb/Ma”[Date - Publication]: “Yb/Ma”[Date - Publication]))\n\nZika: ((ZIKA [Title/Abstract]) AND (virus [Text Word])) AND ((“Yb/Ma”[Date - Publication]: “Yb/Ma”[Date - Publication]))\n\nThe number of publications on each virus was manually recorded on a monthly basis for eleven months for SARS-CoV-2, HCV, and HIV from January to November 2020, and for eighty three months for EVD and ZIKV from January 2014 to November 2020 for further investigation of data. Subsequent nonlinear regression analysis of the PubMed search results was conducted to obtain equation of best fit using SigmaPlot (version 11; Systat Software, Inc., San Jose, CA).\n\n\nResults\n\nWe retrieved monthly publication numbers of the five viruses from the Pubmed database, and obtained the best fitting equation for each virus. Our results are summarized in Figure 1 and Table 1. We identified that temporal dynamics of publications related to the five viruses exhibit four characteristics.\n\nThe solid line in each graph represents the best fit. The corresponding year in the panel B is presented above the x-axis. SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.\n\nFirst, a sigmoidal equation (Equation 1) was found to be the best quantitative description of the publication trend of COVID-19 research:\n\nThe value of each parameter is listed in Table 1. The mathematical meaning of each parameter can be found in our previous publication.4 In brief, the parameter “a” represents an asymptotic maximum value of the function, “b” is related to the shape of the function, and “c” is the year when the value of the function is half of the asymptotic maximum value.4 The sigmoidal kinetics observed in the research trend of COVID-19 (Figure 1) is congruent with other areas of research such as bioinformatics, epigenetics, food sciences, and environmental sciences.4–7\n\nSecond, there was no significant correlation between the temporal point and the number of research publications on HCV and HIV during the time period examined from January to Novmber 2020 (p = 0.240 for HCV, and p = 0.367 for HIV) (Figure 1). This can be attributed to the absence of any significant outbreaks of HCV or HIV during the time period; while these viruses are important in a biomedical sense,10,11 those viruses have likely been endemic.12,13\n\nThird, two examples of outbreaks in the decade of 2010, EVD14 and ZIKV,15 exhibit biphasic kinetics in the publication trend (Figure 1). The phase of sharp increase in number of publications, which overlaps with the time of each outbreak, also follows sigmoidal kinetics (Equation 1 and Table 1) as does COVID-19. The second phase, a decreasing phase, shows a slow and gradual decline that can be described by an exponential decay function (Equation 2):\n\nFourth, the exponential nature of the decay kinetics may be valuable for the prediction of the future of COVID-19 research. In the case of EVD, the publication number started to decrease, when x = 11 (Figure 1), where the publication number is 123 (see underlying data9) corresponding to 82% of the asymptotic maximum value of 150 (Table 1). Zika research started to decrease, when x = 33 (Figure 1), where the publication number is 222 (see underlying data9) corresponding to 101% of its asymptotic maximum value of 219 (Table 1). As of June, 2020, COVID-19 research reached 95% of its asymptotic maximum value of 12900 (Figure 1): 12288/12900 = 0.95 (underlying data9 and Table 1). The quantitative comparison between SARS-CoV-2 and the two viruses suggests that the case of ZIKV is a more appropriate model for the prediction of COVID-19 research. Despite the apparent similarity of the research trend between SARS-CoV-2 and ZIKV, one should note that there is a substantial difference in the asymptotic maximum value (a in Equation 1) between these two areas of research: SARS-CoV-2 has an almost 60 times (≅ 12900/220) larger value of a than ZIKV (Table 1).\n\n\nDiscussion\n\nThe results of our research have implications for three sectors of the global community. One is for the scientific community in that research on COVID-19 is predicted to be active for a long time, even after commencing a downward trend. According to our mathematical model of the research on ZIKV, it will take COVID-19 research approximately 5 years (65.8 months) to reach half of its maximum value: f2(98.8) = f1(33)/2 and 98.8 – 33 = 65.8. While it is not certain when the publications on COVID-19 will start to decline, we expect that it will remain a major topic of research until at least 2025. This prediction may serve as a guide in planning research on COVID-19. The second implication of our results is for researchers in epidemiology as the method introduced in this paper can be easily applied to other epidemics and pandemics. The third implication is for young students. Our analysis of the ongoing research on COVID-19 should show them that science is a valuable way of contributing to humanity by providing solutions for public concerns such as COVID-19.\n\n\nData availability\n\nFigshare: Number of PubMed-indexed articles related to five viruses; SARS-CoV-2, HCV, HIV, Ebola, and Zika. https://doi.org/10.6084/m9.figshare.12958361.v39\n\nThis project contains the following underlying data:\n\n- covid_figshare_kang.csv (spreadsheet of the number of research publications found relating to five viruses).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe would like to thank Kathi Canese, a Program Manager for the support department of PubMed, for the valuable advice in the search of PubMed.\n\n\nReferences\n\nEtienne CF, Fitzgerald J, Almeida G, et al.: COVID-19: transformative actions for more equitable, resilient, sustainable societies and health systems in the Americas. BMJ Glob Health. 2020 Aug; 5(8): e003509. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHsiang W, Forman H, Jain S, et al.: COVID-19 testing capabilities at urgent care centers in states with greatest disease burden [version 2; peer review: 2 approved]. F1000Res. 2020; 9: 328. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang J, Purnell CB: Implications for undergraduate education of two interdisciplinary biological sciences: biochemistry and biophysics. CBE Life Sci Educ. 2011 Summer; 10(2): 111–112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang J, Park S, Venkat A, et al.: Quantitative Analysis of the Trends Exhibited by the Three Interdisciplinary Biological Sciences: Biophysics, Bioinformatics, and Systems Biology. J Microbiol Biol Educ. 2015 Dec 1; 16(2): 198–202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang J, Clifton EC: Quantitative Analysis of Food Science Trends. J Food Sci. 2018 Oct; 83(10): 2405–2406. PubMed Abstract \| Publisher Full Text\n\nKang J, Daines JR, Warren AN, et al.: Epigenetics for the 21st-Century Biology Student. J Microbiol Biol Educ. 2019 Dec 18; 20(3): 20.3.56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang J, Song S, Cowan ML: Comment on Metabolomics for a Millenniums-Old Crop: Tea Plant (Camellia sinensis). J Agric Food Chem. 2020 Jan 15; 68(2): 697–698. PubMed Abstract \| Publisher Full Text\n\nKang J, Kang AM: Trend of the research on rare earth elements in environmental science. Environ Sci Pollut Res Int. 2020 May; 27(13): 14318–14321. PubMed Abstract \| Publisher Full Text\n\nKang J, Kang E, Cowan ML: Number of PubMed-indexed articles related to five viruses; SARS-CoV-2, HCV, HIV, Ebola, and Zika. Figshare, V3. 2020. Publisher Full Text\n\nMukhtar NA, Ness EM, Jhaveri M, et al.: Epidemiologic features of a large hepatitis C cohort evaluated in a major health system in the western United States. Ann Hepatol. 2019 Mar-Apr; 18(2): 360–365. PubMed Abstract \| Publisher Full Text\n\nBuchacz K, Armon C, Palella FJ Jr, et al.: HIV Outpatient Study (HOPS) Investigators. The HIV Outpatient Study-25 Years of HIV Patient Care and Epidemiologic Research. Open Forum Infect Dis. 2020 Apr 11; 7(5): ofaa123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlter MJ: Epidemiology of hepatitis C virus infection. World J Gastroenterol. 2007 May 7; 13(17): 2436–41. Publisher Full Text\n\nAssefa Y, Gilks CF: Ending the epidemic of HIV/AIDS by 2030: Will there be an endgame to HIV, or an endemic HIV requiring an integrated health systems response in many countries? Int J Infect Dis. 2020 Nov; 100: 273–277. PubMed Abstract \| Publisher Full Text\n\nKamorudeen RT, Adedokun KA, Olarinmoye AO: Ebola outbreak in West Africa, 2014-2016: Epidemic timeline, differential diagnoses, determining factors, and lessons for future response. J Infect Public Health. 2020 Jul; 13(7): 956–962. PubMed Abstract \| Publisher Full Text\n\nMadewell ZJ, López MR, Espinosa-Bode A, et al.: Inverse association between dengue, chikungunya, and Zika virus infection and indicators of household air pollution in Santa Rosa, Guatemala: A case-control study, 2011-2018. PLoS One. 2020 Jun 19; 15(6): e0234399. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "97764", "date": "27 Oct 2021", "name": "Ludovico Abenavoli", "expertise": [ "Reviewer Expertise Epidemiology of COVID-19", "COVID-19 clinical aspects", "COVID-19 research trends" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments:\nI read with interest this article. It provides data on the scientific production during the pandemic, with regards to 2020. The background is solid, the results have been discussed, and the conclusion supported the data.\nMinor point:\nI suggest to report the data stratified for original articles, review, brief report and letter to the editor related to COVID-19 topic.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8114", "date": "25 Apr 2022", "name": "Jonghoon Kang", "role": "Author Response", "response": "Dear Dr. Abenavoli We appreciate your time and effort for reviewing our paper. In the revised manuscript, we have included the data you suggested. We believe the added data will enhance the quality of our work and thank you for the suggestions." } ] }, { "id": "100694", "date": "01 Dec 2021", "name": "Mahmoud Nassar", "expertise": [ "Reviewer Expertise Clinical research", "COVID-19", "Cardiovascular Diabetes", "and Organ transplantation." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors Thank you so much for your great effort. This is an interesting topic. Here are some opportunities for improvement:\nPlease justify the selection of the four viruses compared to COVID-19. I propose to make a comparison table with similarities and differences.\n\nPlease comment on why this article did not include other databases like Medline or Embase.\n\nPlease mention other factors affecting that affect the number of publications (direct and indirect).\n\nPlease comment on changes to the journals' publication policies, time, APC, indexing, etc.\n\nPlease comment on the global quarantine, governmental closure, and the negative impact on developed and developing countries' economic and social aspects.\n\nI suggest commenting on the trend or theme1 of research development of the diagnosis, prevention, COVID-19 complication, treatment, vaccination, COVID-19 vaccine-induced complications[ref2],3,4, COVID-19 variants, COVID-19 vaccination booster dose.\n\nThere are many publications as case reports, case series, descriptive, case-control, RCT, meta-analysis, and umbrella review. For each phase and with other comorbidities5,6,[ref7],8. NB: All citations are my own papers and for guidance. You do not need to cite them.\n\nPlease create a limitation section and mention these factors as a study limitation. At least try to mention these factors as limitations in the study.\n\nI suggest creating a diagram to demonstrate the evolution of COVID-19 research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8115", "date": "25 Apr 2022", "name": "Jonghoon Kang", "role": "Author Response", "response": "Dear Dr Nassar, Than you for your thoughtful comments (in plain fonts) on our paper. Here, we respond to your comments (in bold font). Please justify the selection of the four viruses compared to COVID-19. I propose to make a comparison table with similarities and differences. Justification for the selection of the four viruses is comparison of COVID-19 to a negative control group (HCV and HIV) without any recent breaks and to a positive control group (EVD and ZIKV) with recent breaks. We selected those viruses based on our familiarity with them. We have included a comparison table (Table 1) in the revision. Please comment on why this article did not include other databases like Medline or Embase. We have included our justification for the selection of PubMed in our study in Discussion. Please mention other factors affecting that affect the number of publications (direct and indirect). Surely public interests are one of the factors affecting the number of publications as noted in our paper. However, we are not aware of any other factors for sure, even though there must be various factors. For example, we expect that the number of researchers and number of journals in a particular field will affect the number of publications in the field. It will be of great interest recognizing those factors in future research. Please comment on changes to the journals' publication policies, time, APC, indexing, etc. Those factors must play a role in the trend of publications. However, we do not have any explanation for the point. Please comment on the global quarantine, governmental closure, and the negative impact on developed and developing countries' economic and social aspects. Thank you for the additional guidance. However, the nature of this paper is to forecast the number of publications in the future by analyzing the trends and not related to the global quarantine, governmental closure, and the negative impact on developed and developing countries' economic and social aspects. That is not to say these areas are any less important but we are chiefly concerned with the mathematical nature of the trends we saw from our gathering of publication counts on PubMed. I suggest commenting on the trend or theme1 of research development of the diagnosis, prevention, COVID-19 complication, treatment, vaccination, COVID-19 vaccine-induced complications[ref2],3,4, COVID-19 variants, COVID-19 vaccination booster dose. There are many publications as case reports, case series, descriptive, case-control, RCT, meta-analysis, and umbrella review. For each phase and with other comorbidities5,6,[ref7],8. NB: All citations are my own papers and for guidance. You do not need to cite them. Please create a limitation section and mention these factors as a study limitation. At least try to mention these factors as limitations in the study. I suggest creating a diagram to demonstrate the evolution of COVID-19 research. References 1. Nassar M, Nso N, Alfishawy M, Novikov A, et al.: Current systematic reviews and meta-analyses of COVID-19.World J Virol. 2021; 10 (4): 182-208 PubMed Abstract \| Publisher Full Text 2. Nassar M, Chung H, Dhayaparan Y, Nyein A, et al.: COVID-19 vaccine induced rhabdomyolysis: Case report with literature review.Diabetes Metab Syndr. 15 (4): 102170 PubMed Abstract \| Publisher Full Text 3. Nassar M, Nso N, Gonzalez C, Lakhdar S, et al.: COVID-19 vaccine-induced myocarditis: Case report with literature review.Diabetes Metab Syndr. 15 (5): 102205 PubMed Abstract \| Publisher Full Text 4. Kong J, Cuevas-Castillo F, Nassar M, Lei C, et al.: Bullous drug eruption after second dose of mRNA-1273 (Moderna) COVID-19 vaccine: Case report. Journal of Infection and Public Health. 2021; 14 (10): 1392-1394 Publisher Full Text 5. Alfishawy M, Elbendary A, Mohamed M, Nassar M: COVID-19 Mortality in Transplant Recipients.Int J Organ Transplant Med. 2020; 11 (4): 145-162 PubMed Abstract 6. Alfishawy M, Elbendary A, Younes A, Negm A, et al.: Diabetes mellitus and Coronavirus Disease (Covid-19) Associated Mucormycosis (CAM): A wake-up call from Egypt.Diabetes Metab Syndr. 15 (5): 102195 PubMed Abstract \| Publisher Full Text 7. Alfishawy M, Nso N, Nassar M, Ariyaratnam J, et al.: Liver transplantation during global COVID-19 pandemic.World J Clin Cases. 2021; 9 (23): 6608-6623 PubMed Abstract \| Publisher Full Text 8. Nassar M, Nso N, Ariyaratnam J, Sandhu J, et al.: Coronavirus disease 2019 and renal transplantation.World J Clin Cases. 2021; 9 (27): 7986-7997 PubMed Abstract \| Publisher Full Text Thank you for the additional guidance. However, given the nature of this publication is a brief report we feel it would not be necessary to overburden the scope of the paper itself with this addition of the perceived diagram. Brief Reports are small, often preliminary studies, descriptions of unexpected and perhaps unexplained observations or lab protocols that can be described in a short report with a few illustrations (figures/tables), or even a single figure. https://f1000research.com/for-authors/article-guidelines/brief-report. While qualitative investigation on the evolution of COVID-19 research is beyond the scope of our research, your suggestion of including a limitation section is deemed valuable for our research, and we discussed limitation of our approach in the revised edition. We also included two of the references you suggested in our revision. Thank you for suggesting the references." } ] } ]	1	https://f1000research.com/articles/10-283
https://f1000research.com/articles/11-446/v1	21 Apr 22	{ "type": "Research Article", "title": "Predictive scoring system for risk of complications in pediatric dengue infection", "authors": [ "Monisha Bhaskar", "Soundarya Mahalingam", "Harish M M", "Basavaprabhu Achappa", "Monisha Bhaskar", "Harish M M", "Basavaprabhu Achappa" ], "abstract": "Background: Dengue infection has been a worrisome cause of mortality and morbidity in children. Though numerous scoring systems have been developed, they are in the adult population or are too complicated for use in children. Pediatric dengue infection has a wide spectrum from a mild illness to severe complications and an unpredictable course. Hence the need for a predictive scoring system where the possibility of complications can be identified which can contribute to reduction in mortality and morbidity of dengue by prompt referrals and anticipatory management. Methods: Prospective case cohort study of children with confirmed dengue fever. Results: 303 children were included and divided into two groups – the dengue fever group and the complicated dengue group based on the WHO clinical classification. The clinical and laboratory parameters were analysed individually, cut offs identified by ROC curves and compared for significance between the two groups. The parameters that emerged were hypotension, PCV ≥ 42%, platelet count ≤ 75000 cells/cumm, WBC ≥ 7000 cells/cumm, and ALT ≥ 70U/L. Using the adjusted odd’s Ratio, and coefficient, individual predictive scores were tabulated ranging from 0 to 3, with a total score of 0 to 7. A cut-off score of 2 was then identified based upon the sensitivity (84.13%) and specificity (72.50%) as the ideal score to predict complicated dengue. Internal validation of the score was done where the area under the curve for predicting complicated dengue was 0.86 (95% CI 0.8-0.92) with a P value of <0.001. Conclusion: Our dengue predictive scoring system has been developed using five indicators, with a score of two and above, out of seven, suggesting increased risk of developing complications. This has been validated internally and can be used to predict complicated dengue among children.", "keywords": [ "Pediatric Dengue", "Complications", "Predictive scoring" ], "content": "Introduction\n\nDengue fever is a serious public health issue that has resulted in significant mortality and morbidity in both children and adults. The environmental conditions in India, especially South India, play a vital role in the transmission of the infection with multiplication of the vectors, hence the monsoon seasons from June to September here see a surge in the dengue cases. Natural reservoirs of clean water like lakes, back waters as well as artificial water collections like the stagnant water in paddy fields, coconut shells collecting rainwater, village ponds, etc provide fertile grounds for the breeding of the Aedes mosquito.\n\nDengue infection has a myriad of clinical presentations which range from simple viral fevers with myalgia to complicated dengue with Dengue Shock Syndrome (DSS), Dengue Haemorrhagic Fever (DHF), Multiorgan dysfunction and Death. Each year the morbidity of dengue virus has been increasing with statistics from the NVBDCP 2013 (National Vector Borne Disease Control Program) showing that Kerala reported the highest prevalence of dengue (7911) with Karnataka becoming one of the endemic states with an incidence of 6048 cases out of 74454 cases all over India.1 This incidence has only been increasing since then to 1,11,880 cases with 227 deaths in 2016.2 This increasing incidence has reached 188401 cases with 325 deaths in 2017 and in 2018 – 101192 cases with 172 deaths, 2019 – 157315 cases with 166 deaths, 2020 – 39419 cases with 56 deaths all over India.3,4\n\nDengue fever takes a severe toll among high-risk populations like children and elderly and is linked to an increased risk of morbidity, complications, and death. Studies on dengue fever in children have also reported worse outcomes both with respect to complications as well as death in paediatric dengue with an increased incidence of complications among adolescents.4 The clinical features of dengue infection can extend from mild uncomplicated dengue infection with fever, rash, myalgia and arthralgia to gradually worsening complicated dengue. Severe complicated dengue infection implies intractable hypotension and shock, massive bleeding, capillary leak with accumulation of fluid in the third spaces like pleura, peritoneum and pericardium, multiorgan dysfunction due to inflammation causing liver dysfunction, acute kidney injury, cardiac failure following myocardial dysfunction, encephalitis and other neurological complications and eventual death. These complications are worse when the dengue infection affects the same child for the second time implying the extensive activation of the immune system when the infection occurs for the second time causing severe complications.5 In line with this, adolescents have a worse course of the disease as probably they have been exposed to the infection in a mild form earlier in childhood.6 The investigations done to identify dengue infection are the NS1 antigen which is done in the first few days of the illness and the IgM Dengue test which identifies the antibody response to dengue infection after the first 5 days of infection. Other tests are for organ dysfunction which is known to occur with complicated dengue infection which are done regularly as biochemical deterioration precedes clinical deterioration. Treatment in dengue infection is only supportive as there is no cure. It involves administration of iv fluids, vasopressors to increase the blood pressure and other care like mechanical ventilation, dialysis, etc based on the complications that develop. The more the complications that develop, the poorer is the outcome of the disease.7 In children especially the management of fluids and the critical care are a challenge with the rapid progression of the disease as well as the delicate physiological balance in children. In view of this, early identification and watchful expectancy of complications are the only ways to anticipate worsening clinical condition in paediatric dengue.\n\nIt is with these considerations that the present study was conducted to analyse the parameters of pediatric dengue infection and develop a predictive scoring system based upon the laboratory and clinical parameters for the prediction of risks of complications in paediatric dengue.\n\n\nMethods\n\nThis was a prospective case cohort study.\n\nAll children aged 0 – 18 years with dengue infection.\n\nFive years, from Jan 2016 to December 2020.\n\nHospital based study in the two tertiary medical college hospitals associated with our medical college.\n\nSample size: 270 (Calculated based on a previous study by Pongpan et al8)\n\nn = (1.96)2 (0.51) (1-0.51) p = proportion of interest (0.06)2, q = 1-p and d = relative precision.\n\nIn order to cover the time period, all cases in this time interval were included with a minimum of 270. Hence all 303 cases were considered.\n\nAll cases of confirmed dengue cases with NS1 antigen positivity confirmed by IgM dengue ELISA positivity after 5 days.\n\nAll cases who had an acute febrile illness with clinical features of dengue fever but negative diagnostic tests.\n\nThe study was submitted to the ethics committee of the institution (Institutional Ethics Committee, Kasturba Medical College, Mangalore. Ref No ECR/541/Inst/KA/2014/RR – 17) and clearance was obtained. The IEC certification was obtained before initiation of the study (IEC KMCMLR 10-16/497). Informed and written consent of all parents of the children and assent for adolescents was obtained before their inclusion into the study.\n\nDemographic details, mode of presentation, clinical findings, laboratory parameters, treatment parameters and outcomes of all the included children were recorded and taken up for further analysis. The children were classified into two groups – The dengue fever group and the complicated dengue group based upon the WHO classification.\n\nThe following steps were used:\n\n1. Data were entered into Microsoft Excel, and statistical analysis was performed using SPSS 17.0 software. Qualitative variables were reported as frequency and percentages. Depending on the distribution of data, quantitative variables were reported as mean (standard deviation) or median (range).\n\n2. The variables included in the study under demographic details, clinical features, examination findings with laboratory parameters were compared between the 2 groups i.e., Dengue fever group and complicated dengue group using Chi-square test (Bivariate analysis). Individual ROC curves were generated for the variables and cut off values were identified.\n\n3. All the parameters were then compared for statistical significance between the groups using a multivariable regression model by binary logistic regression. This is was done in two steps in order to avoid loss of significance among parameters, hence the first step of the binary logistics was done with a p value less than 0.2. Among the parameters that emerged the next step was to identify statistical significance with the variables that showed statistical significance where p < 0.05. The effect measure was determined using an adjusted odds ratio with a 95% confidence interval.\n\n4. The variables which were significant in the logistic regression model (p<0.05) were used to develop a score. The smallest coefficient from the model was given a score of 1 and scores for other significant variables were derived hence by dividing them by the smallest coefficient and the scores were rounded off to nearest 0.5.\n\n5. The scores were applied for the variables and the total score was calculated by summing the scores. The area under the curve (AUC) with a 95% confidence interval was computed using a ROC curve. When an AUC>0.5, then the test applied is considered as a useful test. The Youden Index (Sensitivity+specificity-1) was computed and a cut-off for the score was chosen. Sensitivity, specificity, and predictive values were calculated with 95% CI.\n\n\nResults\n\nA total of 303 children with pediatric dengue infection were analysed. They were divided into two groups - dengue fever group (240) and complicated dengue group (63) based upon the WHO criteria. A total of two demographic, ten clinical indicators and eight laboratory indicators were computed for each patient. ROC was generated for these individual parameters that were tabulated and cut off points were identified as shown in Table 1.\n\nFollowing this, Multivariate regression analysis was done by binary logistics (Table 2). As an effect metric, the adjusted odds ratio with a 95% confidence interval was determined. Clinical & laboratory characteristics with substantial prediction potential for dengue severity were identified in the multivariable analysis as Hypotension as the strongest predictor with an Odd’s ratio of 20.11, Packed cell volume (>42%), Platelet count (<75,000/cumm), WBC count (>7000/cumm) and ALT (>70 IU/ml). Ferritin was not included in multivariable regression analysis because of missing values.\n\nAmong the parameters that emerged statistically significant, coefficients were calculated with the adjusted Odd’s Ratio and then analysed to develop a score (Table 3). Significant parameter coefficients were converted into item scores by dividing each coefficient by the model's smallest coefficient (0.94) and rounding up or down to the nearest 0.5 integers. Individual predictive scores ranged from 0 to 3, with a total score of 0 to 7 (Table 3). To check the usefulness of the test and score, area under the curve was plotted against sensitivity and 1 – specificity. The area under the curve was calculated as 0.86 with a 95 percent confidence interval (CI) of 0.8-0.92, showing statistical significance with a p value of < 0.001, hence making this scoring a useful predictive test (Figure 1).\n\nThe sensitivity and specificity of the score was calculated at different cut off points and their individual Youden index (Sensitivty+specificity-1) was calculated (Table 4), to identify the ideal score where the sensitivity and specificity would be appropriate. The highest index was found at a score of two (out of seven) with a sensitivity of 84% and a specificity of 72.5%. This was the score chosen so as to not overlook the complications early at admission in pediatric dengue which can progress fast into complications if undetected early.\n\nOur score was then applied to our data and the patients were split into two groups: Scores <2 (dengue fever) and scores ≥2 (complicated dengue) as shown in Figure 2. The scores <2 predicted dengue fever correctly in 174 out of 240 patients and the scores ≥2 predicted complicated dengue correctly in 53 out of 63 patients. The sensitivity of our dengue predictive scoring method is 84.1%, the specificity is 72.5%, the positive predictive value is 44.5 % and the negative predictive value is 94.5%. When the mortality statistics were analysed, there were 4 deaths in the cohort. When the score was applied, all the four deaths had a score of >2.\n\n\nDiscussion\n\nThe complications and unpredictable outcomes of dengue infection in a child necessitates the requirement for early identification and anticipatory management. As we reviewed literature, few scoring systems were available for the prediction of complications in dengue. Most of these scoring systems were in adults and could not be applied to children.\n\nGeneralised scoring systems like the Pediatric Risk of Mortality III (PRISM III) and Pediatric Logistic Organ Dysfunction (PELOD) scores showed good discrimination in predicting mortality and complications when children with dengue are admitted to the PICU.9 However, in such generalised systems, the outcomes of the PICU admission are validated better and these scores require numerous investigations including arterial blood gas analysis. They are not specific to dengue, nor help in early and clinical prediction of development of complications. Dengue infections were also classified as DF, DHF grade I, II and III, with decision tree algorithms which were also used to determine whether or not hospitalisation was required.10,11 These algorithms were more applicable in adults and not specific for pediatric dengue. The algorithm by Lee et al11 shows data mining of the dengue infection by decision tree and this uses numerous calculations and has shown application in engineering mathematics as it is difficult to apply clinically. Scores by Phakhounthong K et al12 showed the development of the score using classification tree analysis, here the algorithm uses the Glasgow Coma Score, hematocrit, creatinine, platelet count and urine protein. This score relies on the clinical judgement of the pediatric GCS which is difficult in smaller children as well as gives the value of S.creatinine which is abnormal as 0.95 mg/dl. These lab parameters are not available immediately and the golden hour at admission gets missed. Hence in our score, we have focussed upon clinical parameters like the vital signs at admission as well as the lab parameters which are easily available even in smaller centres and laboratories. Another scoring system was developed by Huang13 which was to predict mortality in dengue fever but was in adults and as age with comorbidities were significant parameters, this cannot be used in children. All these scoring systems/algorithms11,13,14 had limitations, for example, the study population didn’t include pediatric cases, no specific clinical or laboratory parameters were included related to dengue infection nor such scoring systems were developed in our geographical setting.\n\nThe closest scoring system that we could find in literature was given by Pongpan et al8 here in 198 children in Thailand, they developed a score with six parameters (Age, hepatomegaly, hematocrit, systolic BP, WBC count and platelet counts) with a total score of 18, however in this scoring, clinical judgement of hepatomegaly had the highest weightage of 8.5/18 and this judgement decides the score of the child. Such clinical parameters are not easily applied in a peripheral setting where pediatric examination is difficult by basic medical officers thus making both overdiagnosis and underdiagnosis a problem. Another significant aspect of the score by Pongpan et al was that Systolic BP with a cut off of 90 mmHg was selected. The drawback of this is that, in smaller children where the 50th centile of BP is around 90 mmHg, this score will over diagnose positive markers of complications even if the BP is normal as for age. In our score Hypotension has been given a priority where the detection of hypotension was by taking age dependent cut offs which is easily done even in peripheral centres.\n\nHence to summarise, in consideration of all the above, we created a dengue severity scoring system that uses laboratory indicators and clinical signs to predict the risk of complications and fatality in pediatric dengue infection using binary logistic regression. The final scoring system for predicting complicated dengue comprised five components: PCV, Platelet count, ALT, Highest WBC and Hypotension. All these parameters are easily identified in any setting, tertiary or peripheral. The applicability of the score is easier as well as more relevant. Hypotension was defined by age dependent charts and was found to be the most important predictor of complicated dengue. The laboratory predictors included in our scoring system are simple to perform in outpatient hospital labs, and the scoring method is simple to apply in daily clinical practice. While applying the score, patients with a severity score of zero or one can be treated in the outpatient setting. A patient with a severity score of two may require hospitalisation for close observation. Admission to an intensive care unit, early resuscitation, empiric antibiotics, and other supportive measures are required if a patient's severity score is three or higher.\n\nOne of our study's limitations is the small sample size of the study population as larger samples would give better opportunities to internally validate our scoring system.\n\n\nConclusions\n\nThe significant predictors of complicated dengue were Hypotension, PCV ≥ 42%, Platelet count ≤ 75000 cells/cumm, WBC ≥ 7000 cells/cumm, and ALT ≥ 70U/L. Individual predictive scores ranged from zero to three, with a total score of zero to seven. Score of ≥ two indicates patients going in for dengue related complications. The sensitivity of our dengue predictive scoring method is 84.1%, the specificity is 72.5%, the positive predictive value is 44.5% and the negative predictive value is 94.5%. The dengue prediction score system includes five clinical and laboratory markers that can be used to predict complicated dengue in paediatric patients. Validation of our predictive scoring system is needed before its application in routine clinical practice.\n\n\nData availability\n\nDryad: Mahalingam, Soundarya; Bhaskar, Monisha; Achappa, Basavaprabhu, MM Harish (2022), Predictive Scoring for Risk of Complications in Pediatric Dengue infection, Dryad, Dataset.\n\nDOI: https://doi.org/10.5061/dryad.wpzgmsbpp\n\nLink for data set:\n\nhttps://datadryad.org/stash/share/znfJ64hSeNFEcrPs-PUpcu8VIDOSkEiPmrskbMtagl8.\n\nThis project contains the following underlying data:\n\n• Data file 1. (Excel spreadsheet of all cases)\n\n• Data file 2. (Readme file for coding of the spreadsheet)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nNational Vector Borne Disease Control Programme, 'Dengue/dengue haemorrhagic fever.'.2013. Reference Source\n\nHindustan times-Ministry of Health and Family Welfare, 'National dengue day: Is dengue increasing in India? Am I at Risk?.2018. Reference Source\n\nNational Centre for Vector Borne Disease Control nvbdcp.org India: National Vector borne disease control program; [updated Oct 2021; cited 2011 Mar 14]. Reference Source\n\nMishra S, Ramanathan R, Agarwalla SK: Clinical Profile of Dengue Fever in Children: A Study from Southern Odisha, India. Scientifica. Vol. 2016: Article ID 6391594: Pg no 1–6. Publisher Full Text PubMed Abstract \|\n\nWichmann O, Hongsiriwon S, Bowonwatanuwong C, et al.: Risk factors and clinical features associated with severe dengue infection in adults and children during the 2001 epidemic in Chonburi, Thailand. Tropical Med. Int. Health. 2004; 9(9): 1022–1029. PubMed Abstract \| Publisher Full Text\n\nSharma Y, Kaur M, Singh S, et al.: Seroprevalence and Trend of Dengue Cases Admitted to a Government Hospital, Delhi – 5-Year Study (2006-2010): A Look into the Age Shift. Int. J. Prev. Med. 2012; 3(8): 537–543. PubMed Abstract\n\nFernando S, Wijewickrama A, Gomes L, et al.: Patterns and causes of liver involvement in acute dengue infection. BMC Infect. Dis. 2016; 16(1): 319–319. PubMed Abstract \| Publisher Full Text\n\nPongpan S, Wisitwong A, Tawichasri C, et al.: Development of dengue infection severity score. ISRN Pediatr. 2013 Nov 12; 845876. Publisher Full Text \| PubMed Abstract \| Free Full Text\n\nIskandar HR, Mulyo D, Agnes P, et al.: Comparison of pediatric logistic organ dysfunction (PELOD) score and pediatric risk of mortality (PRISM) III as a mortality predictor in patients with dengue shock syndrome. Pediatrics. 2008; 121(supplement 2): S129. Publisher Full Text\n\nThitiprayoonwongse D, Suriyapkol P, Soonthornphisaj N: Data mining of dengue infection using decision tree. Latest Advances in Information Science and Applications: The 12th WSEAS International Conference on Applied Computer Science.2012; pp. 154–159.\n\nLee VJ, Lye DC, Sun Y, et al.: Decision tree algorithm in deciding hospitalization for adult patients with dengue haemorrhagic fever in Singapore. Trop. Med. Int. Health. 2009; 14(9): 1154–1159. PubMed Abstract \| Publisher Full Text\n\nPhakhounthong K, Chaovalit P, Jittamala P, et al.: Predicting the severity of dengue fever in children on admission based on clinical features and laboratory indicators: application of classification tree analysis. BMC Pediatr. 2018 Mar 13; 18(1): 109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang CC, Hsu CC, Guo HR, et al.: Dengue fever mortality score: A novel decision rule to predict death from dengue fever. J. Infect. 2017 Dec; 75(6): 532–540. PubMed Abstract \| Publisher Full Text\n\nChang K, Lu PL, Ko WC, et al.: Dengue fever scoring system: new strategy for the early detection of acute dengue virus infection in Taiwan. J. Formos. Med. Assoc. 2009 Nov; 108(11): 879–885. PubMed Abstract \| Publisher Full Text" }	[ { "id": "135434", "date": "04 May 2022", "name": "Sanjay Kumar Sahu", "expertise": [ "Reviewer Expertise Pediatrics infectious disease", "Pediatrics Asthma", "Allergy & Immunology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for giving us the opportunity to review this article. It is an excellent article clearly and accurately presented and cites the current literature. However there are few points that needs clarification:\nRegarding inclusion criteria you have included all confirmed dengue cases with NS1 antigen positivity confirmed by IgM dengue ELISA positivity after 5 days. But what about those cases that are IgM negative after 5 days with previous NS1 positivity?\n\nRegarding the cut-off value of serum creatinine (Table 1) in a sample of children aged 0 to 16 years, a single cut-off value of 0.5mg/dl may not be appropriate for all. This can be adjusted to BSA for uniform comparison.\n\nWe are unable to understand the basis of taking 2 cut-off values for serum ferritin of 1000ng/ml and 1500ng/, as greater than 1000ng/ml was statistically significant in your analysis in Table 1. The 2 cut-off values of serum ferritin can be adjusted to a single value.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "135433", "date": "07 Jul 2022", "name": "Farhan Fazal", "expertise": [ "Reviewer Expertise Infectious disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is on the Development of a predictive scoring system for dengue infection among children so as to detect complications early. Thank you for giving me the opportunity to review this article. In the article, the authors have reviewed cases and developed a score that can applied at the point of care to identify the early development of complications.\nOne of the important aspects of the score is that it relies primarily on clinical parameter of hypotension along with basic laboratory parameters that are available in a peripheral lab as well.\n\nThe statistical methods have been done well and explained in much detail for the ease of understanding. The steps outlined can be made concise in a flowchart as well. The Youden index and the multivariate regression is appropriately done as required for such a predictive scoring development.\n\nA good aspect of this study is that the authors have done an internal validation of their scoring with their study sample and have shown that the scoring is appropriate.\n\nThe discussion has compared many scoring systems that have been developed along with the pros and cons of such scoring systems. The study is done on children and there are few studies that are available for comparison which has been done by the authors. Adult scoring systems have also been detailed and their difficulties have been listed out. Hence this is a relevant scoring system for use in children with dengue.\nSome clarifications required are:\nDetails of the serum ferritin as to why two cut offs of 100mg/dl and 1500mg/dl were used needs to be addressed in the methodology and results\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-446
https://f1000research.com/articles/10-1124/v1	08 Nov 21	{ "type": "Research Article", "title": "Determinants of maternal healthcare service utilisation among Indonesian mothers: A population-based study", "authors": [ "Ridwan Setyo Aji", "Ferry Efendi", "Iqlima Dwi Kurnia", "Santo Imanuel Tonapa", "Chong-Mei Chan", "Ridwan Setyo Aji", "Iqlima Dwi Kurnia", "Santo Imanuel Tonapa", "Chong-Mei Chan" ], "abstract": "Background: In Indonesia, maternal health care services are widely available, aiming to improve health and survival among mothers. However, these services remain underutilised, and its determining factor was unknown. This study sought to identify determinant factors of maternal healthcare services utilisation among Indonesian mothers. Methods: This population-based cross-sectional study leveraged the 2017 Indonesia Demographic and Health Survey data. A total of 12,033 mothers aged from 15 to 49 years who had a live birth in the five years preceding the survey were included in the analysis. Multivariable logistic regressions were used to identify the determinant factors. Results: Approximately 93.44% of the mothers had adequate antenatal care, 83.73% had a delivery at the healthcare facility, and 71.46% received postnatal care. The mother’s age and household wealth index were the typical determinants of all maternal healthcare services. Determinants of antenatal care visits were husband’s occupational status, the number of children, and access to the healthcare facility. Next, factors that drive mothers’ delivery at the healthcare facility were the mother’s education level, husband’s educational level, and residential area. The use of postnatal care was determined by the mother’s occupational status, husband’s educational level, number of children, wealth index, access to the healthcare facility, and residential area. Conclusions: Although there were differences in the determinant factors of three key maternal healthcare services, the mother’s age and household wealth index were the typical determinants of all maternal healthcare services utilisation. Providing a tailored programme aligned with these determinant factors may ensure that mothers can access and adequately utilise maternal healthcare services.", "keywords": [ "antenatal care", "institutional deliveries", "maternal health", "postnatal care", "pregnancy" ], "content": "Introduction\n\nMaternal mortality is a serious global health problem that threatens healthcare systems. In 2017, approximately 810 mothers died every day from preventable causes related to pregnancy and delivery around the globe.1 A vast majority of these mortalities occurred in low and middle-income countries, of which Indonesia is one.2,3 In Southeast Asia, the maternal mortality rate among mothers aged 15-49 reached 16,000 per 295,000 mothers death or 5.4% of overall maternal mortality worldwide.1 In Indonesia, the maternal mortality rate was about 305 deaths per 100,000 live births in 2015. This number was still threefold higher than the Millennium Development Goals (MDGs) target set up previously.4,5 The Indonesian government’s challenge will be more demanding as United Nations (UN) shifted to the new goals. The 2030 Sustainable Development Goals (SDGs) launched by the UN in 2016 was aimed to decrease maternal mortality to 70 deaths per 100,000 live births.4 Thus, reaching SDG’s target will be a considerable challenge for Indonesia.\n\nThe utilisation of maternal healthcare services (MHC) among mothers remains a challenge for the health system across the countries. Adequate utilisation of MHC is crucial to reducing maternal mortality and improve pregnant mothers’ well-being. However, maternal healthcare service utilisation among developing countries was relatively inadequate.6–8 Particularly in Indonesia, it was reported that Antenatal care (ANC) visits in 2017 only reached 70%, which was lower than the government’s target of 76%.9 Mothers in favour of Intra-natal care (INC) at the healthcare facility was about 62%, which was below the targeted rate by the government at 85%.10 Moreover, only 48% of mothers used the Postnatal care (PNC) service, where is still far below the expected target at 80%.11 Since the maternal healthcare service has been underutilised among Indonesian mothers and it is much lower than the government target, hence an understanding of its determinant is deserved further investigation.\n\nStudies across the countries have built knowledge for associated factors with the utilisation of MHC. For example, previous works by Kurniati et al. had identified that MHC utilisation among mothers influenced by education, occupation, age at first delivery, contraceptive use, age at first marriage, participation in household decision-making, and attitudes towards intimate partner violence.12 However, a previous systematic review of studies in the lower and middle-income countries underscores that determinants for maternal healthcare service varied greatly in each population.13 Furthermore, numerous studies reported that determinants in three key components of MHC namely ANC visit, institutionalised delivery, and PNC use were considerably different.9,10,12 Nonetheless, none of the studies carried out previously provide the entire picture about the determinants of these three keys to MHC utilisation. Based on these gaps, a study with representative data generated from the population level is deemed necessary.\n\nInvestigating determinants of MHC utilisation among Indonesian mothers requires nationally representative data because it can control ethnic variability among the participants. Drawing from the 2017 Indonesian Demographic Health Survey (IDHS), this population-based study sought to identify determinant factors of MHC utilisation (ANC visit, institutionalised delivery, and PNC use) among Indonesian mothers. An understanding of determinants in each service could provide evidence for the government to develop a personalised intervention for Indonesian mothers based on the type of services.\n\n\nMethods\n\nThis population-based, cross-sectional study used nationally representative data from the IDHS in 2017. This survey is part of the International Demographic and Health Survey (DHS) program, and its details can be found on the DHS program website (see the data availability statement for more detail).\n\nThe population in this study was households from 34 provinces in Indonesia. The IDHS collected data from July 24th to September 30th, 2017, using a two-stage stratified sampling method. The first stage was selecting census blocks based on the wealth index that resulted in 1,970 blocks. In the second stage, 25 ordinary households were chosen from each block with systematic sampling from the list. A total of 49,627 women of childbearing age met the 2017 IDHS criteria to be interviewed. In the present study, the inclusion criteria of respondents included mothers aged 15 to 49 years who had a live birth in the five years preceding the survey. The exclusion criteria were whether the variables incomplete or missing. Of these, a sample of 12,033 mothers from 34 provinces in Indonesia was analysed.\n\nThe outcome variable in this study comprised the three key MHC components, namely ANC visits, institutionalised INC, and PNC after delivery obtained from participants self-reportedly. The ANC visit was categorised under two levels: adequate (4 times or more) and inadequate (fewer than 4 times). Institutionalised INC defined as a delivery that occurred at a healthcare facility regardless of its types (clinic, public health centre, and hospital) and ownership (government-owned and private sector). The use of PNC refers to a health check for the mother after delivery. According to the Ministry of Health Republic of Indonesia, PNC has to be carried out at least three times within the first six hours to the third day after delivery, on the fourth day to 28th after delivery, and the 29th day to the 42nd day after childbirth.\n\nThe explanatory variable in this study consisted of three categories that include mother’s sociodemographic factors, husband’s sociodemographic factors, and household-related factors. Under the category of mother’s sociodemographic factors, there were three variables, namely mother’s age in years (19-24, 25-34, and 35-49), mother’s occupational status (employed and not employed), mother’s educational level (primary, secondary, and higher). Three variables were under the category of husband’s sociodemographic factors consisted of an age gap with husband (younger/older than their husband), husband’s occupational status (employed and not employed), husband’s educational level (primary, secondary, and higher). In regard to household-related factors, in this category, there were three variables comprised the number of children (0, 1-3, and more than 3), household wealth index (richest, rich, middle, poor, and poorest), access to a healthcare facility (problem and not), and residential area (urban and rural).\n\nData obtained from the DHS dataset were analysed using STATA version 16.0 (Stata Corp, College Station, TX, USA). Data were analysed using the descriptive statistics method, and results were presented as weighted frequencies and percentages. The adjusted odds ratios (OR; AOR) with 95% confidence interval (CI) of factors associated with MHC (ANC, INC, and PNC) were estimated by multivariate logistic regressions.\n\nThe DHS program provided approval to use the 2017 IDHS data, and the data set is publicly available on DHS’s website. The 2017 IDHS study protocol has been approved for ethical clearance from the national board review of the Ministry of Health of Republic Indonesia and Inner City Fund (ICF) Macro institutional review board (number 45 CFR 46). Before participants were interviewed, informed consent was sought by each interviewer.\n\n\nResults\n\nA total of 12,033 mothers were eligible for data analyses in this study. Table 1 presents the descriptive characteristics of the study population. A high proportion of mothers were aged from 25 to 34 years (55.26%), employed (50.08%), had secondary education (61.36%), and were predominantly younger than their husbands (81.45%). The majority of husbands were working (99.48%) and had received their secondary education (60.19%). In this study, the mothers had predominantly had 1 to 3 children (95.73%) in their household, considered themselves as rich (21.68%), mostly did not have a problem (89.83%) when accessing health facilities, and resided in the rural area (50.44%).\n\nFigure 1 displays the proportion of Indonesian mothers’ maternal healthcare service utilisation in ANC, INC, and PNC. About 93.44% of mothers had adequate ANC visits (≥4 visits), while only 6.56% had inadequate ANC visits (<4 visits). Regarding institutionalised INC, 83.73% of mothers had delivered at the healthcare facility, whereas about 16.27% did not receive the delivery at the healthcare facility. In terms of the use of PNC, only 71.64% of mothers had the PNC after delivery.\n\nThe multivariate logistic regressions analysis revealed five determinant variables of ANC visits (Table 2). A higher odds of adequate ANC visits was found for mothers aged 35-49 years (AOR = 2.14; 95%CI = 1.55–2.94) and those aged 25-34 years (AOR = 1.48; 95%CI = 1.19–1.84) compared to mothers aged 15-24 years. Mothers who had employed husbands had a 2.91 (95%CI = 1.39–6.09) increased likelihood of having adequate ANC visits than those who were not working. Mothers with 1 to 3 children had 1.66 times (95%CI = 1.12–2.4) greater in having adequate ANC visits than mothers who do not have children. Compared to mothers who were in the poorest category, those who were in richest, rich, middle, and poor had 5.93 (95%CI= 3.81–9.20), 3.04 (95%CI= 2.18–4.25), 2.07 (95%CI = 1.56–2.75), and 1.87 (95%CI = 1.44–2.42) times likely to have adequate ANC visits respectively. Mothers who considered that access to the healthcare facility was not a problem had a 1.55 greater odds (95%CI = 1.20–2.02) of having adequate ANC visits than those who had problem accessing the healthcare facility.\n\n* : p < 0.05.\n\n : p < 0.01.\n\n* : p < 0.001.\n\nRegarding institutionalised INC, five determinant variables were identified as statistically significant (Table 2). A higher odds of delivering at a healthcare facility was found for mothers who aged 35-49 years (AOR = 1.92; 95%CI = 1.52–2.43) and aged 25-34 years (AOR = 1.19; 95%CI = 1.01–1.41) compared to mothers aged 15-24 years. Mothers who had secondary education had 1.57 times higher chances of delivering at healthcare facilities (95%CI = 1.33–1.85) than those with primary education. Also, mothers who had husbands with secondary education had 1.20 times more chances to deliver at healthcare facilities (95%CI= 1.02–1.40) than those with primary education. Compared to mothers who were in the poorest category, those who were in richest, rich, middle, and poor were 9.22 (95%CI = 6.54–13.00), 3.44 (95%CI = 2.71 4.37), 2.57 (95%CI = 2.07–3.20), and 2.14 (95%CI = 1.79–2.56) times likely to deliver at a healthcare facility respectively. Moreover, mothers who resided in urban areas had a 2.44 greater odds (95%CI = 1.96–3.03) of delivering at a healthcare facility than those living in the rural area.\n\nWith regard to the use of PNC, seven determinant variables were identified as statistically significant (Table 2). A higher odds of PNC utilisation was found for mothers aged 35-49 years (AOR = 1.31; 95%CI = 1.09–1.58) and aged 25-34 years (AOR = 1.23; 95%CI = 1.06–1.43) compared to mothers aged 15-24 years. Mothers who were employed had 1.15 (95%CI = 1.03–1.27) times increased likelihood of utilising PNC after delivery than those who were not employed. Mothers who had husbands with secondary education had 1.20 times more chances to utilise PNC (95%CI = 1.05–1.37) than those with primary education. For mothers who had 1 to 3 children had 1.64 times (95%CI = 1.31–2.05) to use PNC than mothers who do not have children. Compared to mothers in the poorest category, those in the middle and rich categories were 1.30 (95%CI = 1.08–1.55) and 1.29 (95%CI = 1.07–1.56) times likely to use PNC. Also, mothers who considered that access to the healthcare facility was not a problem had a 1.23 greater odds (95%CI = 1.03–1.46) utilise PNC than those who thought that access to the healthcare facility had a problem. Moreover, mothers who resided in urban areas had 0.81 times (95%CI = 0.70–0.90) less chances of having PNC after delivery than those in rural areas.\n\n\nDiscussion\n\nThe present study sought to identify determinant factors of MHC utilisation (ANC visit, institutionalised delivery, and PNC use) among Indonesian mothers population using the 2017 IDHS data sets. The results demonstrated that MHC utilisation among Indonesian mothers were relatively high. The main findings from the present study indicated that there were commonalities and differences in the determinant factors of three key MHC, which echoes findings from other studies in the lower and middle-income countries.13–15 Among Indonesian mothers, age and household wealth index were the typical determinants of MHC utilisation. Concerning the ANC visits, husband’s occupational status, the number of children, and access to the healthcare facility were identified as its specific determinants. Next, factors that drove mother’s delivery at the healthcare facility (INC) were the mother’s education level, husband’s educational level, and residential area. In addition, the use of PNC determined by several factors includes the mother’s occupational status, husband’s educational level, access to the healthcare facility, number of children, and residential area. Based on this finding, healthcare professionals should be considering these determinants in providing MHC to Indonesian mothers. Furthermore, a wide-system effort is required from the government sector to develop a programme tailored with these determinant factors that can ensure MHC can be accessed and adequately utilised by mothers.\n\nAge was an essential determinant factor for utilising ANC, institutional delivery, and PNC use among Indonesian mothers. Older mothers tend to utilised MHC correctly than younger mothers. This was thought because older mothers are more mature in appraising the benefit of using MHC. As older mothers are more experienced in pregnancy, they consider ANC service necessary during pregnancy, delivery at a healthcare facility relative safe, and received PNC will be essential for mothers and their babies.16,17 This finding is definitely important because Kurniati et al. found that about 32% of mothers give birth for the first time when they were younger than 19 years old.12 Therefore, developing effective policies for improving MHC among younger mothers is deemed necessary.\n\nThe household wealth index was identified to play a crucial role in determining utilisation of ANC visits, institutionalised delivery, and the use of PNC. Compared to mothers from the lower bound of wealth index, those in high wealth index were prone to have adequate ANC visits, delivery at the healthcare facility, and utilised PNC services, which was in line with findings from the previous study.12,14 The wealth index was well known as a structural factor affecting mothers’ ability to seek healthcare services through multiple mechanisms, including the ability to commute, financial capacity, geographic accessibility, and ability to comprehend the context of such services.18 In doing so, those who are living in poverty condition are at risk for underutilised maternal health services. This finding warrants attention because 16.88% of mothers are in the poorest category, and 20.15% are in the poor at the present study. In order to improve the utilisation of MHC, health professionals need to reach and facilitate mothers who live in poverty.\n\nThe residential area was found as the determinant of institutionalised delivery and the use of PNC services. Mothers who settled in the urban area were more likely to give birth at healthcare facilities and unlikely to utilised PNC services. Regarding institutionalised delivery, those who resided in urban areas had the privilege of a broad range of choices and had better access to the facility that provides delivery care. Unlike the urban area, mothers who settled in the rural or countryside often face difficulties in finding and reaching healthcare facilities because their residence area was relatively least developed.19,20 Apart from that, we discovered that mothers who settled in urban areas were unlikely to utilise PNC services. This might be attributed to mothers' misconceptions about insurance coverage. For instance, a previous qualitative study in Indonesia reported that some mothers thought national insurance cards could be used only for particular health care providers, such as the village midwife, and also they did not think it can be used for PNC.21 This finding is not exclusive to Indonesia, as studies from other countries also demonstrated similar findings.14,15,22 Thus, providing equal services for mothers who settle in urban and rural areas is warranted.\n\nIn favour of access to the healthcare facility, mothers who thought there was no problem tend to have adequate ANC visits and used the PNC service. Mothers may perceive numerous aspects could be causes of accessibility problems to a healthcare facility. However, few elements that often become problems are transportation, distance, medication, and treatment. More importantly, transportation and distance are classical problems in Indonesia, where infrastructures developed disproportionately across the country.23 Such issues significantly hinder mothers from initiating ANC and receiving PNC.24,25 Although approximately 89% of mothers thought there was no problem accessing healthcare facilities in the present study, we thought this remains important because mothers do not often speak up about this matter to the local health authority. Thus, more effort from the health professional to explore the unseen problem in accessing healthcare facilities among mothers is necessary.\n\nWith regard to the number of children, we found it was a determinant of ANC and PNC among Indonesian mothers. Compared to mothers who do not have children, those who had 1 to 3 children were more likely to have adequate ANC visits and PNC use, which was aligned with past works.13,14 This was thought that as mothers became more experienced in motherhood and had appropriate knowledge from previous maternal experiences, they were encouraged to use those services.26,27 In other words, those who were in their first-time pregnancy or delivery were at risk for underutilised ANC and PNC. We recognised that this finding might become an opportunity for health professionals to empower experienced mothers to encourage first-time mothers to use MHC correctly. However, experimental studies are required to prove this idea.\n\nHusband’s educational level revealed as the determinant factor for institutionalised delivery and the use of PNC after delivery. Those with husbands who received secondary education have a greater likelihood to delivery at the healthcare facility and received PNC services. Husband’s education level may be linked with better health awareness that may make the family aware and utilise healthcare services better.28 Also, since the Indonesian culture adopted the patriarchy concept, the husband plays a major role in family decision-making. Subsequently, a husband with better education may lead the family to utilise MHC properly.\n\nThe present study demonstrated that husbands’ work statuses determine the utilisation of ANC service. Husbands’ occupational status is thought to be linked with family income, which may later influence mothers’ ability to access ANC service.29,30 It seems plausible because ANC recommendation in Indonesia is at least four times visits. However, for those financially and geographically disadvantaged may be discouraged from visiting the healthcare facility to receive ANC service. Most probably, the husbands who are working are relatively more economically prepared to face the mother’s needs during the pregnancy period.\n\nThis study identified the mother’s educational level associated with institutionalised delivery that was also supported by other studies.14,20 Mothers who hold secondary education are prone to having delivery at the healthcare facility. This result is plausible since education is a marker for various factors that affect health-seeking behaviours. Compared to those with lower education levels, women with higher levels of education possess the level of health literacy required to make the right choices about their health and are better placed to overcome the cultural barriers to maternal health care use.31,32 Moreover, low education can create a social distance between pregnant women and service providers, leading to poor quality client-provider interactions and discouraging services among women with low education.33\n\nA mother’s occupational status also has a role in determining PNC utilisation. This result aligns with previous studies, where the risk for underutilised PNC among working mothers was lower than a mothers who were not employed.12,14 The possible explanation is that working mothers can control their earnings, preventing them from suffering from financial hardship and increasing their independence to seek healthcare services.34 However, it should be noted that attending PNC requires mothers to spare their time from work which could cost working mothers loss of their incomes. Thus, providing flexible service hours and availability of maternity leave would help working mothers removing their barriers in accessing health services.\n\nThe data of this population-based study were collected by government bodies that have authority in maternal health surveys, namely the Central of Statistics Agency and the National Population and Family Planning Board, Indonesia. Also, study’s findings are generated from a large sample size that tends to provide high statistical power. However, despite the strength of this study, the present study also has some limitations that should be considered. To begin with, the cross-sectional nature adopted in this study cannot be over-interpreted for implying causality. In addition, the answer given by mothers were self-reported and based on their recall preceding survey that has the potential to result in bias. Furthermore, this study used data from a nationally representative sample; hence, this finding may be generalised to Indonesian mothers only.\n\n\nConclusion\n\nThis population-based study revealed that the mother’s age and household wealth index were the typical determinants for each maternal healthcare service utilisation. Additionally, there were variabilities of determinant factors in each service. Provides a programme tailored with these determinant factors may ensure MHC can be accessed and adequately utilised by mothers. Besides, the programme may look into the health care services policy in reaching the underprivileged group and poor accessibility, like mobile health services and peer influence. Furthermore, engaging the father in the issue of MHC should be redesigned and rebranding especially for the young family.\n\n\nData availability\n\nData used in this study are from the standard DHS VII recode dataset of the Indonesian 2017 Demographic and Health Survey (DHS) available from the Demographic and Health Survey (DHS) website. Access to the dataset requires registration and is granted only for legitimate research purposes. A guide for how to apply for dataset access is available at: https://dhsprogram.com/data/Access-Instructions.cfm. Other researchers will be able to access the data set in the same way as the authors and the authors do not have special access rights that others do not have.", "appendix": "References\n\nWorld Health Organization: Trends in maternal mortality 2000 to 2017: estimates by WHO UNICEF, UNFPA, World Bank Group and the United Nations Population Division; 2019.\n\nWorld Health Organization: Maternal mortality.2019. Reference Source\n\nCenters for Disease Control and Prevention: Maternal and child health.2014. Reference Source\n\nMinistry of Health of the Republic of Indonesia: Indonesia Health Profile. Indonesia Agency of Health Research and Development editor. Jakarta: 2019; p. 87.\n\nVictora CG, Requejo JH, Barros AJ, et al.: Countdown to 2015: a decade of tracking progress for maternal, newborn, and child survival. Lancet. 2016; 387(10032): 2049–2059. Publisher Full Text\n\nDingle A, Powell-Jackson T, Goodman C: A decade of improvements in equity of access to reproductive and maternal health services in Cambodia, 2000-2010. Int. J. Equity Health. 2013; 12: 51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMondal D, Karmakar S, Banerjee A: Women’s autonomy and utilization of maternal healthcare in India: Evidence from a recent national survey. PLOS ONE. 2020; 15(12): e0243553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamba M, Attia-Konan AR, Sangaré AD, et al.: Factors associated with the use of maternal health services by mothers in a post-conflict area of western Côte d’Ivoire in 2016. BMC Health Serv. Res. 2020; 20(1): 136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaksono AD, Rukmini R, Wulandari RD: Regional disparities in antenatal care utilization in Indonesia. PLOS ONE. 2020; 15(2): e0224006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSukirman R, Wahyono TYM, Shivalli S: Determinants of healthcare facility utilization for childbirth in Kuantan Singingi regency, Riau province, Indonesia 2017. BMC Public Health. 2020; 20(1): 933. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCentral Bureau of Statistics: The 2012 Indonesia Demographic and Health Survey.2012. Reference Source.\n\nKurniati A, Chen CM, Efendi F, et al.: Factors influencing Indonesian women's use of maternal health care services. Health Care Women Int. 2018; 39(1): 3–18. PubMed Abstract \| Publisher Full Text\n\nBanke-Thomas OE, Banke-Thomas AO, Ameh CA: Factors influencing utilisation of maternal health services by adolescent mothers in Low-and middle-income countries: a systematic review. BMC Pregnancy Childbirth. 2017; 17(1): 65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBabalola SO: Factors associated with use of maternal health services in Haiti: a multilevel analysis. Rev. Panam. Salud Publica. 2014; 36: 1–09. PubMed Abstract\n\nPrithutam B: Factors Associated with Use of Maternal Health Services in Nepal: Analysis of the 2016 Nepal Demographic and Health Survey. J. Nepal Health Res. Counc. 2019; 17(3): 301–307. Publisher Full Text\n\nKebede A, Hassen K, Nigussie TA: Factors associated with institutional delivery service utilization in Ethiopia. Int. J. Women's Health. 2016; 8: 463–475. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaul P, Chouhan P: Socio-demographic factors influencing utilization of maternal health care services in India. Clin. Epidemiol. Glob. Health. 2020; 8(3): 666–670. Publisher Full Text\n\nPeters DH, Garg A, Bloom G, et al.: Poverty and access to health care in developing countries. Ann. N. Y. Acad. Sci. 2008; 1136(1): 161–171. Publisher Full Text\n\nCahyono MN, Efendi F, Harmayetty H, et al.: Regional disparities in postnatal care among mothers aged 15-49 years old in Indonesia. F1000Res. 2021; 10(153): 153.\n\nLaksono AD, Paramita A, Wulandari RD: Socioeconomic disparities of facility-based childbirth in Indonesia.2019.\n\nTitaley CR, Hunter CL, Heywood P, et al.: Why don't some women attend antenatal and postnatal care services?: a qualitative study of community members' perspectives in Garut, Sukabumi and Ciamis districts of West Java Province, Indonesia. BMC Pregnancy Childbirth. 2010; 10(1): 61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamba M, Attia-Konan AR, Sangaré AD, et al.: Factors associated with the use of maternal health services by mothers in a post-conflict area of western Côte d'Ivoire in 2016. BMC Health Serv. Res. 2020; 20(1): 136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBappenas: Local government initiatives in efforts to reduce regional disparities and regional development Jakarta, Indonesia.2018. Reference Source.\n\nMekonnen T, Dune T, Perz J, et al.: Postnatal Care Service Utilisation in Ethiopia: Reflecting on 20 Years of Demographic and Health Survey Data. Int. J. Environ. Res. Public Health. 2021; 18(1): 193.\n\nTeshale AB, Tesema GA, Yeshaw Y, et al.: Individual and community level factors associated with delayed first postnatal care attendance among reproductive age group women in Ethiopia. BMC Pregnancy Childbirth. 2021; 21(1): 1–8. Publisher Full Text\n\nBerhe M, Medhaniye AA, Kahsay G, et al.: Essential neonatal care utilization and associated factors among mothers in public health facilities of Aksum Town, North Ethiopia, 2016. PLoS One. 2017; 12(4): e0175902. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsei CK, Nketiah-Amponsah E, Lambon-Quayefio MP: Household wealth and maternal health: evidence from Ghana. Int. J. Soc. Econ. 2021; 48(1): 63–83.\n\nIslam MA, Barna SD: Concise title: Maternal health service utilization. Clinical Epidemiology and Global Health. 2020; 8(4): 1236–1241. Publisher Full Text\n\nBbaale E: Factors influencing timing and frequency of antenatal care in Uganda. Australas Med J. 2011; 4(8): 431–438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChamileke N: Socio demographic determinants of maternal health service utilization among women 15 to 49 years in Zambezi District in northwestern Zambia. Med. J. Zambia. 2017; 44(3): 149–156.\n\nBabalola S, Fatusi A: Determinants of use of maternal health services in Nigeria-looking beyond individual and household factors. BMC Pregnancy Childbirth. 2009; 9(1): 1–13. Publisher Full Text\n\nGreenaway ES, Leon J, Baker DP: Understanding the association between maternal education and use of health services in Ghana: exploring the role of health knowledge. J. Biosoc. Sci. 2012; 44(6): 733–747. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdamson PC, Krupp K, Niranjankumar B, et al.: Are marginalized women being left behind? A population-based study of institutional deliveries in Karnataka, India. BMC Public Health. 2012; 12(1): 1–6. Publisher Full Text\n\nChama-Chiliba CM, Koch SF: Utilization of focused antenatal care in Zambia: examining individual- and community-level factors using a multilevel analysis. Health Policy Plan. 2015; 30(1): 78–87. PubMed Abstract \| Publisher Full Text" }	[ { "id": "120405", "date": "23 Feb 2022", "name": "Kusrini S. Kadar", "expertise": [ "Reviewer Expertise community health", "family health", "health education", "health promotion" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 an interesting study and it provides new insight to improve the healthcare facilities and services for pregnant mothers. However, the authors need to explain more about what kind of services that the mothers received in the ANC, INC and PNC, especially at the PNC as it is interesting why mothers who live in urban area use the PNC services less. There is no clear explanation as the authors only compare this situation with other countries and due to insurance issues. In the conclusion, it seems that the authors give new ideas but not conclusions from the results; for example using mobile health services and peer influence are not the conclusions of the results. Perhaps this is more a recommendation than conclusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "8108", "date": "21 Apr 2022", "name": "Ferry Efendi", "role": "Author Response", "response": "We appreciate the reviewer’s positive feedback and recommendations. We added the explanations about ANC, INC, and PNC in the introduction to make sure the reader can understand these concepts earlier. Please see the second paragraph. Thank you for your feedback. Regarding “there is no clear explanation as to the authors only comparing this situation with other countries and due to insurance issues”, we have revised and added more explanation in the discussion section. Please see the second paragraph in the sub-section of intermediary determinants. Thank you for your constructive feedback. We have revised our conclusion and made it more coherent with our findings." } ] }, { "id": "121898", "date": "28 Feb 2022", "name": "Danish Ahmad", "expertise": [ "Reviewer Expertise Global health", "maternal health", "health system strengthening." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 findings provide guidance to improve maternal health care seeking in Indonesia. The use of a nationally validated dataset allows findings to be generalisable for the country. The paper's contribution needs to be set against global maternal health research which has conclusively established the determinants of maternal health.\nIn the current format, the paper's readability is affected by inconsistencies in language - this can however be remedied.\nIndonesia as an archipelago provides an opportunity to review maternal health determinants across different provinces and regions in Indonesia. The paper's focus on maternal health determinants needs further clarity in places.\nIn table 2, the access to health services as being categorised as 'no problem' wasn't immediately clear. Please consider explaining how the variable was used.\nThe discussion section is rather wordy and repetitive. The authors may like to use a framework such as the social determinants of health or Andersons health care seeking model to present findings. Another option is to present across individual, household and community/health system headings. The authors may also like to consider the program implications arising from this research.\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? 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": "8109", "date": "21 Apr 2022", "name": "Ferry Efendi", "role": "Author Response", "response": "Many thanks for the reviewer’s suggestion about \"The paper's contribution needs to be set against global maternal health research\". We have added this information. Please see at the end of the first paragraph in the discussion section. Many thanks for your on-point comment regarding the readability. The manuscript has been proofed by an English editor in the current version. Thanks a lot for this query, in this study, we focused on rural and urban areas which comprised of 12033 residential areas in Indonesia. We have added the concept definition of access to healthcare facilities. Thank you very much for bringing this constructive feedback to our study. We have revised the discussion section and organized it based on the social determinant framework. Please see the discussion section. We have revised the conclusion section." } ] } ]	1	https://f1000research.com/articles/10-1124
https://f1000research.com/articles/10-395/v1	18 May 21	{ "type": "Brief Report", "title": "Overlap of vitamin A and vitamin D target genes with CAKUT-related processes", "authors": [ "Ozan Ozisik", "Friederike Ehrhart", "Chris T. Evelo", "Alberto Mantovani", "Anaı̈s Baudot", "Friederike Ehrhart", "Chris T. Evelo", "Alberto Mantovani" ], "abstract": "Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) are a group of abnormalities affecting the kidneys and their outflow tracts, which include the ureters, the bladder, and the urethra. CAKUT patients display a large clinical variability as well as a complex aetiology, as only 5% to 20% of the cases have a monogenic origin. It is thereby suspected that interactions of both genetic and environmental factors contribute to the disease. Vitamins are among the environmental factors that are considered for CAKUT aetiology. In this study, we collected vitamin A and vitamin D target genes and computed their overlap with CAKUT-related gene sets. We observed significant overlaps between vitamin A target genes and CAKUT causal genes, or with genes involved in renal system development, which indicates that an excess or deficiency of vitamin A might be relevant to a broad range of urogenital abnormalities.", "keywords": [ "CAKUT", "vitamin A", "vitamin D", "nutrients", "environmental factor", "renal development" ], "content": "Introduction\n\nCongenital Anomalies of the Kidney and Urinary Tract (CAKUT) are a group of abnormalities affecting the kidneys and their outflow tracts, including the ureters, the bladder, and the urethra. In the European Union, the overall prevalence of CAKUT (in live plus stillbirths) between 2011 and 2018 was approximately 35:10,000.1 The main anomalies observed in CAKUT are hydronephrosis (13.02:10,000) and multicystic renal dysplasia (4.33:10,000); these are followed by posterior urethral valves (1.26:10,000), bilateral renal agenesis (1.27:10,000) and bladder exstrophy/epispadia (0.62:10,000); many other anomalies with low prevalence can also be observed.1 This clinical variability observed in patients is accompanied by a variable severity of the phenotypes.2\n\nApproximately 40 different genes are known to be associated with monogenic causes of CAKUT in humans, but they explain only 5% to 20% of the cases.3,4 The clinical variability and the complex aetiology of CAKUT cases suggest a multifactorial origin with complex interactions of both genetic and environmental factors contributing to the disease.2,3,5\n\nThe role of different environmental factors in CAKUT pathogenesis has been studied previously. It has been shown, for instance, that the drugs inhibiting the angiotensin-converting enzymes cause a specific form of CAKUT, namely tubular dysgenesis.6 Many prenatal and maternal factors were also assessed for their involvement in CAKUT and related anomalies. For example, studies have observed CAKUT associations with pregestational maternal diabetes mellitus,7,8,9 gestational maternal diabetes mellitus,8,10,11 abnormal volume of amniotic fluid,7,10 urogenital infections before the pregnancy,12 any infection during the pregnancy,12 maternal overweight and obesity,7,8,11,13 and lower infant birth weight.7,8,9,10 These maternal and prenatal factors are modulated by environmental risk factors. In particular, maternal diabetes and overweight/obesity as well as low birth weight are related to the nutrition of the maternal-fetal dyad. In this regard, trace nutrients, like vitamins, are likely to play important roles that still await full characterization.14\n\nVitamins are known to regulate renal development.15,16,17,18 The association of maternal vitamin A deficiency with nephron reduction was studied on a rat model.19 The inactivation of the retinoic acid nuclear receptors also resulted in renal malformations in mice.20 An additional study on rats showed that vitamin A deficiency downregulates RET expression which is essential for epithelial-mesenchymal interaction during renal development.21 Goodyear et al. compared a group of pregnant women in Bangalore with a high (55%) prevalence of vitamin A deficiency with a group of pregnant women in Montreal with negligible vitamin A deficiency.22 The authors found that renal volume in newborns, adjusted for body surface area, was significantly lower in the vitamin A-deficient population. While the two populations are different for other genetic and environmental reasons, these results suggest that vitamin A deficiency may have a role in human CAKUT. El-Khashab et al. reported a similar finding in their study on a cohort of Egyptian mother-child pairs;23 children of vitamin A deficient mothers had significantly lower kidney sizes.\n\nThe research on the effects of vitamin D deficiency on kidney development has generated interesting results. Rogers et al.24 observed that incubation of metanephroi with vitamin D3 prior to implantation into adult rats increased the number of glomeruli. Conversely, Maka et al.25 and Nascimento et al.26 observed that maternal vitamin D deficiency stimulated nephrogenesis in rats. Following these studies, Boyce et al. examined the long term effects of maternal vitamin D deficiency and observed that adult male rat offspring of vitamin D deficient dams had reduced creatinine clearance, indicating reduced renal functional capacity.27 They also observed a significant upregulation of renal renin mRNA expression in fetuses and adult male offspring, and reported smaller kidneys in the offspring. Miliku et al. examined the association of 25-hydroxyvitamin D levels during mid-pregnancy with childhood kidney outcomes among 4212 mother-child pairs.28 They observed that children of mothers who were vitamin D deficient during pregnancy had a larger combined kidney volume compared to children of mothers who had optimal vitamin D levels.\n\nIn this study, we assessed whether vitamin A and vitamin D target genes are related to gene sets involved in CAKUT and renal system development. We first created a list of vitamin A and vitamin D target genes, extracting information from the Comparative Toxicogenomics Database (CTD) as well as from two publications.29,30 We then constructed different gene sets relevant to CAKUT, including genes mutated in monogenic forms of CAKUT, genes involved in renal system development, and genes involved in different pathways of interest. Finally, we performed overlap analyses to identify the genes involved in these different gene sets as well as the significant overlaps.\n\n\nMethods\n\nWe first queried vitamin A and downloaded all gene interactions of vitamin A and its descendants from the Comparative Toxicogenomics Database (CTD). We selected only the interactions supported by at least two references. This gave a list of 1086 target genes.\n\nBalmer and Blomhoff29 reviewed published data from 1191 articles to identify genes regulated by retinoic acid (vitamin A acid) in humans and other species. The authors provide a list of retinoic acid target genes split into four categories ranging from strong evidence to indirect regulation through a transcriptional intermediary. We selected the genes from all of these categories, converted gene symbols to human orthologs, and updated gene symbols where necessary using the HUGO Gene Nomenclature Committee (HGNC), Rat Genome Database (RGD), Mouse Genome Database (MGD), BioMart and SynGo. The final list of genes obtained from Balmer and Blomhoff contains 521 target genes, of which 229 are common with genes from CTD.\n\nWe queried vitamin D and downloaded all gene interactions of vitamin D and its descendants from the CTD. We selected only the interactions supported by at least two references, and obtained a list of 263 target genes.\n\nIn Ramagopalan et al.,30 230 genes with significant expression changes in response to vitamin D were identified on lymphoblastoid cell lines using microarrays. We checked the gene names using HGNC and obtained a list of 210 target genes.\n\nThere are only 15 common genes between the list from CTD and the list from Ramagopalan et al. The combined list of vitamin D target genes (458 genes) has 134 genes in common with the combined list of vitamin A target genes (1378 genes).\n\nWe collected complementary gene sets relevant to CAKUT and renal system development from several sources, outlined below.\n\nCAKUT causal genes set\n\nFirst, we created a set of genes known to be mutated in the monogenic form of CAKUT. To do so, we combined two lists of genes provided in the studies of van der Ven et al.3,4 Only six genes are different between these two lists, and their union leads to 42 causal genes (see Extended data55).\n\nGO kidney development gene sets\n\nWe extracted the genes annotated with the three terms below from Gene Ontology (GO).31,32 Please note that these terms have parent-child relationships.\n\n\n\n• GO:0072001 Renal system development (315 genes)\n\n• GO:0001822 Kidney development (306 genes)\n\n• GO:0060993 Kidney morphogenesis (96 genes)\n\nReactome pathways\n\nWe selected different pathways of interest for CAKUT. The renin-angiotensin system is essential for kidney development and mutations in the genes of this system result in CAKUT.33 In addition, Davis et al. discussed the role of RET signaling in kidney development and CAKUT.34 In multiple studies35,36,37,38,39,40 the roles of WNT, NOTCH, and Hedgehog signaling in kidney development and kidney diseases are discussed. Based on these studies we included the following Reactome41 pathways:\n\n• R-HSA-2022377 Metabolism of angiotensinogen to angiotensins (18 genes)\n\n• R-HSA-8853659 RET signaling (38 genes)\n\n• R-HSA-195721 Signaling by WNT (328 genes)\n\n• R-HSA-157118 Signaling by NOTCH (233 genes)\n\n• R-HSA-5358351 Signaling by Hedgehog (149 genes)\n\nWikiPathways\n\nFrom WikiPathways Rare Disease Portal42 we obtained the following four CAKUT-related pathways:\n\n• WP5053 Development of ureteric collection system (47 genes)\n\n• WP4823 Genes controlling nephrogenesis (43 genes)\n\n• WP5052 Nephrogenesis (17 genes)\n\n• WP4830 GDNF/RET signalling axis (23 genes)\n\nWe computed the overlaps between vitamin target gene sets and the gene sets of interest defined previously. After obtaining the overlap results, we calculated the significance of the overlaps (hypergeometric test, p-values adjusted by Benjamini-Hochberg (BH) method) (see Underlying data and Extended data55).\n\n\nResults and discussion\n\nThe results of the overlap analyses between vitamin A and D target genes and CAKUT-related gene sets are presented in Tables 1 and 2, and the corresponding data is available in Underlying data.55\n\nPub: Publications,3,4 GO: Gene Ontology, Reac: Reactome, WP: WikiPathways.\n\nPub: Publications,3,4 GO: Gene Ontology, Reac: Reactome, WP: WikiPathways.\n\nVitamin A target genes obtained from both CTD and Balmer and Blomhoff are significantly enriched in the set of CAKUT causal genes, with an overlap of ten and six genes, respectively (Table 1).\n\nA significant overlap is also observed focusing on GO terms related to renal system development, kidney development, and kidney morphogenesis. These results are expected due to the recognized role of vitamin A in differentiation.43,44 It should be noted that all overlapping CAKUT causal genes except NRIP1 are also part of the kidney development GO term.\n\nConcerning the overlap with Reactome pathways, the vitamin A target genes obtained from CTD are significantly enriched in signaling by NOTCH. The NOTCH signalling pathway is mainly involved in cell-to-cell communication,45 and it plays a role in the development of most organs and tissues.46 Other Reactome terms also have overlapping genes, even if not significant. This includes the WNT signalling pathway (which is mainly involved in developmental processes such as patterning, differentiation/proliferation shift, and migration) and the metabolism of angiotensinogen to angiotensins pathway, from which CTSD, CTSG and MME are vitamin A targets and play roles in the generation of different angiotensin peptides.47,48,49 The impaired conversion of angiotensin I to angiotensin II is the pharmacodynamic adverse mechanism underlying the fetopathy caused by ACE-inhibiting drugs.50 One main feature of this teratogen-induced fetopathy, namely renal tubular dysgenesis, belongs to the CAKUT spectrum. In addition, an overproduction of angiotensin II might have a role in CAKUT anomalies involving renal tubules, as observed in cases of the autosomal recessive polycystic kidney disease.51\n\nFinally, vitamin A target genes extracted from both CTD and Balmer and Blomhoff are significantly enriched in the development of ureteric collection system, genes controlling nephrogenesis, and GDNF/RET signalling axis pathways, as defined in WikiPathways. The target genes extracted from CTD are also significantly enriched in nephrogenesis. Signaling by GDNF through the RET receptor is required for normal growth of the ureteric bud.52 Localized expression of GDNF by the metanephric mesenchyme is important to elicit and correctly position the initial budding event from the Wolffian duct and to promote the continued branching of the ureteric bud. Cells that lack RET are unable to contribute to the tip of the ureteric bud.52\n\nOverall, the significant overlaps observed between vitamin A and the CAKUT gene sets indicate that vitamin A might be relevant to a broad range of urogenital abnormalities.\n\nIn the analyses of vitamin D target gene sets, we observed very few significant overlaps with CAKUT-related gene sets overall. Vitamin D target genes obtained from CTD are only significantly enriched in renal system development, kidney development, and kidney morphogenesis GO terms. It should be noted that in each case 80% of the enriched genes are also vitamin A target genes (according to CTD or Balmer and Blomhoff).\n\nCTSZ, which cleaves angiotensin I to yield angiotensin II,53 is the only vitamin D target in the metabolism angiotensinogen to angiotensins pathway. Thus, within CAKUT, vitamin D might also have a role in renal tubular defects related to anomalies of the renin-angiotensin system.\n\nWNT and NOTCH signaling pathways have genes that are targets of vitamin D but there is a small, non-significant overlap, and the overlapping genes are not the WNT and NOTCH receptors nor ligands.\n\n\nConclusions\n\nIn this study we examined the overlaps of vitamin A and vitamin D target gene sets extracted from different databases and publications with CAKUT-related genes and biological processes. While we only observed significant enrichment of vitamin D target genes in GO kidney development terms, there is significant enrichment of vitamin A target genes in most of the gene sets we tested. Indeed, we observed that vitamin A target genes are enriched in CAKUT causal genes set, kidney development pathways, the signaling by NOTCH pathway, and the GDNF/RET signalling axis pathway. There is a strong overlap with the signaling by the WNT pathway, although it is not statistically significant. Overall, the significant overlaps observed between vitamin A and CAKUT gene sets indicate that vitamin A might be relevant to a broad range of urogenital abnormalities. Several environmental chemicals may impinge on retinoid-regulated pathways,54 and the influence of environment and nutrition cannot be ruled out. Our study is dedicated to the generation of hypotheses. Further study is needed, using up-to-date datasets obtained with new omics techniques, to investigate the fine-grained effects of vitamin excess and deficiency on CAKUT, and to decipher the pathophysiological mechanisms.\n\n\nData availability\n\nZenodo: Overlap of vitamin A and vitamin D target genes with CAKUT-related processes. https://www.doi.org/10.5281/zenodo.4501624.55\n\nThis project contains the following underlying data in the ‘Data’ folder:\n\n• VitA-Balmer2002-Genes.txt (list of vitamin A target genes from Balmer and Blomhoff used for overlap analyses).\n\n• VitA-CTD-Genes.txt (list of vitamin A target genes from CTD used for overlap analyses).\n\n• VitD-Ramagopalan2010.txt (list of vitamin D target genes from Ramagopalan et al. used for overlap analyses).\n\n• VitD-CTD-Genes.txt (list of vitamin D target genes from CTD used for overlap analyses).\n\n• PathwaysOfInterest.gmt (CAKUT-related gene sets used for overlap analyses).\n\n• PathwaysOfInterestBackground.txt (list indicating which background GMT file to be used for the analysis of each CAKUT-related gene set).\n\n• hsapiens.GO:BP.name.gmt (all gene sets from GO:BP used as background information for overlap analyses).\n\n• hsapiens.REAC.name.gmt (all gene sets from Reactome used as background information for overlap analyses).\n\n• hsapiens.WP.name.gmt (all gene sets from WikiPathways used as background information for overlap analyses).\n\nThis project contains the following underlying data in the ‘Result’ folder:\n\n• VitA-CTD-Genes.csv (overlap analysis results for vitamin A targets from CTD).\n\n• VitA-Balmer2002-Genes.csv (overlap analysis results for vitamin A targets from Balmer and Blomhoff).\n\n• VitA-Merged.csv (merged table for the results presented in VitA-CTD-Genes.csv and VitA-Balmer2002-Genes.csv).\n\n• VitA-MergedTexVersion.txt (merged table in LaTeX format for the results presented in VitA-CTD-Genes.csv and VitA-Balmer2002-Genes.csv).\n\n• VitD-CTD-Genes.csv (overlap analysis results for vitamin D targets from CTD).\n\n• VitD-Ramagopalan2010.csv (overlap analysis results for vitamin D targets from Ramagopalan et al.).\n\n• VitD-Merged.csv (merged table for the results presented in VitD-CTD-Genes.csv and VitD-Ramagopalan2010.csv).\n\n• VitD-MergedTexVersion.txt (merged table in LaTeX format for the results presented in VitD-CTD-Genes.csv and VitD-Ramagopalan2010.csv).\n\nZenodo: Overlap of vitamin A and vitamin D target genes with CAKUT-related processes. https://www.doi.org/10.5281/zenodo.4501624.55\n\nThis project contains the following extended data:\n\n• overlapAnalysis.py (Python script used for all the analyses).\n\nThis project contains the following extended data in the ‘Supp’ folder:\n\n• CAKUT-CausalGenesPMC5748921-6115658Comb.txt (list of genes known to be mutated in the monogenic form of CAKUT).\n\n• targetsFromDifferentSourcesOverlap.odt (contains the information on vitamin A and D targets from different sources (CTD, publications), presents source specific genes and common genes).\n\n• VitA-Balmer2002-GeneMapping.csv (detailed table for the gene list from Balmer and Blomhoff).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor contributions\n\nOzisik O: Conceptualization, Data Curation, Formal Analysis, Project Administration, Writing – Original Draft Preparation; Ehrhart F: Conceptualization, Writing – Review & Editing; Evelo C: Conceptualization, Supervision, Writing – Review & Editing; Mantovani A: Conceptualization, Supervision, Writing – Original Draft Preparation; Baudot A: Conceptualization, Data Curation, Project Administration, Supervision, Writing – Original Draft Preparation.", "appendix": "Acknowledgements\n\nWe thank the members of European Joint Programme on Rare Diseases, in particular the members of the Environment/AOP Work Focus.\n\n\nReferences\n\nEU RD Platform prevalence charts and tables: 2020. 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Reprod Toxicol. 04 2020; 93: 250–258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOzisik O, Ehrhart F, Evelo C, et al.: Overlap of vitamin A and vitamin D target genes with CAKUT-related processes. Zenodo. March 2021. Publisher Full Text" }	[ { "id": "85565", "date": "25 Jun 2021", "name": "Elena Menegola", "expertise": [ "Reviewer Expertise Developmental toxicology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Overlap if vitamin A and vitamin D target genes with CAKUT-related processes\" is aimed to indirectly evaluate the possible relationship between Vitamin A and D intake imbalance and congenital anomalies of the kidney and urinary tract (CAKUT). The work considers genes reported as dysregulated in CAKUT and key genes involved in different urinary morphogenetic pathways. Both were overlapped with target genes of Vitamin A and Vitamin D (extracted from CTD and from literature: Balmer and Blomhoff, 2002 for Vitamin A, Ramagopalan et al., 2010 for Vitamin D). As expected considering the biological activity of the Vitamin A morphogenetic derivative (retinoid acid), significant overlaps are reported in the manuscript between Vitamin A target genes and genes involved in several urinary morphogenetic processes and signalling. Interestingly, significant overlaps were observed between Vitamin A target genes and CAKUT-related genes. As far as Vitamin D is concerned, overlappings were limited to some kidney morphogenetic genes but no overlapping was evidenced with CAKUT-related genes.\nIn general, this work can be considered of interest in order to postulate a possible relationship between Vitamin imbalance (mainly Vitamin A) and CAKUT and to suggest that a nutritional evaluation in pregnancy is essential to ensure a physiological development and maternal/fetal health.\nSome suggestions in order to improve the impact of the manuscript:\nAbstract: The abstract should be rewritten in order to better clarify aim and results of the work. In particular, Vitamin D results are lacking.\nIntroduction: The aim should be better explained. A brief description for the reason of the choice of literature (Balmer and Blomhoff, 2002 for Vitamin A, Ramagopalan et al., 2010 for Vitamin D) could enhance the comprehension.\nMethods: Indicate retinoic acid as \"the active metabolite of vitamin A\" instead of \"vitamin A acid\". Better explain the statistical analysis.\nResults: Tables are not fully self explicative. I suggest:\na separate column for p-value; write in bold the genes both overlapping with CAKUT and urinary pathways/signalling.\nMoreover, (1) in table 2 is not explained.\nLooking in detail at Vitamin A table, four CAKUT-causal genes (BMP4, HNF1B, NRIP1 and RET, overlapping both with CTD and Balmer and Blomhoff) appear as a leitmotiv in different vitamin-A related morphogenetic pathways. In my personal opinion, this result should be stressed and a brief description of functions of BMP4, HNF1B, NRIP1 and RET in kidney and urinary tract development introduced in Results and/or Conclusions.\nConclusions: Can be rewritten in order to better focus the relevance of the nutritional management during pregnancy in order to ensure both maternal and fetal health. A general consideration should be introduced, considering also the pleiotropic functions of the involved genes.\nReferences: Please check references, i.e. reference 54 seems not pertinent.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "8103", "date": "25 Apr 2022", "name": "Ozan Ozisik", "role": "Author Response", "response": "We thank the reviewer for the detailed summary and valuable comments. We present the reviewer comments, our answers to them, and quoted texts from the revised manuscript below. Abstract The abstract should be rewritten in order to better clarify aim and results of the work. In particular, Vitamin D results are lacking. We revised the abstract; we clarified our aim and added the results for vitamin D. “...In this study, we aimed to investigate whether vitamin A or vitamin D could have a role in CAKUT aetiology. For this purpose we collected vitamin A and vitamin D target genes and computed their overlap with CAKUT-related gene sets. We observed limited overlap between vitamin D targets and CAKUT-related gene sets. We however observed that vitamin A target genes significantly overlap with multiple CAKUT-related gene sets, including CAKUT causal and differentially expressed genes, and genes involved in renal system development. Overall, these results indicate that an excess or deficiency of vitamin A might be relevant to a broad range of urogenital abnormalities.” Introduction The aim should be better explained. A brief description for the reason of the choice of literature (Balmer and Blomhoff, 2002 for Vitamin A, Ramagopalan et al., 2010 for Vitamin D) could enhance the comprehension. We clarified the aim of the study in the introduction. “In this study, we examined whether vitamin A and vitamin D target genes are related to gene sets involved in CAKUT and renal system development. Our aim is to generate hypotheses for the involvement of these vitamins in the disease aetiology.” We used additional datasets from two publications to have independent sources for vitamin target information. Only a small subset of the articles (27 of 1192) reviewed by Balmer and Blomhoff (2002) are reviewed in CTD, and Ramagopalan et al. (2010) is not reviewed in CTD. We clarified this in the methods section. “As an independent source, we used the data from the study of Balmer and Blomhoff. 30 In this study, the authors reviewed published data from 1191 articles to identify genes regulated by retinoic acid (the active metabolite of vitamin A) in humans and other species. Only a small subset of these articles are part of the CTD curation.” “Ramagopalan et al. 31 identified 230 genes with significant expression changes in response to vitamin D on lymphoblastoid cell lines using microarrays. This publication is not part of the CTD curation, hence it brings additional and independent information.” Methods Indicate retinoic acid as \"the active metabolite of vitamin A\" instead of \"vitamin A acid\". Better explain the statistical analysis. We changed \"vitamin A acid\" to \"the active metabolite of vitamin A\". “In this study, the authors reviewed published data from 1191 articles to identify genes regulated by retinoic acid (the active metabolite of vitamin A) in humans and other species.” We added the description of the statistical analysis. “We computed the overlaps between vitamin target gene sets and the CAKUT gene sets of interest defined previously. After obtaining the overlap results, we calculated the significance of the overlaps using the hypergeometric test . Hypergeometric test calculates the statistical significance of getting k successes in n draws without replacement from a finite population of size N that contains a total of K successes. In our context, k is the number of genes overlapping with a specific gene set, K is the size of that gene set, n is the number of vitamin targets, and N is the background gene set (also known as the reference set). We set N to the number of annotated genes in the database from which we obtained the tested gene set. For the CAKUT causal genes set and the differentially expressed genes set, we used the largest N among the different databases, which corresponds to Gene Ontology. The null hypothesis is that the vitamin target genes are not associated with the gene set and the selection of k genes from that gene set is just a result of simple random sampling. We performed a hypergeometric test for all the gene sets. A consequence of multiple testing is false discoveries in which null hypothesis is rejected erroneously. To control for the false discovery rate, we used the Benjamini-Hochberg correction method (BH adjusted).” Results Tables are not fully self explicative. I suggest: a separate column for p-value; Adding a new column to the table for p-values causes difficulty in layout. We hence added “p-value =” before each value in the parentheses to clarify the table. write in bold the genes both overlapping with CAKUT and urinary pathways/signalling. The overlap of CAKUT causal genes with other gene sets can be interesting for the reader. However, as the aim of the tables in the manuscript is to present overlap of vitamin target genes and CAKUT related gene sets, presenting this extra information might be confusing for the reader. Additionally, the information of overlap between CAKUT causal genes and other pathways will be incomplete and could be misleading as there might be other genes which are common to these but are not vitamin targets. For these reasons, we now provide the overlap of CAKUT causal genes with all the other pathways (without considering whether genes are vitamin targets or not) in a supplementary dataset. This analysis is described in “Supp/SupplementaryAnalyses.odt”, the code for the calculation of this overlap is available as part of overlapAnalysis.py, and the overlap table is presented in “Supp/CAKUTCausalGenesOverlapWithOthers/CAKUTCausalGenesOverlap.csv” in Underlying data and Extended data. Moreover, (1) in table 2 is not explained. “(1)”s in table 2 were p-values of 1. We hope that with the addition of “p-value =”, these p-values are clearer now. Looking in detail at Vitamin A table, four CAKUT-causal genes (BMP4, HNF1B, NRIP1 and RET, overlapping both with CTD and Balmer and Blomhoff) appear as a leitmotiv in different vitamin-A related morphogenetic pathways. In my personal opinion, this result should be stressed and a brief description of functions of BMP4, HNF1B, NRIP1 and RET in kidney and urinary tract development introduced in Results and/or Conclusions. We agree that these are indeed interesting genes to highlight. We stressed that BMP4, HNF1B, NRIP1 and RET, which are CAKUT causal genes, are vitamin A targets according to both CTD and Balmer and Blomhoff. We added a description of their roles. “Four genes (BMP4, HNF1B, RET, NRIP1) appear as particularly interesting as they are CAKUT causal genes and vitamin A targets according to both CTD and Balmer and Blomhoff. BMP4 is a growth factor of the TGF-beta family involved in a wide variety of developmental processes. 48 BMP4 inhibits ectopic budding of the ureteric tips and promotes elongation of the ureter. 49 RET is a tyrosine-protein kinase receptor, involved in a large variety of cellular processes. RET signaling is important, in particular, for the terminal growth of the nephric duct, and for initial formation and outgrowth of the ureteric bud. 50 The Hepatocyte Nuclear Factor 1-beta (HNF1B) is a transcription factor involved in ureteric bud branching and induction of nephrogenesis. It is required for normal S-shaped bodies patterning and subsequent morphogenesis of all nephron segments. 51 Finally, Nuclear receptor interacting protein 1 (NRIP1) modulates the activity of nuclear receptors. It has been shown to play roles in renal malformations via deregulations of retinoic acid signaling. 52” Conclusions: Can be rewritten in order to better focus the relevance of the nutritional management during pregnancy in order to ensure both maternal and fetal health. A general consideration should be introduced, considering also the pleiotropic functions of the involved genes. We added that our findings indicated the importance of nutritional management during pregnancy. “Overall, the significant overlaps observed between vitamin A and CAKUT gene sets indicate that vitamin A might be relevant to a broad range of urogenital abnormalities, pointing out the importance of nutritional management during pregnancy for fetal development.” We thank the reviewer for the suggestion for considering the pleiotropic functions of the involved genes, this can be interesting, however we think that getting into this subject may cause loss of the focus in the conclusion, in which we only state the involvement of vitamin targets in CAKUT related gene sets with the note that our study aims at hypothesis generation. References: Please check references, i.e. reference 54 seems not pertinent. We checked the references and we removed the sentence containing this reference." } ] }, { "id": "95476", "date": "27 Oct 2021", "name": "Matias Simons", "expertise": [ "Reviewer Expertise Human genetics", "developmental biology", "Wnt" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Ozisik et al investigates associations of Vitamin A and D target genes with renal development and CAKUT pathways. They find that a significant overlap for vitamin A target genes but less so for vitamin D target genes. This fits with previous epidemiological studies on vitamin A and renal size. Overall, I find the paper interesting because it provides insights into non-genetic causes of CAKUT. I have only a few minor points:\nGiven that a list of 1086 Vit A target genes but only of 263 Vit D target genes, it is not surprising that less overlap is found for Vit D. This should be commented on.\n\nIt is said in abstract and introduction that only 5% to 20% of CAKUT cases have a monogenic origin. However, it has to be noted that these numbers are only based on whole exome sequencing studies. It is to be expected that whole genome sequencing will discover new monogenic causes.\n\nThe discussion could include more perspectives on how new -omic techniques could be used in the future.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8104", "date": "25 Apr 2022", "name": "Ozan Ozisik", "role": "Author Response", "response": "We thank the reviewer for the valuable comments. We present the reviewer comments, our answers to them, and quoted texts from the revised manuscript below. Given that a list of 1086 Vit A target genes but only of 263 Vit D target genes, it is not surprising that less overlap is found for Vit D. This should be commented on. Considering the overlaps between gene lists, as stated, with less target genes, less genes are expected to overlap with the CAKUT-related gene sets. However, in the manuscript, we do not focus on the exact number of overlapping genes. Indeed, in order to take into account the variable sizes of the different datasets, we used the hypergeometric test. Our focus is hence on the significance of the overlaps considering the dataset sizes, i.e. the observed overlaps as compared to the expected overlaps given the dataset sizes. This means that a small number of overlapping genes could lead to significance for vitamin D overlap with CAKUT-relative genes. However, we did not observe many significant overlaps. The overlaps between vitamin A targets and CAKUT-related gene sets were greater than expectations, which is the reason they are statistically significant. In the revised manuscript we added the explanations for the statistical test. It is said in abstract and introduction that only 5% to 20% of CAKUT cases have a monogenic origin. However, it has to be noted that these numbers are only based on whole exome sequencing studies. It is to be expected that whole genome sequencing will discover new monogenic causes. We used a list of causal genes curated from multiple studies. We are not sure if they are all based on whole exome sequencing. However, we agree that whole genome sequencing will help discovering new monogenic causes. We hence added a statement related to the whole genome sequencing to the conclusion: \"The increased availability of multi-omics technologies, such as whole genome sequencing and metabolomics will certainly open new directions, e.g. by revealing new causal genes or identifying metabolic disturbances due to environmental factors.\" The discussion could include more perspectives on how new -omic techniques could be used in the future. We agree that we do expect a lot of developments in this area, including whole genome sequencing but also single-cell or spatial transcriptomics, on human, animal models or organoids. In order to mention all these potential technologies while keeping the text focused, we added the following sentence to the conclusion: \"The increased availability of multi-omics technologies, such as whole genome sequencing and metabolomics will certainly open new directions, e.g. by revealing new causal genes or identifying metabolic disturbances due to environmental factors.\"" } ] }, { "id": "98474", "date": "15 Dec 2021", "name": "Olivia Angelin-Bonnet", "expertise": [ "Reviewer Expertise Statistics", "Systems Biology", "Biological networks analysis and reconstruction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Overlap of vitamin A and vitamin D target genes with CAKUT-related processes\" investigates the overlap between vitamin A and D target genes and genes associated with congenital anomalies of the kidney and urinary tract (CAKUT). More specifically, the authors assessed the significance of the intersection between sets of genes identified by previous publications or through databases as targets of either vitamin A or D, and sets of genes identified as causal to CAKUT or associated with pathways of interest with respect to CAKUT. This review focuses on the statistical analysis presented in this paper.\nI find the use of a hypergeometric test for this analysis completely appropriate. However, I found that while the Methods section provides a good explanation of the construction of the different gene sets investigated, there is no presentation or discussion of the statistical test used to assess the significance of the overlap between different gene sets. More specifically, I would like to see a brief explanation of the hypergeometric test, in order to justify its use to the user, as well as a statement of its null hypothesis. In addition, there is no mention of the background sets used for the test. I think they should be explicitly mentioned, especially since they differ with the pathway of interest tested (e.g. all GO terms for GO-related gene sets, all Reactome genes for Reactome sets, etc) and are used to constrain the sets of vitamin-related genes (i.e. how many vitamin genes were discarded because they did not appear in the background set?). I also want to note that the availability of the data and the (clearly commented) python script was really appreciated. It ensures that the statistical analysis can be fully reproduced.\nLastly, a minor presentation point: I am not sure that having the p-value between brackets in the same cell as the list of interesting genes in the two tables is very clear, especially when the intersection set is empty and the resulting p-value is 1. I would suggest to add \"p-value = \" before the actual value to make it clearer.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8105", "date": "25 Apr 2022", "name": "Ozan Ozisik", "role": "Author Response", "response": "We thank the reviewer for the valuable comments. We present the reviewer comments, our answers to them, and quoted texts from the revised manuscript below. I find the use of a hypergeometric test for this analysis completely appropriate. However, I found that while the Methods section provides a good explanation of the construction of the different gene sets investigated, there is no presentation or discussion of the statistical test used to assess the significance of the overlap between different gene sets. More specifically, I would like to see a brief explanation of the hypergeometric test, in order to justify its use to the user, as well as a statement of its null hypothesis. In addition, there is no mention of the background sets used for the test. I think they should be explicitly mentioned, especially since they differ with the pathway of interest tested (e.g. all GO terms for GO-related gene sets, all Reactome genes for Reactome sets, etc) and are used to constrain the sets of vitamin-related genes (i.e. how many vitamin genes were discarded because they did not appear in the background set?). I also want to note that the availability of the data and the (clearly commented) python script was really appreciated. It ensures that the statistical analysis can be fully reproduced. We thank the reviewer for pointing out the lack of necessary information on the applied test. We added the explanations for the statistical test, the null hypothesis, and the background sets used in the tests to the “Methods” section under “Overlap analyses” heading. “We computed the overlaps between vitamin target gene sets and the CAKUT gene sets of interest defined previously. After obtaining the overlap results, we calculated the significance of the overlaps using the hypergeometric test . Hypergeometric test calculates the statistical significance of getting k successes in n draws without replacement from a finite population of size N that contains a total of K successes. In our context, k is the number of genes overlapping with a specific gene set, K is the size of that gene set, n is the number of vitamin targets, and N is the background gene set (also known as the reference set). We set N to the number of annotated genes in the database from which we obtained the tested gene set. For the CAKUT causal genes set and the differentially expressed genes set, we used the largest N among the different databases, which corresponds to Gene Ontology. The null hypothesis is that the vitamin target genes are not associated with the gene set and the selection of k genes from that gene set is just a result of simple random sampling. We performed a hypergeometric test for all the gene sets. A consequence of multiple testing is false discoveries in which null hypothesis is rejected erroneously. To control for the false discovery rate, we used the Benjamini-Hochberg correction method (BH adjusted).” I would suggest to add \"p-value = \" before the actual value to make it clearer. As suggested, we added “p-value = ” before the actual value." } ] }, { "id": "100885", "date": "17 Dec 2021", "name": "Sean Eddy", "expertise": [ "Reviewer Expertise Renal systems and computational biology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study involves generating curated vitamin A and D gene sets from the public domain and assessing the relevance of the genes to CAKUT and CAKUT related pathways. The authors meticulously report their results and curation efforts so the findings are readily reproducible and are appropriate for F1000Research.\n\nIs the study design appropriate and is the work technically sound?\nThe addition of targeted vitamin A/retinoic acid and D gene sets is appreciated. Limiting CTD exported gene sets to those with at least 2 references is a reasonable approach to reducing potential false positives. I agree with the authors that this study is currently hypothesis generating. Given that patient CAKUT gene sets already exist and are publicly available (Jovanich et al., 2018, GEO dataset GSE GSE83946)1, the authors should also assess the enrichment of their gene sets against differentially expressed genes from patients with CAKUT and healthy control reference tissue.\nIf applicable, is the statistical analysis and its interpretation appropriate?\nBecause the gene sets are substantially different in size, a direct comparison of enrichment and p-values can be misleading. I would suggest the authors generate a randomized gene set of equivalent size to each of the vitamin A and vitamin D gene sets and perform comparative enrichment analyses on the relevant pathways.\n\nWhile not necessary, the addition of an odds ratio from 2x2 contingency matrices to assess the strength of enrichment would enhance the findings.\n\nThe targeted enrichment analysis of kidney and CAKUT related pathways is a reasonable approach to demonstrating a positive finding, ie. the gene sets identified associated with some of the expected/hypothesized pathways, it would be interesting to see if a more unbiased enrichment approach might also highlight other vitamin A and D relevant pathways, and perhaps additional renal-associated pathways not readily apparent from the targeted approach. This which could also substantiate the authors' vitamin A and D curation efforts.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8106", "date": "25 Apr 2022", "name": "Ozan Ozisik", "role": "Author Response", "response": "We thank the reviewer for the valuable comments. We present the reviewer comments, our answers to them, and quoted texts from the revised manuscript below. Given that patient CAKUT gene sets already exist and are publicly available (Jovanich et al., 2018, GEO dataset GSE GSE83946)1, the authors should also assess the enrichment of their gene sets against differentially expressed genes from patients with CAKUT and healthy control reference tissue. We performed the suggested analysis, described the analysis in the manuscript and presented the results. We observed that vitamin A targets obtained from CTD significantly overlapped with the differentially expressed genes. “CAKUT differentially expressed genes set Jovanovic et al. 33 identified 27 upregulated and 51 downregulated genes as a result of the transcriptome profiling of 15 CAKUT and 7 control ureter samples. We retrieved these differentially expressed genes (DEGs) from the supplementary file they provided. We updated gene names with the help of HUGO Gene Nomenclature Committee (HGNC) and obtained a final list of 74 DEGs (see Extended data 32 ).” Because the gene sets are substantially different in size, a direct comparison of enrichment and p-values can be misleading. I would suggest the authors generate a randomized gene set of equivalent size to each of the vitamin A and vitamin D gene sets and perform comparative enrichment analyses on the relevant pathways. We used hypergeometric tests in order to take into account the sizes of gene sets. Indeed, these tests are used in enrichment analyses to assess sets of genes’ overrepresentation in annotation terms of different sizes. However, we do agree that the experiment proposed by the reviewer is interesting to confirm our observations. We hence additionally performed a randomized sampling analysis. We selected a set of random genes having the same size as the target genes set from the background genes list. We repeated this selection 2000 times. We counted the number of times that the random set had better overlap with the CAKUT-related gene set than the target genes set. The results are consistent with the hypergeometric tests, except for two comparisons in which the overlaps were not significant according to the hypergeometric test but are significant according to the randomized sampling approach. Indeed, the adjusted p-value for the overlap between vitamin A targets retrieved from CTD and Reactome Signaling by WNT term is 0.146 (>0.05, insignificant) in the hypergeometric test but it is 0.0426 (<0.05, significant) in randomized sampling. Additionally, the adjusted p-value for the overlap between vitamin D targets retrieved from CTD and GO kidney morphogenesis term is 0.0507 (>0.05, insignificant) in the hypergeometric test but it is 0.042 (<0.05, significant) in randomized sampling. We should note that the results of the randomized sampling approach may change in different runs. As this is an extra test for confirmation, we provided related documents in Underlying data and Extended data for the interested readers. The analysis process is described in “Supp/SupplementaryAnalyses.odt”. The code of the randomized sampling test is available as part of overlapAnalysis.py; the results are in the “Supp/RandomizedSampling” folder. While not necessary, the addition of an odds ratio from 2x2 contingency matrices to assess the strength of enrichment would enhance the findings. All the numbers necessary to compute the enrichments (gene set sizes, domain sizes, intersection sizes, p-values) are provided in the tables. We think that providing additional tables and metrics might be confusing. The targeted enrichment analysis of kidney and CAKUT related pathways is a reasonable approach to demonstrating a positive finding, ie. the gene sets identified associated with some of the expected/hypothesized pathways, it would be interesting to see if a more unbiased enrichment approach might also highlight other vitamin A and D relevant pathways, and perhaps additional renal-associated pathways not readily apparent from the targeted approach. This which could also substantiate the authors' vitamin A and D curation efforts. We agree that our initial analyses could be extended. As a matter of fact, we are currently extending our approach from CAKUT-associated pathways to the available rare disease pathways. However providing all the enrichment results for all the pathways without any interpretation will not be useful for the audience. As we provide the vitamin target gene lists and the code to reproduce or extend our work, the interested (and expert) researchers with a precise question in mind could perform the analyses." } ] } ]	1	https://f1000research.com/articles/10-395
https://f1000research.com/articles/11-119/v1	31 Jan 22	{ "type": "Research Article", "title": "Assessment of fear, anxiety, obsession and functional impairment of COVID-19 amongst health-care workers and trainees: A cross-sectional study in Nepal", "authors": [ "Alok Atreya", "Samata Nepal", "Ritesh G Menezes", "Qazi Shurjeel", "Sana Qazi", "Muskaan Doulat Ram", "Muhammad Shariq Usman", "Sristi Ghimire", "Anu Marhatta", "Md Nazmul Islam", "Arbin Dev Sapkota", "Chandra Kumari Garbuja", "Samata Nepal", "Ritesh G Menezes", "Qazi Shurjeel", "Sana Qazi", "Muskaan Doulat Ram", "Muhammad Shariq Usman", "Sristi Ghimire", "Anu Marhatta", "Md Nazmul Islam", "Arbin Dev Sapkota", "Chandra Kumari Garbuja" ], "abstract": "Background: The emergence of the COVID-19 epidemic threw the world into turmoil. The medical community bore the brunt of the pandemic's toll. Long work hours, and a lack of personal protective equipment (PPE) and social support all had an influence on mental health. Methods:\nThis cross-sectional study was conducted among Lumbini Medical College Teaching Hospital students and employees in Palpa, Nepal. Data entailing their demographic details, pre-existing comorbidities, or death in the family due to COVID-19 was collected using a self-administered survey. In addition, the level of fear, anxiety, obsession, and functional impairment due to COVID-19 was recorded using previously validated respective scales. Results: In total, 403 health-care workers and trainees participated in our study. The average age of the study participants was 23±4 years, and more than half of them (n=262, 65%) were females. A significant association was found between fear score with age (p-value=0.04), gender (p-value <0.01) and occupation (p-value<0.001). The participants suffering from chronic diseases (p-value=0.36), and those who had experienced a COVID-19 death in the family (p-value=0.18), were not found to be significantly obsessed with COVID-19. However, for those who had experienced a COVID-19 death in the family (p-value=0.51) and age (p-value=0.34), these factors were not found to be significantly associated with higher anxiety levels. Nursing students suffered from a significantly greater functional impairment than other medical professionals (mean score=269.15, p-value < 0.001). A moderately positive correlation was observed between fear, anxiety, obsession, and functional impairment scales. Conclusion: This study revealed various socio-demographic characteristics as risk factors for psychological stress in the people related to the health-care profession of Nepal during the COVID-19 pandemic. A viable answer to this quandary might be adequate psychosocial intervention by health-care authorities, increased social support, and the introduction of better mental health management measures for the front-line medical workers.", "keywords": [ "COVID-19", "SARS-CoV-2", "anxiety", "depression", "health care workers", "Nepal" ], "content": "Introduction\n\nViral pandemics and epidemics are notoriously known in history for their public health risks and widescale destruction. From the influenza pandemic in 1918 and its recent outbreak in 2009,1 severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002–2003 to the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 and many more,2 one thing that has remained constant throughout was the associated high mortality rate and the subsequent social and psychological impact on the general population in the long run.3\n\nThe World Health Organization (WHO) declared COVID-19 as a global emergency in March 2020.4 Countries around the globe imposed several restrictions, including home confinement, social distancing, following proper hand hygiene, use of face masks, and in severe cases, nationwide lockdowns.5 These factors combined with the morbidity and mortality associated with the COVID-19 infection have had detrimental effects on the mental wellbeing of the general population but specifically of the front-line medical workers. Previous literature on the SARS-CoV epidemic has implicated medical staff to be particularly susceptible to anxiety, depression, and stress.6 This may be due to high exposure and, hence, greater risk of contracting the disease or the added workload. These findings can be implicated during the current COVID-19 pandemic because of the same mode of transmission of the infection and a greater patient load than the previous SARS-CoV epidemic.\n\nThe advent of the COVID-19 pandemic sent the world into chaos.5 The medical community suffered the brunt of the pandemic. The lack of medical staff and resources became evident. Long duty shifts, extended work hours, and lack of personal protective equipment (PPE) and social support affected psychological wellbeing.7 A study in Saudi Arabia conducted during the pandemic reported that 73.5% of the health-care personnel suffered from moderate degree fear and anxiety.8 Another study conducted by Labrague et al. reported that 37.8% of the nurses suffered from deteriorating mental health.9 However, the mental health issues experienced by the people related to health-care profession remain the least acknowledged, unaddressed, and untended.10\n\nPrevious literature has focused on estimating the psychological impact of COVID-19 on different sections of the population.8,11,12 However, the current pandemic has brought to light the necessity to screen individuals who are at high risk for developing mental health issues to optimize their productivity. Thus, in the present study, we aim to assess various psychological distress parameters among the health-care personnel of Nepal and identify personal factors and demographics responsible for predisposing them to a higher risk of developing mental health problems.\n\n\nMethods\n\nThis cross-sectional study was carried out among the staff and students of Lumbini Medical College (LMC) Teaching Hospital, Palpa, Nepal. The sample size computed using OpenEpi13 was 384, after considering a confidence level of 95% and a frequency outcome factor of 50%. For more robust results, we included 406 participants in the survey, of which 3 participants who did not consent to participate were excluded from the study. The cohort of health-care workers and trainees in the present study included doctors, nurses, other health-care staff, medical students, and nursing students. The present study was approved by the Institutional Review Committee of LMC vide letter IRC-LMC 06-G/020.\n\nThe questionnaire was in English and disseminated among the medical and nursing students, doctors, nurses, and other health-care staff working at LMC through social media. The first section of the questionnaire was for the consent where it was explained that no financial or material gifts will be provided for completing the questionnaire. The survey did not collect any identifying information of any of the participants and the responses were anonymous. Complete confidentiality of the participants was maintained by not asking them for identifying information like name, working department and post (for employee), year of study (for students) and the email address. Then the participants had an option to choose whether they voluntarily consented to participate or didn’t consent. The second section of the questionnaire was accessible only to those participants who had consented. The survey didn’t continue for the participants who didn’t consent, and the incomplete form were submitted.\n\nThe second section of the questionnaire used in the present study consisted of five parts. The first part of this section of the questionnaire was for demographic data and the remaining 4 parts used four different scales which were previously validated. The first part consisted of demographic information such as gender, age, current occupation, and monthly family income. Information regarding respondent's comorbidity, previous contact with any COVID-19 positive case, and if there was a COVID-19 death in their family was also recorded.\n\nThe second part of the second section of the questionnaire consisted of the fear of the COVID-19 scale adopted from Ahorsu et al.14 Fear of the COVID-19 scale is a unidimensional scale with robust psychometric properties and consists of seven items and assessed via five-point Likert scale method (strongly disagree = 1; strongly agree = 5).\n\nThe third part of the second section of the questionnaire was used to see the obsession of COVID-19 in the participants. The obsession of the COVID-19 scale (OCS) was adapted from Lee.15 There were four items to perceive too much coronavirus thought among the participants over the last two weeks. The participants would rate the items using a five-point time anchored scale (0 = not at all; 4 = nearly every day over the last two weeks). A score of seven or more signified that the person was overthinking of coronavirus.\n\nThe fourth part was the coronavirus anxiety scale. It consisted of five items developed by Lee.16 The participants would rate the items using a five-point time anchored scale (0 = not at all; 4 =nearly every day over the last two weeks). The participant scoring of nine or more in the questions was considered anxious about the coronavirus. The fifth part was the work and social adjustment scale (WSAS), a measure of functional impairment adapted from Mundt et al., where the participant could score on a scale of 0–8, where 0 meant not at all impaired, and eight meant very severely impaired.17 A respondent with a total WSAS score above 20 was considered to have moderately severe or severe psychopathology, scores between 10 and 20 were considered to have a significant functional impairment, but less severe clinical symptomatology, and those who scored less than ten were considered to have subclinical impairments.\n\nData were analyzed using Statistical Package for the Social Sciences (SPSS) version 26 (IBM Corp., Armonk, New York).18 The Shapiro-Wilk test assessed normality. Descriptive statistics were used to report frequencies and proportions for the categorical responses. The disparity between categorical variables was checked using the Chi-square test. The association between age and vaccine acceptance was assessed through binary logistic regression. In the case of continuous data, Mann-Whitney U and Kruskal-Wallis tests were used. Spearman's rho was used to assess the correlation between the scales, and p-value <0.05 was considered significant in all cases. All the underlying data for the present study is available without restriction.19\n\n\nResults\n\nA total of 403 health-care workers and trainees took part in our study. The mean age of the study participants was 23±4 years, and more than half of them were females (n=262, 65%). In terms of the educational level of the participants in the study, nearly half (n=211, 52.4%) of the sample population were medical students. Unmarried individuals constituted a great majority of the sample (n=361, 89.6%). In addition, 166 (41.2%) participants had a monthly family income in the range of 5,000–50,000 Nepalese Rupees. Furthermore, only 13 (3.2%) were suffering from a chronic disease, and just one (0.2%) experienced a COVID-19 related death in the family while a great majority of them (n=376, 93.3%) did not have a positive contact history with a COVID-19 patient as shown in Table 1.\n\nThe fear score on the scale ranged from 7 to 35. A higher fear scale score indicated a greater fear towards COVID-19. The mean fear score was 18.7±5. There was statistically a significant difference between the mean rank scores of males and females (226.02 vs. 157.37), with the males having a higher mean rank score than females. Nursing students had the highest mean scores (mean score= 325.98). A significant association was found between fear score with age (p-value=0.04), gender (p-value <0.01) and occupation (p-value<0.001). The participants with chronic diseases (p-value = 0.28) and those who had experienced a COVID-19 death in the family (p-value= 0.93) did not show a significant level of fear towards COVID-19. Table 2 summarizes these findings.\n\nThe obsession score on the scale ranged from 1 to 16, with 1 being not obsessed and 16 being highly obsessed with COVID-19. The mean obsession score of the study participants was 2.8±2.5. Males had a considerably higher mean rank score than females (215.39 vs. 177.13). Nursing students had the highest mean score compared to people of other occupations (289.99). Those with a positive contact history with COVID-19 scored higher than those who did not (243.78 vs. 199.00). A significant association was found between obsession score with gender (p-value=0.001), occupation (p-value < 0.001), and positive contact history of COVID-19 (p -value= 0.05). It was also observed that age (p-value=0.70), participants suffering from chronic diseases (p-value=0.36), and those who had experienced a COVID-19 death in the family (p-value=0.18) were not found to be significantly obsessed with COVID-19. Table 3 summarizes these findings.\n\nThe anxiety score on the scale ranged from 1 to 20, with 1 being not anxious and 20 being highly anxious about COVID-19. The mean anxiety score of the study participants was 0.88±1.9. Males had a considerably higher mean rank score than females (210.98 vs. 185.32). Nursing students had the highest anxiety score (273.51) compared to other sub-sections of the participants. Health-care workers and trainees suffering from chronic diseases had higher anxiety scores than those without comorbidities (273.38 vs. 199.81). The participants with a positive contact history were more anxious and scored higher than those with no contact history (256.06 vs. 198.12). A significant association was found between anxiety score with gender (p-value=0.009), occupation (p-value <0.001), those suffering from chronic diseases (p-value= 0.008), and those with a contact history (p-value= 0.002). However, factors like age (p-value=0.34) and experience of a COVID-19 death in the family (p-value=0.51) were not significantly associated with higher anxiety levels. Table 4 summarizes these findings.\n\nThe total score ranged from 1–40. A higher score predicted more significant functional impairment. Males had a nominal but significantly greater score than females (212.92 vs. 181.70). Nursing students suffered from a significantly greater functional impairment than other medical professionals (mean score=269.15, p-value<0.001). Factors like age (p-value=0.13), experience of a COVID-19 death in the family (p-value=0.66), contact history (p-value=0.81), other chronic disorders (p-value=0.18) had no significant impact on the functional impairment of the health-care workers and trainees. Table 5 summarizes these findings.\n\nFear, anxiety, obsession, and functional impairment were positively correlated, with Spearman correlation values (rho) ranging between 0.43 and 0.56; this indicated low to moderately positive but significant relationships (p-value < 0.001) as shown in Table 6.\n\n** Correlation is significant at the 0.01 level (2-tailed).\n\n\nDiscussion\n\nThe repercussions the pandemic has on mental health are predictable. Nevertheless, the brunt of the damage endured by people related to the health-care profession is unaccounted for. This study was conducted to evaluate the association of various socio-demographic characteristics of the health-care workers and trainees with various parameters of psychological distress.\n\nIn our study we found, older age to be significantly associated with a greater fear of COVID-19. This finding implicates that participants in the higher age bracket were aware of being at higher risk of contracting a severe symptomatic infection which is plausible considering that health deteriorates with the increasing age. There is increased vulnerability of contracting a fatal disease, high risk of hospitalization, and ICU admissions.20 Our findings concur with the study of Troisi et al., who reported a positive relationship between age and fear level among the health-care personnel of Italy, and the study of Yadav et al. which also reported a greater level of COVID-19 fear among the Nepalese older adults.21,22\n\nMale gender in our study showed a more significant psychological impact. Male participants in our study were found to have significantly altered levels of all four psychological distress parameters assessed in this study compared to their female counterparts. Our findings were in concordance with findings of studies by Alnazly et al. and Majeed et al., who also reported a greater psychological impact of the pandemic on the male health-care personnel in Jordan and older male adults in Pakistan.11,23 However, the extant literature reports women to have higher rates of mental health issues which contradicts the findings of our study.8,24 The differences can be attributed to the study setup, ethnicity, and the cultural norms of the society.\n\nMarital status was not found to influence psychological distress. Alnazly et al. has reported that married individuals have greater levels of fear, stress, and anxiety. Since the majority of the participants in our sample were unmarried, a relationship could not be established.23 Meraya et al. reported higher family income to be inversely associated with psychological distress.25 However, no association in our study was established between family income and psychological distress. The differences can be due to the study setup, as people related to the health-care profession were the least liable group of people to face financial issues during the pandemic.\n\nIt was also observed that among the sample population, nursing students were found to have the highest levels of all four parameters of psychological distress. Our finding was in line with the findings of Alici et al., who found that nursing students of Turkey were suffering from severe anxiety.26 In the same context, Huang et al. reported that generalized anxiety and depressive symptoms were more prevalent in the younger than in, the older population.27 The younger generation fears the pandemic's consequences on their career and has inefficient coping mechanisms. The challenges they face due to distant learning and economic instability contribute to them being more prone to develop psychological distress.27\n\nWe found in our study that a positive contact history rendered the health-care personnel to be more anxious and obsessed, which is plausible because one of the major sources of anxiety among the health-care personnel during the pandemic has been contracting an infection at their workplace and subsequently propagating infection to their families.28 The health-care authorities can overcome this concern of people related to the health-care profession by ensuring the availability of PPE and supporting and fulfilling the financial needs of the families of health-care workers and trainees, in case they get infected and have to take time off from work.\n\nOur study revealed that participants with preexisting chronic illness were significantly more anxious about the COVID-19 crisis. This is a well-established fact that people with co-morbidities have a higher propensity of contracting an infection and have poorer clinical outcomes.29 Our finding is coherent with the preexisting literature that also stated the same finding.30,31\n\nA surprising observation in our study was that, a COVID-19 death in the family was not a contributing factor to psychological distress. This is contrary to the extant literature that reports that a COVID-19 death in the family intensifies psychological distress.32 The probable cause of this difference may be the inadequate number of participants in our sample reporting a COVID-19 death in the family, resulting in inefficient reporting of the relationship.\n\nIn the wake of the pandemic, unpredictability and uncertainty are high. Coupled with the consequences of contracting a severe disease, isolation treatment, and facing the stigma of getting infected, the psychological well-being of the people is bound to suffer. With the health-care workers and trainees in the front line, the stakes for them are even higher. Moreover, a potential for hopelessness, anxiety, and suicide prevails. A possible solution to this dilemma can be appropriate psychosocial intervention by the health-care authorities, enhancing social support, and implementing better mental health management strategies for the people related to the health-care profession.\n\nThere were a few limitations in our study. Due to the cross-sectional design of the survey, causal relationships cannot be inferred. Our study is a single-center study, and the generalization of our results is limited. Our sample population was not equally distributed, and most of our participants belonged to middle age and were medical or nursing students, which could have introduced some biases in the results.\n\n\nConclusions\n\nThis study revealed various socio-demographic characteristics as risk factors for psychological stress in the health-care workers and trainees of Nepal during the COVID-19 pandemic. Enhancing social support and providing a hygienic working environment well-equipped to treat COVID-19 patients and preventing its transmission will prove to be a source of psychological relief for the people related to the health-care profession. Regular psychiatric counseling and an official platform to voice their concerns to the health-care authorities and the government will help mitigate the anxiety and fear of health-care workers and trainees and optimize their productivity.\n\n\nAuthor’s contribution\n\nAtreya A: Project Administration, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; Nepal S: Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; Menezes RG: Conceptualization, Supervision, Methodology, Visualization, Writing – Review & Editing; Shurjeel Q: Formal Analysis, Resources, Visualization, Writing – Review & Editing; Qazi S: Formal Analysis, Resources, Visualization, Writing – Review & Editing; Ram MD: Formal Analysis, Resources, Visualization, Writing – Review & Editing; Usman MS: Formal Analysis, Resources, Visualization, Writing – Review & Editing; Ghimire S: Investigation, Methodology, Writing – Review & Editing; Marhatta A: Investigation, Methodology, Writing – Review & Editing; Islam MN: Formal Analysis, Visualization, Writing – Review & Editing; Sapkota AD: Investigation, Methodology, Writing – Review & Editing; Garbuja CK: Investigation, Methodology, Writing – Review & Editing.\n\n\nData availability\n\nDRYAD: Assessment of fear, anxiety, obsession and functional Impairment of COVID-19 amongst health-care workers and trainees: A cross-sectional study in Nepal. https://doi.org/10.5061/dryad.w0vt4b8sz19\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgement\n\nNone.\n\n\nReferences\n\nHistory of 1918 Flu Pandemic\|Pandemic Influenza (Flu)\|CDC: Accessed September 1, 2021. Reference Source\n\nCascella M, Rajnik M, Aleem A, et al.: Features, Evaluation, and Treatment of Coronavirus (COVID-19).2022. Reference Source\n\nRossi R, Socci V, Talevi D, et al.: COVID-19 pandemic and lockdown measures impact on mental health among the general population in Italy. Front psychiatry. 2020; 11: 790. PubMed Abstract \| Publisher Full Text\n\nMurthy PR, Venkatesha Gupta KV, Ajith Kumar AK: Is anxiety a rising concern during COVID-19 pandemic among healthcare workers? Indian J Crit Care Med. 2020; 24(5): 369–370. PubMed Abstract \| Publisher Full Text\n\nRubin GJ, Wessely S: The psychological effects of quarantining a city. BMJ. 2020; 368: m313. PubMed Abstract \| Publisher Full Text\n\nMahase E: Coronavirus covid-19 has killed more people than SARS and MERS combined, despite lower case fatality rate. BMJ. 2020; 368: m641. PubMed Abstract \| Publisher Full Text\n\nPappa S, Ntella V, Giannakas T, et al.: Prevalence of depression, anxiety, and insomnia among healthcare workers during the COVID-19 pandemic: a systematic review and meta-analysis. Brain Behav Immun. 2020; 88: 901–907. PubMed Abstract \| Publisher Full Text\n\nMohsin SF, Agwan MA, Shaikh S, et al.: COVID-19: Fear and anxiety among healthcare workers in Saudi Arabia: a cross-sectional study. Inquiry. 2021; 58: 004695802110252. PubMed Abstract \| Publisher Full Text\n\nLabrague LJ, Santos JAA: COVID-19 anxiety among front-line nurses: predictive role of organisational support, personal resilience and social support. J Nurs Manag. 2020; 28(7): 1653–1661. PubMed Abstract \| Publisher Full Text\n\nSpoorthy MS, Pratapa SK, Mahant S: Mental health problems faced by healthcare workers due to the COVID-19 pandemic: a review. Asian J Psychiatr. 2020; 51: 102119. PubMed Abstract \| Publisher Full Text\n\nMajeed S, Schwaiger EM, Nazim A, et al.: The psychological impact of COVID-19 among Pakistani adults in Lahore. Front Public Heal. 2021; 9: 578366. PubMed Abstract \| Publisher Full Text\n\nMahmood QK, Jafree SR, Jalil A, et al.: Anxiety amongst physicians during COVID-19: cross-sectional study in Pakistan. BMC Public Health. 2021; 21(1): 118. PubMed Abstract \| Publisher Full Text\n\nDean A, Sullivan K, Soe K: OpenEpi: Open Source Epidemiologic Statistics for Public Health.2013. Reference Source\n\nAhorsu DK, Lin C-Y, Imani V, et al.: The fear of COVID-19 scale: development and initial validation. Int J Ment Health Addict. March 27, 2020; 1–9. PubMed Abstract \| Publisher Full Text\n\nLee SA: How much “thinking” about COVID-19 is clinically dysfunctional? Brain Behav Immun. 2020; 87: 97–98. PubMed Abstract \| Publisher Full Text\n\nLee SA: Coronavirus anxiety scale: a brief mental health screener for COVID-19 related anxiety. Death Stud. 2020; 44(7): 393–401. PubMed Abstract \| Publisher Full Text\n\nMundt JC, Marks IM, Shear MK, et al.: The work and social adjustment scale: a simple measure of impairment in functioning. Br J Psychiatry. 2002; 180(12): 461–464. PubMed Abstract \| Publisher Full Text\n\nIBM SPSS Statistics, RRID:SCR_019096.\n\nAtreya A, Nepal S, Menezes RG, et al.: Dataset: Assessment of fear, anxiety, obsession and functional impairment of COVID-19 amongst health-care workers and trainees: a cross-sectional study in Nepal. Dryad, Dataset. 2021. Publisher Full Text\n\nCOVID-19 Strategy update: Accessed November 1, 2021. Reference Source\n\nTroisi A, Nanni RC, Riconi A, et al.: Fear of COVID-19 among healthcare workers: the role of neuroticism and fearful attachment. J Clin Med. 2021; 10(19): 4358. PubMed Abstract \| Publisher Full Text\n\nYadav UN, Yadav OP, Singh DR, et al.: Perceived fear of COVID-19 and its associated factors among Nepalese older adults in eastern Nepal: a cross-sectional study. PLoS One. 2021; 16(7): e0254825. PubMed Abstract \| Publisher Full Text\n\nAlnazly E, Khraisat OM, Al-Bashaireh AM, et al.: Anxiety, depression, stress, fear and social support during COVID-19 pandemic among Jordanian healthcare workers. PLoS One. 2021; 16(3): e0247679. PubMed Abstract \| Publisher Full Text\n\nAlkhamees AA, Alrashed SA, Alzunaydi AA, et al.: The psychological impact of COVID-19 pandemic on the general population of Saudi Arabia. Compr Psychiatry. 2020; 102: 152192. PubMed Abstract \| Publisher Full Text\n\nMeraya AM, Syed MH, Yasmeen A, et al.: COVID-19 related psychological distress and fears among mothers and pregnant women in Saudi Arabia. PLoS One. 2021; 16(8): e0256597. PubMed Abstract \| Publisher Full Text\n\nKuru Alici N, Ozturk CE: Anxiety and fear of COVID-19 among nursing students during the COVID-19 pandemic: a descriptive correlation study. Perspect Psychiatr Care. May 20, 2021; 58: 141–148. PubMed Abstract \| Publisher Full Text\n\nHuang L, Lei W, Xu F, et al.: Emotional responses and coping strategies in nurses and nursing students during Covid-19 outbreak: a comparative study. PLoS One. 2020; 15(8): e0237303. PubMed Abstract \| Publisher Full Text\n\nShanafelt T, Ripp J, Trockel M: Understanding and addressing sources of anxiety among health care professionals during the COVID-19 pandemic. JAMA. 2020; 323(21): 2133–2134. PubMed Abstract \| Publisher Full Text\n\nZhou Y, Yang Q, Chi J, et al.: Comorbidities and the risk of severe or fatal outcomes associated with coronavirus disease 2019: a systematic review and meta-analysis. Int J Infect Dis. 2020; 99: 47–56. PubMed Abstract \| Publisher Full Text\n\nFeter N, Caputo EL, Doring IR, et al.: Sharp increase in depression and anxiety among Brazilian adults during the COVID-19 pandemic: findings from the PAMPA cohort. Public Health. 2021; 190: 101–107. PubMed Abstract \| Publisher Full Text\n\nÖzdin S, Bayrak ÖŞ: Levels and predictors of anxiety, depression and health anxiety during COVID-19 pandemic in Turkish society: the importance of gender. Int J Soc Psychiatry. 2020; 66(5): 504–511. PubMed Abstract \| Publisher Full Text\n\nJoaquim RM, Pinto ALCB, Guatimosim RF, et al.: Bereavement and psychological distress during COVID-19 pandemics: the impact of death experience on mental health. Curr Res Behav Sci. 2021; 2: 100019. Publisher Full Text" }	[ { "id": "122754", "date": "28 Feb 2022", "name": "Bipin Adhikari", "expertise": [ "Reviewer Expertise Social Science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAtreya and Samata et al. have conducted an important study exploring fear, anxiety, obsession and functional impairment of COVID-19 among health care workers and trainees. The manuscript is very well written and I have few suggestions to improve its scope.\nCan you add rationales for studying about these constructs and the instruments somewhere in methods? This will clarify the need. And few lines on why quantitative measurement was deemed appropriate, and would qualitative measurement be a limitation? If so, please add that in the discussion as well.\n\nWhile the study has delved into the stated constructs among health care workers, it would be good to compare and discuss with the studies from Nepal (or elsewhere) who have explored similar constructs among different population (for e.g. in general population) using other methods, to show how that differ and the implications. A bit more specifically, you could compare the constructs between the health care workers and the non-health care workers as well. Please explore.\n\nYou have discussed quite well the findings and the use of scales in various constructs. I recommend you could relate your findings more at proximal level, e.g. how that contributes (affects) the health care workers’ preparedness or even health system preparedness? Can we stretch the implications to pandemic preparedness or disaster preparedness for current and future as well?\n\nAlso, you can situate your findings in the current context of Nepal’s health system (federal system) how this pandemic or its outcome may have been influenced by lack of clarity and poor delineation in responsibilities between various tiers. These are again implications to enhance the scope of your findings. Please explore more literature around these themes.\n\nSomewhere, can you add a brief explanation about the research site, how is it different to others and what are the social, cultural and even geographical barriers that may (or could) add to the constructs you are measuring?\n\nIs the work 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": "8012", "date": "01 Apr 2022", "name": "Alok Atreya", "role": "Author Response", "response": "Atreya and Samata et al. have conducted an important study exploring fear, anxiety, obsession and functional impairment of COVID-19 among health care workers and trainees. The manuscript is very well written and I have few suggestions to improve its scope. We appreciate the positive comments by the reviewer. Can you add rationales for studying about these constructs and the instruments somewhere in methods? This will clarify the need. And few lines on why quantitative measurement was deemed appropriate, and would qualitative measurement be a limitation? If so, please add that in the discussion as well. We agree with the reviewer that a qualitative study design with in-depth interview would have provided personal experience of the participants and also helped identify the factors affecting mental health, due to norms of social distancing in the middle of the pandemic, this survey was conducted online with the help of pre-validated questionnaires. We have highlighted this in the discussion section is suggested. Discussion, Page 17: “A qualitative study design with in-depth interview would have provided personal experience of the participants and also helped identify the factors affecting mental health. However, due to norms of social distancing in the middle of the pandemic, this survey was conducted online with the help of pre-validated questionnaires.” While the study has delved into the stated constructs among health care workers, it would be good to compare and discuss with the studies from Nepal (or elsewhere) who have explored similar constructs among different population (for e.g. in general population) using other methods, to show how that differ and the implications. A bit more specifically, you could compare the constructs between the healthcare workers and the non-health care workers as well. Please explore. Thank you for the suggestion. We have extensively compared findings from our study with other studies that assessed healthcare workers and other populations. The authors feel that comparison of different constructs/methodologies was not within the scope of the current study – and have thus not discussed that aspect Discussion, Page 15: “Our findings concur with the study of Troisi et al., who reported a positive relationship between age and fear level among the health-care personnel of Italy, and studies conducted amongst healthcare workers and the general population of Nepal – which also reported a positive association between age and fear of COVID-19. Male gender in our study showed a more significant psychological impact. Male participants in our study were found to have significantly altered levels of all four psychological distress parameters assessed in this study compared to their female counterparts. Our findings wer in concordance with findings of studies by Alnazly et al. and Majeed et al., who also reported a greater psychological impact of the pandemic on the male health-care personnel in Jordan and older male adults in Pakistan. However, the extant literature reports women to have higher rates of mental health issues which contradicts the findings of our study. The differences can be attributed to the study setup, ethnicity, and the cultural norms of the society. Marital status was not found to influence psychological distress. Alnazly et al. has reported that married individuals have greater levels of fear, stress, and anxiety. Since the majority of the participants in our sample were unmarried, a relationship could not be established. Meraya et al. reported higher family income to be inversely associated with psychological distress. However, no association in our study was established between family income and psychological distress. The differences can be due to the study setup, as people related to the health-care profession were the least liable group of people to face financial issues during the pandemic. In contrary, low socio-economic status was a driving factor for poor mental health among returning migrant laborers in Nepal. It was also observed that among the sample population, nursing students were found to have the highest levels of all four parameters of psychological distress. Our finding was in line with the findings of Alici et al., who found that nursing students of Turkey were suffering from severe anxiety. In the same context, Huang et al. reported that generalized anxiety and depressive symptoms were more prevalent in the younger than in, the older population. The younger generation fears the pandemic's consequences on their career and has inefficient coping mechanisms. The challenges they face due to distant learning and economic instability contribute to them being more prone to develop psychological distress.” You have discussed quite well the findings and the use of scales in various constructs. I recommend you could relate your findings more at proximal level, e.g. how that contributes (affects) the health care workers’ preparedness or even health system preparedness? Can we stretch the implications to pandemic preparedness or disaster preparedness for current and future as well? Also, you can situate your findings in the current context of Nepal’s health system (federal system) how this pandemic or its outcome may have been influenced by lack of clarity and poor delineation in responsibilities between various tiers. These are again implications to enhance the scope of your findings. Please explore more literature around these themes. We have positively taken this suggestion and have now discussed the poor preparedness of the Nepalese healthcare system in managing natural disasters and disease outbreaks. Discussion, page 16: “Nepal is a lower-middle income country in South Asia with a suboptimal health system preparedness for natural disasters and disease outbreaks.32 There is a lack of robust surveillance system, diagnostic facilities and management infrastructure.32 The lack of coordination in the three tiers of governance was evident during the pandemic, specifically lack of healthcare workers, inadequate supply and management of logistics, and diagnostic facilities.32 The reason for anxiety and depression among the healthcare workers were different from the general public in many aspects. One of the main reasons in the context of Nepal was, the frontline of health-care workers were dutybound amidst the lack of logistics and health safety concerns” Somewhere, can you add a brief explanation about the research site, how is it different to others and what are the social, cultural and even geographical barriers that may (or could) add to the constructs you are measuring? A brief detail to the research site is added. Methods, page 5: “This cross-sectional study was carried out among the staff and students of Lumbini Medical College (LMC) Teaching Hospital, Palpa, Nepal, during the COVID-19 pandemic (August 2020). The sample size computed using OpenEpi 13 was 384, after considering a confidence level of 95% and a frequency outcome factor of 50%. For more robust results, we included 406 participants in the survey, of which 3 participants who did not consent to participate were excluded from the study. The cohort of health-care workers and trainees in the present study included doctors, nurses, other health-care staff, medical students, and nursing students. Although the study site is located in Palpa, which is a mountainous district in Lumbini Province of Nepal, the health-care workers and trainees are from diverse social and cultural backgrounds, and different geographical locations.”" } ] }, { "id": "122749", "date": "07 Mar 2022", "name": "Swapna Talluri", "expertise": [ "Reviewer Expertise 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\nThe authors used the ‘coronavirus anxiety scale’, a tool developed by Lee et al., 2020. Marital status was not found to have any influence on psychological distress in this study. However, majority of the participants in this study are unmarried. So a relationship with marriage could not be established in this study. This explanation is reasonable.\nThe authors state that death in the family is not contributory to psychological distress in contradiction to other studies, the likely explanation for this discrepancy is the low number of participants with deaths in the family. This is a reasonable explanation.\nThe study design and statistical methodology is appropriate. The participants were anonymized which is important for a study of this nature. Appropriate scales for anxiety were utilized.\nThe study results were concordant with other studies except for marital status and death in the family. The authors explanation for the discrepancy is satisfactory.\n\nIs the work 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/11-119
https://f1000research.com/articles/11-444/v1	20 Apr 22	{ "type": "Research Article", "title": "Risk prediction model for 24-hour mortality in preterm infants using lactate and blood gas analysis: A machine learning approach and retrospective cohort study", "authors": [ "Felipe Yu Matsushita", "Vera Lúcia Jornada Krebs", "Werther Brunow de Carvalho", "Vera Lúcia Jornada Krebs", "Werther Brunow de Carvalho" ], "abstract": "Background: This study aimed to evaluate the performance of machine learning algorithms using lactate and arterial blood gas parameters to predict the imminent risk of death in extremely low birth weight infants. Methods: A retrospective cohort study analyzing preterm infants with birth weight less than 1000 grams in a single-center tertiary neonatal intensive care unit in São Paulo, Brazil, between 2012 and 2017 was carried out. We included all infants with at least one arterial blood gas analysis with paired serum lactate. To assess 24-hour mortality risk, we conducted three machine learning algorithms (Logistic Regression, Extreme Gradient Boosting, and AutoML Tables). Results: We analyzed 1932 blood gas samples with matched lactate measurements. Our study population had a median gestational age of 27.1 (26 – 29.1) weeks and a median birth weight of 746 (600 – 880) grams. The Extreme Gradient Boosting model with lactate achieved the highest area under the receiver operating characteristic (AUROC) of 0.898. Base excess, lactate, and pH were, in order of importance, the most important features associated with 24-hour mortality. Conclusions: Incorporating lactate and blood gas samples into real-time mortality predictive models may aid to identify those preterm infants with a higher risk of death.", "keywords": [ "preterm", "machine learning", "artificial intelligence", "prediction", "mortality" ], "content": "Introduction\n\nDespite the fact that child mortality has decreased significantly in recent decades, neonatal mortality remains the major contributor1 and prematurity is the largest direct cause of death in this population.2 More than half of neonatal deaths occur within the first three days of life.3 In this context, several predictive models use prenatal and immediately post-natal factors to predict death. However, in the neonatal intensive care unit (NICU) the patient’s condition changes over time. Moreover, more than 30% of neonatal deaths occur after three days of life.3 Real-time features, rather than non-modifiable baseline variables, may provide a more customized evaluation of mortality risk.\n\nPredictive models are becoming increasingly popular in clinical research. A number of conditions have been predicted using machine learning techniques, from mortality in intensive care units to morbidities, such as acute kidney injury, septic shock, and heart failure.4 The models that are now available for assessing neonatal mortality risk are mostly focused on NICU admission.5–9 Unfortunately, few models use objective criteria to evaluate mortality risk in real-time.\n\nTo address this knowledge gap, we conducted a study to compare the effectiveness of machine learning algorithms for predicting NICU mortality using objective laboratorial features. The objective of this study was to evaluate serum lactate and blood gas analysis as a predictor of mortality in extremely low birth weight infants using machine learning algorithms.\n\n\nMethods\n\nThe study protocol was approved by the institutional ethics committee – Comitê de Ética do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, approval number: CAAE 15762719.6.0000.0068. The requirement for informed consent was waived by the committee given the retrospective nature of the study.\n\nWe analyzed data of extremely low birth weight infants born at a single-center tertiary neonatal intensive care unit in São Paulo, Brazil, between 2012 and 2017. The data collection and the methods have already been published in our previous study.10 All data were obtained from electronic medical records of each patient using specific keywords related to clinical and laboratorial parameters in December 2019 and extracted to a CSV file.22 All neonates with birth weight lower than 1000 grams born between 2012 and 2017 and who had at least one arterial blood gas analysis with paired serum lactate level during neonatal intensive care stay were included. Neonates with severe malformation, complex congenital heart disease, or transferred to another unit before discharge were excluded. Baseline characteristics included birth gestational age (in weeks), birth weight (grams), CRIB II score (Clinical Risk Index for Babies), small for gestational age (defined as birth weight < p10 Fenton growth scale), female gender, 5-minute APGAR score, vaginal birth, twin birth, antenatal corticoid, endotracheal intubation in the delivery room, epinephrine necessity in the delivery room, chorioamnionitis, and the lowest temperature in the first 12 hours of life. Serum lactate levels are presented in mmoL/L.\n\nA total of seven feasible parameters were introduced into the machine learning algorithms. These parameters included blood gas analysis features (pH, pCO2, HCO3, base excess), serum lactate, and general characteristics (days of life and corrected gestational age at the time of laboratory measurement).\n\nWe compared the performance of three machine learning methods to assess 24-hour mortality risk: Logistic Regression,11 Extreme Gradient Boosting,12 and AutoML Tables.13 Patient data were randomly divided into two subsets: a training subset (80%) for hyperparameter tuning to create a plausible model, and a validation subset (20%) for testing the model’s performance. All data with missing values were excluded from the analysis. To select the optimal model, we performed hyperparameter tuning for the Extreme Gradient Boosting model. Hyperparameters are a set of extra parameters that must be established before the learning process to improve the algorithm performance.\n\nLogistic regression classification is a machine learning technique that predicts outcomes using dependent variables and a logit function. It is frequently used as a baseline comparison for binary classifications because it is not only simple to construct but also capable of high performance.11 Extreme Gradient Boosting is an ensemble machine learning technique of weak prediction models. These weak models are added one at a time and fit to correct the prediction errors made by prior models.14 For Extreme Gradient Boosting the following hyperparameters were used: LEARN_RATE = 0.1, BOOSTER_TYPE = ‘GBTREE’, MAX_TREE_DEPTH = 3, SUBSAMPLE = 0.85, EARLY_STOP = TRUE. AutoML Tables is a Google Cloud Platform feature that automatically starts training for multiple model architectures (Linear, Feedforward deep neural network, Gradient Boosted Decision Tree, AdaNet, Ensembles), and it determines the best model. The primary outcome was death within 24 hours after collecting arterial blood gas and lactate.\n\nContinuous variables were tested for normality using Kolmogorov-Smirnov test. To compare demographic characteristics, we used chi-square for categorical variables and Mann-Whitney test for continuous variables. After the optimal hyperparameters were determined for each ML algorithm, we calculated the area under the receiver operating characteristics (AUROC), accuracy, precision, and recall. All analyses were conducted using Python version 3.6.915 and Google Cloud Platform. All patients with missing data were excluded from the study.\n\n\nResults\n\nWe identified a total of 257 neonates who had at least one blood gas analysis and a paired serum lactate during neonatal intensive care unit stay from 2012 to 2017. Eleven patients were excluded because of missing data. Baseline characteristics are presented in Table 1. The median gestational age was 27.1 (26 – 29.1) weeks and the median birth weight was 746 (600 – 880) grams. We found 1932 blood gas samples with corresponding serum lactate levels.\n\nAll models demonstrated good accuracy (91 – 94%) and AUROC results (0.807 – 0.898) when using blood gas measurements with lactate as features. However, their recall (9-29%) was low. The Extreme Gradient Boosting algorithm obtained the highest AUROC score (0.898), accuracy (94.1%), and precision (87.5%) (Table 2). We then used AutoML Tables to determine the importance of each feature associated with 24-hour mortality, and the top three features were, in order: base excess, lactate, and pH levels (Figure 1).\n\nAUROC, area under the receiver operating characteristic.\n\nBE: Base excess; cGA: Corrected gestational age; BIC: HCO3; DOL: Days of life.\n\nWhen lactate was removed as a feature in machine learning models, the AUROC score of the Extreme Gradient Boosting model dropped considerably (0.807) (Table 3).\n\nAUROC, area under the receiver operating characteristic.\n\n\nDiscussion\n\nOur research found that utilizing blood gas samples and lactate, the Extreme Gradient Boosting algorithm may predict 24-hour mortality in extremely low birth weight infants. Lactate can be used to improve predictive models.\n\nIn the NICU, an accurate mortality estimate is a valuable tool that assists healthcare providers. However, most mortality predictive models in preterm infants are limited to NICU admission. For assessing the mortality risk in NICU admission, the Score for Neonatal Acute Physiology Perinatal Extension-II (SNAPPE-II) includes vital signs, laboratory, and baseline characteristics.5 The Clinical Risk for Infants and Babies (CRIB-II) score considers sex, birth weight, gestational age, temperature, and base excess to assess the mortality risk upon NICU admission. TRIPS-II,7 NMR-2000,8 and PISA9 are other recent models that predict mortality after admission. However, dynamic events in the critical care unit may have an impact on the initial risk. Combining baseline characteristics and real-time features provides a more individualized assessment of mortality risk that evolves as the patient’s health changes. To overcome this problem, Jaskari J et al.,16 Lee J et al.,17 and Feng J et al.18 created predictive mortality models based on vital sign data collected during the NICU stay. However, vital signs such as respiratory rate, heart rate, and blood pressure may vary amongst neonates and there is no consensus on what constitutes a normal reference range.19,20 Furthermore, in Lee J et al. study, baseline variables (birth weight and gestational age at birth) were more important than vital sign readings.\n\nLactate and blood gas values can be acquired quickly and is currently a standard clinical practice in intensive care units. The lack of bias from the examiner which contributes to objectivity is an advantage over clinical examination.21 As a result, our research combines the predictive capability of machine learning models with the objectivity of a blood gas analysis, which is a simple readily available, and widely used test. Our findings imply that machine learning models based on lactate and blood gas indicators appears to be superior to logistic regression classifiers in predicting 24-hour mortality in extremely low birth weight infants. To our knowledge, this is the first study to include real-time objective laboratorial features to predict death in preterm infants.\n\nOur research has some limitations, which should be noted. First, this is a single-center retrospective study, and generalizing our findings is challenging. Second, larger datasets assist machine learning predictive models, therefore a larger study is required. Third, it is worth noting that using blood gas analysis to forecast mortality is a highly unbalanced problem; in other words, there are far more blood gas samples than events (death). In unbalanced data, we can acquire a high accuracy and AUROC score by simply predicting that all observations belong to the majority class. Lastly, it is important to note that our models had very high precision with low recall, and it should not be used for screening purposes.\n\n\nConclusions\n\nIncorporating lactate and blood gas measurements into mortality predictive models may improve real-time risk stratification in preterm infants. Traditional logistic classification models appear to be outperformed by more robust machine learning algorithms. Extreme Gradient Boosting models could be used as a support tool for clinical risk stratification of extremely low birth weight infants in the neonatal intensive care unit.\n\n\nData availability\n\nAs this study involved extracting data from patient records, these records/patient files are considered the raw, source data. The raw data (patient files) are not available to readers and reviewers for data protection.\n\nHarvard Dataverse: Blood Gas – Preterm. https://doi.org/10.7910/DVN/LFRNJE.22\n\nThis project contains the following underlying data: Lactate_Mortality – Sheet1.csv (contains 1932 serum lactate and blood gas analysis along with days of life and corrected gestational age at the time of measurement).\n\nHarvard Dataverse: Blood Gas – Preterm. https://doi.org/10.7910/DVN/LFRNJE.22\n\nThis project contains the following extended data: Data key.docx (contains key to make data more accessible)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nUnited Nations Inter-agency Group for Child Mortality Estimation: Levels & Trends in child mortality: 2020 report. Report 2020.2020; 1–56 p.\n\nHug L, Alexander M, You D, et al.: National, regional, and global levels and trends in neonatal mortality between 1990 and 2017, with scenario-based projections to 2030: a systematic analysis. Lancet Glob. Heal. 2019; 7(6): e710–e720. PubMed Abstract \| Publisher Full Text\n\nSankar MJ, Natarajan CK, Das RR, et al.: When do newborns die? A systematic review of timing of overall and cause-specific neonatal deaths in developing countries. J. Perinatol. 2016; 36(S1): S1–S11. PubMed Abstract \| Publisher Full Text\n\nSubudhi S, Verma A, Patel AB, et al.: Comparing machine learning algorithms for predicting ICU admission and mortality in COVID-19. npj Digit. Med. 2021; 4(1): 1–7. Publisher Full Text\n\nHarsha SS, Archana BR: SNAPPE-II (score for neonatal acute physiology with perinatal extension-II) in predicting mortality and morbidity in NICU. J. Clin. Diagnostic Res. 2015; 9(10): SC10–SC12. Publisher Full Text\n\nParry G, Tucker J, Tarnow-Mordi W: CRIB II: an update of the clinical risk index for babies score For personal use. Only reproduce with permission from The Lancet Publishing Group; 2003; vol. 361: 1789–1791.\n\nLee S, Aziz K, Dunn M, et al.: Transport risk index of physiologic stability, version II (TRIPS-II): A simple and practical neonatal illness severity score. Am. J. Perinatol. 2013; 30(5): 395–400. PubMed Abstract \| Publisher Full Text\n\nMedvedev MM, Brotherton H, Gai A, et al.: Development and validation of a simplified score to predict neonatal mortality risk among neonates weighing 2000 g or less (NMR-2000): an analysis using data from the UK and The Gambia. Lancet Child Adolesc. Heal. 2020; 4(4): 299–311. PubMed Abstract \| Publisher Full Text\n\nPodda M, Bacciu D, Micheli A, et al.: A machine learning approach to estimating preterm infants survival: development of the Preterm Infants Survival Assessment (PISA) predictor. Sci. Rep. 2018; 8(1): 1–9.\n\nMatsushita F, Krebs V, Ferraro A, et al.: Early fluid overload is associated with mortality and prolonged mechanical ventilation in extremely low birth weight infants. Eur. J. Pediatr. 2020; 179(11): 1665–1671. PubMed Abstract \| Publisher Full Text\n\nLaValley MP: Logistic regression. Circulation. 2008; 117(18): 2395–2399. Publisher Full Text\n\nChen T, He T, Benesty M: XGBoost: eXtreme Gradient Boosting. R Packag version 071-2.2018; 1–4.\n\nGoogle: Cloud AutoML.Reference Source\n\nFriedman JH: Greedy function approximation: A gradient boosting machine. Ann. Stat. 2001; 29(5): 1189–1232. Publisher Full Text\n\nPedregosa F, Grisel O, Weiss R, et al.: Scikit-learn: Machine Learning in Python. J. Mach. Learn. Res. 2011; 12: 2825–2830.\n\nJaskari J, Myllarinen J, Leskinen M, et al.: Machine Learning Methods for Neonatal Mortality and Morbidity Classification. IEEE Access. 2020; 8: 123347–123358. Publisher Full Text\n\nLee J, Cai J, Li F, et al.: Predicting mortality risk for preterm infants using random forest. Sci. Rep. 2021; 46(1): 1–6. Publisher Full Text\n\nFeng J, Lee J, Vesoulis ZA, et al.: Predicting mortality risk for preterm infants using deep learning models with time-series vital sign data. npj Digit. Med. 2021; 4(1): 1–8. Publisher Full Text\n\nPaliwoda M, New K, Davies M, et al.: Physiological vital sign ranges in newborns from 34 weeks gestation: A systematic review. Int. J. Nurs. Stud. 2018; 77(May 2017): 81–90. PubMed Abstract \| Publisher Full Text\n\nManja V, Lakshminrusimha S, Cook DJ: Oxygen saturation targetrange for extremely preterm infants: A systematic review and meta-analysis. JAMA Pediatr. 2015; 169(4): 332–340. PubMed Abstract \| Publisher Full Text\n\nvon Auenmueller KI , Christ M, Sasko BM, et al.: The Value of Arterial Blood Gas Parameters for Prediction of Mortality in Survivors of Out-of-hospital Cardiac Arrest. J. Emerg. Trauma Shock. . 2017; 10(3): 134–139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu Matsushita F: Blood Gas - Preterm.2022. Harvard Dataverse, V2, UNF:6:cVcTuPkuzNi4QItQBkW1RA==[fileUNF]. Publisher Full Text" }	[ { "id": "201605", "date": "29 Nov 2023", "name": "Thomas Wood", "expertise": [ "Reviewer Expertise Neonatal neuroscience", "neonatal outcome prediction" ], "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 address an important problem, but I have several concerns regarding how the methods were performed/reported:\nThe idea of “real-time” mortality risk is mentioned throughout the manuscript, but it is not clear if this analysis incorporated blood gas/lactate data as a time-dependent component of the prediction models. For example, if 1932 blood gas samples were included from 257 infants, how was the test:train split performed – by infant or by blood gas sample? How were multiple blood gas samples from the same infant handled, how did the prediction update with each new blood gas measurement from the same infant, and how did the prediction window update with each new blood gas? This needs to be more clearly explained. Time series data have been used for mortality risk prediction in preterm infants previously (e.g. Feng et al., npj Digital Medicine 2021), but I’m not sure that’s what was done here.\n\nConsidering the relatively low sample size, why not do cross-validated predictions so that each infant can be used for both training and testing across 5 or 10 folds?\n\nWhy were the most important features only extracted from AutoML Tables? This can also be done with Extreme Gradient Boosting models.\n\nWhy were some of the variables from Table 1 such as sex, multiple gestation, and lowest temperature temperature not included as predictors? Especially considering that they were different between survivors and non-survivors.\n\nHow was chorioamnionitis determined?\n\nSome more discussion of the utility of this kind of model is needed. For instance, it is probably worth mentioning that high precision is much more critical than high recall for this kind of model - if a high predicted risk of mortality is used to redirect care, then the most important thing is to avoid false positives. More detail on the goal of the study, how the methods align with the goal (e.g., is this truly a real-time prediction model?), and the context of the outcome is needed for both the introduction and discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Partly", "responses": [] }, { "id": "211184", "date": "12 Dec 2023", "name": "Fu-Sheng Chou", "expertise": [ "Reviewer Expertise Neonatal respiratory outcome prediction. Growth chart development for preterm infants." ], "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 their research, Matsushita et al. created several machine learning models aiming to predict 24-hour mortality in extremely low birth weight infants, utilizing arterial blood gas and lactate data from 2012 to 2017. While the study addresses a crucial topic, predicting severe outcomes in neonatal care, I find two significant issues with the research:\nLack of Validation and Incomplete Model Performance Reporting: One major concern lies in the absence of validation techniques and the incomplete reporting of model performance. Without validation, the reliability and accuracy of the developed models remain uncertain. Furthermore, the study lacks comprehensive details on how well these models performed, leaving gaps in understanding their effectiveness. Inadequate Rationale for Feature Selection: Another concern pertains to the rationale behind selecting specific features. Notably, the inclusion of calculated values like bicarbonate and base excess in the model raises questions about their necessity. Additionally, the exclusion of demographic and perinatal features lacks explanation. Understanding why certain features were included while others were omitted is crucial for the transparency and credibility of the study. For instance, clarifying why calculated values were preferred over demographic and perinatal data would enhance the study's overall rationale.\nAddressing these concerns would not only strengthen the study's methodology but also contribute significantly to the credibility and applicability of the findings in neonatal care.\n\nIs the work 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/11-444
https://f1000research.com/articles/11-442/v1	20 Apr 22	{ "type": "Brief Report", "title": "High-quality genome assembly of a Pestalotiopsis fungus using DIY-friendly methods", "authors": [ "Joshua L. McGinnis", "Daniel J. Giguere" ], "abstract": "Of the millions of fungal species estimated to exist, about 100,000 have been identified, and only approximately 3000 of those have representative genome assemblies available. Here, we isolated a wild species of Pestalotiopsis from the Los Angeles area, extracted DNA in a low-cost environment (e.g., home lab), and generated a high-quality genome assembly using the low-cost Oxford Nanopore MinION sequencing platform. We found that Pestalotiopsis has a genome composed of 7 nuclear chromosomes, comprising 47.7 megabases. Using this genome, we perform a multi-locus phylogenetic analysis and finally, we discuss how this project (costing $300) demonstrates the increased accessibility of whole genome sequencing.", "keywords": [ "Pestalotiopsis", "genome assembly", "nanopore sequencing", "diy" ], "content": "Introduction\n\nAccessibility to genome sequencing has typically been limited to large, well-funded institutions due to large capital requirements of DNA sequencing platforms, like many high-throughput Illumina platforms that cost hundreds of thousands of dollars to obtain. However, the recently developed MinION nanopore sequencing platform by Oxford Nanopore Technologies provides both the hardware and reagents needed for genome sequencing for $1000 USD, reducing the financial barrier to DNA sequencing. Other equipment like thermocyclers are also becoming more portable and affordable,1 reducing the barrier for “do-it-yourself” (DIY) biologists to perform genetic analysis and engineering projects.2 The distribution of resources and knowledge between DIY biologists and institutional researchers can advance the rate of development to research and provide lower-cost approaches.3\n\nA significant portion of the approximately 22,000 bioactive microbial metabolites are of fungal origin.4 Furthermore, of the estimated three million species of fungi, only about 100,000 species have been identified.5 This poses an opportunity to discover and characterize new metabolite-producing fungi that may lead to the commercialization of novel pharmaceuticals, pigments, enzymes and food-production platforms. Pestalotiopsis, the focus of this study, is a diverse genus of Ascomycete fungi, often pathogenic to plants6 and commonly isolated as endophytes.7 Pestalotiopsis is known to possess potentially valuable metabolic capabilities, including the production of bioactive secondary metabolites like taxol8 and the ability to biodegrade polyurethane.9\n\nThe purpose of this work is 1) to better understand the genome of Pestalotiopsis and 2) to demonstrate that high-quality eukaryotic genome sequencing is now accessible in a DIY environment. To do this, we built a high-quality genome of Pestalotiopsis using nanopore sequencing, highlighting the increased accessibility and affordability of genome sequencing for amateur biologists and early-stage startups.\n\n\nMethods\n\nA wild-type strain of Pestalotiopsis was isolated from a wild-type clone of a Laetiporus sp. found growing on an aged hardwood stump at Whiting Ranch Wilderness Park, Orange County, California, United States.\n\nThe isolate was maintained on Petri dishes (eBay) containing Malt Extract Agar (MEA) (Amazon.com) media at room temperature in natural indoor ambient light. Microscopic images are shown in Figure 1.\n\nA. Conidiomata B. Conidia C. Mature colony on MEA.\n\nA sterile toothpick was used to aseptically transfer a small swath (2-4 mm) of fresh, non-sporulating Pestalotiopsis mycelium from the surface of solid MEA culture into a 0.2 ml microcentrifuge tube containing 30 μl of 0.5 M NaOH (Home Science Tools, SKU: UN1823, MT) lysing buffer.10\n\nA makeshift pestle (Figure 2) was crafted from a 200 μl long pipette tip and quickly melted with the flame from a lighter followed by immediate application to a flat surface sanitized with 70% isopropyl alcohol, in order to form a small flat non-porous tip roughly 1 mm in size. Using the makeshift pestle, the tissue was crushed and macerated in the tube for one to three minutes, or until the tissue was homogeneously suspended in the lysis buffer and no clumps were observed.\n\nA. DIY Magnetic Bead Separator Rack constructed with neodymium magnets; B. Vibrating Back Massager as DIY Vortex Mixer; C. Makeshift Pestle generated by melting a pipette tip with over a flame and applying the melted tip to a sterile solid surface before hardening into a flat blunt-end.\n\nSample 1 (5 μl), 1 kb ladder, Sample 1 (10 μl), 1 kb ladder, Sample 2 (5 μl), 1 kb ladder, Sample 2 (10 μl).\n\nThe tube was allowed to sit for 10 min at room temperature. 150 μl of 100 mM Tris, Boric acid, EDTA (TBE) pH 8.3 buffer (Bioland Scientific, TBE01, CA) was added and the tube was incubated in a thermocycler heat block for 10 minutes at 95 °C. The tissue debris was then pelleted for five minutes at 5000 × g (10,000 rpm) leaving the genomic DNA used as the template for the PCR reaction left remaining in the supernatant.\n\nThe PCR reaction was prepared in a separate 0.2 ml microcentrifuge tube by mixing 9 μl of sterile distilled water, 0.4 μl of 10 μM forward primer, 0.4 μl of 10 μM reverse primer, and 11 μl of 2× Taq PCR Premix (Bioland Scientific, TP01-00, CA). Template DNA was added to the mix by drawing off 1 μl of supernatant from the top of the tube used in the previous lysis step.\n\nCommon fungal DNA barcoding primers ITS1-F (5′-CTTGGTCATTTAGAGGAAGTAA-3′)11 and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)12 were used to amplify the internal transcribed spacer (ITS) region of the fungal ribosomal encoding genes. The cycling conditions were as follows: an initial denaturation step (2 min at 94 °C), 35 cycles of denaturation (94 °C for 30 secs), annealing (55 °C for 2 min) and elongation (72 °C for 1 min), and a final elongation step (72 °C for 10 min).\n\nSuccessful PCR amplification of the barcoding locus was confirmed via electrophoresis using the MiniPCR blueGel system (Amplyus, MA) operating at a fixed 48V.\n\nA total of 5 μl of PCR product was mixed with 1 μl of 6× DNA loading buffer (Bioland Scientific, DSB01-01, CA) and loaded into a 1% TBE gel. The gel was run for approximately 30 minutes until a bright band was visible and a size of 400-700 bp approximated by comparison with a 50 bp DNA ladder (Bioland Scientific, DM02-02, CA).\n\nA total of 15 μl of PCR product was transferred to a new 0.2 ml microcentrifuge, tightly wrapped in parafilm and sent through USPS Priority mail in a small padded envelop for forward-read Sanger sequencing (MCLab, Oakland, CA).\n\nThe resulting chromatogram was analyzed, trimmed and error-corrected by hand using SnapGene Viewer (GSL Biotech LLC, CA). The resulting sequence was then searched against NCBI GenBank using BLAST and aligned against type and ex-type sequences.\n\nFungal tissue preparation\n\nA double-layer media plate was prepared using a 9 mm Petri dish with a 30 ml Malt Extract Agar (MEA) bottom-layer and a 20 ml Potato Dextrose broth top-layer. The top liquid layer was aseptically inoculated with a sterile toothpick by transferring a small amount of conidia from a sporulating culture into the center of the broth and was gently agitated.\n\nThe plate was then covered and left at room temperature in ambient light for several days until a thick mat of fresh non-sporulating mycelium had completely covered the top layer of liquid media.\n\nThe entire mycelial mat was lifted off the plate and transferred to a flat surface using sanitized forceps and patted dry with a paper towel lightly sprayed with 70% isopropyl alcohol. Approximately 1 g of wet mycelium was cut, transferred to a 1.5 ml microcentrifuge tube and macerated with the blunt end of a plastic inoculating loop.\n\nGenomic DNA extraction\n\nDNA extraction was then carried out from the prepared tissue using the Quick-DNA HMW MagBead Kit (Zymo, D6060, CA) with the following modifications to the protocol:\n\n• 200 μl each of DNA Elution Buffer and Biofluid & Solid Tissue Buffer were used to accommodate the increased starting volume of solid tissue. There was no change to the proteinase K volume called for in the protocol.\n\n• The sample was incubated for 24 hours in a water bath at 55 °C).\n\n• The sample was macerated with a plastic micro-pestle to ensure good homogenization and tissue lysis after adding the initial buffers, midway through the initial incubation period, and finally prior to the initial centrifugation steps preceding DNA purification.\n\n• The entire volume of supernatant was used to carry out the DNA purification steps and the volume of Quick-DNA MagBinding Buffer was increased to match the volume of supernatant used.\n\n• DIY-friendly magbead seperator and vortex mixer were used to carry out the remaining steps of the DNA purification protocol.\n\nDIY magnetic bead separator rack\n\nA low-cost diy-friendly magnetic bead separator rack was constructed using packing tape, super strong neodymium disc magnets (32 mm diameter × 3 mm thick) and a cardboard box. A video demonstration is available on Figshare (Extended data31).\n\nDIY vortex mixer\n\nA vibrating back massager with a flat silicon head attachment was wrapped in a piece of rigid cardboard and affixed with tape to provide a contained area for the sample to vibrate. The massager was operated at low-speed for equivalent time periods in all steps requiring vortex mixing. While a lab-grade vortex mixer can be acquired at or below the cost of the massager used in this research, this device was already available to the researchers and further demonstrates the potential viability of alternative tools to those working in DIY lab environments. A video demonstration is available here (Extended data32).\n\nThe initial size and concentration of the extracted genomic DNA was estimated using gel electrophoresis with 1% TBE gel at a fixed 45 V and a 1 kb ladder (Bioland Scientific, DM01-01, CA).\n\nThe microcentrifuge tube containing the extracted DNA was tightly wrapped in parafilm, put in a padded envelop and shipped via USPS Priority mail from Los Angeles, California to Toronto, Canada where it arrived 11 days later.\n\nApproximately 1 μg was brought forward for library preparation. A DNA sequencing library was prepared with the SQK-LSK109 kit (Oxford Nanopore Technologies) according to the manufacturer’s protocol version GDE_9063_v109_revK_14Aug2019. Approximately 5 gigabases of sequencing data was collected using an R9.4.1 flow cell. Basecalling was performed with Guppy v5.015 in superior accuracy mode (Oxford Nanopore Technologies).\n\nSequencing quality was assessed using NanoPlot.13 All reads were used for assembly with Flye v2.914 using the parameters –nano-hq, –genome-size=42m, –threads=16. A 5 kb contig was removed because it was determined it was not part of the genome after performing a web-BLAST search. Polishing was performed with 1 round of Racon v1.4.2215 and one round of medaka v1.4.4 (Oxford Nanopore Technologies), both with default settings. The assembly was annotated using Liftoff v1.6.116 comparing against the Pestalotiopsis sp. NC0098 genome.17 Quality of the genome assembly was evaluated using BUSCO v5.2.2 using the sordiomycetes_odb10 database.18\n\nTo estimate the number of chromosomes, we used a previously described network-based approach.19 We identified the telomere repeat sequence as AACCCT from the initial assembly. All reads containing a minimum of three consecutive repeats of this sequence (or the reverse complement) were extracted using grep. Telomere-containing reads were aligned against each other in all-vs-all mode using minimap2.20 Alignments were then filtered to retain only alignments with more than 95% query coverage using awk. A network graph was generated using the iGraph R package.21 The workflow is available on Zenodo (Analysis code).\n\nA list of type and ex-type sequences containing ITS, TUB and TEF accessions for Pestalotiopsis, including an out-group using the species Neopestalotiopsis saprophytica, was constructed from prior work.22 The resulting phylogenetic analysis is shown in Figure 4.\n\nThe collection of 42 accessions were aligned with MAFFT v7.45323 using the ‘–auto’ configuration command-line argument. The resulting alignments were trimmed and concatenated using Mega 11.24\n\nA Maximum Likelihood (ML) tree was constructed in Mega 11 using the combined ITS+TUB+TEF alignment with default settings and 100 bootstrap replications.\n\nThe final tree was saved in Newick format and visualized by rooting the tree on the Neopestalotiopsis saprophytica outgroup.\n\nThe phylogenetic analysis workflow, scripts and data is available on GitHub (Extended data).\n\n\nResults\n\nThe majority of the DNA obtained from extraction was above 1 kb in length (Figure 3). After sequencing, we obtained a read N50 of 6.6 kb (Table 1), with approximately 105X coverage. Genome completion estimates revealed high completion, low duplication, and low fragmentation of the expected single-copy core genes. The total assembly length was 47.7 megabases, composed of five contigs with telomere repeats present at both the start and end of the contig, four contigs with no telomere repeats present, and a 68-kb circularized contig determined to be the full-length mitochondrial genome (Table 1). The BUSCO scores revealed high completion, with low missing and fragmented expected single-copy core genes.\n\nBUSCO scores (v5.2.2) were calculated using the sordaiomycetes_odb10 lineage, revealing 97.9%C (S:97.5%, D:0.4%, F:0.6%, M:1.5%, n:3817).\n\nAfter all sub-graphs were extracted from the network graph of telomere-containing reads, a total of 14 clusters containing telomere-containing reads were obtained (example of one is shown in Figure 5). Since each cluster represents one telomere and there are two telomeres per chromosome, this results in a total of seven linear nuclear chromosomes.\n\nThis network graph shows the network of long reads that are derived from a single telomere.\n\nThe cost for consumables for this project was determined to be approximately $300 on a per-sample basis (Table 2), however, the total upfront cost would be approximately $3098.\n\nFlow cell cost assumes 1/6 (i.e., 5 Gb of 30 Gigabases) of the throughput capacity was used.\n\nA BLASTn search on the ITS, TUB and TEF regions of the isolate were obtained.\n\nThe ITS region showed similarities to P. grevilleae (NR_147548) with 99.5% (600/603), P. kenyana (NR_147549) with 99.83% (596/597), P. oryzae (NR_147547) with 99.83% (593/594), P. pini (MT374681) with 997% (601/606), and P. knightiae (KM199310) with 99.01% (599/605).\n\nThe TUB region showed similarities to P. photinicola (KY047662) with 98.83% (506/512), P. australasiae (KM199499) with 99.8% (491/492), P. trachicarpicola (JQ845946) with 98.05% (503/513), P. telopeae (KM199500) with 100% (481/481), and P. rosea (JX399069) with 96.88% (496/512).\n\nThe TEF region showed similarities to P. kenyana (KM199395) with 99.87% (774/775), P. oryzae (KM199398) with 99.87% (752/753), P. grevilleae (KM199407) with 97.25% (777/799), P. biciliata (KM199399) with 97.94% (762/778), P. knightiae (KM199408) 97.32% (763/784).\n\n\nDiscussion\n\nHere, we obtained a high-quality genome assembly for Pestalatiopsis sp., comprising five contigs with telomeres at both ends and four contigs without telomere repeats. We also showed that obtaining fungal DNA suitable for Nanopore sequencing in a DIY lab environment is possible by modifying the protocol of a low-cost commercially available extraction kit. Genome sequencing is now becoming more accessible because of the relatively low capital requirements of the Oxford Nanopore MinION platform. In addition, the basecalling and consensus sequence quality are rapidly improving, resulting in high-quality, Nanopore-only genome assemblies for a variety of organisms.25,26\n\nWhile genome assemblers only report on the number and size of contiguous DNA sequences assembled, it is left up to the user to infer further information such as number of chromosomes. We used a previously developed approach19 based on network graphs to determine that 14 unique telomeres were present in the sequencing data. The DNA was extracted during a point in the life cycle where the genome is expected to be haploid. We therefore reason that seven unique telomere-capped chromosomes exist in this species, in addition to the circular mitochondrial genome.\n\nOne known quality concern with nanopore-only genome assemblies is the presence of frame-shifting indels that can affect protein prediction.27 In this assembly, the high BUSCO completion score of 97.9% and low fragmentation score of the single-copy core genes (F:0.6%) over 3817 single-copy core genes suggest minimal frame-shifting indels were present after polishing. Until recently, it was often necessary to polish nanopore genome assemblies with high-quality Illumina reads using a tool like Pilon to obtain high-quality nanopore assemblies.28 However, recent improvements to basecalling models and polishing algorithms enable high-quality genome assemblies from nanopore reads only. This further improves accessibility to high-quality genome assemblies by removing the need to sequence using an expensive platform, typically only found in labs with a large budget or institutional support.\n\nWith the exception of the nanopore library prep and sequencing, all organism isolation and culture work, genomic DNA extraction and microscopy was performed in a residential DIY home lab using consumer-grade or second-hand equipment acquired from consumer-facing online vendors like Amazon.com and eBay.com. This includes pipettors, a water bath, a thermocycler, a microcentrifuge, and a self-built laminar flow hood constructed with plywood, a 24x24x12 HEPA filter and inline duct fan. The cost of sequencing reagents for this genome was around $300; however, many of the reagents require the purchase of six to 24 reactions. All the software used in this project is freely available, thanks to the academic and open-source bioinformatics community.14,15,20,29 Furthermore, all computation (except for basecalling) was performed on a consumer-grade laptop (Lenovo Thinkpad P14s) with 36 GB ram, AMD Ryzen 7 PRO 4750U processor (eight cores, 16 threads) and 1 TB NVMe SSD storage.\n\nPestalatiopsis sp. is known to possess diverse metabolic capabilities, including the production of potentially valuable secondary metabolites such as antitumor taxols,8 polyurethane-degrading hydrolases9 and compounds like alkaloids, polyketides, terpenoids, flavonoids, coumarins, xanthones, quinones, semiquinones, peptides, phenols, phenolic acids, and lactones.30 This additional genome provides a high quality genome for another species, and a workflow to generate low cost genomes to the community for future studies in the metabolic capabilities of this genus.\n\n\nConclusions\n\nHere, we generated a high-quality genome assembly using nanopore sequencing for Pestalatiopsis sp., and conclude that this species possesses seven nuclear chromosomes in addition to a mitochondrial genome. We conclude that genome sequencing for novel fungi species can be performed in a DIY environment for approximately USD$300 on a per-sample basis.\n\n\nData availability\n\nThe strain of Pestalotiopsis used in this research is available upon request from Josh McGinnis (josh@everymanbio.com).\n\nBioProject: High-quality genome assembly of a Pestalotiopsis fungi, https://identifiers.org/bioproject: PRJNA773800\n\nFigshare: DIY Magnetic Bead Separator Rack, https://doi.org/10.6084/m9.figshare.19129856.v2.31\n\nFigshare: Vibrating Back Massager as DIY Vortex Mixer, https://doi.org/10.6084/m9.figshare.19129862.v1.32\n\nData are available under the terms of the Commons Attribution 4.0 International license (CC-BY 4.0).\n\nWorkflows, supporting scripts and accessions used in the phylogenetic analysis are available on GitHub (see below).\n\n\nAnalysis code\n\nAnalysis code available from: https://github.com/EverymanBio/pestalotiopsis\n\nArchived analysis code as at time of publication: https://doi.org/10.5281/zenodo.6028895\n\nLicense: GNU General Public License, version 3 (GPL-3.0).", "appendix": "Acknowledgements\n\nWe would like to acknowledge Damon Tighe for the makeshift-pestle idea, general DNA barcoding troubleshooting and reviewing. We’d also like to thank Kris Tatiossian, Ben Joris and Jos Houbraken for their feedback on this manuscript. We would like to thank Gregory B. Gloor for mentorship and providing funding for publication costs associated with this manuscript.\n\n\nReferences\n\nMarx V: PCR heads into the field. Nat. Methods. May 2015; 12(5): 393–397. 1548-7105. Publisher Full Text\n\nLandrain T, Meyer MAriel Martin Perez, and Remi Sussan. Do-it-yourself biology: Challenges and promises for an open science and technology movement. Syst. Synth. Biol. September 2013; 7(3): 115–126. PubMed Abstract \| Publisher Full Text\n\nRasmussen LM, Guerrini CJ, Kuiken T, et al.: Realizing present and future promise of DIY biology and medicine through a trust architecture. Hastings Cent. Rep. November 2020; 50(6): 10–14. PubMed Abstract \| Publisher Full Text\n\nShin HJ: Natural products from marine fungi. Mar. Drugs. April 2020; 18(5): 230. 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PubMed Abstract \| Publisher Full Text\n\nGiguere DJ, Bahcheli AT, Slattery SS, et al.: Telomere-to-telomere genome assembly of Phaeodactylum tricornutum. bioRxiv : The Preprint Server for Biology. May 2021. Publisher Full Text\n\nLi H: Minimap and miniasm: Fast mapping and de novo assembly for noisy long sequences. Bioinformatics. July 2016; 32(14): 2103–2110. PubMed Abstract \| Publisher Full Text\n\nCsárdi G, Nepusz T: The igraph software package for complex network research. InterJournal. 2006; Complex Systems: 1695. Reference Source\n\nMaharachchikumbura SSN, Hyde KD, Groenewald JZ, Xu J, et al.: Pestalotiopsis revisited.sep 2014; 79: 121–186. Publisher Full Text\n\nKatoh K: MAFFT: a novel method for rapid multiple sequence alignment based on fast fourier transform. Nucleic Acids Res. July 2002; 30(14): 3059–3066. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar S, Stecher G, Li M, et al.: MEGA x: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. May 2018; 35(6): 1547–1549. PubMed Abstract \| Publisher Full Text\n\nHuang Y-T, Liu P-Y, Shih P-W: Homopolish: A method for the removal of systematic errors in nanopore sequencing by homologous polishing. Genome Biol. March 2021; 22(1): 95. PubMed Abstract \| Publisher Full Text\n\nWick RR, Holt KE: Benchmarking of long-read assemblers for prokaryote whole genome sequencing. F1000Res. February 2021; 8: 2138. PubMed Abstract \| Publisher Full Text\n\nWatson M, Warr A: Errors in long-read assemblies can critically affect protein prediction. Nat. Biotechnol. February 2019; 37(2): 124–126. Publisher Full Text\n\nWalker BJ, Abeel T, Shea T, et al.: Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement. PloS One. 2014; 9(11): e112963. PubMed Abstract \| Publisher Full Text\n\nShen W, Le S, Li Y, et al.: SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation. PloS One. October 2016; 11(10): e0163962. PubMed Abstract \| Publisher Full Text\n\nDeshmukh SK, Prakash V, Ranjan N: Recent advances in the discovery of bioactive metabolites from pestalotiopsis. Phytochem. Rev. March 2017; 16(5): 883–920. Publisher Full Text\n\nMcGinnis J: DIY Magnetic Bead Separator Rack. figshare. Media. 2022. Publisher Full Text\n\nMcGinnis J: Vibrating Back Massager as DIY Vortex Mixer. figshare. Media. 2022. Publisher Full Text" }	[ { "id": "135248", "date": "09 May 2022", "name": "Paula Maria Moolhuijzen", "expertise": [ "Reviewer Expertise Genomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis brief report describes the culture, isolation, DNA extraction, genome sequencing and assembly of an unknown Pestalotiopsis species (fungal isolate MUOB 440515). The study utilised Oxford Nanopore Technology (ONT) long read sequence at +100x coverage to obtain telomere-to-telomere contig assembly for 5 of the 7 chromosomes that were estimated also in this study.\nReviewer main comment: This is quite an impressive fungal genome assembly (10 contigs) and a useful resource, especially as only 7 others genomes have been sequenced for this genus. The main focus of the study however appears to be the costing of samples based on a do-it-yourself (DIY) home laboratory, spruiking affordable sample sequencing. I think the authors could improve this report by highlighting more the scientific benefits of affordable sequencing and the quality of this assembly in comparison to what is currently available.\nOther comments and recommendations:\nPlease make it clearer in the methods that the Nanopore library preparation, sequencing and base calling was all done elsewhere. This is not clear until the discussion.\n\nThe authors provide video demonstrations, analysis code and have published the genome in NCBI GenBank. Unfortunately, the published genome is not annotated, can the authors clarify in the manuscript where to find the annotations.\n\nPlease use the isolate identifier “Pestalotiopsis sp. isolate MUOB 440515”. Do not refer to the isolate as the subject.\n\nSection: Abstract:\nThere are over 3,500 genomes in NCBI GenBank. Can this be updated?\n\n“The project (costing $300)”. The low cost of ONT platform sequencing is already mentioned. This number can be misleading, the project costs at least $3000. The cost per sample is potentially $300 if the user multiplexes samples, which is not done here. Can the authors please remove “(costing $300)” from the abstract?\n\nSection: Introduction:\n“recently developed MinION nanopore”. I think the authors can say \"The MinION has been commercially available since 2015\".\n\n“Nanopore Technologies provides both the hardware and reagents needed for genome sequencing for $1000 USD”. This is the cost of the starter kit. Can the authors confirm this here?\n\nSection: Methods:\n“prior to the initial centrifugation steps”. Was a centrifuge used and what does this equipment cost?\n\nSection: Nanopore sequencing and genome assembly:\n“...shipped via USPS Priority mail from Los Angeles, California to Toronto, Canada where it arrived 11 days later”. Who in Toronto, Canada did the ONT sequencing? Please add to methods.\n\n“Basecalling was performed with Guppy v5.015 in superior accuracy mode (Oxford Nanopore Technologies).” What software was used during the sequencing? Was ONT minKNOW used? What were the computer specifications used for the collection of data, base calling and analysis?\n\nFigure 3 and Figure 4 belong in the results section.\n\nSection: Results: Section: The Pestalotiopsis genome contains seven nuclear chromosomes:\nReferring to the title of this section, is this the first time the number of chromosomes has been estimated for this genus? If it is a first finding please highlight this.\n\nDid the authors assign the full length contigs to chromosomes for this isolate? If so, please state the which chromosomes are full length. Can the authors check the genome synteny to the other sequenced species?\n\nHow do the Table 1 assembly metrics compare to other related genomes in the genus? Can the authors please comment on this?\n\nFirst two paragraphs are a bit jumbled. Can the authors describe the genome assembly first and then describe the genes?\n\n“The cost for consumables for this project was determined to be approximately $300 on a per-sample basis (Table 2), however, the total upfront cost would be approximately $3098.” This is only a potential cost as multiplexed samples were not run. The MinION yield of 30 Gb is only an estimate. The yield obtained in this study was 5 Gb, 1/6th of the estimated total yield, but Table 2 has one sample as 1/10th of the consumables cost? Can the authors clarify? Can the authors add a statement that this does not include the actual MinION device?\n\nCan the authors please add a column to Table 2 for item description?\n\nTypo in Table 2. Change “LSK-SQK109” to “SQK-LSK109”.\n\nSection: Phylogenetic analysis:\nNo results are presented for the phylogenetic tree created, only blast results. Can the authors please include the tree results in this section.\n\nCan the authors describe the blast results better? Example: \"The isolate MUOB 440515 ITS region had high nucleotide sequence identity to …\"\n\nTypo, change 997% to 99.7%.\n\nSection: Discussion:\n”However, recent improvements to basecalling models and polishing algorithms enable high-quality genome assemblies from nanopore reads only.” Please add references.\n\n“This further improves accessibility to high-quality genome assemblies by removing the need to sequence using an expensive platform, typically only found in labs with a large budget or institutional support.” But didn’t the authors rely on institutional support for the library preparation, sequencing and base calling? Can the authors please reword this sentence to make it clearer that Illumina sequencing for polishing ONT genomes is not required at xx depth of coverage and include references?\n\n”With the exception of the nanopore library prep and sequencing”. This should be made clearer in the methods section. I am a little surprised the nanopore sequencing wasn’t done in house, as I think it would be doable?\n\n“Furthermore, all computation (except for basecalling) was performed on a consumer-grade laptop (Lenovo Thinkpad P14s) with 36 GB ram, AMD Ryzen 7 PRO 4750U processor (eight cores, 16 threads) and 1 TB NVMe SSD storage.” This should be in the methods, but the authors can still discuss base calling and assembly computational requirements.\n\nLast paragraph of this section. Can the authors discuss this isolate's position in the phylogenetic tree or how it compares to the closest sequenced species?\n\nSection: Conclusion: The conclusion reads like a flyer for a business. In the conclusion can the authors highlight the academic merits provided by this study? For example, maybe describe the scientific benefits that can be derived from affordable genome sequencing?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "136304", "date": "10 May 2022", "name": "Huzefa A Raja", "expertise": [ "Reviewer Expertise My research focuses on freshwater ascomycete fungal diversity", "distribution and phylogeny. I work on isolating and describing these fungi", "thus contributing to the origins and diversity of this important ecological group of fungi. My research findings have important implications in studies of biodiversity", "phylogenetics and taxonomic studies of the kingdom fungi. I also work with natural products chemists on the chemical mycology of freshwater fungi and fungal endophytes." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 seems like a fun project to do. I appreciate the attempts made to isolate a fungus and sequence its genome. I am not sure I am qualified to review the genomics/bioinformatics section of the manuscript. I am not an expert in bioinformatics analysis of fungal genomes. I will focus only on the mycology aspects of this paper, with some comments on the molecular biology section on HMW DNA extraction. I want to provide these comments so the manuscript can be indexed, but it needs some work.\nComments below are on the mycology aspects:\nHow was the fungus isolated? Please give more details.\n\n\"A double-layer media plate was prepared using a 9 mm Petri dish with a 30 ml Malt Extract Agar (MEA) bottom-layer and a 20 ml Potato Dextrose broth top-layer\" – Please can you elaborate more on what was the purpose? Perhaps a figure of this in the Appendix or Supplementary section.\n\nWhy did the authors PCR gene regions again? They could have tried to extract the regions from the whole genome. I can see sequencing the ITS region since it is multi-copy and could be hard to extract from the whole genome. Just wondering. I have no problem with that.\n\nIn the methods, the use of the word “clone” is incorrect in the context used.\n\nFigure 1B can you provide a scale bar.\n\nProvide a citation for the PCR amplification program used.\n\nOverall, I see the manuscript has focused a lot on at-home isolation of DNA and genome sequencing and that is fine. I am not sure the figure of an Agarose gel electrophoresis is necessary in the main manuscript. The modified protocols of the DNA extraction kit might be well suited in an Appendix or Supplementary section.\n\nPhylogenetic analysis needs major work:\na. I cannot tell which isolates of Pestalotiopsis were used from GenBank. All the metadata is missing. The authors should present a table with all of those data and provide the isolate number of the species besides the binomial.\n\nb. Please provide a Maximum Likelihood tree with branch lengths and Bootstrap branch support values ≥ 75 % are shown above the nodes. Bootstrap replicates should be at least 1000, not 100.\n\nc. What was the rationale for using ITS, TUB, and TEF genes?\n\nd. Did the authors do a BLAST search against type data?\n\ne. Did the authors submit the Sanger sequencing data to GenBank? Tree alignment and tree into TreeBase? Was the culture submitted to a culture collection? In the legend for Figure 4, the authors need to give how many bp were used, how many strains, bootstrap values above the nodes, etc.\n\n\"sp.\" in species should not be in italics throughout the manuscript, please check.\n\nMaybe better to give all equipment used and their vendors in a separate table format in the Appendix of the methods? Make sure all specific details are provided.\n\nIn Table 2, the total incurred cost and cost per sample are provided. Can you provide a comparison between the cost incurred for this project and how much an academic lab spends for the same?\n\nI am left wondering after the 3-gene phylogeny and ITS Barcoding work, and genome sequencing of the fungus: did the authors ascertain the species identity of the fungus? Or do they think it is new to science and if so why? Please mention in the manuscript. Also, you have to remember that Pestalotiopsis is a very large genus, with many species with no sequence data. Based on this work I would not be able to call it new unless a thorough species comparison is performed.\n\n\"Sordariomycetes\" is spelled incorrectly in Table 1.\nQuestions/Comments about the genome sequencing:\nDid the authors submit the whole genome data to GenBank?\n\nFor the HMW genomic DNA, what was the Nanodrop or Qubit reading? How much total DNA was obtained and how much per µl? I am curious about how much HMW DNA they obtained and what was the weight of the DNA.\nI have seen the enthusiasm of the first author in working with fungi, and I applaud their attempts to write their workup. Above I try to provide some comments to strengthen the mycology section of the manuscript. It is a good manuscript and I recommend revisions to improve the quality of the work and re-organization of sections to aid in clarity. Remove all detailed methods into the Appendix or Supplementary section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "135251", "date": "12 May 2022", "name": "Sinem Beyhan", "expertise": [ "Reviewer Expertise Fungal genomics 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\nThis is an exciting study of Pestalotiopsis sp. MUOB 440515 genome sequencing and assembly utilizing at home DIY research tools to isolate fungal genomic DNA and Nanopore long-read sequencing in a research lab. This study provides a good resource for the other fungal researchers, as there are only 6 other genomes of Pestalotiopsis species deposited in NCBI. In addition, it is impressive that the researchers were able to obtain a high molecular weight DNA using make-shift tools, and were able to obtain long reads to generate telomere-to-telomere assemblies for most of the chromosomes (5 out of 7). Overall, I think this study would inspire other researchers to try and produce high quality genome assemblies. However, there are a couple of points that authors can improve on to make the manuscript more accessible to the reader.\nIn methods, strain description: Could you please describe how this strain was exactly isolated? Also, what is meant by “a wild-type clone of Laetiporus sp.”? Were these two strains growing together on an aged hardwood stump? In addition, I was able to get the isolate name (MUOB 440515), but it is not obvious throughout the text. Could you please use the isolate name as appropriate?\n\nStrain sharing: The strain needs to be deposited to a publicly accessible strain collection (i.e. ATCC or BEI)\n\nFigure 1: Please include scale bars in all panels.\n\nFigure 4 and related analysis: The “subject” needs to be relabeled as Pestalotiopsis sp. MUOB 440515. All the strains used for this phylogenetic analysis need to be described better in the methods. The previously published work can be cited but a metadata of all the strains needs to be included.\n\nFigure 5 and related analysis: I am wondering if there is a better way of presenting this data. Can you align these telomere containing reads onto the large contigs and see if you can extend the chromosome hands manually?\n\nData sharing: Could the authors include genome annotation in the bioproject?\n\nOverall: As stated multiple times throughout the manuscript, one of the premises here is to provide a low cost sequencing method for other fungal researchers. However, I am not convinced that the manuscript is providing an improvement in lowering the cost. If one were to calculate per sample cost, as the authors did here, I would argue that some researchers would calculate a lower cost depending on the institution. So, the statements about the cost/sample should be revised.\n\nOverall: The methodology as it is described in the abstract and the introduction is misleading. The authors overemphasize the use of DIY tools and at home protocols, but I think the sequencing library preparation and the ONT sequencing were done in a research facility. I am impressed by the quality of the HMW gDNA isolated using a home lab, but I think the statements about a home lab being suitable for this study should be revised and confined to the HMW gDNA isolation portion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-442
https://f1000research.com/articles/11-261/v1	02 Mar 22	{ "type": "Research Article", "title": "Comparison of the clinical effectiveness of self-etch sealant with the conventional acid-etch sealant for the retentive ability and marginal integrity in permanent molars: a split mouth one year randomized controlled trial", "authors": [ "Deveshi Gupta", "Arathi Rao", "Ramya Shenoy", "Baranya Srikrishna Suprabha", "Deveshi Gupta", "Ramya Shenoy", "Baranya Srikrishna Suprabha" ], "abstract": "Background: Self-etching has been shown to be beneficial compared to the other resin sealants especially in pediatric practice. The present in-vivo study was designed to clinically evaluate the sealing ability and retention of the self-etching sealant compared to the conventional resin sealant. The aim was to evaluate and compare the retention and marginal integrity of the self-etch and acid etch sealant materials at three, six and twelve-month follow up. Methods: This was designed a randomized controlled trial, with double blind parallel, split-mouth design. In total, 35 children (70 teeth) between 7 and 10 years of age formed part of the study. Group 1 received acid-etch sealant and Group 2 received self-etch sealants. The study participants and the investigator who performed the statistical analysis were blinded to the treatment allocation. All the samples were evaluated at 3, 6, and 12 months. The inter-group and intragroup comparison were carried out using the Chi-Square test and Friedman test with level of significance set at 5% and the P value less than 0.05. Results: Complete retention of sealants was observed in 34.5% of conventional acid etch (group 1) and 22.9% of self-etch samples (group 2) whereas complete loss of sealants were seen in 11.4% of group 1 and 20% of the group 2 samples and intergroup comparison of retention failure was non-significant (p=0.135). In total, 85.7% of the group 1 and 82.9% of the group 2 samples exhibited good marginal integrity with no clinical changes in the enamel around the margins but this was not statistically significant (p=0.5). Conclusions: Sealants with fewer procedural steps and those which provide adequate retention would be ideal for use in children. Thus, self-etch sealants have been found to be effective and efficient as a sealant in the present in-vivo study. Clinical Trials Registry, India registration: CTRI/2019/03/018343 (29/03/2019).", "keywords": [ "Self-etch", "Acid etch", "Sealants", "Retention", "pit and fissure", "marginal integrity", "discoloration", "ClinPro", "Preventseal" ], "content": "Introduction\n\nOcclusal caries still continue to be the most common form of decay in the permanent dentition due to the presence of deep retentive pits and fissures. Their prevention is a challenge that can be controlled by non-operative measures especially if implemented early.1,2 American Academy of Pediatric Dentistry and Cochrane recommend the application of pit-and-fissure sealants on newly erupted posterior teeth so as to reduce the incidence of pit and fissure caries.3,4 Sealant retention is crucial for it to be effective, and this can be achieved by various factors.5\n\nEtching the enamel with phosphoric acid is a conventional and standard technique practiced globally to improve retention.6 Unfortunately, salivary contamination post etching, decreases the adhesion of the sealant due to the formation of a tenacious surface coating, thus requiring the etching procedure to be repeated. Considering the compliance of the young child, a sealant that provides good retention and having a short procedure of application would be ideal.6\n\nSelf-etching sealants are products that have a one-step application with fluoride releasing properties. They can prove to be beneficial compared to the other resin sealants especially in pediatric practices.2 There have been many in-vitro evaluation studies7,8 conducted comparing the self-etch sealant with conventional acid etch sealants. The present in-vivo study was thus designed with the focus to clinically evaluate the sealing ability and retention of the self-etching sealant compared to the conventional resin sealant.\n\nThe objective of this study was to evaluate and compare the retention of the sealant material and marginal enamel changes at three, six and 12-month follow up following sealant placement. The null hypothesis was that there would be no difference in the retention and marginal enamel structure between the self-etch and conventional acid-etch sealant at three, six and 12-month follow up.\n\n\nMethods\n\nThe study was conducted in the Department of Paediatric and Preventive Dentistry, Mangalore, South India and was a self-funded study.\n\nThis study was a randomized controlled trial, with a double blind parallel, split-mouth study design and 1:1 allocation ratio. The investigation was designed according to the Consolidated Standards of Reporting Trials (CONSORT). Completed CONSORT and TIDieR checklists can be found in the Reporting guidelines section.22–24\n\nAll procedures were performed in conformity with the ethical standards of the institutional research committee and the Helsinki declaration and its amendments. Ethical clearance was obtained from the Institutional Ethics Committee, Manipal College of Dental Sciences, Mangalore prior to the study (Protocol no: 19025, dated 18th February 2019). The trial was registered at the Clinical Trials Registry, India with submission number as CTRI/2019/03/018343 on 22nd November 2019.\n\nWritten informed consent was obtained from the parents and written informed assent was obtained from the individual participants of the study. It was ensured that participation in the study was entirely based on the will of the participants and their parents. They were also assured that their decision to participate or not would not affect the dental treatment services for the child. The participants were free to withdraw from the study at any time. Participation was voluntary and no compensation or incentives were provided to the participants. The information sheet and consent form can be found as Extended data.21\n\nSample size was calculated at 80% power and 90% confidence interval with clinically acceptable margin and proportion of retention of sealants as 50% in each group. Compensating for the loss to follow up at 10%, the minimum final sample size was calculated as 64 teeth, 32 in each group.\n\nOver a span of six-months, 50 children between the ages of 7-10 years, who visited the department for routine dental check-up were examined during their initial clinical examination. Mouth mirrors and probes were used to examine the children’s teeth under good chairside illumination. After screening, the children with fully erupted first permanent molars having deep pit and fissures without any signs of caries on any surface of the same teeth were selected. There was also a need that there should be two teeth that fulfilled the criteria one on each side of the arch. In addition, in order to be included into the study, each child should have presented with Decay, Missing, Filled Surface (DMFS) index of ≥19 in the form of visible cavities, fillings or missing teeth due to caries, with a co-operative behaviour (Frankl score 3 or 4)10 and satisfactory oral hygiene (OHI-S ≤ 3).11 Children with a history of any medical conditions or long-term medications that affected the salivary flow were not included. Additionally, children with pernicious oral habits, undergoing orthodontic treatment or wearing intraoral devices, permanent molars with developmental defects or restorations and/or history of allergies to resins were also excluded.\n\nA coin toss was used to allocate the tooth to each group. Randomization was carried out by an operator who was not involved in any other phases of the clinical trial. The unit of randomization was the individual tooth. A coin was tossed to determine which tooth the conventional acid etch pit and fissure sealant (Group 1: 3M™ ClinPro™, India, 3M ID 70201412429)12 would be applied to. If the coin landed as Heads, the sealant was placed on the right mandibular first permanent molar. Subsequently, the self-etch pit and fissure sealant (Group 2: Preventseal (Itena®, France, PVSEAL-1.2)13 was applied on the left mandibular molar. Similarly, if the coin landed as Tails, the conventional sealant was placed on the left mandibular first permanent molar and the self-etch pit and fissure sealant was applied on the right mandibular molar.\n\nThe group allocation was revealed to the principal investigator who performed the intervention just before the sealant placement procedure began by the operator who had carried out the randomization.\n\nThe study participants and the investigator who performed the statistical analysis were blinded to the treatment allocation. However, the operator (DG) who performed the intervention and the examiner (AR) who evaluated the teeth were not blinded due to the difference in the color of the materials. ClinPro™ Sealant is initially pink and changes to white following curing, while Preventseal is cream white material. A single examiner (AR) evaluated all the teeth at the period of 3, 6 and 12 months.\n\nThe trial commenced from March 2019 to April 2020 and all the follow-up examinations were completed by 15th April 2021. Both the study materials were purchased by the investigators and were used within their labeled shelf life.\n\nThe selected teeth were cleaned with an ultrasonic scaler and then polished with a non-fluoridated prophylactic paste using slow speed hand piece and rubber cup. The teeth were adequately rinsed with water and then air-dried. Cotton rolls and saliva ejectors were used for isolation during the procedure.\n\nThe sealants were applied by a qualified dental surgeon in accordance with the manufacturer’s instructions, on the occlusal pit and fissures of the first permanent molars.\n\nGroup 1 (3M™ ClinPro™, Conventional acid etch sealants): The entire fissure was etched for 30 seconds with 37% phosphoric acid gel using an applicator tip. The teeth were then rinsed using water for 20 seconds and dried with oil free compressed air. A conventional fissure sealant was applied as per the manufacturers’ instructions. A probe was used to remove any air bubbles and the margins were thoroughly checked. The sealant was then polymerized using a visible light curing unit for 30 seconds.\n\nGroup 2 (Preventseal (Itena®, Self-etch sealants): The self-etching sealant was applied to the tooth and allowed to penetrate the fissures for 20 seconds, as per the manufacturers’ instructions. A probe was used to remove any air bubbles and the margins were thoroughly checked. The sealant was then polymerized using a visible light curing unit for 30 seconds.\n\nThe occlusion was verified with an articulating paper and the high points if any, were subsequently reduced using a slow speed finishing bur. The finished sealants were checked for voids using a probe, which if present were refilled. All the sealant applications were crosschecked using a mouth mirror and probe by an experienced examiner. The light intensity output was maintained at 600 mW/cm2 with the help of a radiometer (Demetron 100, Demetron Research Corp, Danbury, CT, USA). The light output was checked after every two patients.\n\n\n\n1. Sealant retention\n\nThe samples in both the group were evaluated at 3, 6, and 12 months from the date of treatment. All examinations were carried out using the visual and tactile method, with the help of mouth mirror and probe under good chair side illumination by a single examiner.\n\nSealant retention was recorded according to Simonsen’s criteria14 as follows: Score 0: Complete loss of sealant; Score 1: Sealant fully retained; Score 2: Partial loss of sealant.\n\n2. Marginal integrity15\n\nEnamel at the sealant margins were closely checked and evaluated. An alteration in colour, loss of translucency and defects along the margins were considered as loss of marginal integrity and given a score of 1. Concurrently, the teeth with no such changes were given a score of 2 and considered as intact marginal integrity.\n\nAll the data was entered and analyzed using Statistical Package for Social Science (SPSS), version 17 (SPSS Inc. Chicago, IL, USA, RRID:SCR_019096) at the completion of the 12-month follow up examination. The frequency distribution for fully-retained, partially retained and complete loss of retention was tabulated. Similarly, the percentage distribution for presence and absence of marginal changes was calculated. The inter-group and intragroup comparison of sealant retention and marginal discoloration at 3 months, 6 months, and 12 months was carried out using the Chi-Square test and Friedman test. For all the tests, the level of significance was set at 5%. The P value was kept less than 0.05.\n\n\nResults\n\nOut of the initial 50 children, 38 participants were selected (76 teeth) and were recruited.20 Out of them 3 children failed to report for sealant placement and did not respond to our calls as depicted in the flow chart (Figure 1).\n\nThe final study samples consisted of 15 males and 20 females with the mean age of 8.5 years.\n\nTeeth with partial/complete loss of sealants were repaired and were then subsequently dropped from the study. Their scores, however, were carried forward into the successive follow up appointments.\n\nComplete retention of sealants was observed in 34.5% of group 1 and 22.9% of group 2 samples, whereas complete loss of sealants was seen in 11.4% of group 1 and 20% of the group 2 samples. Partial loss of sealants was more common in both the groups than complete loss. Group 1 had more teeth (34.3%) with complete retention than group 2 (22.9%) at the end of 12 months. Most of the retention failure was seen in the first evaluation period. Intergroup comparison of Simonsen’s Score for sealant retention between group 1 and 2 at follow-up was done using chi-square test and was found to be non-significant (p=0.135) (Table 1).\n\nChi-Square\n\nDF\n\np Value\n\nChi-Square\n\nDF\n\np Value\n\n2.000\n\n2\n\n0.368\n\n4.00\n\n2\n\n0.135\n\n2.000\n\n2\n\n0.368\n\nChi-Square\n\nDF\n\np Value\n\n1.57\n\n2\n\n0.456\n\n1.57\n\n2\n\n0.456\n\n1.64\n\n2\n\n0.440\n\nOverall, 85.7% of the group 1 and 82.9% of the group 2 samples exhibited good marginal integrity with no clinical changes in the enamel around the margins. Few of the teeth in both the groups exhibited discoloration and roughness but this was not statistically significant (p=0.5). In both the groups all the failures were seen by the first evaluation period at 3 months (Table 2).\n\nChi-Square\n\nDF\n\np Value\n\n0.108\n\n1\n\n0.500\n\n0.108\n\n1\n\n0.500\n\n0.108\n\n1\n\n0.500\n\n\nDiscussion\n\nIn the earlier studies using different products, those sealants that had been applied using self-etch systems had shown lower retention rates than the sealants applied in the conventional approach, regardless of the use of adhesive systems.14,15 In an in vitro study by Garg et al,6 the sealing ability and microleakage of Preventseal and Clinpro™ were evaluated. They documented that the retention and marginal integrity of the self-etching sealant, Preventseal was found to be similar to that of the conventional acid etch sealant.\n\nBased on the above information, we designed our in-vivo study to clinically compare the retention and marginal integrity between Preventseal and Clinpro™ sealants. Clinpro™ Sealant is a light-cured, fluoride releasing pit and fissure sealant with a color-changing feature. It is pink when applied to the tooth surface, and changes to an opaque off-white color when exposed to light. It contains 2, 2-bis[4-(2-hydroxy-3 methacryloxypropoxy)phenyl] propane, tri (ethylene glycol) dimethacrylate, a light cure initiator system based on camphorquinone, a tertiary amine and an iodonium salt.12 Preventseal is a self-etched, light cured, fluoride releasing pit and fissure sealant. As the name suggests it requires no etching, rinsing or drying. It contains Bis-GMA, and triethyleneglycoldimethacrylate - 2-Hydroxyethylmethacrylate.13\n\nA split mouth design was used to reduce the inter-subject variability and ensure that each of the sealants did not influence the outcome of the other segments. It also reduced the sample size and improved our statistical efficiency as each patient served as his/her own control and created a relatively smooth follow-up. Unfortunately, each design has its own merits and demerits. The attrition of even one patient in this trial amounted to loss of data in all the fields and was hence considered when the sample size was calculated.16 Further, this study was double blinded as both the data analyst and the participants were unaware of the type of sealant applied. The coin toss method of randomisation ensured that the children’s teeth were equally allocated to both groups of sealants.17\n\nThis study’s inclusion criteria were drafted keeping the recommendations from AAPD and the British Society of Pediatric Dentistry in mind.3,18 Based on these guidelines, sealants should be applied to the child’s first permanent molar between the ages of 7–10 years.17 This ensures that the newly erupted teeth are protected from demineralization. Additionally, only children with fully erupted first permanent molars were included, to reduce the risk of bias caused by moisture contamination during sealant application.19\n\nAll scientific measurements come with some degree of imprecision, hence the criteria was selected that minimized this to possible extent.\n\nAlthough Clinpro™ exhibited better retention, the results were non-significant. Thus, it can be concluded that the self-etch sealant, Preventseal is as effective as Clinpro™ Sealant with adequate retention and marginal integrity and the null hypothesis is accepted.\n\nA potential trial limitation is that since the two materials have different color, the operator and the clinical evaluator could not be blinded, which could have led to potential bias. To minimize this, the data analyst was blinded to the group allocation.\n\n\nConclusion\n\nSealants with less procedural steps and those that provides adequate retention are ideal to be used in children. Thus, Preventseal has proved to be effective and efficient as a sealant with its self-etch and fluoride releasing ability even in the present in-vivo study.\n\n\nData availability\n\nFigshare: unidentifiable Data. https://doi.org/10.6084/m9.figshare.19207596.20\n\nFigshare: Information sheet, consent form, accent form. https://doi.org/10.6084/m9.figshare.19196297.21\n\nFighsare: CONSORT checklist and flow diagram for ‘Comparison of the clinical effectiveness of self-etch sealant with the conventional acid-etch sealant for the retentive ability and marginal integrity in permanent molars: a split mouth one year randomized controlled trial’.\n\nhttps://doi.org/10.6084/m9.figshare.1913007222 & https://doi.org/10.6084/m9.figshare.19130081.23 TIDieR Checklist. https://doi.org/10.6084/m9.figshare.19130075.24\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nCarvalho JC: Caries process on occlusal surfaces: evolving evidence and understanding. Caries Res. 2014; 48(4): 339–346. PubMed Abstract \| Publisher Full Text\n\nAsefi S, Eskandarion S, Hamidiaval S: Fissure sealant materials: Wear resistance of flowable composite resins. J. Dent. Res. Dent. Clin. Dent. Prospects. 2016; 10(3): 194–199. PubMed Abstract \| Publisher Full Text\n\nWright JT, Crall JJ, Fontana M, et al.: Evidence-based clinical practice guideline for the use of pit-and-fissure sealants: A report of the American Dental Association and the American Academy of Pediatric Dentistry. J. Am. Dent. Assoc. 2016 Aug; 147(8): 672–682.e12. PubMed Abstract \| Publisher Full Text\n\nAhovuo-Saloranta A, Forss H, Walsh T, et al.: Pit and fissure sealants for preventing dental decay in permanent teeth. Cochrane Database Syst. Rev. 2017 Jul 31; 7(7): CD001830. PubMed Abstract \| Publisher Full Text\n\nGarcia-Godoy F, de Araujo FB : Enhancement of fissure sealant penetration and adaptation: the enameloplasty technique. J. Clin. Pediatr. Dent. 1994 Fall; 19(1): 13–18. PubMed Abstract\n\nGarg D, Mahabala K, Lewis A, et al.: Comparative evaluation of sealing ability, penetration and adaptation of a self etching pit and fissure sealant- stereomicroscopic and scanning electron microscopic analyses. J. Clin. Exp. Dent. 2019 Jun 1; 11(6): e547–e552. PubMed Abstract \| Publisher Full Text\n\nBotton G, Morgental CS, Scherer MM, et al.: Are self-etch adhesive systems effective in the retention of occlusal sealants? A systematic review and meta-analysis. Int. J. Paediatr. Dent. 2016 Nov; 26(6): 402–411. PubMed Abstract \| Publisher Full Text\n\nNahvi A, Razavian A, Abedi H, et al.: A comparison of microleakage in self-etch fissure sealants and conventional fissure sealants with total-etch or self-etch adhesive systems. Eur. J. Dent. 2018 Apr-Jun; 12(2): 242–246. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Oral Health Surveys - Basic Methods. Fifth ed.Geneva: World Health Organization; 2013. Last Accessed 25 Jan 2021. Reference Source\n\nFrankl SN, Shiere F: Should the parent remain with the child in the dental operatory?. J. Dent. Child. 1962; 29: 150–153.\n\nGreene JC, Vermillion JR: The simplified oral hygiene index. J. Am. Dent. Assoc. 1964; 68: 7–13. Publisher Full Text\n\n(Accessed on 11th August 2021). Reference Source\n\n(Accessed on 11th August 2021). Reference Source\n\nSimonsen RJ: Retention and effectiveness of dental sealant after 15 years. J. Am. Dent. Assoc. 1991 Oct; 122(10): 34–42. PubMed Abstract \| Publisher Full Text\n\nKhare M, Suprabha BS, Shenoy R, et al.: Evaluation of pit-and-fissure sealants placed with four different bonding protocols: a randomized clinical trial. Int. J. Paediatr. Dent. 2017 Nov; 27(6): 444–453. PubMed Abstract \| Publisher Full Text\n\nMoher D, Hopewell S, Schulz KF, et al.: CONSORT 2010 explanation and elaboration: updated guidelines for reporting parallel group randomised trials. BMJ. 2010; 340: c869. PubMed Abstract \| Publisher Full Text\n\nSuresh K: An overview of randomization techniques: An unbiased assessment of outcome in clinical research. J. Hum. Reprod. Sci. 2011 Jan; 4(1): 8–11. PubMed Abstract \| Publisher Full Text\n\nNunn JH, Murray JJ, Smallridge J: British Society of Paediatric Dentistry: a policy document on fissure sealants in paediatric dentistry. Int. J. Paediatr. Dent. 2000 Jun; 10(2): 174–177. PubMed Abstract \| Publisher Full Text\n\nPalti DG, Machado MA, Silva SM, et al.: Evaluation of superficial microhardness in dental enamel with different eruptive ages. Braz. Oral Res. 2008 Oct-Dec; 22(4): 311–315. PubMed Abstract \| Publisher Full Text\n\nGupta D, Rao A, Shenoy R, et al.: unidentifiable Data. figshare. Dataset. 2022. Publisher Full Text\n\nGupta D, Rao A, Shenoy R, et al.: Information sheet, consent form, accent form. figshare. Dataset. 2022. Publisher Full Text\n\nGupta D, Rao A, Shenoy R, et al.: Consort Checklist. figshare. Dataset. 2022. Publisher Full Text\n\nRao A, Gupta D, Shenoy R, et al.: Fig 1. figshare. Figure. 2022. Publisher Full Text\n\nGupta D, Rao A, Shenoy R, et al.: TIDieR-Checklist. figshare. Dataset. 2022. Publisher Full Text" }	[ { "id": "126007", "date": "07 Mar 2022", "name": "Parajeeta Dikshit", "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\nBased on my expertise I have found the format of the article is well designed. The abstract is concise and has given the needed gist of the article. The study design is transparent with no chances of biases. The authors have discussed and compared the results with other researchers adequately. The article can be indexed as it is.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "126008", "date": "14 Mar 2022", "name": "Muthu Murugan", "expertise": [ "Reviewer Expertise Early Childhood Caries" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 evaluates the clinical performance of self etch versus conventional acid etch sealant by a Split mouth RCT.\n\nAbstract:\nIt has few typos such as - First line in Methods - replace \"a randomized controlled trial\" with \" as a randomized controlled trial\" or \" A double blinded, split mouth randomized controlled trial was carried out\n\nTitle:\nCan be altered to - Comparison of Clinical Effectiveness of Conventional and Self-etch Sealant - A split mouth Randomized Controlled Trial.\nIntroduction:\n\"The need for this research could have been emphasized in the introduction section.\n\nThe heading Objective and Hypothesis to be removed and the last paragraph to be merged with Introduction.\n\nMethods:\nDesign cannot be both - Parallel and Split mouth.\n\nRandomization - Too many repetitions. Was any allocation concealment was done?\n\nPage 4 - Intervention - the statements ‘ The trial commenced from March 2019 to April 2020 and all the follow-up examinations were completed by 15th April 2021. Both the study materials were purchased by the investigators and were used within their labeled shelf life’ cannot be the intervention, please check and rephrase. Page 6, third line -Please mention after how many months in the statement - ‘Complete retention of sealants was observed in 34.5% of group 1 and 22.9% of group 2 samples, whereas complete loss of sealants was seen in 11.4% of group 1 and 20% of the group 2 samples’\nDiscussion:\nIt needs to be re-structured.\n\nThe results need to be compared with previous studies.\n\nPlease mention limitations about carrying out toss coin randomization.\n\nPlease highlight the study limitations of using a split mouth study design - First, the split-mouth design may lead to biased treatment efficacy estimates due to carry-across effects which (most likely) will be low or absent. However, there is even no statistical test that can detect this carry-across effect. Second, bias in recruitment of patients because of the need for symmetrical disease patterns among all segments of the dentition that are randomized and this limits the external validity of the results. Please refer to Lesaffre et al. (20091) for more details.\nConclusion:\nFluoride releasing ability of Preventseal wasn’t assessed in the present study hence in my opinion it shouldn’t be included here.\nLanguage:\nThere is room for improvement. Professional language edit of the entire manuscript can be helpful. Either American or British English to be used throughout.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "7965", "date": "06 Apr 2022", "name": "Arathi Rao", "role": "Author Response", "response": "Abstract: It has few typos such as - First line in Methods - replace \"a randomized controlled trial\" with \" as a randomized controlled trial\" or \" A double blinded, split mouth randomized controlled trial was carried out Response: Correction done. Title: Can be altered to - Comparison of Clinical Effectiveness of Conventional and Self-etch Sealant - A split mouth Randomized Controlled Trial Response: Changes done. Introduction: The need for this research could have been emphasized in the introduction section. Response: It's mentioned as follows: Considering the compliance of the young child, a sealant that provides good retention and having a short procedure of application would be ideal.6 Self-etching sealants are products that have a one-step application with fluoride releasing properties. They can thus prove to be beneficial compared to the other resin sealants especially in pediatric practices.2 There have been many in-vitro evaluation studies6,7 conducted comparing the self-etch sealant with conventional acid etch sealants. The present in-vivo study was thus designed with the focus to clinically evaluate the sealing ability and retention of the self-etching sealant compared to the conventional resin sealant. The heading Objective and Hypothesis to be removed and the last paragraph to be merged with Introduction. Response: Changes done. Methods: Design cannot be both - Parallel and Split mouth. Response: Corrections done. Randomization - Too many repetitions. Response: Repetitions removed. Was any allocation concealment was done? Response: Since the children are selected based on inclusion and exclusion criteria and both the test and control groups are in the given child- the risk of selection bias is minimum. Also the coin was tossed just before the procedure. Page 4 - Intervention - the statements ‘ The trial commenced from March 2019 to April 2020 and all the follow-up examinations were completed by 15th April 2021. Both the study materials were purchased by the investigators and were used within their labeled shelf life’ cannot be the intervention, please check and rephrase. Response: Corrections done. Page 6, third line -Please mention after how many months in the statement - ‘Complete retention of sealants was observed in 34.5% of group 1 and 22.9% of group 2 samples, whereas complete loss of sealants was seen in 11.4% of group 1 and 20% of the group 2 samples’. Response: Changes done and sentence reframed. Discussion: It needs to be re-structured. Response: Changes done. The results need to be compared with previous studies. Response: Similar studies were included as reference 13 and 14 but uses self etch adhesive liners and not self etch sealants. Ref 8 uses self etch sealants but compared microleakage which was beyond the scope of the present study. Ref No 6 is the only in vitro study done comparing the study materials and evaluation criteria. Please mention limitations about carrying out toss coin randomization. Response: Changes made. Please highlight the study limitations of using a split mouth study design - First, the split-mouth design may lead to biased treatment efficacy estimates due to carry-across effects which (most likely) will be low or absent. However, there is even no statistical test that can detect this carry-across effect. Second, bias in recruitment of patients because of the need for symmetrical disease patterns among all segments of the dentition that are randomized and this limits the external validity of the results. Please refer to Lesaffre et al. (20091) for more details. Response: Limitations added. Fluoride releasing ability of Preventseal wasn’t assessed in the present study hence in my opinion it shouldn’t be included here. Response: Changes done. There is room for improvement. Professional language edit of the entire manuscript can be helpful. Either American or British English to be used throughout. Response: Language re checked and changes made." } ] } ]	1	https://f1000research.com/articles/11-261
https://f1000research.com/articles/9-326/v1	04 May 20	{ "type": "Research Article", "title": "Heart rate variability biofeedback intero-nociceptive emotion exposure therapy for adverse childhood experiences", "authors": [ "Stéphanie Hahusseau", "Bruno Baracat", "Thierry Lebey", "Lionel Laudebat", "Zarel Valdez", "Arnaud Delorme", "Stéphanie Hahusseau", "Bruno Baracat", "Thierry Lebey", "Lionel Laudebat", "Zarel Valdez" ], "abstract": "Background: Psychiatric patients with adverse childhood experiences (ACE) tend to be dysfunctional in the interoceptive part of their emotional experience. The integration of interoceptive emotional activity in the insular and cingulate cortices is linked to the regulation of sympathovagal balance. This makes heart rate variability (HRV) an ideal measure for providing feedback on emotion regulation in real time. Methods: A sample of one hundred (n=100) outpatients was evaluated. Participants underwent eight 30-minutes ACE exposure sessions during which patients were guided to experience bodily sensations related to ACE while their HRV was monitored using a commercial biofeedback device. Results: Comparing the results of first to last therapeutic session, a significant decrease in heart rate and an increase in HRV at the onset of the session were observed. Conclusions: This study suggests physiological impact of therapeutic interventions on the autonomic balance and underlines the interest of HRV biofeedback as a clinical practice.", "keywords": [ "Interoception", "Adverse Childhood Experience", "developmental trauma disorder", "Biofeedback", "Retrospective study", "autonomic nervous system", "PTSD" ], "content": "Introduction\n\nHeart Rate Variability reflects an individual's ability to adaptively cope with stress. According to Thayer’s model1 (see also 2,3), orthosympathetic activity is associated with higher central nervous system activity, in particular activity within the limbic system, the amygdala, and the prefrontal and frontal cortex4. One of the roles of these high-level structures is to inhibit the parasympathetic system and activate the sympathetic system. When a person faces a threat, this may elicit a hyperarousal and “flight or fight” response5, which leads to an inhibition of the parasympathetic system and an activation of the sympathetic system. This corresponds to a decrease of HRV and often an increase in Heart Rhythm (HR)6. Emotional events may have an influence on the general stress level7, which in turn is visible in the sympathetic/parasympathetic balance. Generally, these effects are transient because the higher nervous structures (essentially amygdala and prefrontal cortex) inhibit each other and, as soon as the stressor disappears, the system returns to parasympathetic tonus with low HR and high HRV.\n\nFor several decades, autonomic nervous system tests have been used to identify the physiologic correlates of psychiatric illnesses, particularly for affective and anxiety disorders8. In studies of Post-Traumatic Stress Disorder (PTSD) for example, decreased HRV are observed in PTSD patients compared to matched controls9. The HRV of PTSD patients indicates an increase in sympathetic activity and a reduction in parasympathetic activity. Patients suffering from PTSD tend to exhibit hyperactivity of the autonomous nervous system at rest and have been shown to be unable to further mobilize their orthosympathetic system when facing a stressful situation10. In addition, the HRV profile after exposure to a trauma has been shown to be predictive of future traumatic episodes in PTSD11,12. PTSD is associated with the disruption of the autonomic processes that maintain heartbeat regulation13.\n\nResearch in psychiatry indicates that adverse childhood experiences leave durable physiological and neurophysiological traces and that there is a strong relationship between adverse childhood experiences and depression, suicide attempts, alcoholism, drug abuse, and other negative health outcomes14. Adverse childhood experiences (ACEs) are ubiquitous among the adult patient population15. The damaging effects of ACEs are nonspecific, thereby affecting a variety of functions and behaviors. In fact, ACEs have been shown to be negatively correlated with adult mental well-being16. Chronic traumatic experiences in childhood that extend over several years – as in cases with trauma and neglect – impair self-regulation function such as mood regulation and constancy in relations, described in “complex PTSD” and “developmental trauma disorder”.\n\nClinically, autonomic nervous system (ANS) function and emotional well-being are closely related17. Research has shown that having experienced early-life adverse events was associated with lasting effects on Heart Rate Variability (HRV)18, revealing complex interactions between traumatic experiences, ANS functioning and psychopathology19.\n\nIn addition, psychiatric research has shown that having experienced early-life adverse events was associated with altered interoception20. Interoception is crucial for well-being21 as it mediates emotion regulation22. In fact, most psychiatric disorders are sustained by a type of interoceptive phobia23. Interoception require the interplay between perception of body states and cognitive appraisal of these states to inform emotional experience and motivating regulatory behavior24. The insular cortex in humans processes interoceptive activity and integrates and modulates cardiovascular, respiratory and emotional signals in order to create an integrated emotional experience25.\n\nNeurophysiological impairments due to ACEs have been shown to be reversible26,27. Evidence-based psychotherapy for adults with ACE history typically involves a progression through three phases: safety and stabilization; trauma processing; consolidation of therapeutic gains28. The trauma processing phase requires sensitive therapeutic guidance. The other phases are best-practice approaches to all psychotherapeutic treatments, with the focus on the unique impact of ACEs.\n\nEvidence based psychotherapy models for adults with ACEs-related disorders such as emotion-focused trauma therapy and eye-movement-desensitization-reprocessing are useful in the trauma processing phase. The efficacy of these approaches may be related to interoception rather than cognitive focusing29. Efficacy of psychotherapy with trauma patients may depend on the patient being able to face and feel adverse sensory and perceptual stimuli related to trauma-related memories in paced conditions30.\n\nProlonged exposure therapy and cognitive processing therapy have gathered a significant amount of empirical support for PTSD treatment. However, they are not universally effective with patients continuing to struggle with residual post adverse childhood-traumatic symptoms. As such, other type of interventions such as biofeedback may be beneficial. When patients with PTSD were assigned to receive HRV biofeedback plus treatment, the results indicated that HRV biofeedback significantly increased the HRV while reducing symptoms of PTSD31. The present study intends to replicate these results using commercially available biofeedback equipment within an ecological therapeutic environment.\n\n\nMethods\n\nThis procedure was developed by therapist MD SH (first author) for over more than 10 years. The therapeutic protocol comprises two parts. In the first part, which typically comprised eight weekly sessions of half an hour each, the therapist (co-author SH) identified the occurrence of adverse childhood experiences (psychological abuse, physical abuse, contact sexual abuse, or exposure to household dysfunction during childhood, e.g. exposure to substance abuse, mental illness, violent treatment by parent or stepparent, criminal behavior in the household). To do so, the therapist carried out clinical investigations, collected anamnestic and diachronic data, and guided the patient to specific breathing visualization exercises.\n\nIn order to characterize adverse childhood memories responsible for interoceptive phobia, the patient was asked to initially focus his attention on his/her breath, then on nociceptive sensations, and finally on the childhood memories32. The therapist asked the patient to focus his/her attention on his/her breathing while describing the images associated with the memories and specific body sensation or pain that might arise in detail. This exercise was carried out with closed eyes. During this exercise each uncomfortable physical sensation and negative thought was rated in terms of intensity on a 10-point scale. Later, after a meeting devoted to the conceptualization of the selected traumatic memories and their influence on repetitive negative emotions, the therapist helped the patient to establish a coherent narrative within which to frame his/her difficulties. The practitioner explained the therapeutic hypothesis, which would be instantiated in the second phase of the therapy indicated as described below.\n\nThe second phase of the therapy consisted of bi-monthly one-hour therapeutic sessions. In each session, after five minutes of rest, the therapist asked the patient to wear an ear device sensor which is part of a HRV biofeedback device (Emwave2; Heartmath, Inc.). The patient was then asked to focus his/her attention on his/her breathing for two minutes. After two minutes, the evocation of images related to the adverse childhood memory chosen for this session started33. To avoid dissociative processes and develop interoception and parasympathetic activation, the patient was asked to focus his/her attention on the uncomfortable bodily sensations for about 30 minutes34. Feedback on the sympathetic-vagal balance was directly affected by the sound delivered by the biofeedback device. The sound of the biofeedback device is correlated with the low frequency peak in the HRV spectrum (HeartMath Emwave 2 device and associated software; US patent 6,358,201 and Australian patent 770323). The number of sessions depended on the number of adverse childhood experiences to face – in general about 6 sessions. During these meetings, the therapist saved the series of heart beat intervals (R to R intervals) using the biofeedback software. In this study, five minutes of data at the beginning and at the end of the first session of phase 2 (session 1) and the last session of phase 2 (designated as “session 2” even though there might be several sessions between “session 1” and “session 2”) have been analyzed. Each of these 5 minutes comprise 2 minutes of breathing plus the evocation of traumatic imagery.\n\nThe most recent 100 outpatients of therapist SH having experienced at least one type of adverse childhood experience and having used the biofeedback method described above were retained as study population. Only patients for whom more than 3 consecutive sessions were collected were included. These two conditions were the only inclusion criteria. 100 patients was judged appropriate for an HRV study of this nature based on the literature35,36. In general, HRV studies require about of 100 patients or subjects to observe links between mental condition and HRV measures, although some studies have observed significant effects in depressive subjects with group sizes as low as 2737. Inclusion criteria included an history of adverse childhood experience (therapist assessment). Patient who required psychiatric medical treatment (therapist assessment) were excluded from the study. Patients were included regardless of DSM V guidelines for trauma since these do not provide a definition for patients having experienced chronic trauma over several years such as neglect. However, sub-categories in the DSM V were considered as described in a later section. The data was collected over one year. Table 1 summarizes the main features of the data sample.\n\nThe local ethical committee (Comité de protection des personnes Sud Ouest) approved the study and the use of the data for research purposes. Since the study was performed retrospectively, no patient consent was necessary. However, the French national entity for the protection of public and medical digital records (CNIL) authorized the retrospective use of the clinical data for this research (authorization number 1685185). The therapist associated a random number to each patient which was then used to anonymize the questionnaire data, the scanned notes of the therapist and the EKG files of each patient. Except for the therapist (co-author SH), all other investigators were blind to the identity of the patients. The blinding procedure consisted in assigning a randomly generated code to patients, in compliance with CNIL requirements (Commission nationale de l'informatique et des libertés). It was performed at the therapist’s office by the therapist herself to ensure that no identifiable document could inadvertently be lost, stolen, or read by anyone else than the therapist. When a paper form contained identifiable information, it was masked by the therapist, a sticker with the anonymized patient ID was temporarily placed on the form and the form was photocopied for later digital transfer. The questionnaire data was not integrated into the current report to focus on the interpretation of heart beat intervals.\n\nR to R intervals were collected during therapeutic sessions using the biofeedback Emwave2© device. This system uses a photoplethysmographic sensor located on the right ear lobe and series of heart beats are automatically extracted by the biofeedback software. The accuracy of this data was verified in one subject by comparison to a simultaneously recorded real EKG (Biopac MP36 unit and Acqknowledge© using Einthoven Lead II derivation): the heart beats monitored by the biofeedback system were delayed in comparison to the EKG based on the time it takes for blood pressure to build up at the ear lobe. Except for this delay, heart beat measurements were accurate within millisecond precision in comparison to those visible on the EKG. Using heart beat time intervals over 300 seconds, HRV calculations were carried out with the Biomedical Toolkit used on Labview© version 2009. This software performs HRV calculation in the same way as other HRV software packages do – such as the popular Kubios software (Kubios Oy, Finland). R to R intervals were resampled at 8hz, and the power spectrum was calculated over the whole 5-minute record using an FFT decomposition. Power was obtained at each frequency by calculating the square value of the FFT absolute amplitude. In the frequency domain, total HRV was obtained by summing the total spectral power for the low frequency band (LF) 0.05Hz–0.15Hz and the high frequency band (HF) 0.15Hz–0.35Hz. The LF/HF ratio was also calculated. Before performing statistical analyses, a log (Ln) transformation was applied and values were subsequently normalized across subjects. Other heart measures calculated in the time domain were Heart Rate (RR), Root Mean Square of Standard Deviation of R to R intervals (RMSSD), proportion of R to R intervals larger than 50 msec (pNN50), and Triangular Index of R to R intervals.\n\nThe clinical assessment of the therapist led to the creation of the following categories mapped onto DSM V categories: Substance abuse (SA); Somatoform Disorders (SD); Anxious Disorders (AD); Serious Personality Disorder (SPD); Post Traumatic Stress Syndrome (PTSS) (Data not included). All the patients could be diagnosed with trauma complex or developmental trauma disorder38. In addition to these categories, additional independent variables were retained: patient age, patient sex, the number of meetings in phase 2, and the number of days between the first and the last data recording sessions. Data was collected by the therapist on custom forms (available as extended data37) that were later transcribed into digital form after the anonymization process.\n\nChanges in the HRV between the two selected therapy sessions and within each session between the beginning and the end of each recording were analyzed using 2-way repeated measure ANOVA. Measurements related to the first meeting of therapy of phase 2 are indicated by “session 1” and a measurement at the end of the session of phase 2 is indicated by “session 2”. For each of these sessions, a measurement was taken at the beginning of the session (indicated by “Measurement 1”) and another at the end of the session (indicated by “Measurement 2”). There is about 25 minutes delay between “Measurement 1” and “Measurement 2” during which the patient was asked to re-experience traumatizing events – this time frame was not analyzed.\n\nStatistical analyses combine two within-subjects factors with two levels; “Session” and “Measurement”. Additionally, other between-subjects factors and independent variables described in the previous section were included. All the analyses were carried out with General Linear Model (GLM) module of SPSS© (version 17) by using the statistics of Greenhouse-Geisser.\n\nThe existence of corrupted R to R series and/or incomplete data associated with the statistical method used (within-subjects measurement) implies that the number of subjects included in the statistical analyses was lower than 100, and varies depending on the type of analysis. R to R and demographic data are available as underlying data37.\n\n\nResults\n\nSignificant changes in HR and HRV were observed. HR was higher by 3.4 beat per minute (bpm) in session 1 compared to session 2 (D=4.99; DF=1,55; p=0.029). Within sessions HR increased by 1.6 bpm (D=23.53; DF=1,55; p <0.001). There was no interaction between these two factors.\n\nGlobally, total HRV estimated in the frequency domain showed significant changes as well. Within a session, HRV decrease was significant (D=10.97; DF=1,55; p=0.002). The total quantity of transformed HRV decreased by 0.245 points between the beginning and the end of the therapy but failed to reach significance. The interaction between the two factors was significant (D=13.32; DF=1,55; p=0.001). This is due to the fact that of the decrease in HRV between Measurement 1 and Measurement 2 was relatively large during the second session (0.476 points; DF=1,55; p<0.05), but relatively low for session 1 (0.014 points; ns). Table 2 summarizes mean HRV values and standard errors of the mean. Figure 1 summarizes the variations in HRV based on the two factors – the Z score of Ln(HRV) was plotted where the difference were most striking.\n\nAll other analyses of measurements of HRV obtained in the frequency domain (LF, HF, LF/HF) or time domain did not lead to significant differences. Additional inclusion of factors (“Clinical Opinion”, “Sex” as between subject factor, “Age”, “Number of days between Session 1 and Session 2”, “Mean number of meetings” and “Time between the two sessions” as covariates in between-subject factor) in the ANOVA did not lead to significant differences and did not modify the level of significance of the differences mentioned above. Table 3 shows the spectral LF and HF values for the different sessions and measurements.\n\n\nDiscussion\n\nThe present study demonstrates an effect of biofeedback therapeutic interventions both in terms of heart rhythm and heart rhythm variability measurements. Subjects’ HR showed a significant decrease between session 1 and session 2 which could indicate reduced chronic stress. The reduction in the average HR in session 2 compared to session 1 can be interpreted as an effect of therapeutic interventions.\n\nMoreover the patient average HR increased between the beginning and the end of each of the two sessions. This increase in the HR is consistent with the model of Thayer17: the patient experiences a change in emotional state due to the recall of the traumatic experience, and the induced stress leads to an increase in HR.\n\nThe analysis of the modifications of HRV partially confirms this interpretation. At the onset of session 2, patients had higher HRV than at the onset of session 1, which indicates larger parasympathetic influences towards the end of the therapy. Also, in the general population, HRV tends to be lower in patients compared to controls. In the task force of the European Society of Cardiology and the North American Society of Pacing Electrophysiology39, HRV of control subjects in decubitus dorsal at rest over 5 minutes were 3466 ms2/hz (± 1018 ms2/hz). Measurements for the present study were approximately three times lower which could mean that HRV is close to its minimum. 1085 ms2/hz (standard error of the mean (s.e.) 1329 ms2/hz) were calculated for “Session 1-Measurement 1”, 1094 ms2/hz (s.e. 1887 ms2/hz) for “Session 1-Measurement 2”,1195 ms2/hz (s.e. 939 ms2/hz) for “Session 2-Mesurement 1” and 1080 ms2/hz (s.e. 1123 ms2/hz) of “Session 2-Measurement 2”. A possible interpretation for the reduction in HRV within session 2 (and not within session 1), is that the HRV at the onset of session 2 was high enough to allow for a reduction associated with the emotional trauma recall. This was not the case in session 1 where the initial HRV was lower than in session 2 and thus might not have allowed for further reduction in HRV induced by trauma recall.\n\nDifferences in HRV total power but not in the Low Frequency (LF) and High Frequency (HF) of the HRV were observed. The absence of an effect on HF and LF across conditions could be explained by the important inter-individual variability. HF values were weak (average of all conditions 220 ms2/hz) compared to LF values (average of all conditions 1123 ms2/hz). This means that the major part of the total HRV power was due to the LF component and the LF coefficient of variation was large (ranging between 1.07 and 1.94). Finally, differences of HRV between sessions and measurement times, calculated at the individual level, ranged between -1105 ms2/hz and +1151 ms2/hz which means that irrespective of the comparison, there were almost as many patients whose HRV varies in one direction as ones whose HRV varies in the opposite direction.\n\nThe absence of a control group and the naturalistic conditions of this retrospective study, carried out with the constraints imposed by clinical out-patient medical practice, are not ideal, and resulted in large inter-individual measure variances. In this retrospective study, a large number of variables not included in the analysis might also have influenced outcome measures. For example, the decision to follow the therapy could have been accompanied by a change in lifestyle (i.e. general improvement of the hygiene of life), which may affect both HR and HRV measures. In addition, time alone could have been responsible for changes in HRV. As this study has been conceived a posteriori, such variables could not be controlled. However, the absence of statistical effects associated with biographical variables indicates that these types of effects are relatively unlikely.\n\nThe intra-individual differences in the emotional reactivity following the evocation of the traumatic memory were difficult to standardize. One possible solution could be the consideration of an individual cardiovascular reactivity, which may be modeled as influenced by several independent factors40. One of these factors would depend on individual physiological variables and be independent of the nature and the intensity of the emotional trauma evoked during therapy. This factor could be estimated separately using simple tests which have been used to establish relationships between the variations of HRV and the ability to regulate emotions6. Other factors, such as the intensity of the trauma and the type of trauma could also influence cardiac reactivity. This multi-factor type of modeling could potentially help to reduce and understand inter-subject variability and lead to HR and HRV measures with diagnostic and therapeutic value.\n\nIn this protocol which includes two therapeutic components; HRV biofeedback and intero-nociceptive exposure, it is impossible to distinguish the impact of one component versus the other. The hypothesis was that both components are important, and that it is the combination of the two which maximizes the therapeutic effect. Further studies will be necessary to investigate this hypothesis.\n\nThe analysis of HRV is a simple and noninvasive method to quantify the activity of the autonomous nervous system. The sympathetic-parasympathetic balance of patients having undergone important traumas is modified in favor of sympathetic influences. This study shows that interoception exposure therapy – combined with biofeedback - was able to increase parasympathetic influences. Furthermore, progressive reduction in the cardiac rhythm and an increase in HRV at rest over a period of a few months were demonstrated. It is important to note that these variations were independent of the disorder diagnosed by the Psychiatrist, therefore the HRV might be considered as a general indicator of health. These results warrant further investigation of both therapeutic components (HRV biofeedback and intero-nociceptive exposure) and their comparison to other types of interventions.\n\n\nData availability\n\nZenodo: R-R HRV data from Biofeedback on 100 patients. http://doi.org/10.5281/zenodo.370313037\n\nThis project contains the following underlying data:\n\n- Archive_RR_All_subjects (folder containing R – R interval data for all participants as .txt files. Participants can be identified using the ID (e.g. n1799) in the file name)\n\n- biographic_data.txt (Demographic data for participants)\n\nZenodo: R-R HRV data from Biofeedback on 100 patients. http://doi.org/10.5281/zenodo.370313037\n\nThis project contains the following extended data:\n\n- info_sheet.docx (Study data collection form, English)\n\n- info_sheet_fr.docx (Study data collection form, French)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors wish to thank The Research and Editing Consulting Program (RECP) for their editing services.\n\n\nReferences\n\nThayer JF, Lane RD: Claude Bernard and the heart-brain connection: further elaboration of a model of neurovisceral integration. Neurosci Biobehav Rev. 2009; 33(2): 81–88. PubMed Abstract \| Publisher Full Text\n\nBerston GG, Cacioppo JT, Grossman P: Whither vagal tone. Biol Psychol. 2007; 74(2): 295–300. 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PubMed Abstract\n\nBob P, Susta M, Gregusova A, et al.: Dissociation, cognitive conflict and nonlinear patterns of heart rate dynamics in patients with unipolar depression. Prog Neuropsychopharmacol Biol Psychiatry. 2009; 33(1): 141–145. PubMed Abstract \| Publisher Full Text\n\nQuintana DS: Statistical considerations for reporting and planning heart rate variability case-control studies. Psychophysiology. 2017; 54(3): 344–349. PubMed Abstract \| Publisher Full Text\n\nBassett D: A literature review of heart rate variability in depressive and bipolar disorders. Aust N Z J Psychiatry. 2016; 50(6): 511–519. PubMed Abstract \| Publisher Full Text\n\nDelorme A: R-R HRV data from Biofeedback on 100 patients (Version 6) [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3703130\n\nVan der Kolk BA: Developmental trauma disorder: Toward a rational diagnosis for children with complex trauma histories. Psychiatr Ann. 2005; 35(5): 401–408. Publisher Full Text\n\nTask Force of the European Society of Cardiology the North American Society of Pacing Electrophysiology: Heart rate variability: Standards of measurement, physiological interpretation and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Circulation. 1996; 93(5): 1043–1065. PubMed Abstract\n\nStemmler G, Wacker J: Personality, emotion, and individual differences in physiological responses. Biol Psychol. 2010; 84(3): 541–551. PubMed Abstract \| Publisher Full Text" }	[ { "id": "72051", "date": "02 Nov 2020", "name": "Vedat Şar", "expertise": [ "Reviewer Expertise PTSD", "dissociative disorders", "borderline personality disorder", "functional neurological disorders", "epidemiology", "depression" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is based on the hypothesis that psychiatric patients with adverse childhood experiences (ACE) tend to be dysfunctional in the interoceptive dimension of the emotional experience which is characterized by diminished sympathovagal balance. A sufficient number of participants was subjected to exposure related to their ACE while taking the heart rate variability (HRV) to monitor the effect of the intervention. The latter covered also biofeedback sessions. A significant increase in HRV and decrease in heart rate were obtained at the end. The strength of this study is the hypothesis which is based on a firm theoretical basis and the transdiagnostic approach. The weakness of the study is lack of long-term follow up.\nThis is a useful and well written study. To strengthen the paper, the authors should also address polyvagal theory (Porges) shortly to enrich the paper. Another subject to address is the place of HRV in the literature of PTSD that possible differences between effects of adverse childhood experiences and adult types of traumatization on HRV is dealt with.\n\nIs the work 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": "75430", "date": "16 Dec 2020", "name": "Nele De Witte", "expertise": [ "Reviewer Expertise Biofeedback", "emotion regulation", "HRV", "e-mental health", "wearables. I am not an expert in the treatment of ACE and trauma so I would recommend the relevant sections (e.g.", "ACE therapy with interoceptive component) to be read by an expert in this field." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current retrospective non-controlled study aimed to investigate whether the combination of a therapeutic intervention (including emotion exposure and biofeedback) influenced HR and HRV in outpatients with ACE. HR increased during the sessions but decreased from session 1 to session 2. HRV at the beginning of the session increased from session 1 to session 2. While the fact that this is a study in naturalistic circumstances can be a strength, since we are often limited to controlled research in laboratory circumstances, this also causes severe limitations in the design and interpretation of the results. Importantly, we are lacking a control group, which severely impedes interpretation. Additionally, large individual differences occurred and we would need more information about the sample to be able to interpret these. I would also be interested in seeing additional non-physiological outcome measures of the study or information about treatment effectiveness. The manuscripts requires the addition of a lot more information and the writing style and representation of the findings can be improved. However, I do think that the study could provide interesting information to the field if these improvements are made.\nAbstract\n\n“Psychiatric patients with adverse childhood experiences (ACE) tend to be dysfunctional in the interoceptive part of their emotional experience.”\nI would suggest to state that patients or individuals have disfunctions and not that they are dysfunctional. This is a more respectful and correct representation.\n\nIntroduction General flow and readability: I would like to see some clarity in the definitions and presented research findings.The flow can also be improved. Subsections of the introduction appear to stand on their own. Some additional proofreading for the English language is also needed in the whole manuscript (e.g., “Interoception require the interplay between perception of body states and cognitive appraisal of these states to inform emotional experience and motivating regulatory behavior”).\nSpecific comments:\n“Heart Rate Variability reflects an individual’s ability to adaptively cope with stress”\nHRV is a concept that can reflect many things, including emotion regulation, but also stress, general resilience, mental illness, … (also depending on whether you are measuring resting HRV or HRV responses). So please provide some more nuance or turn the sentence around (saying that an individual’s ability to adaptively cope with stress is associated with heart Rate Variability).\n\nPlease add a clear definition of HRV and ACE and add the abbreviation to the word when it is used for the first time. Subsequently, you should just use the abbreviation in the rest of the manuscript (please also do this for any other abbreviations you use). Also specify when you are talking about resting HRV or the HRV response in relation to certain stressors/paradigms/….\n\n“Adverse childhood experiences (ACEs) are ubiquitous among the adult patient population”\nCould you be more specific? Are their prevalence rates available?\n\nPlease define interoception\n\n“In fact, most psychiatric disorders are sustained by a type of interoceptive phobia”\nPlease clarify\n\nNeurophysiological impairments due to ACEs have been shown to be reversible\nWhich neurophysiological impairments are you referring to here?\n\nMethods section Please follow a more traditional sequence in the methods section, starting with the sample. Specific comments:\nWere participants instructed just notice bodily sensation or exert control over them?\n\nConsider to use “their” instead of “his/her”\n\nDo I understand correctly that each session of phase 2 worked with different traumatic experiences? Is it therefore possible to compare the first and last session to one another? Were you able to investigate whether a pattern emerged over different sessions or whether any change between the first and last session was possibly due to the difference in content (perhaps more difficult traumatic instances were also handled in the first session or the other way around?).\n\n“Patient who required psychiatric medical treatment (therapist assessment) were excluded from the study”\nDoes this refer to receiving medication or having a diagnosis other than PTSD. It isn’t entirely clear whether participants have received any formal diagnoses (if so, please specify which) or what the exact treatment context was. I was also wondering whether any underlying details on the ACE were available (how many ACEs were experienced? Severity/burden? Were there any questionnaires used?)\n\nI noticed that the data collection section does have some details on diagnoses. Please add these to the sample section and please included how many individuals were suffering from each disorder. Could you also detail what the “clinical assessment” contains (is it based on a diagnostic interview, questionnaire,…).\n\nCould you specify what individuals were exactly doing during the HRV measurement? Were they sitting, standing, lying down? Were they resting or still actively working with the traumatic experiences?\n\nIs there any way to also report on the subjective effects of the intervention as research shows us that physiological and self-report data doesn’t always agree and it would be interested to have both indicators available.\nResults\nPlease report all results of the statistical analyses correctly (e.g., F (2, 54) = 4.643, p = .014.). You report using different HRV measurements (e.g., RMSSD, HF,…), please report which measurement you are talking about in the results. Be consistent in your terminology.\n\nPlease add significance levels to the figure and explain a bit more what it shows.\n\nWhere there any outliers that influenced results or were there non-responders?\nDiscussion\nHow do you explain that your HRV values were lower than those of the task force of the European Society of Cardiology and the North American Society of Pacing Electrophysiology?\n\nIs there any psychophysiological data from other studies on emotional trauma recall (with or without an intervention) to compare the current data with?\n\nYou mention very large individual differences in the effect of the intervention. Does this allow us to draw general conclusions?\n\n“However, the absence of statistical effects associated with biographical variables indicates that these types of effects are relatively unlikely.”\nPlease explain how you see this?\n\n“This study shows that interoception exposure therapy – combined with biofeedback - was able to increase parasympathetic influences”\nThis is a very strong conclusion, given that it is a non-controlled study with a lot of individual differences. Please add some nuance to this statement.\n\nIn the concluding paragraph, the authors state “HRV at rest” does this refer to the first measurement of each sessions (or both or…?).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "8038", "date": "19 Apr 2022", "name": "Arnaud Delorme", "role": "Author Response", "response": "Reviewer's suggestion: The current retrospective non-controlled study aimed to investigate whether the combination of a therapeutic intervention (including emotion exposure and biofeedback) influenced HR and HRV in outpatients with ACE. HR increased during the sessions but decreased from session 1 to session 2. HRV at the beginning of the session increased from session 1 to session 2. While the fact that this is a study in naturalistic circumstances can be a strength, since we are often limited to controlled research in laboratory circumstances, this also causes severe limitations in the design and interpretation of the results. Importantly, we are lacking a control group, which severely impedes interpretation. Additionally, large individual differences occurred and we would need more information about the sample to be able to interpret these. I would also be interested in seeing additional non-physiological outcome measures of the study or information about treatment effectiveness. The manuscripts requires the addition of a lot more information and the writing style and representation of the findings can be improved. However, I do think that the study could provide interesting information to the field if these improvements are made. Author's comment: We agree that the study lacks a control group. Since it is a retrospective clinical study, a control group was not possible. Still, we believe that the result of changes in patients' HRV over the course of the therapy is of scientific interest. We have clarified the method section and reorganized it. We have also worked on the writing style and remade the figure. Reviewer's suggestion: \"Psychiatric patients with adverse childhood experiences (ACE) tend to be dysfunctional in the interoceptive part of their emotional experience.\" I would suggest to state that patients or individuals have dysfunctions and not that they are dysfunctional. This is a more respectful and correct representation. Author's comment: The text has been changed. Reviewer's suggestion: \"Heart Rate Variability reflects an individual's ability to adaptively cope with stress.\" HRV is a concept that can reflect many things, including emotion regulation, but also stress, general resilience, mental illness, … (also depending on whether you are measuring resting HRV or HRV responses). So please provide some more nuance or turn the sentence around (saying that an individual's ability to adaptively cope with stress is associated with heart Rate Variability). Author's comment: We have changed the text accordingly and provided more nuance to this sentence. Reviewer's suggestion: Please add a clear definition of HRV and ACE and add the abbreviation to the word when it is used for the first time. Subsequently, you should just use the abbreviation in the rest of the manuscript (please also do this for any other abbreviations you use). Also specify when you are talking about resting HRV or the HRV response in relation to certain stressors/paradigms/…. Author's comment: We are now clearly defining HRV and ACE and differentiate between resting HRV and HRV response. In our manuscript, we are dealing with resting HRV, but by HRV response, we mean changes in HRV over the course of the therapy. Reviewer's suggestion: \"Adverse childhood experiences (ACEs) are ubiquitous among the adult patient population\". Could you be more specific? Are their prevalence rates available? Author's comment: We have added the prevalence rate and a citation. Reviewer's suggestion: Please define interoception Author's comment: We are now defining interoception. Reviewer's suggestion: \"In fact, most psychiatric disorders are sustained by a type of interoceptive phobia.\" Please clarify. Author's comment: We have clarified this sentence. Reviewer's suggestion: \"Neurophysiological impairments due to ACEs have been shown to be reversible.\" Which neurophysiological impairments are you referring to here? Author's comment: We were referring to changes in hippocampal volume. We have clarified this sentence. Reviewer's suggestion: Please follow a more traditional sequence in the methods section, starting with the sample. Author's comment: We have reorganized the method section and now start with describing our sample. Reviewer's question: Were participants instructed just notice bodily sensation or exert control over them? Author's comment: They were instructed just to notice them. We have clarified this point. Reviewer's suggestion: Consider to use \"their\" instead of \"his/her\" Author's comment: We have replaced all instances of \"his/her\" with \"their\" Reviewer's question: Do I understand correctly that each session of phase 2 worked with different traumatic experiences? Is it therefore possible to compare the first and last session to one another? Were you able to investigate whether a pattern emerged over different sessions or whether any change between the first and last session was possibly due to the difference in content (perhaps more difficult traumatic instances were also handled in the first session or the other way around?). Author's comment: Usually, patients' adverse experiences revolve around the same theme. The reviewer correctly mentions that there might be some heterogeneity in the ACE reported in each session. We believe this averages out across the patient population, but we have added this limitation to the discussion. Reviewer's question: \"Patient who required psychiatric medical treatment (therapist assessment) were excluded from the study.\" Does this refer to receiving medication or having a diagnosis other than PTSD. It isn't entirely clear whether participants have received any formal diagnoses (if so, please specify which) or what the exact treatment context was. I was also wondering whether any underlying details on the ACE were available (how many ACEs were experienced? Severity/burden? Were there any questionnaires used?) Author's comment: This refers to patients receiving medication. Among others, questionnaires DES (Dissociative Experience Scale) and PCL-S (PTSD checklist – specific) were used with patients, although the diagnosis was based on the expertise of the clinician. We have clarified this section. Reviewer's suggestion: I noticed that the data collection section does have some details on diagnoses. Please add these to the sample section and please include how many individuals were suffering from each disorder. Could you also detail what the \"clinical assessment\" contains (is it based on a diagnostic interview, questionnaire,…). Author's comment: We have moved the section as advised. We have also clarified the clinical diagnosis. Reviewer's suggestion: Could you specify what individuals were exactly doing during the HRV measurement? Were they sitting, standing, lying down? Were they resting or still actively working with the traumatic experiences? Author's comment: All patients were sitting at rest. We have clarified this point. Reviewer's suggestion: Is there any way to also report on the subjective effects of the intervention as research shows us that physiological and self-report data doesn't always agree and it would be interesting to have both indicators available. Author's comment: this is an interesting comment. The current data collection scheme includes the subjective assessment of both the patient and the therapist, who, after a given number of sessions, which might be different for each patient, agreed that the therapy was complete. Reviewer’s suggestion: Please report all results of the statistical analyses correctly (e.g., F (2, 54) = 4.643, p = .014.). You report using different HRV measurements (e.g., RMSSD, HF,…), please report which measurement you are talking about in the results. Be consistent in your terminology. Author's comment: We have changed statistical reporting. We have also clarified the measures being reported. Reviewer's suggestion: Please add significance levels to the figure and explain a bit more what it shows. Author's comment: We have added significance to the figure and additional details to its caption. Reviewer's question: Where there any outliers that influenced results or were there non-responders? Author's comment: This is a detailed analysis we did not perform. We can expect that there were non-responders and outliers, although based on the age of the data, it would take considerable effort to find the number of non-responders, and we do not think it would necessarily be informative – observing no response does not imply that the patient did not respond to the treatment. It might be possible that for a given patient with no increase in HRV, the coupling of emotional traumas with HRV is weaker. ANOVA has poor resistance to outliers, so if there were outliers, their presence was not prominent enough to invalidate our results. We have added a comment to the discussion. Reviewer's question: How do you explain that your HRV values were lower than those of the task force of the European Society of Cardiology and the North American Society of Pacing Electrophysiology? Author's comment: Low HRV is associated with stress, and the data from the European Society of Cardiology and the North American Society of Pacing Electrophysiology were for control participants. It is to be expected that HRV of patients, in general, would be lower than control participants. We have added that interpretation to the manuscript. Reviewer's question: Is there any psychophysiological data from other studies on emotional trauma recall (with or without an intervention) to compare the current data with? Author's comment: We are not aware of studies on emotional trauma recall using HRV that report the measure that we are reporting for detailed comparisons. We have added a citation to a recent study on the subject, but the data is not available. By contrast, we have made our data available. This study is consistent with decreased HRV in the population of ACE patients. Reviewer's question: You mention very large individual differences in the effect of the intervention. Does this allow us to draw general conclusions? Author's comment: HRV is a noisy physiological measure. Large differences in HRV are common in the population, with centenarians having HRV comparable to 20-year-old individuals (despite the well-established decrease in HRV with age). Therefore, large differences in HRV response to treatment are also expected. The origin of HRV inter-individual differences is unknown to our knowledge. Reviewer's question: \"However, the absence of statistical effects associated with biographical variables indicates that these types of effects are relatively unlikely.\" Please explain how you see this? Author's comment: We have rephrased that section to clarify this statement. Reviewer's suggestion: \"This study shows that interoception exposure therapy – combined with biofeedback - was able to increase parasympathetic influences\" This is a very strong conclusion, given that it is a non-controlled study with a lot of individual differences. Please add some nuance to this statement. Author's comment: We have added nuance to that statement. Reviewer's question: In the concluding paragraph, the authors state \"HRV at rest\" does this refer to the first measurement of each sessions (or both or…?) Author's comment: This refers to both measurements. We have clarified this in the method section." } ] } ]	1	https://f1000research.com/articles/9-326
https://f1000research.com/articles/10-466/v1	14 Jun 21	{ "type": "Research Article", "title": "Fermented medicinal herbs improve the hematological and physiological profile of Striped catfish (Pangasionodon hypophthalmus)", "authors": [ "Henni Syawal", "Ronal Kurniawan", "Irwan Effendi", "Brian Austin", "Henni Syawal", "Ronal Kurniawan", "Brian Austin" ], "abstract": "This study sought to determine the effect of fermented medicinal herbs (FMH), i.e. cutchery (Kaempferia galanga), tumeric (Curcuma longa) and curcuma (Curcuma xanthorrhiza) in combination with molasses and probiotic drink (Yakult), administered orally on the hematological and physiological profile of striped catfish (Pangasionodon hypophthalmus). A complete randomized design (CRD) experiment was used with four levels of treatments, namely P0 (control), P1 (FMH 100 ml/kg), P2 (FMH 200 ml/kg) and P3 (FMH 300 ml/kg) of feed. The fish were kept in a farm in cages at 75 fish/m3, and fed with the experimental diets for 60 days. The results revealed that FMH (P2) dietary administration improved hematological and physiological profile of catfish, i.e total erythrocytes of 2.81 x 106 cells/mm3, hematocrit values of 39.00 %, hemoglobin levels of 10.73 g/dl, total leukocytes of 11.41 x 104 cells/mm3, blood glucose 97.33 mg/dl, and total serum protein 4.10 mg/dl compared to controls with 1.89 x 106 cells/mm3, 32.33 %, g/dl, 9.67 x 104 cells/mm3, 67.33 mg/dl, and total serum protein of 3.10 mg/dl, respectively. Moreover, the diet improved special growth rate, feed conversion ratio, feed efficiency and the survival rate of catfish", "keywords": [ "Medicinal herb", "Catfish", "Immunostimulant", "Hematology", "Physiology", "Immunity" ], "content": "Introduction\n\nAttention in aquaculture in developing countries has focused on the use of nonspecific immunostimulants and plant products, which could have a beneficial effect in fish disease control. Interest in medicinal plants for application to aquaculture follows their use in human medicine and agriculture as proven prophylactic and therapeutic agents.1–3 For example, garlic (Allium sativum) and ginger (Zingiber officinale)4 have histories of dietary and medicinal applications as anti-infective agents. Evidence of their value include inhibition towards pathogens of relevance to aquaculture, including bacteria,5–7 viruses,8–10 and protozoa.11–12\n\nSome researchers have reported the impact of herbal supplemented diets on hematology and innate immunity of fish.13–14 For example, Chinese herbs (Lonicera japonica and Ganoderma lucidum) enhanced the non-specific immune response of tilapia (Oreochromis niloticus), leading to protection against Aeromonas hydrophila.15 Similarly, the dietary administration of rose hip and safflower stimulated growth performance, hematological, biochemical parameters and innate immune responses of beluga (Huso huso)16,17 The supplemented probiotic stimulated growth performance and feed utilization of keureling fish Tor tambra. Against this background, fermented medicinal herbs (FMH) in combination with molasses and probiotic drink (Yakult) have been used successfully as dietary supplements on a small number of rural catfish farms in Sumatra, Indonesia, for approximately two years. Anecdotal evidence has suggested that fish grow better and faster, appear to be healthier, and the flesh tastes better after cooking. Fish, which have received these diets, have been examined for potential improvements to hematology and physiology.\n\n\nMethods\n\nThis research was conducted on floating net cages in the Reservoir of the Faculty of Fisheries and Marine Affairs by using a completely randomized design with four treatments and three replications, namely P0 (control), P1 (FMH 100 mL/kg feed), P2 (FMH 200 mL/kg), and P3 (FMH 300 mL/kg). all steps in this experiment was carried out within the international ethical guidelines provided by ARRIVE guidelines.\n\nA total of 2700 fingerling catfish used had an average length of 9 cm and an average weight of 6 g. Catfish were obtained from local farmers in Kampar Regency and distributed randomly into cages measuring 1 × 1.5 × 1 m with a density of 75 individuals/m3. The cages were constructed of polyethylene nets with a mesh size of 7 × 7 mm. Water was free-flowing, and the temperature ranged 27.5–29.5°C.\n\nAll fish were acclimatized for seven days before use. The fish samples were selected based on their performance. The fish that showed active swimming, no wounds or external parasites. To measure the growth of the fish, every 10 days a random weighing of the fish was carried out.\n\nThe dietary supplements were chosen because of their availability, and use in local, Sumatran, aquaculture. Thus, fermented medicinal herbs (FMH) consisting of cutchery (Kaempferia galanga), turmeric (Curcuma longa) and curcuma (Curcuma zanthorrhiza) together with molasses, probiotic drink (Yakult), fresh water and yeast (Saccharomyces cerevisiae) were used (Table 1). Fresh turmeric, cutchery, and curcuma were washed, thinly sliced, mixed and milled. Then, 300-g quantities were transferred to 3 L of water, boiled for 30 min, and cooled to room temperature. The mixture was then squeezed and filtered to obtain the liquid fraction, which totalled 2.7 L. This was mixed with 175 mL of molasses, 65 mL of probiotic drink and 50 g of yeast. Then, the mixture was stirred until homogeneous and poured into 5-L capacity jerry cans, which were tightly closed. Fermentation was allowed to occur for 7–10 days until the aroma changed from a curcuma smell to a strong alcoholic odour, and gas was no longer produced. The gas produced during fermentation was released daily by opening the lids for a few seconds. FMH was added to 1-kg quantities of pelleted feed (Hi-Pro-Vite 781 PT. Central Proteina Prima Tbk) to achieve 0 (control; P0), 100 mL/kg (P1), 200 mL/kg (P2) and 300 mL/kg (P3) The feed was administered three times a day to achieve 5% of body weight for 60 days, which reflected the period that the diets have been used on fish farms. Survival rates were calculated using the following formula, SR = Nt/No×100%, where SR = survival (%), Nt = number of live fish at the end of the study, and No = number of live fish at the beginning of the study.\n\nFish blood was removed at 0, 30 days and 60 days. Thus, the fish were anesthetized with clove oil at a dose of 0.05 mL/L. Blood was withdrawn from the caudal vein using 1-mL syringes that had been rinsed with 10% EDTA, collected in microtubes (Axygon) and stored at room temperature until use. The fish that have been blood drawn are then awakened by placing them in a container filled with aerated water. The fish were returned to the cages after they were seen breathing and swimming normally.\n\nTo determine the number of erythrocytes, blood in 0.1-mL quantities was mixed thoroughly with 1.0 mL of Hayem solution comprising sodium sulfate, sodium chloride and mercuric chloride.19 Then, the number of erythrocytes was determined by use of a hemocytometer at a magnification of ×40 with calculation according to the formula of Blaxhall and Daisley.19 The method of Blaxhall and Daisley19 was used to estimate the total number and types (i.e. lymphocytes, monocytes, neutrophils, and platelets) of leucocytes. For this, blood was dripped onto a hemocytometer slide, covered with a coverslip, and examined microscopically with a magnification of ×40. The total number of leukocytes was calculated using the following formula: ∑Leukocytes = ∑n × 50 cells/mm3, where ∑n = total number of leukocytes in four large boxes, and 50=dilution factor. Calculation of hemoglobin levels was carried out using Sahli’s method.20 To determine hematocrit level, blood was drawn into a hematocrit capillary tube the end of which was blocked with crystoseal.19 Then the capillary tube was centrifuged for 3 min at 11000 rpm in a microhematocrit centrifuge Model SH120-1. The hematocrit was measured as a percentage of the hematocrit value in the microhematocrit reader\n\nBlood glucose was measured using GlucoDr (allmedicus) with a range of 20–600 mg/dL. Glucose testing was carried out in the morning before the fish were fed.21 Total serum protein was measured by the method of Anderson and Siwicki.22 Blood was centrifuged at 3,500 rpm for 15 min to completely separate the serum, which was transferred to a fresh microtube. Then, 20 μL of serum and 1000 μL of protein test reagent (Reiged Diagnostics) were added to each microtube, with mixing. After incubation for 15 min, the absorbance was read at λ 595–610 nm.\n\n\n\na) The absolute weight was measured using the formula AW = Wt − Wo, where AW = absolute weight (g), Wt = average weight at the end of the study (g), and Wo = average length at the beginning of the study (g).\n\nb) Specific growth rates were calculated using the formula, SGR = SGR=Wt−Wot×100, where Wt = larvae weights at the end of the study (g), Wo = larvae weights at the beginning of the study (g), and t = length of study (day).\n\nc) Feed conversion was calculated by using the formula, FCR=ΣFBt+Bm−Bo, where FCR = feed conversion ratio, ΣF = amount of feed fed during experiment (g), Bt = fish biomass weight at the end of maintenance (g), Bo = fish biomass at the beginning of study (g), and Bm = biomass of dead fish during maintenance (g).\n\nd) Feed efficiency was calculated by using formula, FE = (Bt + Bm) − Bo)/F × 100, where FE = feed efficiency, ΣF = amount of feed fed during experiment (g), Bt = fish biomass at the end of experiment (g), Bm = biomass of fish that died during the study (g), and Bo = fish biomass at the beginning of the study (g).\n\nThe experiments employed a completely randomized design (CRD) involving use of SPSS version 22.\n\nAll animals are treated according to animal welfare guidelines that have been established and approved by the Dean of the Faculty of Fisheries and Marine Sciences, Riau University (Prof. Bintal Amin, serves as the ethical committee who approved the use of vertebrate animals in this experiments with number: 346/UN19.5.1.1.4/KP/2020).\n\n\nResults\n\nThe fish consumed the experimental diets better than the controls without any evidence of a period of adjustment. Moreover, compared with the controls, the fish receiving the experimental diets were more active, responding quickly to the arrival of the feed. Overall, the FMH diets improved the growth rate of fish significantly (p < 0.05). In addition, the provision of the supplements maintained the survival rate of catfish at 100% (Table 2).\n\nFMH diets improved the hematological profile of striped catfish significantly (p < 0.05) compared to the experimental controls. Thus, total erythrocyte counts ranged between 2.45–2.81 × 106 cells/mm3, hematocrit values were from 35.67 to 39.00%, and hemoglobin levels ranged from 9.2–10.73 g/dL (Table 3). This compares with 1.89 × 106 cells/mm3, 32.33 %, and 8.80 g/dL, for the controls (Table 3). Consistently, diet P2 exhibited the best level of hematological profiles at both 30 and 60 days after initiating the feeding regime (Table 3). Moreover, the examination of the groups revealed that the fish were in excellent condition, were more agile, and appeared to have a better colour. The internal organs were normal in appearance.\n\nSuperscript letters on the same line show significantly different results between treatments (p < 0.05). RBC: red blood cell (×106 cells/mm3); WBC: white blood cell (×104 cells/mm3).\n\nFMH affected the physiological profile of striped catfish significantly (p < 0.05) compared with the controls. Thus, blood glucose levels and total serum protein increased from 78.67 to 97.33 mg/dL and 3.70 to 4.10 mg/dL; osmoregulation ranged from 288–327 mOsm/L H2O, absolute weight from 89.11–119.08 g, and the survival rate from 96–100% (Table 4).\n\nSuperscript letters on the same line show significantly different results between treatments (p < 0.05). SR: survival rate.\n\n\nDiscussion\n\nThis study has supported the use of FHM feed supplements in Indonesian aquaculture, and provided evidence for the mode of action. Thus, there were notable improvements in the hematological and physiological profiles of catfish. Indeed, similar results have been reported by other researchers. For example, Lee et al. 23 evaluated the dietary supplementation of citrus by-products (CB) fermented with probiotic bacteria on growth performance, feed utilization, innate immune responses and disease resistance of juvenile olive flounder. They noted that innate immunity was significantly enhanced by CBF–BS (CB fermented with Bacillus subtilis) supplementation. This study indicated that the fermentation of CB with probiotic had beneficial effects on innate immunity and thereby increased disease resistance.\n\nThe important components of FMH are considered to be due to the content of secondary metabolites, namely curcuminoids, vitamin C, essential oils, tannins, and flavonoids, which trigger immunostimulation.24 The presence of curcuminoids provide an antioxidant effect on cell membranes reducing erythrocyte cell membrane damage due to oxidation.25 Similarly, flavonoids are natural antioxidants, which are credited with the ability of reducing free radicals and anti-free radicals.26\n\nThe benefit of FMH for improved growth performance mirrors previous work by Hoseinifar et al.,27 who reported that dietary application of the phytoimmunostimulant Persian hogweed (Heracleum persicum) improved growth significantly. Specifically, the final weight, weight gain, specific growth rate and feed conversion ratio were significantly improved in fish that received dietary H. persicum at or above 5 g/kg.27\n\nClearly, this study has demonstrated that dietary FMH, particularly when dosed at 200 mL/kg of feed, improved the hematological and physiological profile of catfish. Also, there was benefit for specific growth rate, feed conversion ratio and survival.\n\n\nData availaibility\n\nAll data underlying the results are available as part of the article and no additional source data are required", "appendix": "Acknowledgements\n\nWe are grateful to University of Riau, Ministry of Research, Technology and Higher Education of Indonesia for financial support.\n\n\nReferences\n\nPandey G, Sharma M, Mandloi AK: Medicinal plants useful in fish diseases. Review Article. Plant Archiv. 2012; 12: 1–4.\n\nPandey G, Sharma M: Immunostimulant effect of medicinal plants on fish. Int. Res. J. Pharm. 2014; 3: 112–114.\n\nGupta A, Mahajan S, Sharma R: Evaluation of antimicrobial activity of Curcuma longa rhizome extract against Staphylococcus aureus. Biotechnol. Rep. 2015; 6: 51–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNya EJ, Austin B: Use of garlic, Allium sativum, to control Aeromonas hydrophila infection in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 2009; 32: 963–970. PubMed Abstract \| Publisher Full Text\n\nChristybapita D, Divyagnaneswari M, Michael RD: Oral administration of Eclipta alba leaf aqueous extract enhances the non-specific immune responses and disease resistance of Oreochromis mossambicus. Fish Shellfish Immunol. 2007; 23: 840–852. PubMed Abstract \| Publisher Full Text\n\nNewaj-Fyzul A, Austin B: Probiotics, immunostimulants, plant products and oral vaccines, and their role as feed supplements in the control of bacterial fish diseases. Fish Dis. 2014; 38: 937–955. PubMed Abstract \| Publisher Full Text\n\nBilen S, Elbeshti HTAG: A new potential therapeutic remedy against Aeromonas hydrophila infection in rainbow trout (Oncorhynchus mykiss) using tetra, Cotinus coggygria. J. Fish Dis. 2019; 42: 1369–1381. PubMed Abstract \| Publisher Full Text\n\nAsely AME, Shaheen AA, Abbass AA, et al.: Immunomodulatory effect of plant-mixed feed in kuruma shrimp, Marsupenaeus japonicus, and its protective efficacy against white spot syndrome virus infection. J. Fish Dis . 2010; 33: 859–863. PubMed Abstract \| Publisher Full Text\n\nYu Q, Liu M, Xiao H, et al.: The inhibitory activities and antiviral mechanism of Viola philippica aqueous extracts against grouper iridovirus infection in vitro and in vivo. J. Fish Dis. 2019; 42: 859–866. PubMed Abstract \| Publisher Full Text\n\nFoysal MJ, Alam M, Momtaz F, et al.: Dietary supplementation of garlic (Allium sativum) modulates gut microbiota and health status of tilapia (Oreochromis niloticus) against Streptococcus iniae infection. Aquacult. Res. 2019; 50: 2107–2116. Publisher Full Text\n\nAmonkar SV, Banerji A: Isolation and characterization of larvacidal principle of garlic. Science . 1971; 174(4016): 1343–1344. PubMed Abstract \| Publisher Full Text\n\nSoffer SA, Mokhtar GM: Evaluation of the antiparasitic effect of aqueous garlic (Allium sativum) extract in Hymenolepiasis nana and giardiasis. J. Egypt. Soc. Parasitol. 1991; 21: 497–502. PubMed Abstract\n\nAwad E, Austin B: Use of lupin, Lupinus perennis, mango, Mangifera indica, and stinging nettle, Urtica dioica, as feed additives to prevent Aeromonas hydrophila infection in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 2010; 33: 413–420. PubMed Abstract \| Publisher Full Text\n\nAwad E, Awad A: Role of medicinal plants on growth performance and immune status in fish. Fish Shellfish Immunol. 2017; 67: 40–54. PubMed Abstract \| Publisher Full Text\n\nYin G, Ardo L, Jeney Z, et al.:Chinese herbs (Lonicera japonica and Ganoderma lucidum) enhance non-specific immune response of tilapia, Oreochromis niloticus and protection against Aeromonas hydrophila. In: Diseases in Asian Aquaculture VI, Fish Health Section. ( Bondad-Reantaso MG, Mohan CV, Crumlish M, Subasinghe RP eds.). Manila, Philippines: Asian Fisheries Society; 2008; 269–282.\n\nDadras H, Hayatbakhsh MR, Shelton WL, et al.: Effects of dietary administration of rose hip and safflower on growth performance, haematological, biochemical parameters and innate immune response of Beluga, Huso huso (Linnaeus, 1758). Fish Shellfish Immunol. 2016; 59: 109–114. PubMed Abstract \| Publisher Full Text\n\nMuchlisin ZA, Murda T, Yulvizar Y, et al.: Growth performance and feed utilization of keureling fish Tor tambra (Cyprinidae) fed formulated diet supplemented with enhanced Probuitic. F1000Res . 2017; 6: 137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAustin B, Austin DA: Methods for the Microbiological Examination of Fish and Shellfish . Chichester, England: Ellis Horwood; 1989.\n\nBlaxhall PC, Daisley KW: Routine haematological methods for use with fish blood. J. Fish Biol. 1973; 5: 577–581. Publisher Full Text\n\nPatil PJ, Thakare GV, Patil SP: Variability and Accuracy of Sahli’s Method in Estimation of Haemoglobin Concentration. Natl J Integr Res Med. 2013; 4(1).\n\nPhilipson HL, Stefani CE, Victoria EP, et al.: Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis. Zebrafish. 2010; 7(2). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson DP, Siwicki AK:Basic hematology and serology for fish health programs. Asian Fisheries Society; Fish Health Section. Symposium 2, Phuket; Thailand. In: Diseases In Asia Aquaculture . 1995; 2. : 185–202.\n\nLee BJ, Kim SS, Song JW, et al.: Effects of dietary supplementation of citrus by-products fermented with a probiotic microbe on growth performance, innate immunity and disease resistance against Edwardsiella tarda in juvenile olive flounder, Paralichthys olivaceus (Temminck & Schlegel). J. Fish Dis. 2013; 36: 617–628. PubMed Abstract \| Publisher Full Text\n\nIkawati Z, Yuniarti N, Margono SA: The analgesic effect of a curcumin analogue 1,5-bis (4’-hydroxy-3’-metahoxphenyl)-1,4-pentadien-3-on (Gamavuton-0) in acute and persistent pain. J. Appl. Pharmaceut. Sci. 2014; 4: 48–51.\n\nMaribito R, Falliti G, Geraci A, et al.: Curcumin protects –sh group and sulphate transport after oxidative damage in human erythrocytes. Cell Physiol Biochem . 2015; 36: 345–357. PubMed Abstract \| Publisher Full Text\n\nNimse SB, Pal D: Free radicals, natural antioxidants, and their reaction mechanisms. RSC Adv . 2015; 5: 27986–28006. Publisher Full Text\n\nHoseinifar HS, Zoheiri F, Lazado CC: Dietary phytoimmunostimulant Persian hogweed (Heracleum persicum) has more remarkable impacts on skin mucus than on serum in common carp (Cyprinus carpio). Fish Shellfish Immunol. 2016; 59, 77–82. PubMed Abstract \| Publisher Full Text" }	[ { "id": "87441", "date": "22 Jul 2021", "name": "S. M. Sharifuzzaman", "expertise": [ "Reviewer Expertise Aquaculture health and nutrition" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSome specific comments:\n1) Please revise title as \"Fermented medicinal herbs improve hematological and physiological profiles of Striped catfish (Pangasionodon hypophthalmus)\"\n2) \"Some researchers have reported the impact of herbal supplemented diets on hematology and innate immunity of fish\" - so why were you combining molasses and probiotics? Please explain this in introduction section.\n3) What were the probiotics cell numbers in different experimental diets?\n\n4) An additional experiment with medicinal herbs, i.e. cutchery (Kaempferia galanga), turmeric (Curcuma longa) and curcuma (Curcuma zanthorrhiza) devoid of molasses, probiotic drink (Yakult) and yeast (Saccharomyces cerevisiae) would be interesting to determine the role of probiotics and prebiotics - the authors are requested to include 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "92787", "date": "31 Aug 2021", "name": "Saurav Kumar", "expertise": [ "Reviewer Expertise Aquatic Environment and Health 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\nTitle:\n\nCorrect Pangasianodon spelling.\n\nAbstract:\nWrite only significant results and no need to mention values of estimated parameters in this section.\n\nIntroduction:\n\nAuthor needs to highlight the researchable problem with normal herbal immunostimulant, and why is the fermentation process required for preparing the product?\n\nMethodology:\n\nFish acclimatization and standard procedure of FHM product preparation need to be mentioned with suitable references.\n\nStatistical analysis of data needs to be mentioned.\n\nResults:\nWhy do the significant change in the control group observe in RBC of 30 days sampled fish from 60 days?\n\nDo the experimental periods have a significant effect on observed parameters?\n\nSimilar changes are also observed in haemoglobin, hematocrite values...\n\nDiscussion:\nThe section is well written however the mechanism need to be illustrated.\n\nReferences:\n\nVery few recent references and authors need to cite the recent findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-466
https://f1000research.com/articles/11-426/v1	14 Apr 22	{ "type": "Research Article", "title": "TReNCo: Topologically associating domain (TAD) aware regulatory network construction", "authors": [ "Christopher Bennett", "Viren Amin", "Daehwan Kim", "Murat Can Cobanoglu", "Venkat Malladi", "Christopher Bennett", "Viren Amin", "Daehwan Kim", "Murat Can Cobanoglu" ], "abstract": "Introduction: There has long been a desire to understand, describe, and model gene regulatory networks controlling numerous biologically meaningful processes like differentiation. Despite many notable improvements to models over the years, many models do not accurately capture subtle biological and chemical characteristics of the cell such as high-order chromatin domains of the chromosomes. Methods: Topologically Associated Domains (TAD) are one of these genomic regions that are enriched for contacts within themselves. Here we present TAD-aware Regulatory Network Construction or TReNCo, a memory-lean method utilizing epigenetic marks of enhancer and promoter activity, and gene expression to create context-specific transcription factor-gene regulatory networks. TReNCo utilizes common assays, ChIP-seq, RNA-seq, and TAD boundaries as a hard cutoff, instead of distance based, to efficiently create context-specific TF-gene regulatory networks. Results: We used TReNCo to define the enhancer landscape and identify transcription factors (TFs) that drive the cardiac development of the mouse. Conclusion: Our results show that we are able to build specialized adjacency regulatory network graphs containing biologically relevant connections and time dependent dynamics.", "keywords": [ "GRN", "gene regulatory network", "epigenomics", "TAD" ], "content": "Author summary\n\nThe regulation of genes is the basis of all biological processes. Gene regulatory networks (GRN) are powerful tools for understanding complex biological connections between interacting genetic elements. They have proven invaluable in understanding driving forces in normal development and differentiation of cells and in understanding the factors involved in cancer progression. Despite the improvements in network construction, we lack a comprehensive method for constructing highly personalized networks to the genetic background of the cells used and validating the findings. Furthermore, as the complexity of the network graphs increases, we need more computationally efficient ways of representing and exploring the networks. To this end, we developed a method which utilizes epigenetic marks of enhancer/promoter activity, gene expression, and Transcriptionally Active Domains (TADs) to rapidly construct accurate context-specific GRN. Our results show that we are able to expand current methods for generating regulatory networks to take advantage of transcriptionally active chromatin domains. The networks produced in this way contain an expanded set of potentially relevant biological connections that can be explored. We believe this method opens the possibility to understand deeper connections and new possibilities for biological discovery.\n\n\nIntroduction\n\nIt is of critical importance to understand, model, and describe gene regulatory networks (GRN) that control diverse cellular functions of interest like those that drive differentiation or transitions from one development stage to another (Lee et al. 2002; DeRisi et al. 1997; Goode et al. 2016). With the advent of next-generation sequencing technologies, it is now commonplace to reconstruct these networks to connect transcription factors (TFs) to the genes they regulate (Karlebach and Shamir 2008). One classic method is integration of cis-regulatory elements, like enhancers, and gene expression via matrix factorization to form network graphs between genes and TFs (Marbach et al. 2016). Generally, this is done using Chromatin Immunoprecipitation (ChIP) for H3K27ac to identify enhancers and RNA-seq to identify controlled genes. In many cases connections are determined through perturbations in upstream components like TFs and observing resultant changes in downstream expression levels (Gasperini et al. 2019). This method works exceptionally well for certain classes of TF and for closely linked enhancer-gene interactions. However, it commonly uses arbitrary length cut offs to prevent enhancers from erroneously influencing genes in distant parts of the genome. This can lead to enhancers having shorter or broader ranges of influence than what occurs biologically. As many recent chromosome-confirmation-capture (e.g. 5C, Hi-C and ChIA-PET) experiments have shown, there can be very broad and dynamic interactions made between different parts of a chromosome (Branco and Pombo 2007; McCord et al. 2020). Thus, it is more relevant to dynamically limit enhancers range of influence to only the topologically linked portions of the genome an enhancer is confined to, also known as Topologically Associated Domains (TADs). These regions are highly conserved across cell types and are known to limit the influence of cis-regulatory elements by physically separating them (Beagan and Phillips-Cremins 2020). Thus, it is critical that these cutoffs are included in the model to fully represent and capture the true biological processes occurring.\n\nHere we present TAD-aware Regulatory Network Construction or TReNCo, a powerful, memory efficient tool for constructing regulatory networks from enhancer, promoter, and gene expression data without the need for perturbations. We designed TReNCo to construct a graph of interaction weights between TFs and the genes that they control using TAD boundaries to dynamically limit the range of enhancer influence. We utilize dynamic programming to factor matrices within TADs and combine network into a full adjacency matrix for a regulatory graph. With this method, we are able to capture biologically relevant interactions between known TFs and their gene targets. We show that this network contains many subtle interactions that could be a treasure trove of novel or uncharacterized interactions. We believe this method opens the possibility for understanding deeper mechanistic connections and new possibilities for identifying biological targets for drug discovery.\n\n\nMethods\n\nTReNCo begins by generating distinct transcription start sites (TSSs), using a parsing tool, MakeGencodeTSS, for protein-coding genes from Gencode annotation files: Mouse Gencode version 4 by default. Promoters are then constructed using bedtools (2.29) slop (Quinlan and Hall 2010) to extend 1000 nucleotides upstream and 200 nucleotides downstream of the TSS in a strand specific extension. Enhancer boundaries are then generated by using bedops (2.4) merge (Neph et al. 2012) to merge the user defined H3K27ac ChIP peak bed files and excluding overlaps with promoter regions.\n\nA transcript expression matrix with normalized log2 transcripts/fragments per kilobase million (TPM) is generated from the provided RNA-seq expression tables with each row corresponding to a gene and the columns corresponding to a sample. The same is done for enhancers with bedtools coverage (Quinlan and Hall 2010) being used to calculate the coverage for each enhancer for each sample using the enhancer regions defined previously.\n\nLog-odds ratio for TF (TF) binding to promoters and enhancers is calculated with MEME-suite software, FIMO (Grant et al. 2011), using cis-bp motif database on promoter and enhancer sequences extracted from the genome, default mm10 (GRCm38), using bedtools getfasta (Quinlan and Hall 2010) and the bed files generated previously. TF matrices are constructed by reformatting the native output of FIMO and converting TF names to gene symbols.\n\nGene expression and H3K27ac ChIP data was selected from ENCODE. Data was chosen using a script to select mouse heart data corresponding to embryonic day 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, postnatal day 0 and 8 weeks old and had a matched set of gene expression and ChIP-seq. In total every sample had at least technical duplicates with embryonic day 14.5, postnatal day 0, and 8 weeks time points having 4 replicates of gene expression and embryonic day 14.5 having 4 ChIP-seq replicates. Two of the embryonic day 14.5 gene expression data were dropped due to poor correlation (average R2 less than 0.7) with the rest of the data set (S1 Table, for accession numbers).\n\nPlots and graphs were built using seaborn for python and ggplot2 for R scripts. All heatmaps were built in python and analyzed with scikit-learn. Gene networks from previous studies were downloaded from the corresponding journals and converted to a list. Genes networks for Gata4, Srf, Mef2a, and Nfx2-5 were subset from our networks and targets for these TFs were converted to lists. Venn-diagrams of gene list overlaps were built in python using venn2 package. Analysis of GO-terms was performed in R using enrichGO in clusterProfiler with org. Mm.eg.db database. Plots for GO-terms were built using the built in dotplot function in clusterProfiler.\n\n\nResults and discussion\n\nWe designed TReNCo utilizing a previously reported core matrix factorization method with a distance-based scoring system broken down into subunits based on TAD boundaries (Marbach et al. 2016; Cuellar-Partida et al. 2012). In brief, our algorithm uses normalized gene expression count tables from RNA-seq (tsv files) and H3K27ac ChIP-seq read alignments (bam files), peaks (bed files), and TAD boundaries (a bed file) (Figure 1). Though the source of these data can vary, we designed TReNCo with ENCODE uniform processing pipelines in mind. We first generated initial expression matrices for gene expression (G) and enhancer expression (E) by sample. This was accomplished using the count tables from RNA-seq and building count tables for all enhancers merged into non-overlapping segments from the ChIP-seq data. These counts were used to calculate the TPM which are then log-scaled. A key file, provided by the user, is used to link related files to build a full expression matrix and, secondarily, serves to reduce memory usage by allowing batch processing of data.\n\nA) Diagram with required inputs, gene expression, enhancers, and TAD boundaries input into a model using the basic equations shown leading to a gene interaction graph result. ‘+’ and ‘x’ indicate standard matrix additions and multiplications, respectively. The other operations such as ⊙, log, square root, and arctan are all element-wise. B) Pseudo-code for constructing a Gene Interaction Graph.\n\nWe next worked to establish TF-gene linkages by identifying TF binding sites in promoters and enhancers using a program, FIMO (a part of the MEME software suite and report the log-odds score of TF binding) a major weight needed for establishing interaction (Grant et al. 2011). We designed a simple pipeline to generate promoter and enhancer master bed files and remove any potential overlaps between promoters and enhancers to ensure that TFs are not double counted to a gene. Furthermore, these files contained the union of all promoters and enhancers between the samples in order to streamline the identification of TFs. This was a critical time saving step as FIMO cannot be multithreaded and can take upward of 24 hours to run. By using master files, we could run FIMO only once per process leading to a huge performance boost.\n\nWith these core datasets, for each sample we were able to select sample-specific genes, enhancers, and TF interactions. To ensure proper TAD boundaries were followed and to improve speeds through multithreading, we designed a dynamic programming algorithm to process these datasets by TADs and generate TAD-specific distance weight matrices (Dt) for each set. These matrix subsets were factored with the square-root of a TAD-specific interaction matrix produced via vector multiplication between the gene (gk,t) and enhancer (ek,t) expression profiles resulting in a TAD-weight matrix (Wk,t). To generate an enhancer-specific graph edges, the weight matrix was factored with the TAD-specific enhancer-TF by gene matrix (Mt) normalized to the maximum value of the matrix. This was done to set a standard scale of log-odds that was comparable between enhancers and promoters. We designed this component with the assumption that enhancer-TF binding should be similar in promoters and should be weighted the as a log-odds scale in the network. A promoter-TF by gene specific subnetwork (Pt) was produced in a similar manner as the enhancer-specific network with weighing done using a TAD-specific gene expression vector since all distances between promoters and genes are 1. Arctangents were applied to both matrices due to the properties of the transformation where larger values approach an asymptote of π/2 while smaller values are approximately scaled linearly. This scaling draws larger value outliers into a tighter range without heavily influencing lower values and assumes a maximum impact a TF can have on a gene. The resulting matrices were added together and further weighted by normalized TF gene expression to lower the influence of lowly expressed TFs while minimally changing the effects from highly expressed TFs. The resulting TAD subgraphs were concatenated together into a full network adjacency graph matrix. Since this was a sparse matrix, TReNCo represents it as an adjacency array allowing us to store the information in much less space than is needed for a matrix.\n\nTo validate the model, we used the extensive cohort of matched gene expression and H3K27ac ChIP-seq analyses in ENCODE (Davis et al. 2018) (S1 Table) and used TAD boundaries generated from HI-C data from Mouse ES (Gorkin et al. 2020; Dixon et al. 2012). We decided to use mouse heart data due to the abundance of well correlated time point data spanning embryonic day 10.5 to 8 weeks after birth, highly characterized heart developmental processes, and the availability of previously documented TF-gene networks (Akerberg et al. 2019; Schlesinger et al. 2011) (Figure 2). While the ChIP-seq data is not highly correlated across all the sample types, the gene expression data has an R-squared of at least 0.7 between different biological samples. One e14.5 experiment set had an average R-squared of approximately 0.6 with all other biological samples. To remove this potentially problematic dataset in this analysis before the larger more computationally expensive processes occur, we added an optional soft filter in TReNCo to automatically remove any samples with an average R-squared less than 0.7 across all samples, for gene expression data. We were left with a set of highly correlated data that led us to conclude that this dataset was sufficient to use to TReNCo.\n\nA) Timeline of basic mouse cardiovascular development with life stage on top and developmental stages on the bottom; B) Number of samples for each timepoint and data type; C) Correlation heatmap between samples and replicates at each time point.\n\nPrevious studies of the mouse heart have identified Gata4, Mef2a, Nkx2-5, Tbx5, and Srf as important embryonic lethal TFs critical for development (Gittenberger-De Groot et al. 2005). When looking at the distribution of these TFs over time, we observed that there are many subtle dynamics in how the TFs’ weights shift. Gata4, Mef2a, Tbx5, and Nkx2-5 show a multimodal distribution with three major peaks and varying differences between time points though mostly the distributions overlapped (Figure 3A, S2 Figure, S3 Figure, S4 Figure). We found that the weight distribution followed a similar trend; the dominant population of edge weights appears less than 0.1, a second mid population is between 0.1 and 0.3, and a final population above 0.3 that stretches up to 1. Another TF, Foxs1, demonstrated a more pronounced time point dependent change in addition to a tri-modal edge score (Figure 3B). Interestingly, Srf did not show this trend and tended to have lower weight edges throughout the distributions. To visualize the timepoint dynamics more clearly, we generated a heatmap of the distributions with inflection points added to determine changes in gene weights that may occur (Figure 3C and D). Inflection points, in this case, are simple differences in weights between each time point and the previous time point. These data are ideal for highlighting changes between each time point. An additional differential heatmap of all weight differences with respect to the embryonic day 10.5 point was generated to visualize change from a central time (S1 Figure). It was clear that these TF’s have time dependent dynamics in our model. Gata4, Nkx2-5, and Tbx5 appear to interact with most of their targets constantly throughout early development as indicated by a mostly yellow (no change) inflection point heat map until adult heart. These TF’s have been shown to be important in normal cardiac development (Misra et al. 2014) and act as potential as cardiac reprogramming factors from embryonic fibroblast (Hashimoto et al. 2019). At this time, we observed a net decrease in the Gata4 network weights as observed by an increase in negative inflection points and a decrease in positive values. Mef2a showed a similar trend as Gata4 with a minor increase in network weights leading up to birth, which has been previously shown to be important in postnatal heart development and regulation (Desjardins and Naya 2016). Srf shows a different trend with most of the weights being relatively low until P0 where there is a minor but noticeable uptick in the network weights. This observation matches the biological importance of Srf in early cardiac development and its critical role in maintaining adult heart function (Mokalled et al. 2015). Foxs1 demonstrates the most profound change over time with the initial weights being very low and increasing over time until embryonic day 16.5. After this time the weights begin to decrease into adulthood but never go away completely. This may be due to the role of Foxs1 as a key factor in vascular development (De Val 2011) which in important in earlier development.\n\nA) Histograms of TF-gene interaction weights for 5 different genes, separated by developmental time points; B) Histogram as in A, for Foxs1 TF separated into individual developmental time point plots; C) Heatmap of TF-gene interaction weights sorted by time points; D) Heatmap as in C, showing gene inflection points calculated by log2 the ratio of gene weights. Green indicates increase gene weight from the last time point while Red indicates a decrease.\n\nThere have been a number of studies on mouse cardiac TF regulatory networks with one study looking at the regulatory networks of Gata4, Mef2a, Nkx2-5, and Srf and providing the interactions they identified (Schlesinger et al. 2011). We extracted the interactions of the aforementioned TFs from our network and compared it with the previously identified interactors (Figure 4A and S8). We found that our networks contain over 10,000 putative interactions (weight edge weight greater than 0) that were not reported previously. Interestingly, regardless of the timepoint, our networks captured only about 63% of Gata4 targets, 61.5% of Mef2a targets, and 57% Nkx2-5 and Srf targets of the previous network’s interactions leaving a large portion of their networks unique to their analysis (Figure 4B). We speculate there are two likely explanations for the absence of a 100% overlap: 1) the previous network established interactions using the canonical distance-based cutoff leading to some genes being added or removed erroneously if cutoffs differed from our TAD boundaries; or 2) while our TAD boundaries are more accurate than distance-based cutoffs, the TADs we use are not fully representative of cardiac specific TADs leading to loss of some connections in our network. Regardless of the reason, we wanted to understand if the main overlap between our networks was due to the previous study finding the strongest interactors of the TFs. To test this, we performed the Kolmogorov–Smirnov test (KS-test) on the cumulative distribution between the overlap edge weights and the full edge weights (Figure 4C and S2 Table). We found that in all time points the overlapping genes identified have higher mean edge weights than the total (Figure 4D). This implies that we are identifying true strongly interacting targets and a broad set of possible true but weakly interacting targets.\n\nA) TF-gene interaction Venn Diagram overlap between TReNCo model and previous study; B) Line plot showing percent TF-gene interactions captured from a previous study with the TReNCo model; C) CDF plot with weight of overlapping interactions vs background in TReNCo model; D) Bar plot of mean TF-gene weights for Specific/overlapping interactions (red), all other background interactons (green) and all interactions (blue).\n\nTo further support the biological relevance of our networks, we selected the full TF network, the overlapping network, and the connections unique to our network, and performed GO-term enrichment analysis (Figure 5, S9 Figure). We see that in the case of Mef2a, there is a similar core of regulatory processes that are maintained from 10.5e and 8w (Figure 5). Of interest, we found that in younger 10.5e hearts there was significant enrichment for development related genes as opposed to older 8w hearts which had T cell activation terms enriched. This makes sense when considering recent studies showing Mef2a involvement in inflammation-related processes and the interaction of T cell activation and inflammation (Skapenko et al. 2005; Xiong et al. 2019). Furthermore, we found that overlapping targets between our data and previous data contained terms enriched for cardiac development while full and unique networks showed enrichment for cardiac related and general biological terms. Thus, it is reasonable to conclude that our network contains true biologically relevant interactions in cardiac tissue throughout development.\n\nEnrichment for gene terms between embryonic day 10.5 and 8 week old adult heart. Highlighted terms show first difference in term list.\n\nThe TReNCo model is powered by explicitly modeling the enhancer landscape for each cell type, detecting enhancer activity changes and TF-enhancer relationships across all cell types, limited by TAD boundaries. Our results show that this method can identify cell type–specific TFs that are biologically relevant while also providing potential candidates for further biological study and validation.\n\nIn addition, the model enables further analysis of key TFs using a limited amount of data. This allows the model the be easily be applied to any organisms, tissues and cell types with at least H3K27ac ChIP-seq and RNA-seq data and TAD boundaries. We recognize that H3K27ac is not the only available enhancer mark and feel that other marks (e.g., H3K4me1, ATAC-seq) (Heintzman et al. 2007; Davie et al. 2015) could be substituted into the model with little change necessary. The integration of additional data types would allow for the extension and increase the versatility of the model, providing increased confidence in the gene regulatory networks. Genomic data indicating open regions of chromatin (e.g., ATAC-seq, DNase-seq) (Crawford et al. 2006; Davie et al. 2015) could be added to the enhancer and promoter signal (Figure 1) and help increase the dynamic range of weight score and provide insight into interactions that are poised in earlier time points or cell types versus true interactions. Additionally, integrating other histone modifications (Heintzman et al. 2007; Rajgopal et al. 2014), may provide a filter for enhancer identification, which would reduce the false positives. We believe that adding or substituting any of the data described above would allow for greater use and would improve the model, which is all made easier due to the flexibility of the model.\n\n\nConclusions\n\nOur results show that we were able to expand current methods for generating regulatory networks to take advantage of TADs to limit the predicted influence of enhancers. In this way, we were able to produce highly similar results as reported previously with the added benefit of the networks containing an expanded set of potentially relevant biological connections that can be explored. Additionally, we have developed a framework that can be exploited for a diverse array of species and cell types requiring only two experimental assays, H3K27ac ChIP-seq and RNA-seq. We believe this method opens the possibility for understanding deeper connections and new possibilities for biological discovery.\n\n\nAuthors’ contributions\n\nC.B., V.A., D.K., M.C., and V.S.M. performed validation analysis and discussed the results of TReNCo. C.B., V.A., M.C., and V.S.M. designed and implemented TReNCo. C.B., V.A., D.K., M.C., and V.S.M. wrote the manuscript.\n\n\nData availability\n\nZenodo: TReNCo: Topologically associating domain (TAD) aware regulatory network construction (extended data). https://doi.org/10.5281/zenodo.6392155\n\n• The ENCODE data are available from ENCODE data portal:\n\no S1 Table. ENCODE Data for Validation.\n\nZenodo: TReNCo: Topologically associating domain (TAD) aware regulatory network construction (extended data). https://doi.org/10.5281/zenodo.6392155 (Bennett et al. 2022)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nProject name: TReNCo\n\nProject home page: https://git.biohpc.swmed.edu/BICF/Software/trenco\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.6394452 (Bennett et al. 2021)\n\nOperating system(s): Linux, Mac OS X and Windows\n\nProgramming language: Python\n\nLicense: MIT", "appendix": "References\n\nAkerberg BN, Gu F, VanDusen NJ, et al.A reference map of murine cardiac transcription factor chromatin occupancy identifies dynamic and conserved enhancers. Nat. Commun. 2019; 10: 4907. (Accessed July 16, 2020). PubMed Abstract \| Publisher Full Text Reference Source\n\nBeagan JA, Phillips-Cremins JE. On the existence and functionality of topologically associating domains. Nat. Genet. 2020; 52: 8–16. (Accessed March 24, 2021). PubMed Abstract \| Publisher Full Text\n\nBennett C, Amin V, Kim D, et al.: TReNCo (1.0.0). Zenodo. 2021. Publisher Full Text\n\nBennett C, Amin V, Kim D, et al.: TReNCo: Topologically associating domain (TAD) aware regulatory network construction (extended data) [Data set]. Zenodo. 2022. Publisher Full Text\n\nBranco MR, Pombo A. Chromosome organization: new facts, new models. Trends Cell Biol. 2007; 17: 127–134. (Accessed March 24, 2021). PubMed Abstract \| Publisher Full Text Reference Source\n\nCrawford GE, Holt IE, Whittle J, et al.: Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Res. 2006 Jan; 16(1): 123–131. Publisher Full Text \| PubMed Abstract \| Free Full Text\n\nCuellar-Partida G, Buske FA, McLeay RC, et al.: Epigenetic priors for identifying active transcription factor binding sites. Bioinformatics. 2012; 28: 56–62. (Accessed September 5, 2019). PubMed Abstract \| Publisher Full Text\n\nDavie K, Jacobs J, Atkins M, et al.: Discovery of transcription factors and regulatory regions driving in vivo tumor development by ATAC-seq and FAIRE-seq open chromatin profiling. PLoS Genet. 2015 Feb 13; 11(2): e1004994. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavis CA, Hitz BC, Sloan CA, et al.The Encyclopedia of DNA elements (ENCODE): Data portal update. Nucleic Acids Res. 2018; 46: D794–D801. (Accessed March 12, 2021). PubMed Abstract \| Publisher Full Text Reference Source\n\nDe Val S: Key transcriptional regulators of early vascular development. Arterioscler. Thromb. Vasc. Biol. 2011; 31: 1469–1475. (Accessed March 29, 2021). PubMed Abstract\n\nDeRisi JL, Iyer VR, Brown PO: Exploring the metabolic and genetic control of gene expression on a genomic scale. Science (80-). 1997; 278: 680–686. (Accessed March 24, 2021). Reference Source\n\nDesjardins C, Naya F: The Function of the MEF2 Family of Transcription Factors in Cardiac Development, Cardiogenomics, and Direct Reprogramming. J. Cardiovasc. Dev. Dis. 2016; 3: 26. (Accessed March 29, 2021). PubMed Abstract\n\nDixon JR, Selvaraj S, Yue F, et al.: Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012 Apr 11; 485(7398): 376–380. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGasperini M, Hill AJ, McFaline-Figueroa JL, et al.A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens. Cell. 2019; 176: 377–390.e19. (Accessed November 11, 2019). PubMed Abstract \| Publisher Full Text Reference Source\n\nGittenberger-De Groot AC, Bartelings MM, Deruiter MC, et al.: Basics of cardiac development for the understanding of congenital heart malformations. Pediatr. Res. 2005; 57: 169–176. (Accessed March 24, 2021). PubMed Abstract \| Publisher Full Text Reference Source\n\nGoode DK, Obier N, Vijayabaskar MS, et al.: Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation. Dev. Cell. 2016; 36: 572–587. PubMed Abstract \| Publisher Full Text\n\nGorkin DU, Barozzi I, Zhao Y, et al.: An atlas of dynamic chromatin landscapes in mouse fetal development. Nature. 2020 Jul; 583(7818): 744–751. Erratum in: Nature. 2020 Oct; 586(7831): E31. Erratum in: Nature. 2021 Jan; 589(7842): E4. 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: 1017–1018. (Accessed June 18, 2019). PubMed Abstract\n\nHashimoto H, Wang Z, Garry GA, et al.: Cardiac Reprogramming Factors Synergistically Activate Genome-wide Cardiogenic Stage-Specific Enhancers. Cell Stem Cell. 2019; 25: 69–86.e5. (Accessed March 29, 2021). PubMed Abstract\n\nHeintzman ND, Stuart RK, Hon G, et al.: Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 2007 Mar; 39(3): 311–8. Epub 2007 Feb 4. PubMed Abstract \| Publisher Full Text\n\nKarlebach G, Shamir R: Modelling and analysis of gene regulatory networks. Nat. Rev. Mol. Cell Biol. 2008; 9: 770–780. (Accessed March 24, 2021). Publisher Full Text Reference Source\n\nLee TI, Rinaldi NJ, Robert F, et al.: Transcriptional regulatory networks in Saccharomyces cerevisiae. Science (80-). 2002; 298: 799–804. (Accessed March 24, 2021). PubMed Abstract \| Publisher Full Text Reference Source\n\nMarbach D, Lamparter D, Quon G, et al.: Tissue-specific regulatory circuits reveal variable modular perturbations across complex diseases. Nat. Methods. 2016; 13: 366–370. (Accessed September 5, 2019). Reference Source\n\nMcCord RP, Kaplan N, Giorgetti L: Chromosome Conformation Capture and Beyond: Toward an Integrative View of Chromosome Structure and Function. Mol. Cell. 2020; 77: 688–708. PubMed Abstract \| Publisher Full Text\n\nMisra C, Chang SW, Basu M, et al.: Disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. Hum. Mol. Genet. 2014; 23: 5025–5035. (Accessed March 29, 2021). PubMed Abstract\n\nMokalled MH, Carroll KJ, Cenik BK, et al.: Myocardin-related transcription factors are required for cardiac development and function. Dev. Biol. 2015; 406: 109–116. (Accessed March 29, 2021). PubMed Abstract\n\nNeph S, Kuehn MS, Reynolds AP, et al.: BEDOPS: high-performance genomic feature operations. Bioinformatics. 2012; 28(14): 1919–1920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajagopal N, Ernst J, Ray P, et al.: Distinct and predictive histone lysine acetylation patterns at promoters, enhancers, and gene bodies. G3 (Bethesda). 2014 Aug 12; 4(11): 2051–2063. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchlesinger J, Schueler M, Grunert M, et al.: The Cardiac Transcription Network Modulated by Gata4, Mef2a, Nkx2.5, Srf, Histone Modifications, and MicroRNAs ed. D. Schübeler. PLoS Genet. 2011; 7: e1001313. (Accessed December 12, 2019). PubMed Abstract \| Publisher Full Text\n\nSkapenko A, Leipe J, Lipsky PE, et al.: The role of the T cell in autoimmune inflammation. Arthritis Res. Ther. 2005; 7 Suppl 2: S4–14. (Accessed April 21, 2021). PubMed Abstract \| Publisher Full Text\n\nXiong Y, Wang L, Jiang W, et al.: MEF2A alters the proliferation, inflammation-related gene expression profiles and its silencing induces cellular senescence in human coronary endothelial cells. BMC Mol. Biol. 2019; 20: 8. (Accessed April 21, 2021). PubMed Abstract \| Publisher Full Text" }	[ { "id": "136507", "date": "05 Aug 2022", "name": "Junbai Wang", "expertise": [ "Reviewer Expertise Bioinformatics in gene regulatory networks" ], "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\nBennett et al. proposed a method to build gene regulatory networks by integrating gene expression profiles (RNA-seq), enhancer marker (H3K27ac, ChIP-seq), and TAD boundary information. The authors tested the method on mouse data by using public data in ENCODE. The authors claim that their method \"makes sense\" and network target genes have around 60% of overlapping to the previous study.\nHowever, there are four major problems in the manuscript:\nThe description of their method/algorithm for integrating gene expression, enhancer marker, and TAD boundary information is very poor. It is not clear to me how the final gene regulatory network is built.\n\nThe authors used FIMO to predict TF binding on gene promoters, this may not be correct because many TFs share the same binding motifs, how can we distinguish them from such in silico predictions without looking at in vivo TF-DNA binding data such as Chip-seq et al?\n\nThe results presentation also confuses me, especially the quality of Figures 3, 4, and 5 are poor and there is very little description of these figures in the main text; therefore, the conclusions are not supported by the results.\n\nAuthors have to reorganize their manuscript in a better way that presents the methods, results, and discussion sections clearly in the manuscript for readers to understand it.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "181405", "date": "12 Oct 2023", "name": "Sonika Tyagi", "expertise": [ "Reviewer Expertise Bioinformatics", "Data Science", "Machine Learning" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a workflow called TAD-aware Regulatory Network Construction or TreNCo that combines gene expression data, histone capture of Enhances/promoters (ChIP-seq) to TAD boundaries information generated at genome scale- claimed to build GRNs. RNA-seq and ChIP-seq data was matched and separately available genomic TAD data was used.\nExisting approaches to define ehancer and promoter distances are ad-hoc. Recently, availability of genome wide interaction data using assays like HiC are a more accurate substitute to include dynamic enhancer/promoter interactions with transcriptional signals to study biological networks. Therefore, the authors fill a significant gap in the field by attempting to streamline this process. However, it needs to be noted that TADs may change depending on the development stage of the cells. Hence, using matched HiC data is ideal when performing given integrative analyses. Authors should discuss this in the manuscript.\nKnown gene annotations are used identify TSS and promoters. Histone marks data was used to define enhancer boundaries and coverage. Similarly, RNAseq data yield the gene expression table. TF motifs were located on promoter and ehancer sequences using MEME suite.\nMajor comments:\n\nIn the methods section versions of software and databases used are missing, this is required for reproducibility of analysis.\n\nThe workflow has been tested only on 4 heart development TFs. It is also not clear how the TF weights were calculated as shown in the Figure 3-C.\n\nAuthors should justify why capture data for TFs was not used instead of making predictions themselves.\n\nHow many genes are we looking at here after merging data for TFs and Enhancers?\n\nThe low overlap of authors results with previously published data indicates the limitation of using static TAD data that’s not matched. This should be emphasised when discussing the limitations of the approach in the discussion section. Could authors not find matched RNAseq, ChIPSeq and HiC data? That would be a good way to confirm these speculations of low mapping of interactions predicted by trenco pipeline.\n\nThe title of the paper suggests that the method is to build network at the end. But authors are only generating a list of genes that is then mapped to find pathway enrichments.\n\nThe authors have used GO enrichment analysis to associate predicted interactions to biological function. I would highly recommend presenting a GSEA analysis here since multiple genes may be linked to a GO category and usually genes within a TAD domain tend to be co-regulated.\n\nThe authors have provided the source code via Git and instructions to do a conda install of the workflow. MIT user license applies. This page can benefit by demonstrating a step by step analysis of an example data. I could not test the software myself as the conda env set up step resulted in an error ('Solving environment: failed') and requires further debugging.\n\nAuthors should include information on how automated the pipeline is. Some guidance must be provided to prepare the mapping files as described in the methods section.\n\nAuthors have pointed out that other type of regulatory element capture data can be included in the workflow- I think this will be an important extension to the tool to provide a comprehensive regulatory integration approach. ATAC data for example, is more readily available than HiC data.\n\nHow easy is it to include a new assay data in this integrative approach?\n\nFigure resolutions are poor.\n\nMinor\n\nIts much easier to provide feedback with line numbers in the manuscript.\n\nIn the author summary:Transcriptionally Active Domains (TADs) should be “Topologically associated domains”.\n\nIn 2023, the use of term “next gen sequencing” doesn’t really hold true. We are now past the third generation of sequencing. I would recommend the term “high throughput sequencing” instead.\n\nThis sentence has an extra “the” in it: “We designed this component with the assumption that enhancer-TF binding should be similar in promoters and should be weighted the as a log-odds scale in the network”\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": [] } ]	1	https://f1000research.com/articles/11-426
https://f1000research.com/articles/10-973/v1	27 Sep 21	{ "type": "Study Protocol", "title": "A study protocol for the cardiac effects of a single dose of either oxytocin 2.5 IU or carbetocin 100 µg after caesarean delivery: a prospective randomized controlled multi-centre trial in Norway", "authors": [ "Maria Bekkenes", "Marte Morin Jørgensen", "Anne Flem Jacobsen", "Morten Wang Fagerland", "Helene Rakstad-Larsen", "Ole Geir Solberg", "Lars Aaberge", "Olav Klingenberg", "Trude Steinsvik", "Leiv Arne Rosseland", "Maria Bekkenes", "Marte Morin Jørgensen", "Anne Flem Jacobsen", "Morten Wang Fagerland", "Helene Rakstad-Larsen", "Ole Geir Solberg", "Lars Aaberge", "Olav Klingenberg", "Trude Steinsvik" ], "abstract": "Background: Both oxytocin and carbetocin are used to prevent uterine atony and post-partum haemorrhage after caesarean delivery in many countries, including Norway. Oxytocin causes dose-dependent ST-depression, troponin release, prolongation of QT-time and arrythmia, but little is known about myocardial effects of carbetocin. We have previously demonstrated comparable vasodilatory effects of oxytocin and carbetocin and are now undertaking a Phase 4 trial to investigate whether carbetocin causes similar changes to myocardial markers compared with oxytocin. Methods: Our randomized controlled trial will be conducted at three obstetrics units at Oslo University Hospital and Akershus University Hospital, Norway. Planned enrolment will be of 240 healthy, singleton pregnant women aged 18 to 50 years undergoing planned caesarean delivery. Based on pilot study data, each participant will receive a one-minute intravenous injection of either oxytocin 2.5 IU or carbetocin 100 µg during caesarean delivery. The prespecified primary outcome is the change from baseline in high-sensitive troponin I plasma concentrations at 6–10 hours after study drug administration. Secondary outcomes include uterine tone grade at 2.5 and five minutes after study drug administration, adverse events for up to 48 hours after study drug administration, estimated blood loss within eight hours of delivery, need for rescue treatment and direct/indirect costs. Enrolment and primary analysis are expected to be completed by the end of 2021. Discussion: Women undergoing caesarean delivery should be assessed for cardiovascular risk particularly as women with an obstetric history of pregnancy induced hypertension, gestational diabetes mellitus, preterm birth, placental abruption, and stillbirth are at increased risk of future cardiovascular disease. Any additional ischaemic myocardial risk from uterotonic agents will need to be balanced with the benefit of reducing the risk of postpartum haemorrhage. Any potential cardiotoxicity difference between oxytocin and carbetocin will help inform treatment decisions for pregnant women. Registration: Clinicaltrials.gov NCT03899961 (02/04/2019).", "keywords": [ "Oxytocin", "carbetocin", "troponin I", "anaesthesia", "caesarean delivery", "uterine atony" ], "content": "Abbreviations\n\nAE: adverse event\n\nAHUS: Akershus University Hospital\n\nCI: confidence interval\n\nCTCAE: Common Terminology Criteria for Adverse Events\n\nIU: international unit\n\nIV: intravenous\n\nmmHg: millimetre of mercury\n\nOUH: Oslo University Hospital\n\nPMI: perioperative myocardial injury\n\nSAP: statistical analysis plan\n\n\nIntroduction\n\nWomen who undergo caesarean delivery are at risk of excessive postpartum bleeding caused by uterine atony. Prophylactic administration of intravenous (IV) oxytocin to prevent uterine atony is standard first-line practice after caesarean delivery in many countries, including Norway.1,2 Nevertheless, serious cardiovascular adverse events, including ST segment depression, hypotension and tachycardia have been reported after IV oxytocin.2 Women with long QT syndrome 2 have a particularly high postpartum risk of lethal arrhythmias.3 Oxytocin dose reduction and/or increased infusion duration may reduce risk of some cardiac-related adverse effects and increase patient safety.2\n\nCarbetocin, a synthetic oxytocin receptor agonist with a significantly longer half-life than oxytocin, reduces postpartum blood loss by stimulating uterus contraction.4-6 Carbetocin has been in clinical use for the prevention of postpartum haemorrhage in Europe since 1999, and a room-temperature stable formulation has been available since 2015.7 The efficacy is documented in several randomized controlled trials and meta-analyses.5,8-10 Although both oxytocin and carbetocin have similar haemodynamic effects,11-13 there is insufficient evidence of their effects on the myocardium. Cardiac troponin I is a protein that is released by myocardial myocytes when starved of oxygen and can be used as an indirect measure of ischaemic heart damage. Perioperative myocardial injury can be defined as elevated or increased cardiac troponin I plasma concentration, with or without additional ischaemic signs or symptoms.14 High-sensitivity detection assays can be used to monitor normal ranges of plasma troponin I concentrations.15\n\n\nAims and objectives\n\nThe aim of this Phase 4 trial is to determine any potential differences between oxytocin and carbetocin in their myocardial effects by measuring plasma troponin I using a high-sensitivity assay in healthy women with a singleton pregnancy undergoing a planned caesarean delivery. Plasma concentrations will be collected before caesarean delivery and 6–10 hours after study drug administration. Other endpoints relating to uterus tone, blood loss, blood pressure, heart rate, post-operative pain and side effects will also be assessed.\n\n\nProtocol\n\nThe study is a parallel group, randomized, patient- and investigator-blinded Phase 4 study. Enrolment of participants started in April 2019 and is anticipated to be completed by the end of 2021. The protocol has been approved by the Regional Committee for Medical Research Ethics and the Norwegian Medicines Agency, and is being conducted according to the Good Clinical Practice principles that have their origins in the Declaration of Helsinki. The trial was registered at Clinicaltrials.gov (NCT03899961) in April 2019.\n\nThe project has approval from the Regional Committee for Medical and Health Research Ethics (REC 2014/1210), The Norwegian Medicines Agency and the Institutional Data Protection officer at Oslo University Hospital. Signed informed consent form and expected cooperation of the participants for treatment and follow-up will be obtained and documented according to the International Council of Harmonisation-Good Clinical Practice (ICH GCP), and national/local regulations.\n\nWomen will be recruited from the general population at the three birth clinics at Oslo University Hospital (two clinics) and Akershus University Hospital in Norway.\n\nAll participants will have a normal singleton pregnancy at gestational age of 36 weeks or more, and will be able to read and understand Norwegian. Women with common comorbid diagnoses (diabetes, hypothyreosis, hypertension, etc) and pregnancy after in vitro fertilization will also be eligible for enrolment. Women will be excluded from enrolment if they have any of the following: placenta praevia or invasive placenta; pre-eclampsia; a bleeding disorder, such as von Willebrand disease type I; current treatment with low-molecular-weight heparin or other anticoagulation medication (not including aspirin); any known intolerance to either of the study drugs; prolonged QT-time or other serious cardiac disease; liver or kidney failure; epilepsy; or any medical reason why, in the opinion of the investigator, the patient should not participate.\n\nA total of 240 healthy pregnant women aged between 18 and 50 years will be included in our trial. Signed informed consent form and expected cooperation of the participants for treatment and follow-up will be obtained and documented according to the International Council of Harmonisation-Good Clinical Practice (ICH GCP), and national/local regulations. All data pertaining each enrolled participant will be entered into the electronic clinical report file (CRF; Viedoc®, Uppsala, Sweden).\n\nScreening\n\nPotentially eligible participants will be screened by the principal investigator for inclusion after their last consultation before their scheduled delivery. Oral and written information about the trial will be provided to each woman at least 24 hours before delivery and written informed consent obtained before randomization. Consent, participation and redraw of consent will be documented in electronic patient record. All screened women, including those who do not give consent to participate in the study, will be registered by number. Screened women who are not enrolled due to exclusion criteria or non-fulfilment of inclusion criteria will be registered by number and the reason for not participating in the study will be recorded.\n\nParticipants will be randomized 1:1 to receive either carbetocin 100 μg or oxytocin 2.5 IU after caesarean delivery. The patients will be randomized according to a computer-generated list of random numbers, with block sizes unknown to the researchers as an integral part of Viedoc – the eCRF solution. Although the standard procedure according to its label is to administer 1 mL of carbetocin (100 μg/mL),7 to maintain treatment masking, both study drugs will be diluted to 5 mL using normal saline by a trained member of staff otherwise uninvolved with the trial, and labelled with the trial identification and randomization number according with ICH GCP and local regulations.\n\nA single dose of either oxytocin 2.5 IU (Syntocinon®, Swedish Orphan Biovitrum, Stockholm, Sweden) or carbetocin 100 μg (Pabal®, Ferring Pharmaceuticals, St-Prex, Switzerland) will be administered by the investigator (a trained anaesthetist) as a one-minute IV injection immediately after delivery of the baby’s head and shoulders. IV oxytocin 2.5 IU is standard dose used prophylactically at our institutions. Both oxytocin and carbetocin are used prophylactically after delivery to prevent uterine atony and excessive blood loss.\n\nPrimary endpoint\n\nThe primary endpoint is the difference between the oxytocin 2.5 IU and carbetocin 100 μg treatment groups in change from baseline in high-sensitive troponin I plasma concentrations 6–10 hours after study drug administration.\n\nSecondary outcomes\n\nUterine tone will be assessed at 2.5 minutes and five minutes after study drug administration, using a numerical rating scale 0–10, where 0 = no tonus and 10 = maximum tonus, and 7 = clinically satisfactory tonus.13 Blood loss will be estimated by volume during the surgical procedure as well as calculating estimated blood loss based on haematocrit percentage within 24 hours after delivery, height and weight prior to caesarean delivery.16\n\nPostoperative pain during the first 48 hours after delivery will be assessed in a subgroup of women at one centre. For these women, pain intensity (numerical rating scale 0–10) and opioid consumption (time and dose) will be recorded. In addition to the standard pain-relief treatment, these patients will have a patient-controlled intravenous morphine pump.\n\nDirect and indirect healthcare costs will be assessed. Direct costs will include administered drugs related to prophylaxis and treatment of uterine atony, blood loss, and side effects of the therapeutic interventions. Indirect costs include the number of hours staff spend with patients in theatre and in post-anaesthesia care unit.\n\nVital signs monitoring\n\nVital signs and baseline blood tests (haemoglobin and sodium concentrations) of participating women will be recorded prior to administration of anaesthesia. Throughout each caesarean delivery, there will be continuous monitoring of vital signs, including echocardiogram, blood pressure and heart rate.\n\nRoutine assessments of neonatal status will be recorded (Apgar 1 and 5 minutes, umbilical vein and artery acid-base status).\n\nThe participants will be informed about the expected adverse events (AEs) prior to study enrolment and instructed to score grade of AEs at 0–2, >2–5 and >5–10 minutes after the start of study drug administration, when the majority of AEs are expected to occur. Each participant will be instructed to inform the investigator immediately if they manifest any signs or symptoms they perceive as AEs the following 48 hours (duration of the trial). Unexpected serious adverse events will be reported, also after this period, until discharge from the hospital. All AEs will be recorded according to the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE).\n\nSuspected unexpected serious adverse reactions that result in death, are immediately life-threatening, require hospitalisation, result in persistent or significant disability or incapacity, will be reported immediately to the health authorities. Other suspected serious unexpected adverse reactions will be reported in an unblinded manner to the health authorities as soon as possible but within a maximum of 15 days of first knowledge by the Sponsor (Oslo University Hospital). In order to keep the Investigator and other persons generating data to the study blinded to treatment group, the unblinding and the reporting will be performed by the clinical trial unit at Oslo University Hospital, who is also responsible for monitoring the trial and randomization.\n\nExpected adverse events\n\nExpected AEs are feeling of warmth, chest pain, shortness of breath, palpitations, flushing, headache, nasal congestion, xerostomia, and metallic taste. Both drugs in the study will lead to vasodilatation with a decrease in blood pressure and an increase in heart rate. The causal relationship of each AE to the study medication will be assessed by the investigators as either unrelated, unlikely to be related, possibly or probably related or definitely related.\n\nRescue medication\n\nIn case of uterus atony, patients will be treated with rescue oxytocin 1 IU every 2 minutes up to maximum 5 IU. Time of rescue medication administration will be recorded. Any additional treatment required, whether medical or surgical, will be decided by the attending obstetrician and anaesthesiologist according to local guidelines.\n\nAnaesthesia and pain medication\n\nSpinal anaesthesia will be given according to study procedure (2 mL bupivacaine [5 mg/mL] + 0.4 mL fentanyl [50 μg/mL]), hypotension prophylaxis (0.5 μg/kg IV phenylephrine [0.1 mg/mL] followed by infusion rate 0.25 μg/kg/min) and IV volume (isotonic saline) 10 mL/kg starting concomitantly with spinal anaesthesia. Spinal induced hypotension (systolic arterial pressure <90 mmHg) will be treated with an extra IV bolus of phenylephrine if the heart rate is above 60 beats/min or with IV ephedrine 5–10 mg if 60 beats/min or below. Analgesic medication will be administered as required by the participants and will include oral paracetamol 1 g and oral ibuprofen 400–600 mg four times per day. Patients may be given one IV bolus ketorolac trometamol 30 mg and IV bolus or oral oxycodon administered according to local guidelines and as required by the participants.\n\nAll medication interventions, including drug dose and time of administration will be recorded.\n\nPatients may be discontinued from study treatment and assessments at any time. Specific reasons for discontinuing a patient for this study are as follows: (1) Voluntary discontinuation by the patient who is at any time free to discontinue her participation in the study, without prejudice to further treatment; (2) Severe non-compliance to protocol as judged by the principal investigator; (3) Incorrect enrolment i.e., the patient does not meet the required inclusion/exclusion criteria for the study. Patients who withdraw or are withdrawn from the study, will stop further treatment. Reasons for discontinuation will be documented.\n\nBlood samples from participating women will be collected at baseline (prior to caesarean delivery) and between six and 10 hours after study drug administration. Haemoglobin and sodium concentrations will be assessed using the point-of-care blood gas analyser(s) (ABL 800 Analyzer®, Radiometer, Copenhagen, Denmark). Blood plasma samples, stored in a certified research biobank freezer (–70 °C), will be analysed centrally for troponin I concentration as one batch at the Vestre Viken Trust, Drammen Hospital, Norway, once the last participant has completed the study. Plasma Troponin I concentrations for each participant will be measured using high-sensitive detection with a chemiluminiscent microparticle immunoassay (Alinity i®, Abbott, Illinois, U.S.A.). Normal values of troponin I in a female population (age 18 to 50 years) should be <15 ng/L, with the cut-off corresponding to the 99 percentile of a healthy reference population.17\n\nEvery investigator responsible for entering data into the eCRF will be provided with a unique identity and password, thereby creating an electronic signature of the investigator attesting the accuracy of the data on each eCRF. If any assessments are omitted, the reason for such omissions will be noted on the eCRFs. Corrections, with the reason for the corrections if applicable, will be dated and signed by the investigator in the eCRF. Electronic files, including the data from CRF entered into an electronic database software will be stored at the research server at Oslo University Hospital. This data storage is administered by the Data Inspectorate’s local representative. The patient identification and the code list will be kept by the principal investigator in a locked office, ensuring confidentiality. Investigators involved with data analysis will have access to the study data base. Patient files will be kept for the maximum period of time permitted by each hospital.\n\nThe Clinical Study Monitor, independent from the investigators, will visit each investigator regularly to will check and collect completed CRFs, discuss the progress of the study and monitor drug usage according to ICH GCP guidelines. The monitoring will also include source data verification (data audit). A data monitoring committee will not be used as the data collection is expected to be straightforward. Authorized representatives of a regulatory authority and Ethics Committee may visit the centre to perform inspections, including source data verification.\n\nThe sample size required for our current study was calculated using data from a pilot study of 40 patients, in which the largest difference in plasma troponin I concentration was found at 10 hours, with a mean ± standard deviation change from baseline of 0.41 ± 0.79 ng/L in the carbetocin group versus 1.78 ± 4.48 in the oxytocin group. The sample size calculation was thus based on 80% power to detect a between-group difference in change from baseline to 10 hours of 1.37, to be analysed using a two-sample T-test with adjustments for unequal variances. With a significance level of 5%, our study will need to include 178 patients (89 in each treatment group) in this confirmatory trial. To adjust for loss of information from missing values and patient drop outs, 240 women will be enrolled. The drop-out rate after enrolment is expected to be low as the duration of the study is short.\n\nThe primary endpoint and all other continuous endpoints that include baseline and ≥1 follow-up measurement(s) will be analysed with linear regression models, with the follow-up measurement defined as the dependent variable and treatment group and baseline measurement defined as independent variables. Based on the fitted models, we will estimate treatment group differences in changes from baseline with 95% confidence intervals (CIs), together with a p-value for the null hypothesis of no treatment group difference. We expect at least some degree of skewness in the primary and some of the secondary endpoints, and maybe in the residuals from the linear regression models. The amount of skewness will be assessed with histograms and descriptive statistics, such as mean, median, variance, and the skewness index. In cases where the distribution of the residuals deviates markedly from the normal distribution, or when the endpoints themselves are too skewed to use means as measures of central tendency, we will use median regression models instead of linear regression models, thus analysing between-group differences in median changes from baseline instead of mean changes from baseline. Standard errors and CIs in the median regression models will be obtained via bootstrapping with 100 replications.\n\nContinuous endpoints measured at a single time point will be analysed with two-sample T tests and 95% CIs for the difference between means based on the t distribution, with adjustment for unequal variances. Median regression will be used for variables with highly skewed distributions.\n\nBinary outcomes will be analysed with Fisher mid-P tests and Newcombe hybrid score confidence intervals for the difference between probabilities.18 Ordered categorical outcomes will be analysed with score tests for effect in a proportional odds model (the Wilcoxon-Mann-Whitney test).18\n\nAll analyses will be performed on the intention-to-treat population. AEs and vital signs will be presented with descriptive statistics. In addition, the primary endpoint will also be analysed using the per protocol population. Further details of the statistical methods, including definitions of the intention-to-treat and per protocol populations, how to handle missing data, and sensitivity and exploratory analyses are available in the Statistical Analysis Plan (SAP) available on the trial registration page.\n\nThe results of the study will be published in an open-access peer-reviewed journal and deposited at the trial registration page. Requests for data sharing/case pooling may be directed to the project Principal Investigator: Professor Rosseland on email: lrossela@ous-hf.no.\n\nThe trial is actively recruiting at all sites.\n\n\nDiscussion\n\nBoth oxytocin and carbetocin are used routinely after caesarean delivery to prevent uterine atony and excessive blood loss.5,8 We know that oxytocin causes dose-dependent ST-depression, troponin release, prolongation of QT-time and arrythmia, but little is known about myocardial effects of carbetocin.19 Our ongoing study has been designed to assess high-sensitivity troponin I plasma concentrations after the prophylactic administration of either oxytocin or carbetocin to determine whether there are any differences in their myocardial effects. The protocol of this parallel, randomized, blinded phase 4 study has been approved by the Regional Committee for Medical Research Ethics and the Norwegian Medicines Agency, and follows the principles for Good Clinical Trial practice. In addition, the biobank has been approved by the Regional Committee for Medical Research Ethics for storage of blood plasma from the participating women. All enrolled women will have the same techniques performed for their caesarean delivery, comprising a Pfannenstiel skin incision. Once the baby and placenta have been delivered, exteriorization of the uterus will be performed according to local guidelines. This will limit confounding variables of different surgical techniques interference with assessment of uterine tone. The primary endpoint of change in troponin I (high-sensitive detection method) will be analysed after the final participant has completed the trial as one batch, therefore the results will not influence any participant’s treatment, including rescue treatment with oxytocin, or follow-up.\n\nAlthough the use of oxytocin has reduced the risk of postpartum haemorrhage, its haemodynamic effects may cause myocardial ischaemia.20 Carbetocin, a synthetic derivate of oxytocin but with a longer half-life (median terminal elimination half-life is 33 minutes after intravenous administration and 55 minutes after intramuscular administration7) shows similar haemodynamic effects to oxytocin.11-13 Like oxytocin, carbetocin binds to the oxytocin receptor therefore affects the same tissues.7 Oxytocin has been shown to increase cardiac output by decreasing vascular tone in small and peripheral arteries, resulting in a lower blood pressure and a compensatory increase in heart rate, implying increased myocardial oxygen demand.21 Carbetocin also increases cardiac output.13 Minor differences have been detected in the recovery times for heart rate and blood pressure changes, with heart rate elevations lasting slightly longer with carbetocin, possibly related to the longer half-life of carbetocin compared with oxytocin.11,13\n\nPerioperative myocardial injury (PMI) is an important but often undetected complication on noncardiac surgery as it rarely is accompanied by typical symptoms of myocardial ischaemia, such as chest pain, or dyspnea.14 Although PMI is common after noncardiac surgery (approximately 16% of patients with a median age of 74 years),14 little is known about the risk of PMI in women undergoing caesarean delivery. One small study of 26 women showed troponin I plasma concentrations suggestive of myocardial ischaemia 12-hours post caesarean delivery.22 All 26 women had received postpartum intravenous oxytocin (10 IU, over 30 seconds),22 which is a much higher dose than our current study, in which women will be randomized to receive a one-minute intravenous infusion of either oxytocin 2.5 IU or carbetocin 100 μg. Nevertheless, the clinical relevance is that PMI appears to increase post-operative mortality risk. In a prospective cohort study of nearly 22,000 people undergoing in-patient noncardiac surgery, a peak postoperative high sensitivity troponin T increase of at least 5 ng/L during the first three days after surgery was significantly associated with 30-day mortality even if there were no ischaemic signs or symptoms (adjusted hazard ratio, 4.69; 95% CI, 3.52–6.25).23 A smaller study of 2018 patients undergoing nearly 3000 surgical procedures also showed that a preoperative to postoperative increase of ≥14 ng/L of high-sensitivity troponin T (detected in 285 patients) was associated with increased mortality at 30-days (9.8% of patients with PMI versus 1.6% without PMI) and at one-year (22.5% of patients with PMI versus 9.3% without PMI).14 Our study will assess high-sensitivity troponin I levels as we expect very small levels of troponin release in response to either oxytocin or carbetocin. Currently, the Abbott troponin I immunoassay has a lower limit of detection of 0.1 ng/L whereas the Roche troponin T immunoassay has a higher lower limit of detection of 5.0 ng/L.24 Our baseline assessments will help provide a reference range of troponin I for pregnant women at term. All troponin I measurements below 0.1 ng/L will be designated as 0 ng/L.\n\nThe study design is robust, the sample size based on a priori power calculations with parameters estimated from recent, relevant pilot data, and the trial will be conducted in a representative sample of pregnant women at three Norwegian birth clinics. Our study is enrolling women from general obstetric practice, including those with comorbidities such as gestational diabetes and hypertension. There is no upper limit for body mass index for participating women, and with an upper age limit of 50 years, nearly all women with singleton pregnancies at the three clinics will be eligible for inclusion. Women aged between 18 and 50 years make up approximately 25% of the Norwegian population.25 Although this is not an international study, many other countries in Europe have similar obstetric practices.\n\nIn 2019, a core outcomes set was published for prevention of post-partum haemorrhage to aid better comparison of clinical trials ensuring a minimum of outcomes to be reported.26 Our protocol was approved prior to its publication. Nevertheless, our study will assess seven out of nine of the core outcomes (blood loss, shock, transfer to higher level of care, use of additional haemostatic interventions and adverse events). We will not assess breastfeeding or patient satisfaction with treatment, but will report pain and pain relief medication.\n\nAll caesarean deliveries will be performed with standardized anaesthesia and surgery, including the meticulous blood pressure control to reduce cardiac troponin I due to spinal anaesthesia–induced hypotension and/or tachycardia. Both intraoperative tachycardia and hypotension are associated with myocardial injury after noncardiac surgery,27 thereby, we are limiting an important confounding factor that may complicate the interpretation of our results.\n\nAs all participating women have to be able to read and understand Norwegian in order to provide informed consent, this may limit generalizability to diverse ethnic background to some extent. We will provide detailed demographic and baseline characteristics of all participating women.\n\nIn the analyses of direct and indirect costs, any potential drug specific long-term cardiac adverse events rate will not be included. We expect differences in health care costs between oxytocin and carbetocin cost to be minor.\n\nWomen with an obstetric history of pregnancy induced hypertension, gestational diabetes mellitus, preterm birth, placental abruption, and stillbirth are at increased risk of future cardiovascular disease.28,29 Women undergoing caesarean delivery should be assessed for cardiovascular risk. Any additional ischaemic myocardial risk from uterotonic agents administered postpartum need to be balanced with the benefit of reducing the risk of postpartum haemorrhage. We anticipate that data from our study will help future clinical management decisions around planning delivery in pregnant women, including those with heart disease. As far as we can tell, no other study has compared the myocardial effects of oxytocin and carbetocin when used as postpartum uterotonic agents after caesarean delivery. We currently know little about troponin release after vaginal delivery and the role of uterontonics on troponin release in this population, which would require further investigation.\n\n\nConclusion\n\nThe results of our trial will help inform treatment decisions around preventing uterine atony in women undergoing caesarean delivery.\n\n\nData availability\n\nZenodo: Supporting material for article ‘A study protocol for the cardiac effects of a single dose of either oxytocin 2.5 IU or carbetocin 100 μg after caesarean delivery: a prospective randomized controlled multi-centre trial in Norway’, https://doi.org/10.5281/zenodo.5217789.30\n\nThis project contains the following extended data:\n\n- Ethics approval CMT2014 REC English translation.pdf\n\n- Norwegian Medicines Agency approval SLV-godkjenning CMT-studien English included.pdf\n\n- Pasientinformasjon - v3.pdf\n\n- Protocol v8 signed.pdf\n\nZenodo: SPIRIT and TIDieR checklists for ‘A study protocol for the cardiac effects of a single dose of either oxytocin 2.5 IU or carbetocin 100 μg after caesarean delivery: a prospective randomized controlled multi-centre trial in Norway’, https://doi.org/10.5281/zenodo.5519494.30\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors wish to thank the members of the study group at Akershus University Hospital (AHUS) and Oslo University Hospital (OUH) for their excellent support which enables data collection and completion of this study; Hanne Helene Johnsen, Hanne Wickstrøm, Thomas Günther, Ellen Støvland Fjuk and Else-Marie Ringvold at AHUS, Eldrid Langesæter, Anders Krogh, Anders Aasheim and Ida Kristin Larsson Gerø at OUS-Rikshospitalet and Charlotte Loennechen, Thomas Heyerdahl, Bjarne Røed, Tomas Drægni and Camilla Smith at OUH-Ullevål. The persons acknowledged have given their permission hereto.\n\nMedical writing support was provided by Celia J Parkyn, PhD, and funded by Ferring Pharmaceuticals, St-Prex, Switzerland. Ferring Pharmaceuticals did not take part in the study design, data collection, interpretation of the data or manuscript preparation.\n\n\nReferences\n\nPrevention and Management of Postpartum Haemorrhage. BJOG: Int J Obstetrics Gynaecol. 2017; 124(5): e106–e49.\n\nHeesen M, Carvalho B, Carvalho JCA, et al.: International consensus statement on the use of uterotonic agents during caesarean section. Anaesthesia. 2019 Oct; 74(10): 1305–19. Epub 2019/07/28. eng. PubMed Abstract \| Publisher Full Text\n\nBodi I, Sorge J, Castiglione A, et al.: Postpartum hormones oxytocin and prolactin cause pro-arrhythmic prolongation of cardiac repolarization in long QT syndrome type 2. Europace. 2019 Jul 1; 21(7): 1126–38. Epub 2019/04/03. PubMed Abstract \| Publisher Full Text\n\nWidmer M, Piaggio G, Nguyen TMH, et al.: Heat-Stable Carbetocin versus Oxytocin to Prevent Hemorrhage after Vaginal Birth. N Engl J Med. 2018 Aug 23; 379(8): 743–52. Epub 2018/06/28. PubMed Abstract \| Publisher Full Text\n\nGallos ID, Williams HM, Price MJ, et al.: Uterotonic agents for preventing postpartum haemorrhage: a network meta-analysis. Cochrane Database Syst Rev. 2018 Apr 25; 4: CD011689. Epub 2018/04/26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJin XH, Li D, Li X: Carbetocin vs oxytocin for prevention of postpartum hemorrhage after vaginal delivery: A meta-analysis. Medicine (Baltimore). 2019 Nov; 98(47): e17911. Epub 2019/11/26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPabal 100 micrograms in 1 ml solution for injection. Summary of product characteristics. Accessed March 18, 2021.Reference Source\n\nSu LL, Chong YS, Samuel M: Carbetocin for preventing postpartum haemorrhage. Cochrane Database Syst Rev. 2012 Feb15(2): CD005457. Epub 2012/02/18. PubMed Abstract \| Publisher Full Text\n\nPizzagalli F, Agasse J, Marpeau L: [Carbetocin versus Oxytocin during caesarean section for preventing postpartum haemorrhage]. Gynecol Obstet Fertil. 2015 May; 43(5): 356–60. Epub 2015/04/22. PubMed Abstract \| Publisher Full Text\n\nMannaerts D, Van der Veeken L, Coppejans H, et al.: Adverse Effects of Carbetocin versus Oxytocin in the Prevention of Postpartum Haemorrhage after Caesarean Section: A Randomized Controlled Trial. J Pregnancy. 2018; 2018: 1374150. Epub 2018/02/28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoertl MG, Friedrich S, Kraschl J, et al.: Haemodynamic effects of carbetocin and oxytocin given as intravenous bolus on women undergoing caesarean delivery: a randomised trial. BJOG. 2011 Oct; 118(11): 1349–56. Epub 2011/06/15. PubMed Abstract \| Publisher Full Text\n\nPisani I, Tiralongo GM, Gagliardi G, et al.: The maternal cardiovascular effect of carbetocin compared to oxytocin in women undergoing caesarean section. Pregnancy Hypertens. 2012 Apr; 2(2): 139–42. Epub 2012/04/01. PubMed Abstract \| Publisher Full Text\n\nRosseland LA, Hauge TH, Grindheim G, et al.: Changes in blood pressure and cardiac output during cesarean delivery: the effects of oxytocin and carbetocin compared with placebo. Anesthesiology. 2013 Sep; 119(3): 541–51. Epub 2013/04/20. PubMed Abstract \| Publisher Full Text\n\nPuelacher C, Lurati Buse G, Seeberger D, et al.: Perioperative Myocardial Injury After Noncardiac Surgery: Incidence, Mortality, and Characterization. Circulation. 2018 Mar 20; 137(12): 1221–32. Epub 2017/12/06. PubMed Abstract \| Publisher Full Text\n\nVenge P, Johnston N, Lindahl B, et al.: Normal plasma levels of cardiac troponin I measured by the high-sensitivity cardiac troponin I access prototype assay and the impact on the diagnosis of myocardial ischemia. J Am Coll Cardiol. 2009 Sep 22; 54(13): 1165–72. Epub 2009/09/19. PubMed Abstract \| Publisher Full Text\n\nStafford I, Dildy GA, Clark SL, et al.: Visually estimated and calculated blood loss in vaginal and cesarean delivery. Am J Obstet Gynecol. 2008 Nov; 199(5): 519 e1–7. Epub 2008/07/22. PubMed Abstract \| Publisher Full Text\n\nBahadur K, Ijaz A, Salahuddin M, et al.: Determination of high sensitive cardiac troponin I 99th percentile upper reference limits in a healthy Pakistani population. Pak J Med Sci. 2020 Sep-Oct; 36(6): 1303–7. Epub 2020/09/25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFagerland MW, Lydersen S, Laake P: Statistical Analysis of Contingency Tables: Chapman and Hall/CRC; 2017.\n\nJonsson M, Hanson U, Lidell C, et al.: ST depression at caesarean section and the relation to oxytocin dose. A randomised controlled trial. BJOG. 2010 Jan; 117(1): 76–83. Epub 2009/09/29. PubMed Abstract \| Publisher Full Text\n\nSvanström MC, Biber B, Hanes M, et al.: Signs of myocardial ischaemia after injection of oxytocin: a randomized double-blind comparison of oxytocin and methylergometrine during Caesarean section. Br J Anaesth. 2008 May; 100(5): 683–9. Epub 2008/04/04. PubMed Abstract \| Publisher Full Text\n\nRabow S, Hjorth U, Schönbeck S, et al.: Effects of oxytocin and anaesthesia on vascular tone in pregnant women: a randomised double-blind placebo-controlled study using non-invasive pulse wave analysis.BMC Pregnancy Childbirth.2018 2018/11/22; 18(1): 453. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoran C, Ni Bhuinneain M, Geary M, et al.: Myocardial ischaemia in normal patients undergoing elective Caesarean section: a peripartum assessment. Anaesthesia. 2001 Nov; 56(11): 1051–8. Epub 2001/11/13. PubMed Abstract \| Publisher Full Text\n\nWriting Committee for the VSIDevereaux PJ, Biccard BM, Sigamani A, et al.: Association of Postoperative High-Sensitivity Troponin Levels With Myocardial Injury and 30-Day Mortality Among Patients Undergoing Noncardiac Surgery. JAMA. 2017 Apr 25; 317(16): 1642–51. Epub 2017/04/27. PubMed Abstract \| Publisher Full Text\n\nLippi G, Sanchis-Gomar F: “Ultra-sensitive” cardiac troponins: Requirements for effective implementation in clinical practice. Biochem Med (Zagreb). 2018 Oct 15; 28(3): 030501. Epub 2018/11/16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStatistisk sentralbyrå, Statistics Norway. Key figures for the population. Accessed May 6, 2021.Reference Source\n\nMeher S: How should we diagnose and assess the severity of PPH in clinical trials? Best Pract Res Clin Obstet Gynaecol.2019 Nov; 61: 41–54. Epub 2019/06/04. PubMed Abstract \| Publisher Full Text\n\nAbbott TEF, Pearse RM, Archbold RA, et al.: A Prospective International Multicentre Cohort Study of Intraoperative Heart Rate and Systolic Blood Pressure and Myocardial Injury After Noncardiac Surgery: Results of the VISION Study. Anesth Analg. 2018 Jun; 126(6): 1936–45. Epub 2017/10/28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrandi SM, Filion KB, Yoon S, et al.: Cardiovascular Disease-Related Morbidity and Mortality in Women With a History of Pregnancy Complications. Circulation. 2019 Feb 19; 139(8): 1069–79. Epub 2019/02/20. PubMed Abstract \| Publisher Full Text\n\nParikh NI, Gonzalez JM, Anderson CAM, et al.: Adverse Pregnancy Outcomes and Cardiovascular Disease Risk: Unique Opportunities for Cardiovascular Disease Prevention in Women: A Scientific Statement From the American Heart Association. Circulation. 2021 May 4; 143(18): e902–e16. Epub 2021/03/30. PubMed Abstract \| Publisher Full Text\n\nRosseland LA: Supporting material for article ‘A study protocol for the cardiac effects of a single dose of either oxytocin 2.5 IU or carbetocin 100 μg after caesarean delivery: a prospective randomized controlled multi-centre trial in Norway’. Zenodo. 2021. Publisher Full Text" }	[ { "id": "95511", "date": "26 Oct 2021", "name": "Pamela J. Angle", "expertise": [ "Reviewer Expertise Anesthesiology", "Health Research Methodology", "Obstetrical anesthesia" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 describes a parallel, blinded, two-arm randomized controlled safety trial which aims to examine the myocardial effects of intravenous carbetocin (100mcg ) versus oxytocin (2.5units), given over 1 minute immediately after elective cesarean delivery. Rescue therapy will include the use of oxytocin as necessary in both groups (up to 5 units). The study will recruit 240 women, with single gestation pregnancies greater than 36 weeks, and without medical comorbidities (e.g. hypertension, diabetes), from three university-affiliated clinics in Norway. Women will receive standardized management of spinal anesthesia, the fluid co-load, and vasopressors. The primary endpoint will be changes in high sensitivity Troponin I levels, measured at baseline preoperatively and 6-10 hours after drug administration between groups as well as other important secondary outcomes, including severe adverse events, measured up to 48 hours after delivery. Differences in pain between groups will be assessed at one study site. Women will receive multimodal analgesia and intravenous patient-controlled opioid analgesia. Given that oxytocin is a first line drug for the prevention of uterine atony and postpartum hemorrhage, the variability with which it is currently dosed, and the rising age and co-morbidities found in childbearing women in developed countries, study findings may have important implications for the management of patients at higher cardiac risk during cesarean section.\n\nThe Spirit checklist was previously (2018) completed by the authors during protocol development but some elements of the trial design and conduct remain unclear. Protocols available on other trial registry websites provide much of the additional information required. The study is funded, in part, by an unrestricted grant from the makers of Carbetocin. The authors note that funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.\nIt should be made clear early in the protocol that the current safety Trial builds on the authors’ previous pilot work (randomized controlled trial, n=40) to provide the reader with more context. The pilot study demonstrated a reduction in changes in high sensitivity Troponin I levels from baseline with Carbetocin( 0.41+/-0.79ng/L ) compared with oxytocin (1.78 +/-4.48 ng/L) after elective cesarean delivery. It also showed that changes in high sensitivity Troponin I peaked 10 hours after drug administration when the primary outcome of the current trial is to be measured. [Protocol Synopsis, The Clinical Carbetocin Myocardium Trial- Part II1]. Information provided in the sample size estimation section suggests that the study is powered as a superiority trial aimed at confirming findings of the previous pilot work.\nAdditional details and clarification related to trial organization and how the trial is run from day to day at the three sites are required. The role of the clinical trials unit mentioned seems to be limited to the generation of the randomization schedule and oversight/monitoring of the trial itself.\n\nPlease provide additional information related to randomization (e.g. were random permutated blocks of different sizes used? As reported, the person making up study drugs was the only person who knew treatment assignments. As reported, the PI screened potential candidates across all three sites for eligibility. Who consented patients? How exactly were patients allocated to treatment arms? Was this internet-based using Viedoc also? Were baseline Troponin I levels available for any of the patients prior to treatment allocation or were these also batched with the 6-10 hour samples for testing? Is there any plan to assess the blinding of the anesthetist involved in patient care during the c-section?\nThe primary analysis will be by modified intention to treat. The primary outcome will be assessed using an independent student t-test followed by regression modeling. It appears the authors assume that randomization will balance any known confounders (e.g. women with hypertension, diabetes) between treatment arms. It will be important to assess whether any significant difference found in the primary outcome holds up after adjusting for imbalances.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "8040", "date": "14 Apr 2022", "name": "Leiv Arne Rosseland", "role": "Author Response", "response": "PROTOCOL MANUSCRIPT - Review 26 Oct 2021 \| for Version 1 Pamela J. Angle, Sunnybrook Research Institute, Toronto, Canada Reviewer comments are in italicized text. Author responses are in bold text. Comment 1 The protocol describes a parallel, blinded, two-arm randomized controlled safety trial which aims to examine the myocardial effects of intravenous carbetocin (100mcg ) versus oxytocin (2.5units), given over 1 minute immediately after elective cesarean delivery. Rescue therapy will include the use of oxytocin as necessary in both groups (up to 5 units). The study will recruit 240 women, with single gestation pregnancies greater than 36 weeks, and without medical comorbidities (e.g. hypertension, diabetes), from three university-affiliated clinics in Norway. Women will receive standardized management of spinal anesthesia, the fluid co-load, and vasopressors. The primary endpoint will be changes in high sensitivity Troponin I levels, measured at baseline preoperatively and 6-10 hours after drug administration between groups as well as other important secondary outcomes, including severe adverse events, measured up to 48 hours after delivery. Differences in pain between groups will be assessed at one study site. Women will receive multimodal analgesia and intravenous patient-controlled opioid analgesia. Given that oxytocin is a first line drug for the prevention of uterine atony and postpartum hemorrhage, the variability with which it is currently dosed, and the rising age and co-morbidities found in childbearing women in developed countries, study findings may have important implications for the management of patients at higher cardiac risk during cesarean section. The Spirit checklist was previously (2018) completed by the authors during protocol development, but some elements of the trial design and conduct remain unclear. Protocols available on other trial registry websites provide much of the additional information required. The study is funded, in part, by an unrestricted grant from the makers of Carbetocin. The authors note that funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. It should be made clear early in the protocol that the current safety Trial builds on the authors’ previous pilot work (randomized controlled trial, n=40) to provide the reader with more context. The pilot study demonstrated a reduction in changes in high sensitivity Troponin I levels from baseline with Carbetocin( 0.41+/-0.79ng/L ) compared with oxytocin (1.78 +/-4.48 ng/L) after elective cesarean delivery. It also showed that changes in high sensitivity Troponin I peaked 10 hours after drug administration when the primary outcome of the current trial is to be measured. [Protocol Synopsis, The Clinical Carbetocin Myocardium Trial- Part II1]. Information provided in the sample size estimation section suggests that the study is powered as a superiority trial aimed at confirming findings of the previous pilot work. Author response We have added text to the ‘Aims and objectives’ to state that the protocol of this current study has followed a pilot study. The pilot study is not currently published as it is undergoing journal review. Comment Additional details and clarification related to trial organization and how the trial is run from day to day at the three sites are required. The role of the clinical trials unit mentioned seems to be limited to the generation of the randomization schedule and oversight/monitoring of the trial itself. Author response We have amended the text to state that the principal investigator from each study site screened women for inclusion in the study. Comment Please provide additional information related to randomization (e.g. were random permutated blocks of different sizes used? As reported, the person making up study drugs was the only person who knew treatment assignments. As reported, the PI screened potential candidates across all three sites for eligibility. Who consented patients? How exactly were patients allocated to treatment arms? Was this internet-based using Viedoc also? Were baseline Troponin I levels available for any of the patients prior to treatment allocation or were these also batched with the 6-10 hour samples for testing? Is there any plan to assess the blinding of the anesthetist involved in patient care during the c-section? Author response We have amended the article to better describe the organization of randomization in Viedoc and assessment of blinding and allocation of patients. We have clarified that test assessing each responsible investigator will be performed in the ‘Randomization and blinding’ subsection. Baseline troponin I levels were not assessed prior to randomization. All troponin I levels were batch assessed and none of the investigators will have access to these results until after the last patient has completed the study. No changes have been made with regard to assessment of troponin I levels as these are explained in the subsection ‘Laboratory tests’ Comment The primary analysis will be by modified intention to treat. The primary outcome will be assessed using an independent student t-test followed by regression modeling. It appears the authors assume that randomization will balance any known confounders (e.g. women with hypertension, diabetes) between treatment arms. It will be important to assess whether any significant difference found in the primary outcome holds up after adjusting for imbalances. Is the rationale for, and objectives of, the study clearly described? Partly Is the study design appropriate for the research question? Yes Are sufficient details of the methods provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Not applicable Author response We have added a few sentences at the end of the ‘Introduction’ to provide more clearly the rationale for conducting the study. In addition, we have provided more information about the statistical analysis as we will conduct additional prospective sub-analysis of patients who did not receive rescue treatment in cases of uterine atony as this may change the results." } ] }, { "id": "96674", "date": "01 Nov 2021", "name": "Kim Ekelund", "expertise": [ "Reviewer Expertise Obstetric anesthesiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 #2 Thank you for the opportunity to review this manuscript. The main objective of this study was to compare the cardiac effect, measured as the increased level of Troponin I, after intravenous administration of Oxytocin 2.5IU or Carbetocin 100µg during cesarean section (CS) in healthy, singleton pregnant women aged 18 to 50 years undergoing planned caesarean. The prespecified primary outcome was change from baseline in high-sensitive troponin I plasma concentrations at 6–10 hours after study drug administration. Secondary outcomes include uterine tone grade at 2.5 and five minutes after study drug administration, adverse events for up to 48 hours after study drug administration, estimated blood loss within eight hours of delivery, need for rescue treatment, and direct/indirect costs.\nOverall, the manuscript is well written, easy to read, and thoroughly describes a protocol for a Norwegian potentially clinically relevant RCT. I have a few general comments. The protocol is registered on Clinicaltrials.gov as NCT03899961 and is referred to as Carbetocin Myocardium Trial 2014 Part 2 (CMT2014/2). The authors have also registered The Clinical Carbetocin Myocardium Trial (CMT) as NCT02528136. This study is in the manuscript described as a pilot study including 40 patients. Moreover, the is a study funded by the manufacturer of Carbetocin: Ferring Pharmaceuticals, including funding of medical writing support for this manuscript. Although it is stated “The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript”, I will as a reader be more skeptical compared to a non-funded protocol. However, the protocol describes this openly and no conflicts of interest can be identified.\nFinally, I understand the study already are up and running. (“Study status: The trial is actively recruiting at all sites.”, and on Clinicaltrials.gov: September 30, 2021, is registered as Final data collection date for primary outcome measure). This makes me wonder, why I am asked to review a protocol now, and not in 2019 before recruitment began?\nJust more wondering: Has the cardiac effect of Oxytocin / Carbetocin been studied in an animal model? Has it been studied in non-pregnant adults? Can Troponin I be measured after physical activity (walking staircases, jogging, or similar?\nI have more additional specific comments and questions:\n“Protocol. The study is a parallel-group, randomized, patient- and investigator-blinded Phase 4 study. Enrolment of participants started in April 2019 and is anticipated to be completed by the end of 2021…”\nDoes this mean, that we are reviewing a protocol already in action?\n\n“Eligibility criteria. All participants… (but not) prolonged QT-time or other serious cardiac diseases.\nDo you do a pre-CS echocardiography before inclusion? Or just during CS?\n\n“Randomization and blinding. Participants will be randomized 1:1… to a computer-generated list of random numbers, with block sizes unknown”…\nCan you provide us with a more detailed description of the randomization? It sounds like a Viedoc@ is a black box? No description can be found regarding the “integral part of Viedoc – the eCRF solution”.\n\n“Study drug dosage and administration A single dose of either Oxytocin 2.5 IU (Syntocinon®, Swedish Orphan Biovitrum, Stockholm, Sweden) or Carbetocin 100 μg … will be administered by the investigator (a trained anesthetist) as a one-minute IV injection.\nAdministering 2.5 IU of Oxytocin as a one-minute injection seems to be fast, and faster than the recommendations. We recommend an injection of 1IE/minute, or slower.\n\nAdministering 100mikrog carbetocin diluted to 5ml as a one-minute iv injection also seems too fast. Should be administered slower than 1 minute.\n\nIf the approximate ED95 for Oxytocin for elective CS is 0.35 IU and 15 µg for Carbetocin, shouldn't we test these dosages?\n\nIn Ref 4 (Widmer et al.)1 Oxytocin 10IU with Carbetocin 100µg is compared, They find 0.04 vs 0.09% that complains of chest pain, respectively, i.e., after a smaller dose of Carbetocin compared to what is used in this study. Have the authors considered reducing the Carbetocin dose?\n\n“Prespecified analyses. Secondary outcomes Uterine tone will be assessed at 2.5 minutes and five minutes after study drug administration, using a numerical rating scale 0–10, where 0 = no tonus and 10 = maximum tonus, and 7 = clinically satisfactory tonus.”\nDo the obstetricians get any training in using this scale? Do they have a Gold Standard to compare with?\n\n“Postoperative pain during the first 48 hours after delivery will be assessed in a subgroup of women at one centre.”\nWhy is this relevant? And if relevant, why is this only done in one centre?\n\n“Direct and indirect healthcare costs will be assessed”.\nWill the costs of 10 IU Oxytocin (approx. 2 €) and 100 µg Carbetocin (approx. 40€) be included in the assessment?\n\n“Concomitant medication Rescue medication In case of uterus atony, patients will be treated with rescue oxytocin 1 IU every 2 minutes up to maximum 5 IU.”\nIs it max 5 IU in total (ie 2.5 + 1 + 1 + 0.5 ??) or is it in addition to the 2.5?\n\nWhat happens after the maximum 5 IU is administered and there still is some atony?\n\nWill the additional Oxytocin not disturb the picture when comparing to Carbetocin – are these women excluded from getting Troponin I measured after 10 hours?\n\n“Laboratory tests.…Normal values of troponin I in a female population (age 18 to 50 years) should be <15 ng/L, with the cut-off corresponding to the 99 percentile of a healthy reference population. 17”\nIn ref. 17 (Bahadur et al)2, the Pakistan population had a median age of the included female on 56 years old with the oldest being 86 and youngest being 21 years old. Most of the people 246 (82.3%) were above 40 years old. This means that they were older than the Norwegian CS population, and Troponin I increase with age, according to ref. 15, Venge et al. 3 Do the authors have any comments on this?\n\nDo we know if the Pakistan women have a similar Troponin level as Norwegian?\n\nWhat was the baseline level in the pilot study?\n\nIs Troponin I the best measurement for myocardial injury?\n\nWhat about proBNP?\n\nIf all the levels are < the 99th percentile i.e. normal, can we then state that Carbetocin and Oxytocin are safe (regarding cardiac side-effects)?\n\nIf there are minor differences but still within normal ranges, then this might not be clinically relevant? Like studies showing significant difference in blood loss on i.e., 178mL between treatment A and B... Significant - yes. Relevant - no! Any comments?\n\n“Power and sample size considerations. The sample size required for our current study was calculated using data from a pilot study of 40 patients, in which the largest difference in plasma troponin I concentration was found at 10 hours, with a mean +/- standard deviation change from baseline of 0.41+/-0.79 ng/L in the Carbetocin group versus 1.78+/-4.48 in the Oxytocin group”.\nI admit I am not used to doing statistical analysis, but does this mean that the change from the baseline can be 0.4ng/L – 0.8ng/L = -0.4ng/L? Because then Carbetocin improves the cardiac effect, reducing the Troponin I level? And similar for Oxytocin?\n\nOr does this mean that the data is skewed? And is “Median” not preferred instead of “Mean”?\n\nWhat is the baseline for Oxytocin and Carbetocin, respectively?\n\nIs this because the authors suspect the results to be clinically not relevant, although detecting significant differences?\n\nAnd is this because Ferring is funding the project? (As I mentioned in the beginning, the reader should be more suspicious).\nAnalysis plan\nI believe I do not have enough knowledge to comment relevantly on the analysis plan.\n\nStrength and Limitations. In 2019, a core outcome set was published for the prevention of postpartum hemorrhage to aid better comparison of clinical trials ensuring a minimum of outcomes to be reported…We will not assess breastfeeding or patient satisfaction with treatment, but will report pain and pain relief medication.”\nWhy is this relevant to the study?\n\nIs the dose of Oxytocin 2.5IU used elsewhere?\n\nThe cardiovascular events that are described after oxytocin iv dose (ST-changes, hypotension, tachycardia), have they been found after 2.5 IU?\n\nWill data be released at the person level (for i.e. reviewer) in case of potential cardiac damage, and high Troponin I level during the CS?\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "8041", "date": "14 Apr 2022", "name": "Leiv Arne Rosseland", "role": "Author Response", "response": "01 Nov 2021 \| for Version 1 Kim Ekelund, Department of Obstetrics, Juliane Marie Centre, Rigshospitalet, Copenhagen, Denmark Kim Lindelof, Department of Anesthesiology, Juliane Marie Centre, Rigshospitalet, Copenhagen, Denmark Reviewer comments are in italicized text. Author responses are in bold text. Comment Thank you for the opportunity to review this manuscript. The main objective of this study was to compare the cardiac effect, measured as the increased level of Troponin I, after intravenous administration of Oxytocin 2.5IU or Carbetocin 100µg during cesarean section (CS) in healthy, singleton pregnant women aged 18 to 50 years undergoing planned caesarean. The prespecified primary outcome was change from baseline in high-sensitive troponin I plasma concentrations at 6–10 hours after study drug administration. Secondary outcomes include uterine tone grade at 2.5 and five minutes after study drug administration, adverse events for up to 48 hours after study drug administration, estimated blood loss within eight hours of delivery, need for rescue treatment, and direct/indirect costs. Overall, the manuscript is well written, easy to read, and thoroughly describes a protocol for a Norwegian potentially clinically relevant RCT. I have a few general comments. The protocol is registered on Clinicaltrials.gov as NCT03899961 and is referred to as Carbetocin Myocardium Trial 2014 Part 2 (CMT2014/2). The authors have also registered The Clinical Carbetocin Myocardium Trial (CMT) as NCT02528136. This study is in the manuscript described as a pilot study including 40 patients. Moreover, the is a study funded by the manufacturer of Carbetocin: Ferring Pharmaceuticals, including funding of medical writing support for this manuscript. Although it is stated “The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript”, I will as a reader be more skeptical compared to a non-funded protocol. However, the protocol describes this openly and no conflicts of interest can be identified. Author response Studies require funding and we have been transparent about where our funding has come from. We are ensuring that the safety profiles in relation to the release of cardiac markers of two commonly used drugs are characterized fully so that clinicians will be able to make informed treatment decisions. We will report the results of this trial so that clinicians will be able to use our findings in order to improve standard of care for all women requiring prophylaxis treatment to prevent post-partum haemorrhage. Our pilot study manuscript is currently undergoing peer-review at another journal. Comment Finally, I understand the study already are up and running. (“Study status: The trial is actively recruiting at all sites.”, and on Clinicaltrials.gov: September 30, 2021, is registered as Final data collection date for primary outcome measure). This makes me wonder, why I am asked to review a protocol now, and not in 2019 before recruitment began? Author response We decided to write a protocol manuscript describing the Carbetocin Myocardium Trial 2014 part 2 (CMT 2) in January 2021 when we started writing our manuscript for the Pilot study. Our rationale for publishing the protocol is to increase awareness that oxytocin and carbetocin may have slightly different effects on the myocardium. At the point we decided to publish the protocol in more detail we had already started enrollment. The enrollment progress has been delayed due to the COVID-19 pandemic. Ideally this protocol article would be published prior to recruiting patient. We hope that readers will find the protocol and intention of our clinical trial valuable when making assessments of which uterotonic agent to use. Comment Just more wondering: Has the cardiac effect of Oxytocin / Carbetocin been studied in an animal model? Has it been studied in non-pregnant adults? Can Troponin I be measured after physical activity (walking staircases, jogging, or similar? Author response The potentially harmful effects of oxytocin on heart tissue and circulation have been known for decades. The risk of such effect can to some extent be reduced by lowering the dose and increasing duration of IV injection. Notwithstanding, oxytocin receptor agonists are the recommended drug of choice for prevention and treatment of PPH. Indeed, PPH remains the leading cause of maternal death. Carbetocin has gained popularity during the recent years due to the prolonged duration of action. To our knowledge, little data exist on the effect of carbetocin on the myocardium in animal models. As both oxytocin and carbetocin are used daily at the described doses in obstetric units all over the world, we find it compelling to investigate whether the myocardial effects of oxytocin and carbetocin are comparable in healthy women undergoing ceasarean delivery of their baby, even if the myocardial effects of carbetocin are not well described in animal models. We have amended the last paragraph of the Introduction to state more clearly that although there are no animal models, the myocardial effects of oxytocin and carbetocin warrant further investigation. Although animal models are lacking, there are studies that assessed the effects of oxytocin on heart tissue in pregnant women. The effect of oxytocin was studied in a clinical trial conducted by Svanström et al., including women undergoing cesarean delivery, investigating differences in the effect of oxytocin 10 IU and methylergometrine 0.2 mg on ECG changes related to myocardial ischemia. Clinically relevant occurrence of ECG changes were found in the oxytocin group, but not in the methylergometrine group. A control group of 10 non-pregnant patients receiving 10U of oxytocin also developed similar ECG changes. The authors conclude that the effects are related to oxytocin administration and not to pregnancy, surgical procedure, delivery, or sympathetic block from spinal anaesthesia (Svanström et al. 2008). The clinical relevance of transient troponin release is still unknown. High-sensitive troponins, both troponin I and troponin T, can be measured after physical activity like prolonged endurance activities as marathon but also after short-term and intermittent exercise like 30min of running and basketball (Gresslien and Agewall 2016). There is some uncertainty on the clinical implication of these transient troponin release. However, there is increasing concern that exercise-induced increases in troponin may not be a benign physiological response to exercise, but an early marker of future mortality and cardiovascular events (Aengevaeren et al. 2019). We have amended the manuscript (second paragraph of the ‘Discussion’) to include the above information. Comment I have more additional specific comments and questions: “Protocol. The study is a parallel-group, randomized, patient- and investigator-blinded Phase 4 study. Enrolment of participants started in April 2019 and is anticipated to be completed by the end of 2021…” Does this mean, that we are reviewing a protocol already in action? Author response Yes, we have started enrollment of the study. Please see the response to a similar comment above. Comment “Eligibility criteria. All participants… (but not) prolonged QT-time or other serious cardiac diseases. Do you do a pre-CS echocardiography before inclusion? Or just during CS? Author response In our clinic screening the patients with electrocardiogram is not a routine part of the preparation of patients for caesarean delivery. Patients with known cardiac disease or prolonged QT-time will be considered non-eligible for study participation. Thus, the participants are not screened with a pre-CS echocardiography to be included in the study. We have added that ECG was not part of the screening process to the section ‘Eligibility criteria’. Comment “Randomization and blinding. Participants will be randomized 1:1… to a computer-generated list of random numbers, with block sizes unknown”… Can you provide us with a more detailed description of the randomization? It sounds like a Viedoc@ is a black box? No description can be found regarding the “integral part of Viedoc – the eCRF solution”. Author response We have amended the article to include a better description of the randomization process. Comment “Study drug dosage and administration A single dose of either Oxytocin 2.5 IU (Syntocinon®, Swedish Orphan Biovitrum, Stockholm, Sweden) or Carbetocin 100 μg … will be administered by the investigator (a trained anesthetist) as a one-minute IV injection. Administering 2.5 IU of Oxytocin as a one-minute injection seems to be fast, and faster than the recommendations. We recommend an injection of 1IE/minute, or slower. Administering 100mikrog carbetocin diluted to 5ml as a one-minute iv injection also seems too fast. Should be administered slower than 1 minute. Author response Administration of oxytocin analogues is performed with great variety both nationally and internationally. There has been a shift from bolus dosing toward short infusion in case of IV administration. Thomas et al. showed that 5U oxytocin as a rapid bolus (approximately 1 second) induce more powerful hemodynamic effects compared to 5U administered as a 5-minute infusion (Thomas et al. 2007). However, little data exist comparing a 1-minute injection with a 5-minute infusion. In our clinic the current practice is to administer either oxytocin 2.5 IU or carbetocin 100 µg as a 1-minute IV infusion. This is well within the recommended dosage regimen suggested in the guidelines published by Heesen et al in Anaesthesia in 2019, recommending that doses of oxytocin up to 3 IU or carbetocin up to 100 mg should be administered over a time period of more than 30 seconds (Heesen et al. 2019). Moreover, Dell-Kuster et al proved no advantage on haemodynamic stablity of administering 100mg Carbetocin IV as a 5-minute infusion compared to a 1-minute injection (Dell-Kuster et al. 2017). We have amended the article to include a statement that the duration of the injection of the study drugs was in line with current guidelines. Comment If the approximate ED95 for Oxytocin for elective CS is 0.35 IU and 15 µg for Carbetocin, shouldn't we test these dosages? Author response It is well known that ED95 (the dose required to achieve the desired effect in 95% of the population) for oxytocin analogues are well below doses used in clinical practice for prevention of PPH in elective caesarean delivery. We still think it is important to investigate the difference in haemodynamic and myocardial effects of oxytocin and carbetocin at doses used in current clinical practice. Comparing myocardial effects of 0.35 IU of oxytocin and 15 mg of carbetocin will be an interesting topic for further studies. We have not made any amendments with regard to this comment. Comment In Ref 4 (Widmer et al.) Oxytocin 10 IU with Carbetocin 100 µg is compared. They find 0.04 vs 0.09% that complains of chest pain, respectively, i.e., after a smaller dose of Carbetocin compared to what is used in this study. Have the authors considered reducing the Carbetocin dose? Author response To our knowledge, Widimer et al. are comparing the effect of oxytocin 10 IU administered intramuscularly (IM) with carbetocin 100 mg IM for the prevention of PPH after vaginal delivery. The dose of 100 mg of carbetocin is identical to the dose used in our study, the only difference is the route of administration, IV instead of IM. Widimer et al. found that 0.04% of patients in the oxytocin group complained of chest pain compared to 0.09% in the carbetocin group, however the group differences were not significant. We have not made any amendments with regard to this comment. Comment “Prespecified analyses. Secondary outcomes Uterine tone will be assessed at 2.5 minutes and five minutes after study drug administration, using a numerical rating scale 0–10, where 0 = no tonus and 10 = maximum tonus, and 7 = clinically satisfactory tonus.” Do the obstetricians get any training in using this scale? Do they have a Gold Standard to compare with? Author response The obstetricians are presented with the 11-point numerical rating scale of uterine tone prior to the operation. A similar scale has been used in several clinical studies to determine level of uterine tone numerically (Sarna et al. 1997, Rosseland et al. 2013, Tabl et al. 2019). In our experience a uterine tone of 6 or less correlate well with the need for rescue uterotonics. Recently Cole et al. published an article concluding that the 0 to 10 numerical rating scale for uterine tone may be a reliable, standardized tool for research in reporting degree of uterotonic contraction during cesarean delivery (Cole et al. 2021). We have added a statement to the article to include the Cole et al 2021 citation in the section ‘Secondary outcomes’ Comment “Postoperative pain during the first 48 hours after delivery will be assessed in a subgroup of women at one centre.” Why is this relevant? And if relevant, why is this only done in one centre? Author response Group differences in reported pain and opioid consumption will be further investigated in a sub-study including 80 patients. We have amended the manuscript to make it clearer that there is a pain sub-study. 80 patients were deemed sufficient to see if there is a difference in post-operative pain experienced by participating women. Pain experienced by people is very subjective, and many factors affect the levels of pain a person may experience. We decided to conduct the pain sub-study just in one centre to remove as many confounding variables as possible. If the 80 women in the sub-study were distributed across the three centres, we would not be sure whether there were other external factors at play, not just the study drugs. Comment “Direct and indirect healthcare costs will be assessed”. Will the costs of 10 IU Oxytocin (approx. 2 €) and 100 µg Carbetocin (approx. 40€) be included in the assessment? Author response For estimation of direct and indirect healthcare costs, the costs of oxytocin and carbetocin will be included in the assessment along with the costs of rescue uterotonics, need for transfusions and duration of patient occupation of the operating room and the post-anaesthesia care unit after administration of study drug. We have made some minor edits to the article to clarify the direct and indirect healthcare costs. Comment “Concomitant medication Rescue medication In case of uterus atony, patients will be treated with rescue oxytocin 1 IU every 2 minutes up to maximum 5 IU.” Is it max 5 IU in total (ie 2.5 + 1 + 1 + 0.5 ??) or is it in addition to the 2.5? What happens after the maximum 5 IU is administered and there still is some atony? Will the additional Oxytocin not disturb the picture when comparing to Carbetocin – are these women excluded from getting Troponin I measured after 10 hours? Author response Rescue medication with 1 IU of oxytocin for treating uterine atony will be administered every 2 minutes up to a maximum of 5 IU, in addition to either study drug (2.5 IU oxytocin or 100 mg carbetocin). In case there is still a need for additional uterotonic treatment, this will be provided according to departmental procedures, including administration of prolonged oxytocin infusion, methylergometrine, prostinphenem, misoprostol or surgical or mechanical intervention as required. We have amended the article under the section ‘Rescue medication’ to clarify this point. The analysis of group differences in troponin I release will be performed in the modified intention-to-treat population, comprising all participants receiving allocated test drug. In addition, we will conduct a pre-planned sub-analysis including all participants receiving allocated test drug but not requiring rescue drugs. We have added the sub-analysis to the fourth paragraph of the ‘Analysis plan’ as this was previously omitted by mistake. Comment “Laboratory tests.…Normal values of troponin I in a female population (age 18 to 50 years) should be <15 ng/L, with the cut-off corresponding to the 99 percentile of a healthy reference population. 17” In ref. 17 (Bahadur et al)2, the Pakistan population had a median age of the included female on 56 years old with the oldest being 86 and youngest being 21 years old. Most of the people 246 (82.3%) were above 40 years old. This means that they were older than the Norwegian CS population, and Troponin I increase with age, according to ref. 15, Venge et al. 3 Do the authors have any comments on this? Do we know if the Pakistan women have a similar Troponin level as Norwegian? What was the baseline level in the pilot study? Is Troponin I the best measurement for myocardial injury? Author response It is well known that level of high-sensitive troponins in healthy patients vary with age and sex. During recent years, the 99th percentile cut-off for normal circulating concentration of high-sensitive troponin I (cTnI) has been lowered to <15 ng/L. This corresponds well to the findings of Kimenai et al. investigating 1540 individuals in a healthy European reference population (age 57±8 years, 52.4% women). Overall 99th percentile upper reference limits of cTnT and cTnI were 15 and 13 ng/L, respectively (Kimenai et al. 2016). Bossard et al published 99th percentile cut off for normal high-sensitive troponin I in a young healthy European population of 2077 adults from the general population aged 25–41 years without cardiovascular disease. The 99th percentile of cTnI was 15.79 ng/L in men and 5.11 ng/L in women (Bossard et al. 2017). Kimenai et al. also found that cardiac troponin concentrations differ in women and men and are stronger predictors of cardiovascular events in women (Kimenai et al. 2021). The baseline level of cTnI in the pilot study was detected within the normal limits in all but one patient. Recently Furenäs et al. published a study investigating dynamics of NTproBNP and cTnT in 196 healthy pregnant women. They found that existing cut-off values for ruling out heart failure (NTproBNP <300 ng/L) and myocardial ischaemia (hs-cTnT <14 ng/L) are applicable during pregnancy and after delivery, and that elevated levels mandate further attention on cardiac symptoms and renal function (Furenäs et al. 2020). We have amended the text in section ‘Laboratory tests’ to explain these points and added additional supporting references. High-sensitive troponin is the preferred tool for detecting myocardial stress and injury. In our clinic the lower detection limit of cTnT is 5 ng/L whereas levels of cTnI down to 0.1 ng/L were detected in our material. By choosing cTnI we are able to describe even small changes in high-sensitive troponin more accurately in a healthy population of young women. Comment What about proBNP? Author response Dynamics of NTproBNP was investigated in the pilot trial. We found no group differences in the development of NTproBNP and for this reason we decided not to proceed with measurements of NTproBNP in the CMT2 trial. Comment If all the levels are < the 99thpercentile i.e. normal, can we then state that Carbetocin and Oxytocin are safe (regarding cardiac side-effects)? If there are minor differences but still within normal ranges, then this might not be clinically relevant? Like studies showing significant difference in blood loss on i.e., 178mL between treatment A and B... Significant - yes. Relevant - no! Any comments? Author response We have reason to believe that even small elevations of high-sensitive troponins in relation to non-cardiac surgery are of clinical interest. Devereaux et al. investigated 21,842 adult patients undergoing non-cardiac surgery, mean age 63.1±10.7 years and 49.1% were female. They found that an absolute hsTnT change of 5 ng/L or higher was associated with an increased risk of 30-day mortality (Devereaux et al. 2017). Although in this study participants were much older than in our study, we believe that transient elevations may indicate subclinical myocardial injury that may result in a higher risk of future cardiovascular event that may be treatable or preventable. We have not made any specific amendments to this comment, but refer to our earlier response that transient elevations in troponins after physical activity may be an early marker for cardiovascular events. Comment “Power and sample size considerations. The sample size required for our current study was calculated using data from a pilot study of 40 patients, in which the largest difference in plasma troponin I concentration was found at 10 hours, with a mean +/- standard deviation change from baseline of 0.41+/-0.79 ng/L in the Carbetocin group versus 1.78+/-4.48 in the Oxytocin group”. I admit I am not used to doing statistical analysis, but does this mean that the change from the baseline can be 0.4 ng/L – 0.8ng/L = -0.4 ng/L? Because then Carbetocin improves the cardiac effect, reducing the Troponin I level? And similar for Oxytocin? Or does this mean that the data is skewed? And is “Median” not preferred instead of “Mean”? Author response The measurements of cTnI in our pilot study were skewed, and therefore best described by median and range, however for sample size calculations mean values and standard deviations are used. Comment What is the baseline for Oxytocin and Carbetocin, respectively? Author response The baseline cTnI values for both the oxytocin and carbetocin groups in the Pilot study were similar. Comment Is this because the authors suspect the results to be clinically not relevant, although detecting significant differences? Author response We started the pilot study not knowing whether there would be any difference between oxytocin and carbetocin in their cardiac effects but as oxytocin is known to affect the myocardium, we thought it was worth investigating given that carbetocin has a longer duration of action than oxytocin. As both oxytocin and carbetocin are used for PPH prophylaxis treatment, if it turns out that one has a less pronounced effect on the myocardium, this will help make clinical decisions for pregnant women with heart disease. Comment And is this because Ferring is funding the project? (As I mentioned in the beginning, the reader should be more suspicious). Author response We have been transparent in the study’s funding and Ferring’s role in the study. Comment Analysis plan I believe I do not have enough knowledge to comment relevantly on the analysis plan. Strength and Limitations. In 2019, a core outcome set was published for the prevention of postpartum hemorrhage to aid better comparison of clinical trials ensuring a minimum of outcomes to be reported…We will not assess breastfeeding or patient satisfaction with treatment, but will report pain and pain relief medication.” Why is this relevant to the study? Author response The core outcome set for the prevention of PPH has been established to allow easier comparison of clinical trials studying prevention of PPH. The core outcome set was published after our protocol was approved. The relevance to our study is that we are assessing seven out of nine recommended core outcomes, except for breastfeeding and patient satisfaction. The core outcome set includes intention and maintenance of breastfeeding as important patient-centred outcome as PPH may trigger a series of events that prevent or reduce a mother’s ability to fully breastfeed, particularly if the mother and baby are separated. It is also worth noting that breastfeeding releases oxytocin and therefore affects uterus tone but there is insufficient evidence for whether breastfeeding reduces PPH risk. Comment Is the dose of Oxytocin 2.5 IU used elsewhere? The cardiovascular events that are described after oxytocin iv dose (ST-changes, hypotension, tachycardia), have they been found after 2.5 IU? Author response No one has yet published the cardiovascular effects of oxytocin 2.5 IU IV. We have investigated this dose in our pilot study and will add the citation once it has been published. Comment Will data be released at the person level (for i.e. reviewer) in case of potential cardiac damage, and high Troponin I level during the CS? Author response We are batch assessing high-sensitivity troponin I levels after the last patient has completed the trial, which means that all patients will received standard of care. Follow-up is for 48 hours after their caesarean delivery. We will not know whether individual patients have high Troponin I levels during their hospital stay. Further studies will be required to determine risk of cardiovascular events due to PPH prophylaxis treatment, if there is indeed any future risk. Comment Is the rationale for, and objectives of, the study clearly described? Partly Is the study design appropriate for the research question? Yes Are sufficient details of the methods provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Not applicable Author response No response necessary. References Aengevaeren, V. L., M. T. E. Hopman, P. D. Thompson, E. A. Bakker, K. P. George, D. H. J. Thijssen and T. M. H. Eijsvogels (2019). \"Exercise-Induced Cardiac Troponin I Increase and Incident Mortality and Cardiovascular Events.\" Circulation 140(10): 804-814. Bossard, M., S. Thériault, S. Aeschbacher, T. Schoen, S. Kunz, M. von Rotz, J. Estis, J. Todd, M. Risch, C. Mueller, L. Risch, G. Paré and D. Conen (2017). \"Factors independently associated with cardiac troponin I levels in young and healthy adults from the general population.\" Clin Res Cardiol 106(2): 96-104. Cole, N. M., I. Abushoshah, K. G. Fields, D. A. Carusi, J. N. Robinson, B. T. Bateman and M. K. Farber (2021). \"The interrater reliability and agreement of a 0 to 10 uterine tone score in cesarean delivery.\" Am J Obstet Gynecol MFM 3(3): 100342. Dell-Kuster, S., I. Hoesli, O. Lapaire, E. Seeberger, L. A. Steiner, H. C. Bucher and T. Girard (2017). \"Efficacy and safety of carbetocin given as an intravenous bolus compared with short infusion for Caesarean section - double-blind, double-dummy, randomized controlled non-inferiority trial.\" Br J Anaesth 118(5): 772-780. Devereaux, P. J., B. M. Biccard, A. Sigamani, D. Xavier, M. T. V. Chan, S. K. Srinathan, M. Walsh, V. Abraham, R. Pearse, C. Y. Wang, D. I. Sessler, A. Kurz, W. Szczeklik, O. Berwanger, J. C. Villar, G. Malaga, A. X. Garg, C. K. Chow, G. Ackland, A. Patel, F. K. Borges, E. P. Belley-Cote, E. Duceppe, J. Spence, V. Tandon, C. Williams, R. J. Sapsford, C. A. Polanczyk, M. Tiboni, P. Alonso-Coello, A. Faruqui, D. Heels-Ansdell, A. Lamy, R. Whitlock, Y. LeManach, P. S. Roshanov, M. McGillion, P. Kavsak, M. J. McQueen, L. Thabane, R. N. Rodseth, G. A. L. Buse, M. Bhandari, I. Garutti, M. J. Jacka, H. J. Schünemann, O. L. Cortes, P. Coriat, N. Dvirnik, F. Botto, S. Pettit, A. S. Jaffe and G. H. Guyatt (2017). \"Association of Postoperative High-Sensitivity Troponin Levels With Myocardial Injury and 30-Day Mortality Among Patients Undergoing Noncardiac Surgery.\" Jama 317(16): 1642-1651. Furenäs, E., P. Eriksson, U. B. Wennerholm and M. Dellborg (2020). \"Pregnancy in a healthy population: dynamics of NTproBNP and hs-cTroponin T.\" Open Heart 7(2). Gresslien, T. and S. Agewall (2016). \"Troponin and exercise.\" Int J Cardiol 221: 609-621. Heesen, M., B. Carvalho, J. C. A. Carvalho, J. J. Duvekot, R. A. Dyer, D. N. Lucas, N. McDonnell, S. Orbach-Zinger and S. M. Kinsella (2019). \"International consensus statement on the use of uterotonic agents during caesarean section.\" Anaesthesia 74(10): 1305-1319. Kimenai, D. M., R. M. Henry, C. J. van der Kallen, P. C. Dagnelie, M. T. Schram, C. D. Stehouwer, J. D. van Suijlen, M. Niens, O. Bekers, S. J. Sep, N. C. Schaper, M. P. van Dieijen-Visser and S. J. Meex (2016). \"Direct comparison of clinical decision limits for cardiac troponin T and I.\" Heart 102(8): 610-616. Kimenai, D. M., A. S. V. Shah, D. A. McAllister, K. K. Lee, A. Tsanas, S. J. R. Meex, D. J. Porteous, C. Hayward, A. Campbell, N. Sattar, N. L. Mills and P. Welsh (2021). \"Sex Differences in Cardiac Troponin I and T and the Prediction of Cardiovascular Events in the General Population.\" Clin Chem 67(10): 1351-1360. Rosseland, L. A., T. H. Hauge, G. Grindheim, A. Stubhaug and E. Langesaeter (2013). \"Changes in blood pressure and cardiac output during cesarean delivery: the effects of oxytocin and carbetocin compared with placebo.\" Anesthesiology 119(3): 541-551. Sarna, M. C., A. K. Soni, M. Gomez and N. E. Oriol (1997). \"Intravenous oxytocin in patients undergoing elective cesarean section.\" Anesth Analg 84(4): 753-756. Svanström, M. C., B. Biber, M. Hanes, G. Johansson, U. Näslund and E. M. Bålfors (2008). \"Signs of myocardial ischaemia after injection of oxytocin: a randomized double-blind comparison of oxytocin and methylergometrine during Caesarean section.\" Br J Anaesth 100(5): 683-689. Tabl, S., M. Balki, K. Downey, G. Tomlinson, D. Farine, G. Seaward and J. C. A. Carvalho (2019). \"Uterotonics in elective caesarean delivery: a randomised non-inferiority study comparing carbetocin 20 μg and 100 μg.\" Anaesthesia 74(2): 190-196. Thomas, J. S., S. H. Koh and G. M. Cooper (2007). \"Haemodynamic effects of oxytocin given as i.v. bolus or infusion on women undergoing Caesarean section.\" Br J Anaesth 98(1): 116-119." } ] } ]	1	https://f1000research.com/articles/10-973
https://f1000research.com/articles/11-423/v1	14 Apr 22	{ "type": "Case Report", "title": "Case Report: Natal tooth in a four-day old newborn", "authors": [ "Nabeela Fatima", "Saurab Karki", "Subashchandra Pokharel", "Samikshya Basnet", "Nabeela Fatima", "Saurab Karki", "Samikshya Basnet" ], "abstract": "Background: A natal tooth is a tooth that is present at the time of birth. Eruption of teeth in infants is a rare phenomenon. Though rare, natal and neonatal tooth must be looked upon with great importance because they may lead to many complications. Case report: We present a case of a four-day old baby presenting with a mandibular natal tooth, which was successfully extracted without any complications. The mother had presented with a single umbilical artery during her pregnancy, which might be the proposed etiology of the natal tooth as the exact etiology is unknown. Conclusion: The exact etiology of natal tooth is unknown but there are a lot of factors that might be a cause. For the majority of cases, extraction is the mainstay of therapy as the risk associated is higher and can be life-threatening in most cases.", "keywords": [ "natal tooth", "congenital teeth", "predecidious teeth", "precocious dentition", "extraction", "case report" ], "content": "Introduction\n\nEruption of teeth in infants is a rare phenomenon. When present at birth they are known as natal teeth and if present during the first 30 days are known as neonatal teeth. After six months, the natural eruption of teeth occurs in infants.\n\nThe other terms used for natal and neonatal teeth from the literature are ‘congenital teeth’, ‘dentition praecox’, ‘fetal teeth’. Also, terms previously used were ‘dens connatalis’, ‘predecidious teeth’, ‘precocious dentition’.1–4\n\nThe incidence of natal and neonatal teeth is 1:716 to 1:30,000 as reported by Zhu and King,2 and 1:2000 to 1:3800 as reported by Chow.5 The general ratio for natal:neonatal teeth is 3:11. In most cases, these teeth are present as lower primary incisors.6 A study by Bodenhoff reported 85% of these are mandibular incisors; 11% are maxillary incisors; 3% are mandibular canines and 1% are maxillary canines or molars.7\n\nThe clinical categories of these teeth include:\n\ni) A shell-like crown structure that is loosely attached to the alveolus by gingival tissue with no root.\n\nii) A solid crown attached loosely to the alveolus by gingival tissue, without or little root.\n\niii) Eruption of the incisional margin of the crown through gingival tissue.\n\niv) Edema of the gingival tissue with an un-erupted but palpable tooth.1,8\n\nThe causative factors include infection, febrile states, trauma, malnutrition, superficial position of the tooth germ, hormonal stimulation, and maternal exposure to environmental toxins.9,10 The environmental predisposing factors include polychlorinated biphenyls (PCB); polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), which are usually believed to cross the placenta and the concentrations of these are found in adipose tissues of newborns, which are then correlated with those present in the mother’s milk.11,12\n\nThe complications might include discomfort during suckling, laceration of the mother’s breast, sublingual ulceration (Riga-Fede disease) with feeding refusal, ulceration on the ventral surface of the tongue caused by the tooth’s sharp incisal edge and swallowing or aspiration of teeth.10,13\n\nIf extraction is considered as a treatment plan, the clinician should assess the risk of hemorrhage and wellbeing of the baby especially in the case of supernumerary teeth. For mature teeth of normal dentition, it is crucial to take measures to preserve the tooth in healthy condition in the baby’s mouth using appropriate clinical resources.\n\n\nCase report\n\nA four-day old male baby born through lower segment cesarean section (LSCS) weighing 2.89 kilograms presented with a gum-like appearance at birth on the lower jaw. On noticing the projection, he was brought to medical attention on day four after birth. Eruption of a tooth started from the gum like projection from day eight and the tooth fully erupted a month after his birth. His maternal records showed a single umbilical artery (SUA) in targeted imaging for fetal anomalies (TIFFA). He was otherwise an active baby with adequate feeds. The diagnosis made by the pediatric team was natal tooth.\n\nHis birth records showed a bleeding time of two minutes 45 seconds and a clotting time of four minutes. In view of a single umbilical artery, the baby was referred for ultrasonography (USG) abdomen and pelvis which was normal. He was vaccinated as per immunization protocols.\n\nThe gum-like appearance further protruded and presented as a tooth over time (Figure 1).\n\nThe baby was then referred for a dental consultation. The dentist made a note of the complaints as hard tissue in the lower front teeth region and natal tooth in the lower gum pad in the anterior region (Figure 1). The plan of treatment was the extraction of the tooth (Figure 2). Post extraction was uneventful without any complications. The parents were counseled for post-operative instructions and advised to maintain oral hygiene and review after one week.\n\nThe timeline of important events is as follows:\n\nJanuary 2, 2022: The child was born.\n\nJanuary 6, 2022: Gum like projection on the anterior region of lower jaw.\n\nJanuary 11, 2022: Notable eruption of tooth in the anterior region of lower jaw.\n\nFebruary 1, 2022: Dental consultation and extraction of tooth.\n\n\nDiscussion\n\nNatal or neonatal teeth are conical or yellowish with hypoplastic enamel and dentin with poor or absent development of the root. In most cases, these teeth are mobile.2,14,15 The predisposing factors include dominant autosomal traits, endocrine disturbances (pituitary, thyroid, gonads), excessive or increased resorption of overlying bone, poor mental health, and congenital syphilis.16 A study by Gladen et al. reported 13 out of 128 infants having natal or neonatal teeth and showed that the mother was heavily exposed to polychlorinated biphenyls and benzofurans in Taiwan.11 On the other side, a study by Alaluusua et al. showed no association between milk levels of polychlorinated biphenyls and benzofurans with the occurrence of a natal tooth.12\n\nNo intervention is necessary if the tooth doesn’t interfere with breastfeeding and is asymptomatic. Extraction is recommended if the tooth is supernumerary and consultation with a pediatrician is strongly recommended.10\n\nThe treatment options include:\n\n• Maintenance of tooth in the mouth, unless this would cause injury to the baby.\n\n• When the tooth is not mobile it should be left unless it interferes with feeding.\n\n• When highly mobile, the risk of aspiration is high. So, extraction might be the mainstay for treatment.7,10\n\nThe factors that need to be considered include implantation and degrees of mobility, inconveniences during suckling, interference with breastfeeding and possibility of traumatic injury.17\n\nA study by Allwright reported extraction of 25 natal teeth with no complications of hemorrhage due to the risk of hypothrombinemia.18 All the extraction procedures were done in babies who were older than 20 days and it is recommended that the child is at least 10 days old for safer outcomes.18,19 If it is not possible to wait, then evaluation of the need and administration of vitamin K should be performed to prevent hemorrhage. The advisable dose is 0.5–1 mg, which is given intramuscularly.20\n\nMost importantly, the decision of retaining or extracting the tooth should be carefully evaluated for three factors: scientific knowledge, clinical common sense, and parenteral concern. It is very important to give complete information to the parents before letting them decide.\n\n\nConclusion\n\nNatal tooth is presented as a rare indication in infants. The exact etiology is unknown but there are a lot of factors that might be a cause. For the majority of cases, extraction is the mainstay of therapy as the associated risk of retaining the tooth is higher and can be life-threatening in most cases. A study on the accelerated or premature pattern of dental development will provide more insights.\n\n\nData availability\n\nAll the 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 the clinical details and clinical images was obtained from the parents of the patient.", "appendix": "Acknowledgements\n\nWe are grateful to the Peer Research Mentorship Program (PRMP) started by the International Society for Chronic Illness (ISCI) for their support.\n\n\nReferences\n\nTo EWH: A study of natal teeth in Hong Kong Chinese. Int. J. Paediatr. Dent. 1991; 1(2): 73–76. PubMed Abstract \| Publisher Full Text\n\nZhu J, King D: Natal and neonatal teeth. ASDC J. Dent. Child. 1995; 62(2): 123–128.\n\nMayhall JT: Natal and Neonatal Teeth Among the Tlinget Indians. J. Dent. Res. 1967 Jul 1; 46(4): 748–749. PubMed Abstract \| Publisher Full Text\n\nKates GA, Needleman HL, Holmes LB: Natal and neonatal teeth: a clinical study. J. Am. Dent. Assoc. 1984 Sep; 109(3): 441–443. PubMed Abstract \| Publisher Full Text\n\nChow MH: Natal and Neonatal Teeth. J. Am. Dent. Assoc. 1980 Feb 1; 100(2): 215–216. Publisher Full Text\n\nKing NM, Lee AM: Prematurely erupted teeth in newborn infants. J. Pediatr. 1989 May; 114(5): 807–809. PubMed Abstract \| Publisher Full Text\n\nBodenhoff J, Gorlin RJ: Natal and neonatal teeth: Folklore and fact. Pediatrics. 1963 Dec 1; 32(6): 1087–1093. Publisher Full Text\n\nHebling J, Zuanon ACC, Vianna DR: Dente Natal—a case of natal teeth. Odontol. Clin. 1997; 7: 37–40.\n\nCunha RF, Boer FAC, Torriani DD, et al.: Natal and neonatal teeth: review of the literature. Pediatr. Dent. 2001; 23(2): 158–162. PubMed Abstract\n\nLeung AKC: Natal Teeth in American Indians-Reply. Am. J. Dis. Child. 1986 Dec 1; 140(12): 1214. Publisher Full Text\n\nGladen BC, Taylor JS, Wu Y-C, et al.: Dermatological findings in children exposed transplacentally to heat-degraded polychlorinated biphenyls in Taiwan. Br. J. Dermatol. 1990; 122(6): 799–808. Publisher Full Text\n\nAlaluusua S, Kiviranta H, Leppäniemi A, et al.: Natal and neonatal teeth in relation to environmental toxicants. Pediatr. Res. 2002; 52(5): 652–655. PubMed Abstract\n\nBuchanan S, Jenkins CR: Riga-Fedes syndrome: Natal or neonatal teeth associated with tongue ulceration. Case report. Aust. Dent. J. 1997; 42(4): 225–227. Publisher Full Text\n\nGalassi MS, Santos-Pinto L, Ramalho LTO: Natal maxillary primary molars: case report. J. Clin. Pediatr. Dent. 2004; 29(1): 41–44. PubMed Abstract\n\nZiai MN, Bock DJ, Da Silveira A, et al.: Natal Teeth: A Potential Impediment to Nasoalveolar Molding in Infants With Cleft Lip and Palate. J. Craniofac. Surg. 2005 Mar; 16(2): 262–266. PubMed Abstract \| Publisher Full Text\n\nŠtamfelj I, Jan J, Cvetko E, et al.: Size, ultrastructure, and microhardness of natal teeth with agenesis of permanent successors. Annals of Anatomy - Anatomischer Anzeiger. 2010 Aug 20; 192(4): 220–226. PubMed Abstract \| Publisher Full Text\n\nKhandelwal V, Nayak UA, Nayak PA, et al.: Management of an infant having natal teeth. BMJ Case Rep. 2013 Jun 3; 2013: bcr2013010049. Publisher Full Text\n\nAllwright WC: Natal and neonatal teeth: a study among Chinese in Hong Kong. Br. Dent. J. 1958; 105: 163–172.\n\nRusmah M: Natal and neonatal teeth: a clinical and histological study. J. Clin. Pediatr. Dent. 1991 Jan 1; 15(4): 251–253. PubMed Abstract\n\nRyba GE, Kramer IRH: Continued growth of human dentine papillae following removal of the crowns of partly formed deciduous teeth. Oral Surg. Oral Med. Oral Pathol. 1962 Jul 1; 15(7): 867–875. PubMed Abstract \| Publisher Full Text" }	[ { "id": "184917", "date": "31 Jul 2023", "name": "Maria Alzira Cavacas", "expertise": [ "Reviewer Expertise 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\nDear Authors,\nCongratulations for your work!\nThe topic is interesting and, despite not being very common, it can help to articulate the way of conduct between clinicians/pediatricians and dentists when confronted with these clinical cases.\nHowever, I think the authors should explain better the title or even change it because they call it \"Natal tooth\" and, as the literature show and the authors mentioned : \"When present at birth they are known as natal teeth and if present during the first 30 days are known as neonatal teeth\".\nDoubt emerges about the true reason for the extraction....like the authors and literature affirm: \"No intervention is necessary if the tooth doesn’t interfere with breastfeeding and is asymptomatic.\" Did the tooth cause pain to the mother without the opponent? Did the tooth have mobility? It seems to me it did not because figure 2 shows a considerable root portion (about 2/3).\nOr was it made just to prevent future mobility and risk of aspiration? I recommend more clarification of this point in the paper. Finally, I wonder exactly what anesthesia was used in the procedure?\nBest regards, Maria Alzira Cavacas\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": "198647", "date": "09 Oct 2023", "name": "Arlette Suzy Setiawan", "expertise": [ "Reviewer Expertise Pediatric 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\nOverall, this article offers a comprehensive insight into the topic of natal and neonatal teeth. Here’s a critical appraisal:\nStrengths:\nIn-depth Introduction: The introduction comprehensively introduces the reader to the topic, offers definitions, alternative terminologies, and statistics about the incidence.\n\nRich Literature Review: The article quotes and references numerous studies, providing a solid base of background knowledge.\n\nClear Clinical Categories: The categorization of the clinical presentations of natal/neonatal teeth is clear and concise.\n\nCase Study: Including a case study brings in real-world relevance and helps readers understand the topic with a practical example.\n\nComprehensive Treatment Options: The discussion section offers a broad range of treatment options along with factors to consider when choosing them.\nAreas for Improvement:\nConfusing Statistics: In the introduction, the statement, \"Eruption of teeth in infants is a rare phenomenon,\" might mislead since eruption of teeth is a normal process in infants. It’s the timing (natal/neonatal) that’s rare.\n\nInconsistent Information: The article starts by stating that the eruption of teeth after six months is natural. However, typically, the first tooth might emerge as early as four to six months.\n\nContradictory Conclusion: The conclusion states that extraction is the primary therapy due to the high risks associated with retaining the tooth. Yet, the discussion mentions that extraction is only necessary if the tooth interferes with breastfeeding or is symptomatic. This is a little conflicting.\n\nRecommendation Age Inconsistency: The article states that the child should be at least 10 days old for safer extraction outcomes, but it later mentions extraction procedures in babies older than 20 days. This might confuse readers about the ideal age for extraction.\n\nLack of Discussion on Etiology: The exact cause of natal or neonatal teeth isn't discussed in-depth, even though it's mentioned in the conclusion.\n\nEditorial Improvements: Certain parts could be clearer with better phrasing. For instance, \"Natal tooth is presented as a rare indication in infants.\" The word \"indication\" seems out of place.\nSuggestions:\nClear up any contradictory statements or clarify them further for the reader.\n\nConsider expanding on the etiological factors that could lead to natal or neonatal teeth.\n\nThe discussion could also benefit from an exploration of any genetic factors or patterns observed in the presence of natal or neonatal teeth.\n\nInclude potential long-term implications or follow-ups necessary for infants with natal or neonatal teeth.\nThis article provides a valuable overview of natal and neonatal teeth but could benefit from further clarity in certain areas and a deeper dive into the etiology.\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/11-423
https://f1000research.com/articles/9-1446/v1	11 Dec 20	{ "type": "Research Article", "title": "Effectiveness of 36 hospital learning centers in Thailand: continuation of child patient education, parent attitudes toward child’s illness and service satisfaction", "authors": [ "Adidsuda Fuengfoo", "Kim Sakulnum", "Sumitra Owjinda" ], "abstract": "Background: This study aimed to determine the effectiveness of 36 hospital learning centers for the continued education of sick children using electronic distance learning television (eDLTV), parents’ attitudes toward their child’s illnesses and education, and service satisfaction of the centers. Methods: The sample included 4,430 children aged 4-18 years old with common illnesses, chronic illnesses and developmental disorders, as well as 4,430 parents who had taken care of the child for at least 6 months. The methods included attitude surveys, which were analyzed using chi-square tests and t-tests.\nResults: The factors associated with education continuation of the children were illness types (parents were less worried about children with common illness and more concerned about education of children with chronic diseases and children with disabilities), distance from home to school, transportation type, parents’ education level, marital status, and family income. About 99.8% of patients with common illnesses continued their education, followed by 99.3% of disabled children, and 95.9% of chronic patients. Satisfaction score towards the services at the learning centers were high (mean scores: 4.28 and 4.43 respectively, out of 5 = strongly satisfied). Conclusion: After completing an education program through eDLTV at a center, a total of 97.7% of children continued their education and were highly satisfied with the service at the center. Parents had positive attitudes towards their child’s illnesses and education.", "keywords": [ "child illness", "education service", "school engagement", "satisfaction", "hospital learning center" ], "content": "Introduction\n\nAs a crucial foundation for development, education should be equally accessible to all children, regardless of differences in social or physical status. Today medical advancements have increased the survival rate of children with chronic diseases to 90%, reducing mortality rates and complications1, and increasing life expectancy by as much as 20 years longer2. The prevalence of children with chronic diseases differs depending on definition. A survey in the United States indicated that approximately 20% of children had chronic illnesses3. In Thailand, the number is still unclear because chronic illness is defined as a long-term illness involving treatments that might affect the lifestyles of children and families4–6. Such changes can include absence from school, which could affect learning and social activities7,8.\n\nThe UN Convention of the Rights of the Child states that every child has the right to be protected and receive equal education9. As a member of the UN, Thailand implemented a law stipulating that “education is not only limited to the classroom”. Therefore, medical institutes have merged with special education centers to establish “learning centers in the hospital” so that pediatric patients can continue to have equal access to education. Making use of information technology to support education, the project is called “The Information Technology Project under the Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn”. Similar learning centers in other countries involve the coordination of multidisciplinary teams to enhance social and learning activities or sick children.\n\nThe Queen Sirikit National Institute of Child Health (QSNICH) Learning Center in Thailand has been operating for 20 years. In the past10, education for sick children supported academic knowledge and daily living skills which made them feel included in society, and reflected educational and social perceptions of the related life adjustments and psychological conditions. QSNICH has previously conducted research11 on education for sick children, aiming to determine the effectiveness of learning centers in 36 provinces in Thailand. With official ministry-level collaboration among various parties to obtain operational results regarding continued learning for sick children and the associated factors12, QSNICH has provided a model for other hospitals across Thailand to ensure that all sick children have equal access to education.\n\nThis study has been extended from previous research13 on the effectiveness of the learning center at QSNICH, which investigated the associated factors affecting further education of children with special needs. The aims of this study were to determine the effectiveness of 36 other network learning centers across Thailand for the continued education of pediatric patients, as well as the factors associated with the patients’ further education. It also evaluated parents’ attitudes toward their children’s illness and education, as well as service satisfaction of the center.\n\n\nMethods\n\nA cross-sectional study was conducted to collect information from the target population at 36 learning centers in the hospitals across Thailand under the supervision of Thai Ministry of Public Health. The study used a survey to collect information (Extended data14). The study was conducted from January 1, 2018, to December 31, 2018.\n\nThe target population included 4,430 child patients aged 4-18 years as well as 4,430 parents who had taken care of the child for at least six months, meaning that the total study population included 8,860 individuals. The sample size calculation was based on Wayne15. The total number of pediatric patients from 36 learning centers at the hospitals was 30,000 (n=30,000), and the incidence of children who went back to school was 80% (p=0.8))\n\nSystematic random sampling was used to select parents and children from the lists of patients at the 36 learning centers. If any individual refused to participate, the next name would be selected.\n\nThe survey was conducted by the researchers after the parents gave written informed consent for their child to participate. For children aged 4–7 years, parents were asked to participate in the survey on behalf of the child. The session was audio recorded for data analysis.\n\nInclusion criteria: 1) Pediatric patients age between 4–18 years who are attending learning centers at the hospital; 2) Pediatric patients who have attended learning centers more than 2 times; 3) pediatric patients with chronic illnesses who are admitted at the hospital or needed consecutive follow-up.\n\nExclusion criteria: pediatric patients with serious illness and life-threatening conditions.\n\nThe survey was divided into four parts: (1) demographic information of the children and parents (gender, age, cause of admission and length of stay (Table 1)), (2) information about the patient-parent relationship, and (3) satisfaction with service at the learning centers (measured using Likert scales of 1=unsatisfied, 2=poor, 3=fair, 4=satisfied, 5=very satisfied).\n\nSAS 9.4 software was used for data management. Descriptive statistics, including percentages, means, and standard deviations, were used to analyze the population data and SAS 9.4 software was used to calculate frequency, percentage, mean, and standard deviation to measure the attitudes and satisfaction of the parents and children. Satisfaction data were divided into five levels, from strongly agree to strongly disagree, based on the statistical test results. Chi-square tests and t-tests were used to assess the variables associated with education continuance at the learning center.\n\nThis study and the selection of the participating hospitals were approved by the Office for Ethics in Human Research, Ministry of Public Health, Thailand in 2018 (ref. REC.024/2561), and was conducted in accordance with the Declaration of Helsinki.\n\n\nResults\n\nThe patient population was classified into three groups: those with common illnesses, chronic illnesses, and developmental disorders (n=4,430). There were a total of 4,430 caregivers: 82.94% were caregivers for patients with common illnesses, 82.68% chronic illnesses, and 72.34% developmental disorders. Biological parents or relatives comprised 17.07% of the common illnesses group, 17.28% chronic illnesses, and 26.95% development disorders; foster parents or teachers accounted for 0.06%, 0.04%, and 0.71%, respectively. Most caregivers were women (75.74%, 80.83%, and 82.62%, respectively), of Thai nationality (99.26%, 99.33%, and 99.65%, respectively), and Thai ethnicity (97%, 98.83%, and 99.65%, respectively). Other ethnicities included Akha, Lahu, and Burmese. The main religion was Buddhism (92.01%, 94.51%, and 97.87%, respectively), followed by Islam and Christianity. Most caregivers were aged 31–40 years (43.71%, 39.89%, and 34.40%, respectively), followed by those aged 41–50 years (23%, 39%, and 40%, respectively) and 21–30 (17.29%, 13.7%, and 18.86%, respectively). Most parents had completed vocational level, or high -school level education, followed by those with a primary or secondary educational level. Most parents were employed in labor (41.5%), agriculture (23.09%), and sales (11.22%). Most parents were married, separated, or divorced and had monthly household incomes of 10,0000–30,000 baht, followed by 5,000-10,000 baht; only a small number had incomes of >30,000 baht or <5,000 baht. Most families owned a house, while some rented a house or room. Only a small number lived with a relative. Most repondents lived in the northeastern part of Thailand.\n\nOf the 4,430 patients, 1,764 patients had common illnesses, 2,384 chronic illnesses, and 282 developmental disorders. In general, the caregivers of all three types of patients rated the following issues similarly. Regarding illness severity, 29.9% rated it at the minimum level, 33.6% moderate, and 20% somewhat high. Regarding when the child became ill, 43.8% of caregivers always informed teachers while 24.02% often did so. Approximately 35.7% were somewhat highly confident that their child could get along with their peers, while 24.0% were somewhat confident and 24% were highly confident. Likewise, 37.8% of caregivers put somewhat high effort to into helping their child, while 25.0% made a high-level effort. Approximately, 37.6% frequently explained the assignments to their child, and 27.5% always did so. About 37.6% had high confidence that the school provided appropriate study plans for their child, 25.5% were moderately confident, and 26.1% were extremely confident. About 34.6% of patients discussed what happened in school with their child, and 23.3% did so all the time. Regarding caregivers’ confidence in encouraging their child to control their emotions, 37.6% were somewhat highly confident, 23.5% moderately confident, and 19.6% extremely confident. About 84% of caregivers sent their child to school every day.\n\nDuring hospitalization, 25.1% of the patients were in kindergarten, 58.7% in primary school, and 13.8% in secondary school. After discharge, 22.3% in kindergarten, 57.5% in primary school, and 15.6% in secondary school. About 20.9% of the patients lived <1 km from school, 53.4% lived 1–5 km away, and 25.3% lived the same distance as before joining the learning center. About 57.0% traveled to school by bicycle/motorcycle, 17.3% by private car, and 20.0% by public transportation; the means of transportation remained almost the same before and after being treatment at the center.\n\nAs shown in Table 2, 28.5% of common illness patients were in kindergarten, 57.1% in primary school, and 13.2% in secondary school. After discharge, 28.0% were in kindergarten., 57.3% in primary school, and 13.3% in secondary school. Approximately, 15.7% lived 1 km or less from school, 56.8% 1– 5 km, and 27.5% the same distance as before entering the center. About 56.4% traveled to school by bicycle/motorcycle, 16.9% by personal vehicle, and 21.5% by public transportation. Transportation type remained largely the same before and after the treatment at the center.\n\naDid not study = 34 cases, Study drop = 70 cases\n\nBefore entering the center, 23.3% were in kindergarten, 59.6% in primary school, and 14.9% in secondary school. After discharge, 18.5% were in kindergarten., 57.1% in primary school, and 18.0% in secondary school. About 24.3% of patients lived less than 1 km from school, 51.3% 1–5 km, and 23.7% the same distance as before entering the center. About 58.3% traveled to school by bicycle/motorcycle, 17.5% by personal car vehicle, and 18.4% by public transportation. Means of transportation were mostly the same before and after treatment.\n\nBefore entering the center, 19.5 % of patients were in kindergarten, 61.4% in primary school, and 9.6% in secondary school. After discharge, 18.4% were in kindergarten, 62.4% in primary school, and 9.6% in secondary school. About 24.1% of the patients lived less than 1 km from school, 50.4% 1–5 km. away, and 24.8 the same distance as before. About 50.4% traveled to school by bicycle/motorcycle, 18.4% by private vehicle, and 23.1% by public transportation. The means of transportation were slightly different before and after entering the center.\n\nAs shown in Table 3, satisfaction surveys were distributed to two groups of patients: those who continued their education and those who did not. Scores were classified into five ranges (1.00-1.80 = strongly disagree, 1.81-2.60 = disagree, 2.61-3.40 = uncertain, 3.41-4.20 = agree, and 4.21-5.00 = strongly agree). The average satisfaction score of the two groups was 4.21-5.00. Patients who continued their education thought that the lessons in the classroom were interesting; they scored this item higher than the other group did. Entertainment had been used to provide amusing and relaxed learning for these children. The patients who did not continue their education were those in final stage of life.\n\nTable 4 shows the factors associated with education continuation. About 99.8%, 95.9%, and 99.3% of common, chronic, and development disorder patients continued their education, respectively. Significantly fewer chronic patients continued their education compared to common illness patients (OR=0.05; 95% CI: 0.02-0.14). About 97.8%, 97.5%, and 97.9% of kindergarten, primary school, secondary school students continued their education, respectively. There were no statistically significant differences between the three groups. Regarding distance from home to school, 97.1% <1 km away, 97.7% 1–5 km, and 98.7% >5 km; there were no statistically significant differences among the three groups. Regarding transportation to school, about 95.0% walked, 97.7% traveled by bicycle/motorcycle, 97.5% used a personal car, and 99.0% used public transportation. For those who continued their education, there were significant differences between patients who traveled by bicycle/motorcycle (OR=2.292; 95% CI: 1.152-4.560) and public transportation (OR=5.144; 95% CI: 2.062-12.833).\n\nMost students (97%) continued their education regardless of who they lived with. About 98.1% of students who continued their education lived with male primary caregivers, whereas 97.5% lived with female primary caregivers. A total of 97.6% of Thai patients and 100% of non-Thai patients continued their education. The age, education, and occupation of caregivers were not statistically associated with the decision of the patient to continue their education. Regarding marital status, most parents were married, followed by divorced parents (OR=0.470; 95% CI: 0.223-0.990), while there were significantly fewer widowed parents (OR=0.323; 95% CI: 0.145-0.718).\n\nAbout 96.9% of households had an average monthly income of <5,000 baht, 97.2% made 5,001-10,000 baht, and 98.3% made >10,000 baht. There were significant differences between households making <5,000 baht and those making >10,000 baht (OR=1.8; 95% CI: 1.032-3.184). Income was not significantly related to education continuation.\n\nFactors associated with education continuation were illness type (more chronic patients discontinued their education), hospitalization period (the longer the stay, the greater the chance of discontinuing education), distance from home to school, transportation type, and factors related to caregivers (i.e., education level, occupation, and marital status).\n\n\nDiscussion\n\nThe 36 learning centers in this study were located in primary or secondary hospitals that had accepted patients from local hospitals in the area. Most patients had chronic illnesses, mostly related to hematology, cancer, and heart disease. This study’s results align with those of previous studies10,11,13,16,17 of tertiary hospitals. About 45% of patients had common illnesses associated with gastroenterology, respiratory disorders, and accidents. The 282 children in the developmental disorder group had attention deficit hyperactivity disorder, intellectual disability, autism, and learning difficulties.\n\nCompared to research10 conducted in 2000, the number of children with developmental disorders at the learning centers had increased. This is perhaps attributable to patients with physical and developmental disabilities now having better access to education. The average ages of patients with chronic and common illnesses, and developmental disorders were 9.65 years (7.10-12.44), 8.69 years (6.17-11.19), and 8.85 years (6.90-11.11), respectively. Children with chronic illnesses were more likely to discontinue education because of limitations imposed by their health conditions, namely, a high risk of becoming infected or receiving treatment during the academic year. In such cases, modified education plans were introduced. Distance learning, facilitated by technology, is recommended to help patients to keep up with classes, and stay in touch with friends, which would reduce awkwardness when they returned to school. Mobile education can overcome teacher-related limitations, and it has the advantage of allowing children to select lessons for themselves. Other factors that deprived children to attend school in this study were distance from home to school, type of transportation, education level of parents, marital status, and income. However, other factors such as nationality, ethnicity or religion had no relationship with the continuation of education statistically. The results of this study was different from the a previous study11; travel distance and types of transportation in the city did not affect education as much transportation in the country.\n\nTeachers’ roles must be adjusted in the assessment guidelines18–20 and they must be able to work with patients who have different physical and psychological conditions and education. Teachers should facilitate extra lessons for patients through special classes or e-learning, and help them pass their exams. International research studies13,21,22 have found that “bibliotherapy” is an effective method to for reducing anxiety, and developing skills related to learning, emotion regulation, and social interaction.\n\nWe found that parents perceived their child’s chronic illnesses as moderately severe to seriously severe. For children with common illnesses, parents found that these illnesses were not moderately severe. Most parents of children with chronic and common illness highly recognized the importance of social modification, they always kept the school updated and were highly confident that the school could provide an appropriate plan for their children. Parents of children with developmental disorders were moderately to highly confident that their child was able to build relationship with friends. Teaching and psychological skills of the teachers were needed because each patient had a different physical condition, education level and psychological condition. They must be able to help the patients reduce anxiety, adjust to their health condition and give appropriate advice. Learning activities could be given anywhere outside the classroom to encourage a learning atmosphere.\n\nIn this study, the satisfaction survey of children at the learning center gave a high score of 4.21-5.00 (mean = 4.26, SD = 0.65) for every dimension. The item that was rated the highest score was that the children enjoyed participating in the activities. The item with the second highest score was that there was interesting and up-to-date content (for example, EDLTV is used in teaching according to international standard of the Ministry of Education). The third highest scoring item was that the activities were appropriate for learning (mean = 4.18, SD =0.79). Satisfaction in being part of fun and appropriate activities was rated the fourth highest item, while the item with the lowest score was that there was a variety of material and adequate facilities used in the activities (mean = 4.11, SD = 0.74).\n\nThe collaboration between learning centers and schools to monitor and refer patients, establishes a long-term plan and provide appropriate support help the patients a lot. The Cochrane library13,21 suggests promoting a law that would foster combined work by multidisciplinary teams from hospitals, families, schools, and communities. This way, services could be developed holistically22, and children would be encouraged to continue their education. Social and society family burdens would be reduced, which could achieve the objective of children growing up to be valuable assets of the country.\n\n\nConclusion\n\nMost child patients in the learning centers assessed in this study in Thailand decided to continue their education. Factors that were statistically related to discontinuing education were illnesses type, distance from home to school, transportation type, education level, marital status and family income. Appropriate educational management plans are needed to provide for these children. Parents had positive attitudes about illness and education. Children’s satisfaction with the learning center was high.\n\nA good education system should allow all children to have equal access to education, including vulnerable children.\n\nImprovement in technology supports distance learning, which combines patient ‘healthcare, education, and social life’, thus enhancing learning equity, and helping them to return to a normal life after reentering society.\n\nThe factors affecting education continuation should be recognized and considered in education planning for sick children.\n\nSatisfaction should include every dimension of services for quality improvement.\n\nThe support from Special Education Act and multi-disciplinary team will lead to successful hospital learning centers\n\nThe study population was limited to children at 36 hospital learning centers in Thailand. Respondents from various institutes in other contexts should be included to increase diversity. Since different hospitals provide different types and level of services for patients with different needs, future studies should include learning centers in different types of hospitals throughout Thailand such as primary, secondary and tertiary care facilities.\n\n\nData availability\n\nThis project contains the following underlying data:\n\nFigshare: Survey data record of 36 learning centers, Thailand https://doi.org/10.6084/m9.figshare.13140659.v114\n\nFigshare: English survey.pdf, https://doi.org/10.6084/m9.figshare.8208338.v223\n\nThis project contains the following extended data:\n\n- Copy of the survey with English translation.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nOn behalf of the authors, a deep appreciation shall be sent to 36 hospital learning centers across Thailand, Ministry of Public Health and Ethic Committee, and Queen Sirikit National Institute of Child Health for supporting this study.\n\n\nReferences\n\nAnnunziato RA, Rakotomihamina V, Rubacka J, et al.: Examining the effects of maternal chronic illness on child well-being in single parent families. J Dev Behav Pediatr. 2007; 28(5): 386–91. PubMed Abstract \| Publisher Full Text\n\nAtheros T, Arpaichiratana C: Model of continuing care foe chronically ill children. Thai Journal of Nursing Council. 2011; 26(special issue): 112–25.\n\nEnderlein G: Daniel, Wayne W.: Biostatistics – A Foundations for Analysis in the Health Sciences. Wiley & Son New York – Chichester – Brisbane – Toronto – Singapore, 6th ed. 1995, 780 S., 58. - , ISBN 0 – 471 – 58852-0 (cloth). Biom J. 1995; 37(6): 744. Publisher Full Text\n\nLaw Office of the Basic Education Commission Ministry of Public Health: Education Provision for Persons with Disabilities Act B.E. 2551 and 12 subordinate legislations. Special Education Bureau 2008; (Accessed: 27 January 2020). Reference Source\n\nPompimon J: A study of the organization programs for young chronically ill in-patient children. Bangkok: Chulalongkorn University. 2000.\n\nZuckerman B: Developmental and Behavior Pediatrics: a handbook for primary care. 2nd edn. Philadelphia: Lippincott William & Wilkins, 2005; 152–7. Reference Source\n\nWijlaars LPMM, Gilbert R, Hardelid P: Chronic conditions in children and young people: learning from administrative data. Arch Dis Child. 2016; 101(10): 881–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLum A, Wakefield CE, Donnan B, et al.: Understanding the school experiences of children and adolescents with serious chronic illness: a systematic meta-review. Child Care Health Dev. 2017; 43(5): 645–62. PubMed Abstract \| Publisher Full Text\n\nCouncil for Exceptional Children Division of Physical, Health an Multiple Disabilities: School Success for children experience chronic illness: national recommendations for addressing global barriers. Physical Dis. 2017; 36(2): 8–15. Publisher Full Text\n\nStoll BJ, Hansen NI, Bell EF: Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network. Pediatrics. 2010; 126(3): 443–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYunak R: Interviewing children with chronic illness in qualitative research. J Nurs Health Sci. 2012; 6: 1–11.\n\nLoCasale-Crouch J, Johnson B: Transition from pediatric to adult medical care. Adv chronic kidney Dis. 2005; 12(4): 412–7. PubMed Abstract \| Publisher Full Text\n\nFuengfoo A, Leelathanaporn S, Mekrungcharas T, et al.: Effectiveness of the Hospital Learning Center (Queen Sirikit National Institute of Child Health): Satisfaction with service and parents’ attitudes towards children’s illness [version 1; peer review: 2 approved, 1 approved with reservations] F1000Res. 2019; 8: 1616. (Accessed: 15 March 2020). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFuengfoo A: Survey data record of 36 learning centers, Thailand. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13140659.v1\n\nDaniel WW: Biostatistics:A Foundation of Analysis in the Health sciences. (6th ed.)John Willey & Sons,Inc. 1995. Reference Source\n\nCenter for Chronic Disease Prevention and Control: What are chronic and noncommunicable diseases? Ottawa, ON: Public Health Agency of Canada. (Accessed: 18 August 2018). Reference Source\n\nSpecial Education Bureau, Ministry of Education: Guidelines for Performance Evaluation of Learning Center in Hospital for Chronically Ill Patients. Bangkok: Office of the Basic Education Commission 2015; (Accessed: 13 January 2020). Reference Source\n\nBell MF, Bayliss DM, Glauert R, et al.: Chronic Illness and Developmental Vulnerability at school Entry. Pediatric. 2016; 137(5): e20152475. PubMed Abstract \| Publisher Full Text\n\nSpecial Education Bureau, Ministry of Education: ‘Practice Guideline for Teachers in Chronic Children Learning Center in Hospital Bangkok: Office of the Basic Education Commission. 2015; (Accessed: 13 January 2020). Reference Source\n\nVan Cleave J, Gortmaker SL, Perrin JM: Dynamic of obesity and chronic health conditions among children and youth. JAMA. 2010; 303(7): 623–30. PubMed Abstract \| Publisher Full Text\n\nPugbunmee R: Chronically ill adolescents: impact on self-care. Rama Nurse. 1997; 3: 103–9.\n\nWorld Health Organization: innovative care for chronic conditions: building blocks for action. Geneva, Switzerland: world Health Organization 2002; (Accessed: 15 October 2018). Reference Source\n\nFuengfoo A: English Survey.pdf. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.8208338.v2" }	[ { "id": "78813", "date": "17 Feb 2021", "name": "Issarapa Chunsuwan", "expertise": [ "Reviewer Expertise Developmental and behavioral pediatrician" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 continuing research to find ways to promote education for children in hospitals. The hospital school model was further implemented in 36 centers in Thailand in which many children have received services. In this paper, the authors aim to evaluate the effectiveness of the learning centers. I have some suggestion as following:\nIntroduction part\nFor a clearer understanding, the Authors should give more information about the 36 learning centers, such as how they are settled in hospitals (place, operational time), who are teachers in the centers, how they select pediatric patients to attend the centers (by doctor referral or who else) and how they provide educational service (curriculum, activity)? And also, more information about eDLTV may help readers who are not in Thailand understand it. The Authors may add information about how the QSNICH learning center supervises the other 36 learning centers.\n\nMethodology\nA flow chart that shows how 4430 participants in this study were recruited from 30,000 pediatric patients of the 36 centers could be helpful to show a clear overview of the recruitment process.\n\nThere are 4430 data in the research rather than 8860. The author can explain that 4430 children included in the research and 4430 parents were interviewed.\n\nDefinition for classifying common illness from chronic illness should be mentioned (depend on the type of disease or hospital day)\n\nThe questionnaires used in the study should be defined in detail for better understanding, such as numbers of items in the satisfactory scales and how is the total score obtained, what are questions asked in the demographic data and validation process of the questionnaire.\n\nResults\nThe main research findings on effectiveness may not yet clear to readers. The authors may adjust or add a table that defines the effectiveness of the program. For example, in Table 3, statistical analysis of differences in satisfaction between 2 groups could add more information about effectiveness.\n\nIn table 4, Is not clear that the heading \"continue education\" means continue education at a learning center or at a formal school. and also the distance from school means the formal school or learning center.\n\nKey messages\nThe main result of this study about effectiveness should be added as the key message.\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": [] }, { "id": "118876", "date": "17 Jan 2022", "name": "Shervin Assari", "expertise": [ "Reviewer Expertise I am an expert of child development", "social inequalities", "and longitudinal data analysis. I also work related to chronic disease and mental health problems." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a single-group interventional study. Here are areas for improvement:\nThe abstract and also the paper should make the design much more clear. Is this a longitudinal one-arm intervention (designed by authors) or is this a natural experiment designed by a system and the authors just conducted the evaluation?\n\nThe result is not just about attitudes. Many other factors such as socioeconomic status predict child educational success, but the title only mentions attitude.\n\nOne major problem is that all samples are analyzed together. However, some of these children have physical health problems and some have disabling developmental challenges. These two groups are qualitatively different and cannot be combined. Just controlling for this type as a nominal variable (0/1) does not solve the problem. I think at least for sensitivity analysis, all results should be replicated in a stratified fashion, separately for each of the groups mentioned.\n\nSES and social determinants of health are root causes of health and well-being. The paper, however, does not mention these concepts despite that it measures them.\n\nOverall, I think the data are much stronger than the discussion section. Many more areas could be discussed which are left untouched.\n\nThese children are educationally heterogeneous. This includes children at kindergarten to much higher levels. Progress in educational programs is highly dependable on the workload required. So, simply controlling for the educational level does not solve this problem. Overall, the high heterogeneity of the sample is a major problem here.\n\nSex/gender is beyond a control variable. Parents may differently invest in their boys and girls (gender norms). Similarly, a child may get different attention and investment depending on the number of siblings. Thus, more attention is needed on the composition of the household beyond marital status.\n\nAnother important area in this paper is poor eligibility. These poor selection criteria have made it very difficult to know who was in the study. I think the authors need to replicate their findings in groups of patients with more homogenous conditions.\n\nMore information is needed on the variation of the adherence to the curriculum across learning centers. How do various centers do their job? Was there any effect on centers?\n\nParticipants were nested to centers so they are not independent samples. Please discuss your solution to address this data challenge.\nOverall, despite the paper having great potential and focusing on a novel topic that is rarely done by others, authors could do a much better job with inclusion, statistics, and discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "8020", "date": "14 Apr 2022", "name": "adidsuda fuengfoo", "role": "Author Response", "response": "1. This is a single-group interventional study. Here are areas for improvement: The abstract and also the paper should make the design much more clear. Is this a longitudinal one-arm intervention (designed by authors) or is this a natural experiment designed by a system and the authors just conducted the evaluation? This research was conducted in a form of natural experimental study in which variables are influenced by nature or factors outside of the researchers’ control. A cross-sectional design has been implemented in this study where a group of the population is selected based on the inclusion criteria set for the study: types of illnesses, patient’s and parent’s demographic information in order to measure the effectiveness of the project and to examine the factors correlating with continuing education of children under HSH project, patient’s attitudes and patient’s satisfaction towards HSH project. However, a limitation of a cross-sectional study is that it is difficult to identify causal effects and only describe associations between variables. 2. The result is not just about attitudes. Many other factors such as socioeconomic status predict child educational success, but the title only mentions attitude. Assessment of the Learning Center in Hospital Program was assessed on the function and effectiveness of children with health problems participating in the Learning Center in Hospital Program. Another angle, besides taking into account accuracy, should also consider preferences along the way. Therefore, it is assessed in various dimensions, such as assessing the perspectives of service users in terms of parental attitudes and the satisfaction of sick children participating in the Hospital Learning Center project, which is a secondary objective of this research and is discussed in the information found for the purpose. 3. One major problem is that all samples are analyzed together. However, some of these children have physical health problems and some have disabling developmental challenges. These two groups are qualitatively different and cannot be combined. Just controlling for this type as a nominal variable (0/1) does not solve the problem. I think at least for sensitivity analysis, all results should be replicated in a stratified fashion, separately for each of the groups mentioned. The research team would like to accept and appreciate the advice that is very helpful. In the issue of data analysis should be separated into groups, diseases because each group of diseases has a different nature and affects learning. The researcher, therefore, brought this issue to further improve in the analysis of the data to separate the groups into three groups: common disease children, chronic disease children, and children with developmental problems, as shown in the table. 4. SES and social determinants of health are root causes of health and well-being. The paper, however, does not mention these concepts despite that it measures them. Socioeconomic status is a very important part of healthcare and is the foundation for proper health management and lifestyle. It is a valuable recommendation when further analyzed separately. The disease group found that social socioeconomic status affecting admission to continuing education after discharge from the hospital was associated with all disease groups. When analyzed, it was found that there was still a relationship between chronic disease and chronic disease groups with developmental problems because it is a long continuous treatment, and with costs that depend on severity, complexity, and advances in technology, children with chronic and developmental diseases have a better chance of living longer. 5. Overall, I think the data are much stronger than the discussion section. Many more areas could be discussed which are left untouched. The researcher agreed and used the data for further analysis, separating the disease groups. We have added more discussion on factors that remained related to children's continued return to school when analyzing disease groups: parents' education, socioeconomic status, family income, number of siblings, etc. 6. These children are educationally heterogeneous. This includes children at kindergarten to much higher levels. Progress in educational programs is highly dependable on the workload required. So, simply controlling for the educational level does not solve this problem. Overall, the high heterogeneity of the sample is a major problem here. We agree on the issue of diversity in the classroom as one of the limitations of education in nature experimental study. In a future study, we will include prospective follow-up studies and segmented samples for cross-sectional study to reduce such limitations. 7. Sex/gender is beyond a control variable. Parents may differently invest in their boys and girls (gender norms). Similarly, a child may get different attention and investment depending on the number of siblings. Thus, more attention is needed on the composition of the household beyond marital status. Since gender is an uncontrolled variation, the researcher has selected the factors that are more likely to affect the continuation of children which are the number of siblings and family income. After analyzing separately, the study found that parents with more than 2 children are likely to discontinue education of the child with chronic diseases or with developmental problems. At the same time, families with a monthly income lower than 5,000 baht considered stopping further education of the sick child. With economic constraints, parents may need to think about the cost-effectiveness of sending their child to school. It must be decided that delivering a child without chronic illnesses or developmental delay may be more cost-effective than children with chronic illnesses or developmental delay since education cost is not the only expense under parents’ responsibility, but also healthcare costs or medication costs for the sick child. In contrast, the study found that the number of siblings and hospital length of stay do not correlate with children without chronic illnesses or developmental delay to further education because the normal school system allows them to go back to school after they recover. 8. Another important area in this paper is poor eligibility. These poor selection criteria have made it very difficult to know who was in the study. I think the authors need to replicate their findings in groups of patients with more homogenous conditions. It is a valuable suggestion that gives the researcher a clearer perspective. Therefore, the researcher has divided the groups to select the pediatric patients by separate disease groups and analyze the factors affecting admission to further education in order to create more homogeneous conditions as shown in the table. 9. More information is needed on the variation of the adherence to the curriculum across learning centers. How do various centers do their job? Was there any effect on centers? Hospital Learning Centers for sick children use a standardized curriculum established by the Ministry of Education applicable for children at all education levels: pre-kindergarten, primary, secondary, tertiary levels, as well as vocational education. This curriculum is a collaboration between MOU partners: the Ministry of Education, Ministry of Public Health, and Ministry of Science and Technology. All of the operational processes need to follow the Clinical Practice Guideline (CPG) and pass an external audit from a third party to ensure that the education that is delivered to sick patients in every hospital learning center meets the standard. 10. Participants were nested to centers so they are not independent samples. Please discuss your solution to address this data challenge. The selection of a sample in this study was a voluntary response sample from all 36 learning centers in Hospitals across Thailand. That is, after the instruction was given to the participants, they were able to participate in the study voluntarily. If the number of participants was more than the expected population, a random sample selection would be implemented. In order to access the data, permission from the Human Ethics Committee of the Ministry of Public Health that oversees all 36 network hospitals must be approved." } ] } ]	1	https://f1000research.com/articles/9-1446
https://f1000research.com/articles/11-178/v1	14 Feb 22	{ "type": "Research Article", "title": "Association of women’s literacy and children’s mortality rate among countries in southeast Asia 2015-2019: a cross-sectional study", "authors": [ "Fauzi Budi Satria", "Rahayu Lubis", "Yi-Hsin Elsa Hsu", "Usman Iqbal", "Fauzi Budi Satria", "Yi-Hsin Elsa Hsu", "Usman Iqbal" ], "abstract": "Background: Women’s literacy is often associated with the health status of family members, especially children. Unfortunately, in some regions of Southeast Asia, the rates of women’s literacy are still very low, and in these areas, children’s mortality rates are also very high. This study aims to identify the association of women's literacy and children’s mortality rates among countries in the Southeast Asian region from 2015 to 2019. Methods: In this cross-sectional study, children’s mortality rates were defined as mortality rates in newborns and under-fives. Besides women’s literacy (WL), we also assessed the Human Development Index (HDI), Freedom Status (FS), Government Effectiveness (GE), and the proportion of births assisted by skilled health staff (BASHS) as the independent variables in this study. First, we describe the profiles of WL as well as newborn and under-five mortality rates among 11 countries in Southeast Asia from 2015 to 2019, and then we assess the correlations. Results: Between 2015 to 2019, Myanmar, Lao, and Timor-Leste had the highest child mortality rates. In addition, these four countries were the countries with the lowest WL rates compared to other countries. Children’s mortality rates were significantly associated with WL, GE, HDI, and BASHS. Meanwhile, this study found that the FS of a country was not significantly associated with the children’s mortality rate of countries in the Southeast Asia region. Conclusion: This study found that there’s an indication that the better the WL rate, as well as the governance, the HDI, and maternity services in a country, the lower its children’s mortality rates. Therefore, improvement in multiple sectors such as governance, economy, health system, education, and gender equality is required to help countries in this region achieve the United Nations’ Sustainable Development Goals target by 2030.", "keywords": [ "women’s literacy", "child mortality", "newborn mortality", "under-five mortality", "Southeast Asia", "Regional health", "Sustainable Development Goals", "gender equality", "social determinants of health" ], "content": "Introduction\n\nGlobally, the under-five mortality rate declined by 59% between 1999 and 2018; from 93 deaths per 1000 live births to 39 per 1000 live births.1 However, this number needs to be improved as the United Nations’ Sustainable Development Goals (SDGs) target for 2030 is to reduce preventable under-five mortality to at least as low as 25 per 1000 live births. Moreover, achieving this goal becomes more challenging as most under-five mortality occurs in low and lower-middle-income countries, especially countries within Africa and South Asia,1,2 where children’s health can be affected by several factors such as healthcare service quality, family wealth status, and social status, including mother's education level.3\n\nMany kinds of research had been conducted to identify the association between a mothers’ education level and their children’s health status. The evidence has shown that the mother’s education is an important determinant of the health of children.4 It’s stated that even after accounting for household income, the number of siblings, health environments, and other socioeconomic variables, the mother’s literacy is still a major factor that influences children’s health status.4 A study conducted in the Kashmir valley has shown an inverse relationship between women’s literacy rate and children’s mortality rate. This study found that women’s literacy has an immense contribution in declining the infant mortality rate (IMR) and maternal mortality rate (MMR) and thus can help in improving the health status of both women and children.5 In sub-Saharan Africa, a study found that the decline in mortality rates of children under five years was much higher among the children born to mothers who have never received formal education.6 In Indonesia, child vaccination rates were increased from 19% when mothers have no education, compared to 68% when mothers have at least secondary school education.7 A study in India also showed that the female literacy rate is a good predictor of infant mortality rate in India. The study’s results showed that infant mortality rate was inversely related to women’s literacy rate, while men’s literacy was not.8\n\nProper education is the first step in empowering women and young girls and providing women with the best possible chance for a prosperous and healthy life.9 For women who go on to have families, education can also aid in household and family management.10 Lastly, educating girls saves lives and builds stronger families, communities, and economies. In addition, an educated female population increases a country's productivity and promotes economic growth.11 According to an International Labor Organization report, educating girls has proven to be one of the most important ways of breaking poverty cycles and is likely to have significant impacts on access to formal jobs in the longer term. Moreover, it’s reported that some countries lose more than $1 billion a year by failing to educate girls to the same level as boys. Therefore, it’s believed that educating women will increase their opportunity to get a better job and receive a higher wage. Furthermore, this also may address gender imbalances especially in the labor force.11 By looking at the importance and benefits of women’s literacy in non-health sectors and regions, therefore, we conducted this study to identify the association between women’s literacy and children’s mortality rates among countries in Southeast Asia.\n\n\nMethods\n\nThis cross-sectional study aimed to identify the association between women’s literacy and children’s mortality rate in Southeast Asia from 2015 to 2019. There are eleven countries in the region and all of them are members of the Association of Southeast Asian Nations (ASEAN); Brunei Darussalam, Myanmar, Cambodia, Timor-Leste, Indonesia, Laos, Malaysia, the Philippines, Singapore, Thailand, and Vietnam.\n\nIndependent variables in this study were the children’s mortality rates. It was defined as newborns’ mortality rate (NMR) and under-five mortality rate (UFMR). The NMR is the number of neonates dying before reaching 28 days of age, per 1000 live births each year. Meanwhile, the UFMR refers to the probability of a child born in a specific year or period dying before reaching the age of five and is defined as a probability of death derived from a life table and expressed as a rate per 1000 live births. We collected the data about the NMR and the UFMR of these 11 countries during the 2015 to 2019 period from the website of Our World in Data and World Health Organization (WHO) respectively.12,13\n\nNext, for the dependent variables in this study, besides women’s literacy (WL), we also employed other variables for comparison such as Human Development Index (HDI), Freedom Status (FS), Government Effectiveness (GE), and the percentage of births assisted by skilled health staff (BASHS). We selected these variables to align with the SDGs’ targets on child mortality rates, but also because these indicators not only measure child survival, but also reflect the social, economic, and environmental conditions in which children live. We collected the data from various online databases from 2015 to 2019.\n\nThe WL is the percentage of women aged 15 years and over who can read and write by understanding simple short statements about everyday life. The literacy rate is an outcome indicator to evaluate educational attainment. Therefore, literate women are considered capable of understanding written information and using it to improve the health, nutrition, and education of household members. We collected the data from the current World Bank published report and then calculated the average WL rate of each country during the observed period.14\n\nThe BASHS are defined as the proportion of deliveries attended by personnel trained to give the necessary supervision, care, and advice to women during pregnancy, labor, and the postpartum period; to conduct deliveries on their own, and to care for newborns. We collected the BASHS data from the WHO website and calculated the average of BASHS among countries in Southeast Asia between 2015 and 2019.15\n\nThe HDI is a summary measure of average achievement in three key dimensions of human development: a long and healthy life, level of education, and having a decent standard of living. The health dimension is assessed by life expectancy at birth, while the education dimension is measured by the mean of years of schooling for adults aged 25 years and expected years of schooling for children of school entering age. The standard of living is measured by gross national income (GNI) per capita. We selected the HDI as one of the variables in this study because it can also be used to question national policy choices, asking how two countries with the same level of GNI per capita can end up with different HDI outcomes. We collected the HDI data from the Human Development Report 2020 report on the UNDP website, and then calculate the average HDI score of the eleven study countries in the period between 2015 to 2019 period.16,17\n\nThe FS described the freedom status of countries based on the annual global report on political rights and civil liberties composed by Freedom House. There are seven main topics covered by this report, including freedom of expression and belief (media freedom, religious freedom, academic freedom, and free private discussion). The score ranges from 0 to 100, where the county with a score of 70 to 100 is labeled as a “free” country. Meanwhile, the country with scores 40 to 69 will be labeled as “partially free (PF)” and score 0 to 39 as “not free (NF)”. In this study, we calculated the average freedom scores of all eleven countries between 2015 to 2019.18–20\n\nThe GE was selected as a variable in this study since it may reflect perceptions of the quality of public services, the quality of the civil service and the degree of its independence from political pressures, the quality of policy formulation and implementation, and the credibility of the government's commitment to such policies. The score of GE ranges from -2.5 (weak) to 2.5 (strong). We collected the GE data from the Worldwide Governance Indicators (WGI) project 2020 reports and calculated the average GE scores of the selected countries from 2015 to 2019.21\n\nFirst, we described the profiles of children’s mortality rates and women’s literacy among the eleven countries in Southeast Asia region from 2015 to 2019. Next, after conducting a normality test, we conducted paired t-test to assess the association between our independent (NMR and UFMR) and dependent variables (WL, GE, FS, HDI, and BASHS) to identify which independent variables were mostly associated with the mortality rates.\n\n\nResults\n\nFirst, we developed Figures 1 and 2 to describe the children’s mortality rates and women’s literacy rates across the eleven countries in Southeast Asia during 2015 to 2019 period. Figure 1 showed that three countries with highest newborn mortality rates (NMR) was the Myanmar (2.36), Lao (2.31), and Timor-Leste (2.05). Meanwhile, the countries with the lowest NMR in the region were Singapore (0.1), Malaysia (0.44), and Brunei Darussalam (0.56). Similar with NMR, Lao (4.94), Myanmar (4.82), and Timor-Leste (4.74) were also the three countries with the highest UFMR in the Southeast Asia. Meanwhile, the countries with the lowest UFMR were Singapore (0.26), Malaysia (0.83), and Thailand (0.99). Then, as shown in Figure 2, we found that the Philippines (97.55), Brunei Darussalam (96.3), and Singapore (95.68) were the three countries with the highest rate of WL across countries in the Southeast Asia region. Meanwhile, the WL rates in this region were the lowest in Timor-Leste (64.21), Cambodia (75.03), Myanmar (79.06), and Lao (79.39).\n\nNext, we developed Table 1 to describe the profile of the eleven study countries from 2015 to 2019. The weakest GE was found in Cambodia (-0.64), Lao (-0.55), Myanmar (-1.1), and Timor-Leste (-0.98). Meanwhile, the three countries that have the strongest GE were Singapore (2.22), Brunei Darussalam (1.17), and Malaysia (0.94). Next, none of the countries in this region were labeled as free countries, with six countries labeled as not-free (FS<40), and the remaining five countries labeled as partially free (FS=40-69). Lao was the country with the lowest (12.67) freedom score, while Timor-Leste was the highest (67.5). Moreover, among the eleven countries, HDI was very high in Singapore (0.93), Brunei Darussalam (0.84), and Malaysia (0.8), while Myanmar (0.57) and Cambodia (0.58) were the lowest. Then, in this region, the proportion of births attended by skilled health staff (BASHS) was the highest in Brunei Darussalam, Malaysia, Singapore, and Thailand (BASHS>99). Meanwhile, BASHS was the lowest in Timor-Leste (56.7), Myanmar (60.2), and Lao (64.4).\n\nLastly, Table 2 was developed to describe the results of the correlation analysis. From the five variables, the FS of a country was the only variable that was not significantly associated with children’s mortality rates in the countries studied. Meanwhile, out of the four variables that were significantly associated with children’s mortality rates, all of them were negatively associated. The association between women’s literacy and children’s mortality rates was (-0.73 and -0.76), while the association between other variables and children’s mortality rate was more than 90%.\n\n\nDiscussion\n\nBased on the results of our study, we found that no country in the ASEAN region has a children’s mortality rate above the global average during the 2015 to 2019 period. This finding is in line with the previous report which showed that children’s mortality rates above the global average were more commonly found in the African region22 and not in Southeast Asia. Even Myanmar, which had the highest average newborn mortality rate, and Lao which had the highest average of under-five mortality rate in this region, had only 2.36 deaths and 4.82 per 1,000 live births from 2015 to 2019 respectively, compared to 17 deaths22 and 38 deaths per 1000 live births23 globally. Furthermore, our study found that the three countries (Myanmar, Lao, and Timor-Leste) with the highest children’s mortality rate in this region were lower-middle-income countries.24 This finding is also coherent with the previous report that showed that the highest burden of under-five mortality is reported to be mostly found in low-income or lower-middle-income countries.23 In addition, previous evidences had shown that the income level of the country was extremely correlated with the children’ mortality rate. The poorest countries have the highest levels of child mortality, and the countries with the highest income have the lowest rates.12 The results of our study showed that countries like Singapore, Brunei, and Malaysia were among the countries with the lowest children mortality rates. Moreover, our study also found that the correlation between HDI and children’s mortality rates was very high (NMR: -0.93, UFMR: -0.90)\n\nNext, our study found that out of eleven countries in the region, the majority (eight countries) had BASHS coverage above the global average (83%).25 Moreover, even the BASHS coverage in Brunei, Malaysia, Singapore, and Thailand reached more than 99%. On the other hand, our results showed that three other countries whose BASHS coverage was below the global average also had the highest children’s mortality rates in the region. Those three countries were Myanmar (60.2%), Lao (64.4%), and Timor-Leste (56.7%). This study results did not only show disparities in the Southeast Asia region but also confirm the United Nations International Children's Emergency Fund (UNICEF) report which states that although BASHS coverage continues to increase in 2020 globally, the coverage continues to be uneven with significant disparities between regions.25 In our study, the correlation between BASHS and children mortality rate were significantly high (NMR: -0.91, UFMR: -0.95).\n\nWomen who have literacy are believed to be able to use their knowledge and understanding to improve the welfare of their families.14 Our study showed that the WL rates of seven countries in the Southeast Asia region from 2015 to 2019 were above the global average (83%) in 2019. Moreover, this study also found that the children’s mortality rates among these seven countries were much lower than the global average.22,23 In addition, the previous study had been proven that better education of women might reduce mortality among children.26 However, our result showed the association between WL and children’s mortality rates among countries in Southeast Asia was only around 70% (NMR: -0.73, UFMR: -0.76). The implication of this finding can be seen from the low children’s mortality rates in Cambodia. While the WL there was the second lowest (75%) in the region after Timor-Leste (64.2%) and also below the global average,23,22 the mortality rates among children in Cambodia were only slightly different from Indonesia and the Philippines which had much higher WL rates than Cambodia in the same period.\n\nThe governance of a country may have widespread effects on the health of its population.27 A previous study showed that the better the governance, the lower children’s mortality rates. In another study, it’s mentioned that the quality of governance is even more critical in determining a good outcome for both mother and child.28 This evidence was coherent to our finding that showed the strong negative association between governance and mortality rate among newborns (-0.92) and children under-five (-0.89). In our study results, Myanmar, Lao, Timor-Leste, and Cambodia were the four countries with the lowest government effectiveness, and also the countries with the highest children’s mortality rates in the Southeast Asia region during the 2015 to 2019 period.\n\nFinally, our study showed a consistent negative association between all of our independent variables (WL, GE, FS, HDI, and BASHS) and both of our dependent variables (NMR and UFMR). However, we found that the relationship between the freedom status of a country (FS) and children’s mortality rates among countries in Southeast Asia from 2015 to 2019 was not statistically significant. Then, we also highlighted the association between WL and children’s mortality rates in this study which was only around 70%. This association is quite high but relatively low compared to other variables that we assessed in this study which correlate around 90% and above. Based on this study results, we assume that children’s mortality rates can be reduced and potentially achieve the SDGs target by 2030 with the improvement in multiple sectors such as governance, the economy, health care, and education, as well as by strengthening gender equality. Accelerated improvement in various sectors is urgently needed, considering that the latest SDG report mentions that many SDG indicators have experienced setbacks due to the impact of the COVID-19 pandemic.29\n\n\nConclusion\n\nThe results of our study found that there’s a significant association between women’s literacy and children’s mortality rates among countries in the Southeast Asia region during the 2015 to 2019 period. Moreover, the HDI, GE, and BASH also had a significant correlation with children’s mortality rates in the same period. However, among eleven countries in the Southeast Asia region from 2015 to 2019, there were 3 countries whose indicators were all below the global average, while there were also many countries whose all indicators were much better than the global average. In order to achieve the SDGs 2030 target which does not want to leave anyone behind, this kind of disparity must be addressed immediately. Therefore, we consider that it is necessary to re-strengthen the ASEAN as a binding organization for countries in the Southeast Asia region, by expanding the field of cooperation in ASEAN by including the health sector. The goal is that countries in the region will work together to achieve the SDGs targets by 2030.\n\n\nData availability\n\nThis project contains the following underlying data from the sources linked below:\n\n• Newborn mortality rate (Our World in Data)\n\n• Under-five mortality rate (WHO)\n\n• Women’s literacy (The World Bank)\n\n• Births attended by skilled health staff (WHO)\n\n• Human development index (UNDP)\n\n• Freedom status (Freedom House)\n\n• Government effectiveness (The World Bank)\n\nFigshare: Underlying Data-Association of Women’s Literacy and Children’s Mortality Rate Among Countries in Southeast Asia 2015-2019.XLSX30\n\nhttps://doi.org/10.6084/m9.figshare.18904481.v1\n\nThis project contains the following extended data:\n\n• Master Data.XLSX\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nWHO: Child health.2021 [cited 2021 Nov 8]. Reference Source\n\nWHO: SDG Target 3.2\|Newborn and child mortality: By 2030, end preventable deaths of newborns and children under 5 years of age, with all countries aiming to reduce neonatal mortality and under-5 mortality.2021 [cited 2021 Nov 8]. Reference Source\n\nWHO: Child health.2021 [cited 2021 Nov 8]. Reference Source\n\nChen Y, Li H: Mother’s education and child health: Is there a nurturing effect?. J. Health Econ. 2009 Mar 1; 28(2): 413–426. PubMed Abstract \| Publisher Full Text\n\nBatool N, Shah SA, Dar SN, et al.: Impact of female literacy on infant mortality and maternal mortality in Kashmir valley: a district level analysis. GeoJournal. 2020 Dec 1; 85(6): 1545–1551. Publisher Full Text\n\nBado AR, Sathiya SA: Women’s Education and Health Inequalities in Under-Five Mortality in Selected Sub-Saharan African Countries, 1990–2015. PLoS One. 2016 Jul 21; 11(7): e0159186. PubMed Abstract \| Publisher Full Text\n\nPRB: The Effect of Girls’ Education on Health Outcomes: Fact Sheet. PRB; 2015 [cited 2021 Nov 8]. Reference Source\n\nSaurabh S, Sarkar S, Pandey DK: Female Literacy Rate is a Better Predictor of Birth Rate and Infant Mortality Rate in India. J. Family Med. Prim. Care. 2013; 2(4): 349–353. PubMed Abstract \| Publisher Full Text\n\nEduvoice India: Women Literacy and Empowerment: A Review\|Eduvoice. The Voice of Higher Education.2021 [cited 2021 Nov 14]. Reference Source\n\nKaur R: Why to educate women? – Advantages. MyIndia; 2013 [cited 2021 Nov 14]. Reference Source\n\nTheirworld: Girls’ education. Theirworld. Theirworld; 2021 [cited 2021 Nov 14]. Reference Source\n\nRoser M, Ritchie H, Dadonaite B: Child and Infant Mortality. Our World in Data.2013 May 10 [cited 2021 Nov 7]. Reference Source\n\nWHO: Indicator Metadata Registry Details.2021 [cited 2021 Nov 8]. Reference Source\n\nThe World Bank: Literacy rate, adult female (% of females ages 15 and above)\|Data.2021 [cited 2021 Nov 3]. Reference Source\n\nBirths attended by skilled health personnel (%).[cited 2022 Jan 31]. Reference Source\n\nUNDP: Human Development Index (HDI)\|Human Development Reports.2021 [cited 2021 Nov 19]. Reference Source\n\nUNDP.\|Human Development Reports: 2020 [cited 2021 Nov 19]. Reference Source\n\nFreedom House: FAQ - Freedom in the World. Freedom House; 2021 [cited 2021 Nov 19]. Reference Source\n\nFreedom House: Freedom in the World Research Methodology. Freedom House; 2021 [cited 2021 Nov 19]. Reference Source\n\nFreedom House: Freedom in the World. Freedom House; 2021 [cited 2021 Nov 19]. Reference Source\n\nThe World Bank: Worldwide Governance Indicators. Worldwide Governance Indicators.2021 [cited 2021 Nov 19]. Reference Source\n\nUNICEF: Neonatal mortality. UNICEF DATA; 2021 [cited 2021 Nov 14]. Reference Source\n\nUNICEF: Child Mortality. UNICEF DATA; 2021 [cited 2021 Nov 14]. Reference Source\n\nLower middle income\|Data: [cited 2022 Feb 2]. Reference Source\n\nDelivery care: UNICEF DATA.[cited 2022 Feb 2]. Reference Source\n\nMondal MNI, Hossain MK, Ali MK: Factors Influencing Infant and Child Mortality: A Case Study of Rajshahi District, Bangladesh. J. Hum. Ecol. 2009 Apr 1; 26(1): 31–39. Publisher Full Text\n\nLin R-T, Chien L-C, Chen Y-M, et al.: Governance matters: an ecological association between governance and child mortality. Int. Health. 2014 Sep; 6(3): 249–257. PubMed Abstract \| Publisher Full Text\n\nHall S, Lopez M, Murray S, O’Hare B: Government revenue, quality of governance and child and maternal survival. Appl. Econ. Lett. 2021 Sep 9; 0(0): 1–6. Publisher Full Text\n\nChild mortality (under 5 years): [cited 2022 Feb 2]. Reference Source\n\nUnderlying Data-Association of Women’s Literacy and Children’s Mortality Rate Among Countries in Southeast Asia 2015-2019.XLSX. figshare.2022 [cited 2022 Feb 3]. Reference Source" }	[ { "id": "123550", "date": "28 Mar 2022", "name": "Siow Li Lai", "expertise": [ "Reviewer Expertise Population studies", "public and reproductive health", "gender 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\nThe paper requires major changes before it can be indexed. Overall, the analysis is too simple. Use the term \"neonatal\" mortality instead of \"newborn\" mortality. The authors should consider using infant mortality along with the other two mortality measures. More relevant references are required to strengthen the case study, particularly why this study focused on Southeast Asian countries, despite the relatively low child mortality rate? Please proofread the paper carefully (the authors even turn over the dependent and independent variables).\n\nSome other comments are listed below.\nThe following citation is misleading. It sounds like child mortality is lower among uneducated than the educated ones, but the actual fact is the other way round, even though the former has experienced a slipper decline in child mortality. Since the authors are looking at a cross-sectional study, they should look at the level rather than the trend -> In sub-Saharan Africa, a study found that the decline in mortality rates of children under five years was much higher among the children born to mothers who have never received formal education.\n\nProvide a citation for the following: According to an International Labor Organization report, educating girls has proven to be one of the most important ways of breaking poverty cycles and is likely to have significant impacts on access to formal jobs in the longer term.\n\nProvide the present situation/statistics of child mortality and the levels of social and economic development in Southeast Asia in the Introduction section.\n\nThe number of observations (n=11) is too small for a firm conclusion. The authors may consider using longer time-series data.\n\nThe authors have proposed a normality test and paired t test in the Methodology section, but these analyses are missing in the Results section. The type of correlation test (parametric or non-parametric) in table 2 is not mentioned in the Methodology section. Please address these issues, and provide the software used to perform the analyses.\n\nThe Discussion and Conclusion sections should focus on the association between women’s literacy and child mortality, as proposed in the title of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Partly", "responses": [ { "c_id": "8052", "date": "29 Apr 2022", "name": "Fauzi Budi Satria", "role": "Author Response", "response": "Thank you for your review I do agree with most of your suggestions and comments. That’s why we did major changes to our study which we will briefly explain below. “Use the term \"neonatal\" mortality instead of \"newborn\" mortality” “The authors should consider using infant mortality along with the other two mortality measures.” Previously I only had 2 variables, newborn and under-five mortality rate. I have changed the term of the newborn to neonatal. In the current version, instead of two, I have 3 dependent variables (infant mortality rate/IMR, neonatal mortality rate/NMR, and under-five mortality rate/UFMR). “More relevant references are required to strengthen the case study, particularly why this study focused on Southeast Asian countries, despite the relatively low child mortality rate?” I had added references to strengthen the background. In brief, countries in this region were so varied in terms of economic level, the number of populations, culture, governmental system, etc. “The following citation is misleading. It sounds like child mortality is lower among uneducated than the educated ones, but the actual fact is the other way round, even though the former has experienced a slipper decline in child mortality. Since the authors are looking at a cross-sectional study, they should look at the level rather than the trend -> In sub-Saharan Africa, a study found that the decline in mortality rates of children under five years was much higher among the children born to mothers who have never received formal education.” I have revised and rephrased most of the result and discussion parts “Provide a citation for the following: According to an International Labor Organization report, educating girls has proven to be one of the most important ways of breaking poverty cycles and is likely to have significant impacts on access to formal jobs in the longer term.” Yes, we have done it “Provide the present situation/statistics of child mortality and the levels of social and economic development in Southeast Asia in the Introduction section.” We modified the design of the study. The current version is only focused on women’s literacy, and it’s the only independent variable in this study. Previously, there were 5 independent variables (women’s literacy, Human Development Index, Government Effectiveness, Birth attended by skilled health staff, and freedom status). “The number of observations (n=11) is too small for a firm conclusion. The authors may consider using longer time-series data.” We extended the study period from 2015-2019 to 1991-2020, from previously only 5 years of data for each country, we have 30 years. So, in the current version, instead of 55 observations (11 countries in 5 years) now it has 330 observations (11 countries in 30 years). “The authors have proposed a normality test and paired t test in the Methodology section, but these analyses are missing in the Results section. The type of correlation test (parametric or non-parametric) in table 2 is not mentioned in the Methodology section. Please address these issues, and provide the software used to perform the analyses.” We provided table 2 to describe our Shapiro-Wilk normality test result. For the correlation test, we run the Pearson correlation test as our sample size is large enough and the average is more accurately represents the center of the distribution of the data than the median. “The Discussion and Conclusion sections should focus on the association between women’s literacy and child mortality, as proposed in the title of this study.” As we redesign our study, currently we only have one independent variable (women’s literacy/WL) and three dependent variables (IMR, NMR, UFMR). We rewrote the discussion part and make it more focused to highlight how WL correlated to and affected IMR, NMR, and UFMR." } ] } ]	1	https://f1000research.com/articles/11-178
https://f1000research.com/articles/11-416/v1	13 Apr 22	{ "type": "Brief Report", "title": "Analyses within risk strata overestimate gain in discrimination: the example of coronary artery calcium scores", "authors": [ "Lin Zhu", "Katy JL Bell", "Anna Mae Scott", "Paul P Glasziou", "Katy JL Bell", "Anna Mae Scott", "Paul P Glasziou" ], "abstract": "Risk prediction models are potentially useful tools for health practitioners and policy makers. When new predictors are proposed to add to existing models, the improvement of discrimination is one of the main measures to assess any increment in performance. In assessing such predictors, we observed two paradoxes: 1) the discriminative ability within all individual risk strata was worse than for the overall population; 2) incremental discrimination after including a new predictor was greater within each individual risk strata than for the whole population. We show two examples of the paradoxes and analyse the possible causes. The key cause of bias is use of the same prediction model as for both stratifying the population, and as the base model to which the new predictor is added.", "keywords": [ "ROC curve", "C-statistic", "risk prediction models", "heart disease risk factors" ], "content": "Introduction\n\nSeveral new biomarkers, including coronary artery calcium scores (CACS), have been proposed to improve cardiovascular (CVD) risk prediction models, such as the Framingham Risk Score (FRS) or Pooled Cohort Equations (PCE). Their incremental value is usually judged by any improvement discrimination, using measures such as the C-statistic - the area under the receiver operating characteristic curve (AUC) - despite some limitations.1,2\n\nSeveral recent assessments of CACS added value to CVD risk models were done within CVD risk-strata specific gain but restricting the study population in this way may inflate the apparent gain.\n\n\nMethods\n\nIn the process of a systematic review to assess the incremental value of CACS beyond traditional CVD risk assessment, we identified two studies that report the change in C-statistic from adding CACS within CVD risk strata as well as for the overall cohort.3 We used these two studies to illustrate observed paradoxes, and then explore possible reasons using a simple simulation. All analyses were performed with R Project for Statistical Computing (version3.6.3, RRID: SCR_001905).\n\n\nResults\n\nTwo studies provided sufficient data - the Heinz Nixdorf Recall (HNR) and Multi-Ethnic Study of Atherosclerosis (MESA) studies.4,5 Both compared the C-statistics of base models to C-statistics of extended models (including CACS) in sub-groups defined by CVD risk scores. The apparent increase in C-statistic from adding CACS was greater within every risk sub-group that for the overall cohort (Figure 1) – so all strata gains were above average. There are two paradoxes, the first explaining the second.\n\nData of Panel A extracted from Geisel 2017, Table 3; Data of Panel B extracted from Blaha 2021, Figure 2. Both studies compared the C-statistics of base models (FRS in HNR, PCE in MESA) to C-statistics of extended models (including CACS) in sub-groups defined by CVD risk scores. CACS: coronary artery calcium scores.\n\nThe first paradox is that the discriminative ability of the CVD risk score within individual CVD risk strata is worse than for the overall population. This surprising “finding” is a statistical artefact: the discriminative ability of a variable will always appear to be less if its range is limited (or within a more homogeneous population), than within the full (more heterogeneous) population.6\n\nThe second paradox is the apparent gain in C-statistic for CACS added to the base model is greater within each individual risk strata than for the whole study population. This is not a true “gain”: within each CVD risk stratum the “discrimination” is artificially reduced, and hence the “gain” from CACS artefactually increased. This results in overestimation of the improved discrimination provided by CACS.\n\n\nDiscussion\n\nThese two paradoxes related to stratification may seem somewhat surprising but may be more readily understood with other examples. Intelligence quotient (IQ) might be predictive of a young person’s future income level, but any discrimination is weakened by assessment within 10-unit IQ strata. Similarly, blood pressure predicts future stroke, but this prediction is weakened if examined within 10 mmHg bands. This apparent weaker predictive ability is due to the artificial constriction of the predictor and the nature of the discrimination measure.\n\nFigure 2 provides a hypothetical example to help explain these paradoxes. Figure 2A shows 42 people - 21 who have an event and 21 do not - grouped into low, moderate, and high risk according to a risk score. The C-statistic is good for the overall cohort (0.78), but lower in the narrower risk subgroups of 14 people (low risk: 0.61, moderate risk: 0.57, high risk: 0.61), because some of the “discrimination” is already used in separating into these groups. Figure 2B adds a second prognostic factor which “improves” the C-statistic more within each of the risk subgroups (low risk: 0.02, moderate risk: 0.03, high risk: 0.03) than in the overall cohort (0.01).\n\nFigure 2A shows 21 who had event (red dots) 21 do not (blue dots) - The C-statistic for the overall cohort (0.78) is higher than in any of three risk subgroups (low risk: 0.61, moderate risk: 0.57, high risk: 0.61). Figure 2B – For a second indicator (crosses; Odds Ratio of ~2.0) added to model the C-statistic “improves” more in each of the risk subgroups (low risk: 0.02, moderate risk: 0.03, high risk: 0.03) than in the overall cohort (0.01).\n\nGiven the increasing use of risk stratified analyses of prognostic gain, we recommend the incremental discrimination provided by a new biomarker should not be analysed within risk stratified subgroups based on the CVD risk score. Authors, reviewers, and editors should be aware of this flawed analysis and avoid it. More generally, the limitations of discrimination measures1 mean we should consider alternative measures to assess the incremental value of new biomarkers7 and be wary of stratified analyses, particularly when the stratification and the base CVD risk score are the same.\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\nAuthor contributions\n\nL.Z.: Methodology, Software, Writing - Original Draft. K.B.: Methodology, Supervision, Writing - Reviewing and Editing. A.S.: Supervision, Writing - Reviewing and Editing. P.G.: Methodology, Conceptualization, Supervision, Writing - Reviewing and Editing.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nKB is supported by NHMRC Investigator grant 1174523. PG is supported by NHMRC Australian Fellowship grant 1080042. This study was funded by NHMRC Centre of Research Excellence grant 2006545.", "appendix": "Acknowledgments\n\nNHMRC had no role in study design, data analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nCook NR: Use and Misuse of the Receiver Operating Characteristic Curve in Risk Prediction. Circulation. 2007; 115: 928–935. PubMed Abstract \| Publisher Full Text\n\nWare JH: The Limitations of Risk Factors as Prognostic Tools. N. Engl. J. Med. 2006; 355: 2615–2617. Publisher Full Text\n\nBell KJL, White S, Hassan O, et al.: Evaluation of the Incremental Value of a Coronary Artery Calcium Score Beyond Traditional Cardiovascular Risk Assessment: A Systematic Review and Meta-analysis. JAMA Intern. Med. 2022. (In process).\n\nGeisel MH, Bauer M, Hennig F, et al.: Comparison of coronary artery calcification, carotid intima-media thickness and ankle-brachial index for predicting 10-year incident cardiovascular events in the general population. Eur. Heart J. 2017; 38: 1815–1822. PubMed Abstract \| Publisher Full Text\n\nBlaha MJ, Whelton SP, Al Rifai M, et al.: Comparing Risk Scores in the Prediction of Coronary and Cardiovascular Deaths. JACC Cardiovasc. Imaging. 2021; 14: 411–421. PubMed Abstract \| Publisher Full Text\n\nDebray TPA, Damen JAAG, Riley RD, et al.: A framework for meta-analysis of prediction model studies with binary and time-to-event outcomes. Stat. Methods Med. Res. 2019; 28: 2768–2786. PubMed Abstract \| Publisher Full Text\n\nPencina MJ, D’Agostino RB, D’Agostino RB, et al.: Evaluating the added predictive ability of a new marker: From area under the ROC curve to reclassification and beyond. Stat. Med. 2008; 27: 157–172. PubMed Abstract \| Publisher Full Text" }	[ { "id": "192871", "date": "08 Aug 2023", "name": "Jonathan D Mosley", "expertise": [ "Reviewer Expertise Genetic 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\nZhu et al. have written a brief report highlighting the consequences to c-statistic estimates when risk models are secondarily stratified by the modeled predicted risk. In this case, stratification can lead to inflated c-statistic estimates within strata. This is an important artifact to highlight, as it is common to see post-hoc stratification in risk prediction studies to identify subgroups where a biomarker is purported have a greater benefit. While the overall message is clear and well-written, there are some details missing in the methods, particularly with respect to the simulation. It would also be useful to highlight other scenarios where the issue would manifest.\nMy specific comments are:\nMethods: The authors should clarify whether the data from figure 1 are re-calculated estimates or whether these were the original data presented in the referenced papers. How was the c-statistic calculated (either in the original papers of in this study).\n\nMethods: More details are needed regarding the simulation. It is not clear what was done. Does figure 2 show the results of the simulation study? or is this just an illustrative figure?\n\nDiscussion: Will the c-statistics always be inflated with stratification? Are there scenarios where it could go down?\n\nDiscussion: It is common to see post-hoc stratification performed using one variable that is a component of a prediction model. For instance, this is often seen with stratification by age (which is a component of most prediction models) to show that a biomarker has greater effect in younger individuals. Does this scenario lead to the same inflated c-statistic estimates?\n\nIs the work 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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "284210", "date": "07 Jun 2024", "name": "Abhaya Indrayan", "expertise": [ "Reviewer Expertise Medical Biostatistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOpen Peer Review\nParadox in the Values of C-index\nAbhaya Indrayan Max Healthcare Institute, New Delhi\nZhu et al.1 have highlighted an important paradox with the values of C-index when the aggregate picture is not the sum of its parts. They demonstrated this with the help of two real-life examples. In both, the gain in discrimination measured by the change in C-statistic is smaller in aggregate but larger in each of the risk-score stratum when the CAC Extended Model is compared with the Base Model in Heinz Nixdorf Recall study2 and Multi-Ethic Study of Atherosclerosis3. The second anomaly they pointed out is that the C-index is higher for aggregate than for each risk score stratum.\nGood to see such anomalies have been detected and highlighted. These anomalies add to the one earlier discussed by Cook4 inadequacy of C-index as a measure of discrimination that it compromises the contribution of individual biomarkers when used for assessing the performance of a model. In addition, the one recently discussed by Indrayan et al5 is the inadequacy of C-index for assessing predictivity as this index is based on sensitivity and specificity which are measures of discrimination and classification of the known outcomes and not the prediction of the unknown outcomes. In the present paper also, Zhu et al. have used the term ‘predictive ability’, which is inappropriate for C-index.\nWhereas the authors have successfully demonstrated the paradox, the explanation provided by them requires more elaboration. They attribute this paradox to the homogeneity of values within each stratum which may have ‘artificially’ reduced the discrimination ability. A more detailed explanation would have helped the readers to appreciate this paradox. For example, it is not mentioned that this would always happen and whether the anomaly increases with the decrease in the stratum size. Secondly, it would have been helpful if properly framed advice was included for the researchers regarding explaining or getting over this paradox. They advise that incremental discrimination by an added marker should avoid the analyses within subgroups. This advice may not be applicable when subgroup analysis is required in a clinical context. My suggestion is that subgroups may be analysed where needed but the anomaly be fully explained. It would have been more useful to the readers when the alternatives meausres6 they suggest were fully explained regarding how they can be used to study or explain the paradox.\n\nIs the work 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? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/11-416
https://f1000research.com/articles/10-1258/v1	08 Dec 21	{ "type": "Brief Report", "title": "Interplay among positive and negative symptoms, neurocognition, social cognition, and functional outcome in clinically stable patients with schizophrenia: a network analysis", "authors": [ "Thammanard Charernboon" ], "abstract": "Background: Schizophrenia has a broad range of interrelated symptoms and impairment in functioning. The objective of the study was to explore the interplay between positive symptoms, negative symptoms, neurocognition, social cognition and functional outcome in patients with schizophrenia using network analysis. Methods: Participants were 64 clinically stable patients with schizophrenia. Psychopathologic, neurocognition, social cognition, and functional outcome were measured using the Scale for the Assessment of Positive Symptoms, Scale for the Assessment of Negative Symptoms, Addenbrooke’s Cognitive Examination III, Faces test, Reading the Mind in the Eyes test, and Personal Social Performance scale. Results: The network analysis suggested that functional outcome was the most central in the network followed by avolition and asociality. Functioning was directly connected to avolition, asociality, blunted affect, neurocognition and emotion recognition. The positive symptoms were the most remote and therefore the least important node. Conclusion: The high centrality of functioning suggests the need for improving of everyday life skills for patients with schizophrenia. Moreover, treatment of specific negative symptoms, neurocognition and emotion recognition could also enhance functional outcome.", "keywords": [ "Negative symptoms", "network analysis", "neurocognition", "schizophrenia", "social cognition" ], "content": "Introduction\n\nSchizophrenia is associated with a broad range of symptoms including positive symptoms, negative symptoms and neurocognition impairments. Recently, social cognition deficits also have been increasingly reported.1,2 Despite the increase in new medications and treatments, functional outcome is not as good as expected. Functional recovery is observed in less than 25% of patients with schizophrenia.3\n\nWhile all of the symptoms are believed to effect functional outcome to some extent, negative symptoms, neurocognitive, and social cognition seem to have the highest impact on functioning.4 However, the interplay between these symptoms and functioning is highly complex. Since not only can negative symptoms, neurocognition, and social cognition predict functional outcome, all of these symptoms are also interrelated to each other. For example, previous studies demonstrated that negative symptoms were closely associated with social cognition.5,6 Social cognition and neurocognition are also usually correlated to some degree.7 Moreover, some studies suggest that social cognition might be a mediator between neurocognition and symptomatology.8 Therefore, as noted before, a traditional statistical approach might not be suited for examining complex relationships of interconnected variables. For instance, a simple correlation approach could not control for the influence of other variables. Linear regression and structural equation models require a priori assumptions regarding the selection of predictors, mediators, precursors, and outcomes. Furthermore, traditional analysis could not demonstrate which variables connect to other variable more often and which ones play a more or less central role than others.\n\nNetwork analysis is a relatively new and powerful methodological approach to investigate complex relationship patterns. It is a data-driven technique that does not require a priori assumption of relationships among variables. With a network analysis approach, all phenomena are conceptualized as systems of causally connected signs and symptoms. The system can be analysed and presented in its full complexity.9 In the network, the key variables will be located at the center of the network, while less important and connected variables will be in the periphery. The aim of this study was to explore the interplay of positive symptoms, five groups of negative symptoms, neurocognition, two forms of social cognition (theory of mind, emotion recognition), and functioning in patients with clinically stable schizophrenia using network analysis.\n\n\nMethods\n\n64 outpatients with schizophrenia were recruited from a mental health clinic at Thammasat University Hospital, Thailand between February 2018 and August 2019. They were between 20 and 60 years old and had at least four years of educational level. All patients met criteria for schizophrenia based upon the Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5) criteria10 and were in a clinically stable phase, as defined by no changes in the treatment or symptoms for the previous three months. Exclusion criteria were having a major neurological disorder or other psychiatric disorders (i.e., intellectual disability, active major depressive disorder and substance dependence excluding smoking). The participants were invited to participate in this study by our research assistants during outpatients visit. The study was approved by the Human Research Ethics Committee of Thammasat University (No. MTU-EC-PS-0-191/60). Written informed consent forms were obtained from all participants. Participants have a right to withdraw from the study at any time.\n\nPositive symptoms\n\nPositive symptoms were measured using the Scale for the Assessment of Positive Symptoms (SAPS).11,12 Positive symptoms were presented as a single unitary construct using the sum of global ratings. The global ratings range from 0 to 20 with the higher score indicating more severe positive symptoms.\n\nNegative symptoms\n\nThe Scale for the Assessment of Negative Symptoms (SANS) was administered to assess negative symptoms.13,14 In this study, negative symptoms were classified into five subdomains i.e., blunted affect, alogia, anhedonia, avolition, and asociality. Each subdomain has a maximum score of five, which is computed from the average scores of the relevant items of each subdomain. It should be noted that inattention subdomain was not included in the analysis because of its overlap with neurocognitive assessment.\n\nNeurocognition\n\nThe Addenbrooke’s Cognitive Examination III (ACE-III) was used to assess neurocognitive function. It assesses five neurocognitive domains: attention, verbal fluency, memory, language, and visuospatial ability with a maximum score of 100 points.15,16 A previous study demonstrates that ACE-III was sensitive to detect cognitive impairment in patients with schizophrenia.17\n\nSocial cognition\n\nThe social cognition assessments included tests of emotion recognition (Faces test),18 and theory of mind (Reading the Mind in the Eyes test: RMET).19,20 The Faces test consists of 20 photographs of people faces showing a variety of emotions. The participants were required to match the emotion of a person. The maximum score is 20 with a higher score suggesting better emotion recognition ability.18 The RMET includes 36 pictures of persons’ eye regions where participants must select which of four words best describes the mental state of a target person. The RMET has a score range of 0-36 with a higher score indicating better theory of mind capability.19,20\n\nFunctional outcome\n\nThe functioning of the participants was measured using the Personal Social Performance scale (PSP).21 It assessed the patients’ functioning based on four dimensions: useful activities, social relationships, self-care and disturbing/aggressive behaviors. The score ranges from 1 to 100. The score of 91-100 indicates excellent function, while 1-10 suggests lack of autonomy in basic function.21\n\nCharacteristics and clinical data were retrieved from medical records. Psychiatrists interviewed the patients and rated the SAPS, SANS and PSP. Then, the ACE-III, Faces, and RMET tests were administered by independent clinical psychologists on the same day. All measures were paper versions.\n\nDescriptive statistic and partial correlation were analyzed using STATA version 14.0. Network analysis was conducted using R version 4.0.5 (R Foundation for Statistical Computing). 10 variables were selected for the network analysis. The least absolute shrinkage and selection operator network (LASSO) was used as type of network. The centrality measures of the network were also analyzed. The network was visualized using qgraph package.\n\nA network comprising variables was created, which are presented by ‘nodes’ (circle), and the links between the nodes called ‘edges’ (solid line). Thicker edges represent stronger relationships. Blue edges represent positive correlation, and red edges represent negative correlations. The algorithm places strongly associated nodes at the center of the network and the weakly associated variable at the periphery.\n\nThe centrality indices are the method to examine the relative centrality of constructs within the network. They reveal which is the most important variable in the network. The common centrality measures are ‘strength’, ‘betweenness’ and ‘closeness’. Node strength reflects how strongly a node is directly connected to other nodes. It was determined by the sum weighted associations to other nodes. Betweenness of a node is defined as the number of times that a node is part of the shortest path between two other nodes. The closeness of the node implies how easy it is to reach all other nodes. A high closeness index indicated a short average distance of an interest node to all other nodes. For each measure, higher values indicated more centrality in the network.\n\n\nResults\n\n64 patients with schizophrenia were recruited into the study. Characteristics and descriptive statistics of the network analysis variables are reported in Table 1. The patients had a mean age of 37 (standard deviation (SD) 12.6) years and level of education of 13.3 (SD 3.4) years. The average duration of illness was 8 (SD 9) years. Table 2 presents the partial correlation matrix between 10 network analysis variables. There was no missing data in all variables.\n\nThe network structure and centrality measures are demonstrated in Figures 1 and 2. The negative symptoms and cognitive function (neurocognition and social cognition) variables seem to be as two separate communities on upper and lower side of the network. The positive symptoms node appears as an isolate and farthest node from the central.\n\nACE: Addenbrooke’s Cognitive Examination III; Asocial: asociality; Blunt: blunted affect; Eyes: Reading the Mind in the Eyes test; Faces: Faces test; PSP: Personal and Social Performance scale; SAPG: global Scale for the Assessment of Positive Symptoms score.\n\nACE: Addenbrooke’s Cognitive Examination III; Asocial: asociality; Blunt: blunted affect; Eyes: Reading the Mind in the Eyes test; Faces: Faces test; PSP: Personal and Social Performance scale; SAPG: global Scale for the Assessment of Positive Symptoms score.\n\nThe functional outcome was found to be the most central node and is displayed in the center of the network structure. It connected and demonstrated inverse correlations to three subdomains of negative symptoms (asocial, avolition and blunt affect) and positive correlations to emotion recognition and neurocognition performance.\n\nIn the social cognition domain, only emotion recognition was directly connected to functioning, whereas theory of mind was connected to emotion recognition and neurocognition but not directly connected to functional outcome. Among cognitive function variables, neurocognition had the highest centrality index and was the strongest connection to functional outcome.\n\nAmong negative symptoms variables, the highest centrality index variables were asociality and avolition subdomains. Except for blunt affect, the other four negative symptoms nodes were interconnected to all other negative symptoms’ nodes.\n\nBetween negative symptoms and cognitive function, only alogia and blunt affect showed association with emotion recognition and neurocognition, whereas asociality, avolition and anhedonia showed no direct association to cognitive function.\n\n\nDiscussion\n\nThis study used a network analysis technique to explore the complex relationship among positive symptoms, negative symptoms, neurocognition, social cognition and real-life functioning. To our knowledge, there are few studies examining this association with network approach.\n\nOur results illustrate that functional outcome is the most central and key role in the network. It has the highest centrality index in strength, betweenness and closeness. It is also connected to negative symptoms, neurocognition, and social cognition. This finding supports the notion that real-life function should serve as the main target of treatment and clinical trials of schizophrenia; and rehabilitation for patients with schizophrenia should focus on and involve training of everyday real-life skills.22 Besides functioning, avolition and asociality appear to be the second most central and important symptoms. These two negative symptoms also firmly correlate to functional outcome and connect to other subdomains of negative symptoms. These results confirm the previous findings that, generally, negative symptoms and cognitive symptoms are the strongest predictors of functional outcome in schizophrenia.4,23\n\nThe network also shows that neurocognition and emotion recognition are interconnected and close to real-life functioning. Therefore, neurocognition and emotion recognition trainings could be implemented and would benefit patients with schizophrenia. It is interesting that emotion recognition might directly improve functional outcome or could have an indirect effect by decreasing blunt affect symptoms. This association is in line with previous studies that blunted affect seems to be closely related to emotion recognition.5,24\n\nConforming to previous studies, our network analysis confirms that positive symptoms are less important and less influential on functioning in clinically stable patients with schizophrenia.25,26 The result underlines the importance of evaluating and treating negative symptoms and cognitive symptoms, as positive symptoms alone have only minimal affect on real-life function. Furthermore, medications that specifically target negative symptoms, social cognition, or neurocognitive symptoms are urgently needed for patients with schizophrenia.\n\nRegarding cognitive function and in line with previous studies on schizophrenia, this study shows that social cognition is highly correlated with neurocognition.5,6 The social cognition node that directly links to functioning is emotion recognition. This connection highlights the important role of emotion recognition in patients’ real-life functioning. The result supports previous studies that found social cognition to be a strong prediction of function outcome in patients with schizophrenia.4,27 On the other hand, though, theory of mind is strongly correlated to neurocognition and emotion recognition; it is not directly connected to functioning and is the most distant cognitive function node.\n\nOur study also supports a multidimensional model of negative symptoms,28,29 that, although most of the five subdomains are correlated to each other, they demonstrated a different pattern of relationships. For example, blunt affect is strongly correlated with alogia, but not directly connected to asociality.\n\nThe strength of this study is that we used network analysis to examine the complex relationship among functional outcome, clinical symptoms and cognitive functions. In addition, we also used five dimensions of negative symptoms instead of a unidimensional approach which could avoid losing information relevant to the connection with others factors and the ability of each subdomain to predict functional outcome.30\n\nOur study has some limitations. First, the sample size was relatively modest. Therefore, positive symptoms and neurocognition were constructed as single-measured variables to minimize the number of parameters. Second, on account of the inclusion of only clinically stable patients with schizophrenia, this means that most had none or very few positive symptoms, therefore, the results might not be able to be generalized to patients with actively psychotic episodes.\n\n\nData availability\n\nThere are restrictions on publicly sharing the dataset, as a result of the confidential nature of the data and the informed consent given by the study participants, which has been approved by the Human Research Ethics Committee, Faculty of Medicine, Thammasat University. However, thenetwork analysis dataset (ten variables without demographic data) may be requested by contacting the corresponding author (TC) and access will be granted to researchers affiliated with an accredited institution, and reviewers Full name, title, institution, and purpose for using the dataset should be included in an email.", "appendix": "Acknowledgements\n\nThe author would like to thank Tiraya Lerthattasilp, MD and our psychologists for help in collecting the data.\n\n\nReferences\n\nCharernboon T: Negative and neutral valences of affective theory of mind are more impaired than positive valence in clinically stable schizophrenia patients. Psychiatry Investig. 2020; 17(5): 460–464. PubMed Abstract \| Publisher Full Text\n\nSavla GN, Vella L, Armstrong CC, et al.: Deficits in domains of social cognition in schizophrenia: a meta-analysis of the empirical evidence. Schizophr. Bull. 2013; 39(5): 979–992. PubMed Abstract \| Publisher Full Text\n\nHaro JM, Novick D, Bertsch J, et al.: Cross-national clinical and functional remission rates: Worldwide Schizophrenia Outpatient Health Outcomes (W-SOHO) study. Br. J. Psychiatry. 2011; 199(3): 194–201. PubMed Abstract \| Publisher Full Text\n\nFett AK, Viechtbauer W, Dominguez MD, et al.: The relationship between neurocognition and social cognition with functional outcomes in schizophrenia: a meta-analysis. Neurosci. Biobehav. Rev. 2011; 35(3): 573–588. PubMed Abstract \| Publisher Full Text\n\nCharernboon T: Different subdomains of negative symptoms in clinically stable patients with schizophrenia: Determining the nature of their relationships with emotion recognition, theory of mind and neurocognition. Cogent Psychology. 2020; 7(1): 1849892. Publisher Full Text\n\nSergi MJ, Rassovsky Y, Widmark C, et al.: Social cognition in schizophrenia: relationships with neurocognition and negative symptoms. Schizophr. Res. 2007; 90(1-3): 316–324. Publisher Full Text\n\nCharernboon T, Patumanond J: Social cognition in schizophrenia. Ment. Illn. 2017; 9(1): 7054. PubMed Abstract \| Publisher Full Text\n\nLam BY, Raine A, Lee TM: The relationship between neurocognition and symptomatology in people with schizophrenia: social cognition as the mediator. BMC Psychiatry. 2014; 14: 138. PubMed Abstract \| Publisher Full Text\n\nBorsboom D, Cramer AO: Network analysis: an integrative approach to the structure of psychopathology. Annu. Rev. Clin. Psychol. 2013; 9: 91–121. PubMed Abstract \| Publisher Full Text\n\nAmerican Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders. Washington DC: American Psychiatric Publishing; 2013. Publisher Full Text\n\nAndreasen NC: Scale for the Assessment of Positive Symptoms (SAPS). University of Iowa Iowa City; 1984.\n\nCharernboon T: Preliminary study of the Thai-version of the Scale for the Assessment of Positive Symptoms (SAPS-Thai): content validity, known-group validity, and internal consistency reliability. Arch. Clin. Psychiatry (São Paulo). 2019; 46(1): 5–8. Publisher Full Text\n\nAndreasen NC: Scale for the Assessment of Negative Symptoms (SANS): Department of Psychiatry. College of Medicine, The University of Iowa; 1984.\n\nCharernboon T: Preliminary Study of the Thai Version of the Scale for the Assessment of Negative Symptoms (SANS-Thai). Global J. Health Sci. 2019; 11(6): 19. Publisher Full Text\n\nCharernboon T, Jaisin K, Lerthattasilp T: The Thai version of the Addenbrooke's Cognitive Examination III. Psychiatry Investig. 2016; 13(5): 571–573. PubMed Abstract \| Publisher Full Text\n\nHsieh S, Schubert S, Hoon C, et al.: Validation of the Addenbrooke's Cognitive Examination III in frontotemporal dementia and Alzheimer's disease. Dement. Geriatr. Cogn. Disord. 2013; 36(3-4): 242–250. PubMed Abstract \| Publisher Full Text\n\nCharernboon T, Chompookard P: Detecting cognitive impairment in patients with schizophrenia with the Addenbrooke's Cognitive Examination. Asian J. Psychiatr. 2019; 40: 19–22. PubMed Abstract \| Publisher Full Text\n\nCharernboon T: Validity and reliability of the Thai version of the Faces Test. J. Med. Assoc. Thail. 2017; 100(6): 42–45.\n\nBaron-Cohen S, Wheelwright S, Hill J, et al.: The “Reading the Mind in the Eyes” Test revised version: a study with normal adults, and adults with Asperger syndrome or high-functioning autism. J. Child Psychol. Psychiatry. 2001; 42(2): 241–251. Publisher Full Text\n\nCharernboon T, Lerthattasilp T: The Reading the Mind in the Eyes Test: Validity and reliability of the Thai version. Cogn. Behav. Neurol. 2017; 30(3): 98–101. PubMed Abstract \| Publisher Full Text\n\nMorosini PL, Magliano L, Brambilla L, et al.: Development, reliability and acceptability of a new version of the DSM-IV Social and Occupational Functioning Assessment Scale (SOFAS) to assess routine social functioning. Acta Psychiatr. Scand. 2000; 101(4): 323–329. PubMed Abstract \| Publisher Full Text\n\nFarkas M: The vision of recovery today: what it is and what it means for services. World Psychiatry. 2007; 6(2): 68–74. PubMed Abstract\n\nChue P, Lalonde JK: Addressing the unmet needs of patients with persistent negative symptoms of schizophrenia: emerging pharmacological treatment options. Neuropsychiatr. Dis. Treat. 2014; 10: 777–789. PubMed Abstract \| Publisher Full Text\n\nDitlevsen JV, Simonsen A, Bliksted VF: Predicting mentalizing deficits in first-episode schizophrenia from different subdomains of negative symptoms. Schizophr. Res. 2020; 215: 439–441. PubMed Abstract \| Publisher Full Text\n\nVentura J, Hellemann GS, Thames AD, et al.: Symptoms as mediators of the relationship between neurocognition and functional outcome in schizophrenia: a meta-analysis. Schizophr. Res. 2009; 113(2-3): 189–199. PubMed Abstract \| Publisher Full Text\n\nGalderisi S, Rucci P, Kirkpatrick B, et al.: Interplay Among Psychopathologic Variables, Personal Resources, Context-Related Factors, and Real-life Functioning in Individuals With Schizophrenia: A Network Analysis. JAMA Psychiat. 2018; 75(4): 396–404. PubMed Abstract \| Publisher Full Text\n\nIrani F, Seligman S, Kamath V, et al.: A meta-analysis of emotion perception and functional outcomes in schizophrenia. Schizophr. Res. 2012; 137(1-3): 203–211. PubMed Abstract \| Publisher Full Text\n\nStrauss GP, Nunez A, Ahmed AO, et al.: The Latent Structure of Negative Symptoms in Schizophrenia. JAMA Psychiat. 2018; 75(12): 1271–1279. PubMed Abstract \| Publisher Full Text\n\nStrauss GP, Ahmed AO, Young JW, et al.: Reconsidering the latent structure of negative symptoms in schizophrenia: A review of evidence supporting the 5 consensus domains. Schizophr. Bull. 2019; 45(4): 725–729. PubMed Abstract \| Publisher Full Text\n\nMarder SR, Galderisi S: The current conceptualization of negative symptoms in schizophrenia. World Psychiatry. 2017; 16(1): 14–24. PubMed Abstract \| Publisher Full Text" }	[ { "id": "102245", "date": "17 Dec 2021", "name": "Pichai Ittasakul", "expertise": [ "Reviewer Expertise Electroconvulsive therapy", "schizophrenia", "mood disorders" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments to the Author, I would like to clarify the following:\nHow did the investigator obtain informed consent from the participants in this study? This should be stated in the methods.\n\nIn the methods, the measurement (SAPS, SANS, and PSP, ACE-II, Faces, RMET were administered by psychiatrists and clinical psychologists. How is the degree of agreement (interrater reliability) among independent raters?\n\nThe investigators should consider mentioning the limitation of this study: a cross-sectional nature of the study does not allow appropriate testing of the direction of the effect.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7925", "date": "11 Mar 2022", "name": "Thammanard Charernboon", "role": "Author Response", "response": "We appreciate the time and efforts of the editor and reviewers in reviewing this manuscript. We have addressed all issues indicated in the review report and believed that the revised version can meet the journal publication requirements. Reviewer 1 How did the investigator obtain informed consent from the participants in this study? This should be stated in the methods. We have added the suggested information as follows: “The participants were invited to participate in this study by our research assistants during outpatient visits. The study was approved by the Human Research Ethics Committee of Thammasat University (No. MTU-EC-PS-0-191/60). Written informed consent forms were obtained from all participants. Participants had a right to withdraw from the study at any time.” In the methods, the measurement (SAPS, SANS, and PSP, ACE-III, Faces, RMET were administered by psychiatrists and clinical psychologists. How is the degree of agreement (interrater reliability) among independent raters? We did not evaluate inter-rater reliabilities in our study. However, a previous study has explored the interrater reliability of the Thai version of the ACE-III. [Charernboon T, Jaisin K, Lerthattasilp T. The Thai version of the Addenbrooke’s Cognitive Examination III. Psychiatry Investigation, 2016; 13: 571-573.] A Thai version of the ACE-III had an excellent inter-rater reliability with a Pearson’s correlation of 1.0 (p < 0.001). Srisurapanont et al. also evaluate the inter-rater reliability of the PSP, Thai version. The intraclass correlation coefficients of the PSP total score was 0.75 (p<0.001). [Srisurapanont M, et al. Cross-cultural validation and inter-rater reliability of the Personal and Social Performance Scale, Thai version. J Med Assc Thai 2008; 91: 1603-8.]. These properties have been added to the manuscript. On the other hand, the Faces and RMET are self-rated, so inter-rater reliabilities are not available. The investigators should consider mentioning the limitation of this study: a cross-sectional nature of the study does not allow appropriate testing of the direction of the effect. Thank you for the recommendation. We have added this limitation as follows: “... Lastly, the cross-sectional nature of the data could not allow proper testing of the direction of the causes and effects. A longitudinal study with long term follow-up period would be more appropriate for evaluating the effects of predictors to future functional outcome.”" } ] }, { "id": "122301", "date": "21 Feb 2022", "name": "Haruo Fujino", "expertise": [ "Reviewer Expertise Research on cognitive and social functioning in schizophrenia" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Interplay among positive and negative symptoms, neurocognition, social cognition, and functional outcome in clinically stable patients with schizophrenia: a network analysis” focuses on the relationships among psychiatric symptoms, cognitive function, and social functioning in patients with clinically stable schizophrenia using network analysis. The application of the analysis in these data is interesting and would have potential contributions to the literature.\nI have the following comments for the first version of the paper. I hope that these comments could help improve the reporting of the paper.\nMajor comments:\nThe variable selection is a critically important factor that characterizes the correlation network. Please include the justification for the selected variables, particularly negative symptoms, in this study. The explanation would facilitate understanding of the focus and context of the study.\n\nInformation about the stability and accuracy of the network should be included in the Methods and Results sections. The information would provide useful information for interpreting the strength and instability of the network.\n\nThe sentence in the Discussion section, \"To our knowledge, there are few studies examining this association with network approach.\" does not provide information or citations. I recommend including recent studies using the network approach (as shown in the references below.) The inclusion of the recent papers and discussion on the current and previous findings would enhance the significance and clarify the nature of the research:\nHajdúk M, Penn DL, Harvey PD, Pinkham AE. Social cognition, neurocognition, symptomatology, functional competences and outcomes in people with schizophrenia - A network analysis perspective1.\n\nPena-Garijo J, Monfort-Escrig C. The centrality of secure attachment within an interacting network of symptoms, cognition, and attachment dimensions in persons with schizophrenia-spectrum disorders: A preliminary study2.\n\nLui SSY, Zhang RT, Lau WYS, et al. Prospective Memory Influences Social Functioning in People With First-Episode Schizophrenia: A Network Analysis and Longitudinal Study3.\n\nIn the Discussion section, the sentence “Our study also supports a multidimensional model of negative symptoms…”, was not supported by the results. In the Results section, the sentence “The negative symptoms and cognitive function variables seem to be as two separate communities…”, suggested that negative symptoms constructed a cluster. Please remove this part.\n\nRegarding the strengths and limitations, please discuss how sample size limits the validity of the study or leads to potential bias, including the results of network stability and accuracy.\n\nMinor comments:\nPlease revise the word “functional outcome” in the title because the outcome implies a longitudinal outcome.\n\nIn the Discussion, the sentence “The finding supports the notion that real-life function should serve as the main target of treatment…”, needs revision because the network analysis only showed the cross-sectional associations between the variables. A more careful statement is preferable.\n\nThe sentence “positive symptoms are less important and less influential on functioning in clinically stable…” requires revision. Although this is the reviewer’s opinion, positive symptoms are clinically important targets in schizophrenia. It may have less influence on social functioning in groups. However, it is an essential treatment target in clinical practice.\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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7926", "date": "07 Mar 2022", "name": "Thammanard Charernboon", "role": "Author Response", "response": "We appreciate the time and efforts by the editor and reviewers in reviewing this manuscript. We have addressed all issues indicated in the review report, and believed that the revised version can meet the journal publication requirements. Reviewer 2 Major comments: The variable selection is a critically important factor that characterizes the correlation network. Please include the justification for the selected variables, particularly negative symptoms, in this study. The explanation would facilitate understanding of the focus and context of the study. We greatly appreciate the reviewer’s efforts to carefully review the paper and the valuable suggestions offered. As suggested, we have added more explanations in the introduction and methods sections as follows: Introduction “… While all of the symptoms are believed to affect functional outcome to some extent, negative symptoms, neurocognitive, and social cognition seem to have the highest impact on functioning. 4 For example, previous studies generally demonstrate that negative symptoms have associations with poorer levels of functioning. 5 Neurocognitive impairment is a predictor for low levels of a real-world functioning. 6 Moreover, in the last decade, social cognition, a relatively new concept, appears to be a strong predictor of social abilities and real-world function. Theory of mind and emotion recognition are the most extensively studied domains in social cognition study, and impairment in these tasks are risk factors for poor social and functional outcome in patients with schizophrenia.” 4,6 Methods “… In this study, negative symptoms were classified into five subdomains i.e., blunted affect, alogia, anhedonia, avolition, and asociality as suggested by the NIMH-MATRICS consensus statement on negative symptoms. 17 Each subdomain has a maximum score of five, which is computed from the average scores of the relevant items of each subdomain. It should be noted that inattention subdomain was not included in the analysis because of its overlap with neurocognitive assessment. We used five dimensions of negative symptoms instead of a unidimensional approach because several recent factor analysis studies suggest that a unidimensional model of negative symptoms does not adequately capture the complexity of negative symptoms. 17, 18 Furthermore, previous study showed that negative symptoms subdomains seem to have a different relationship with functioning. For example, avolition and apathy seem to be a stronger predictor of poor outcomes than blunted affect 4, 19. Therefore, utilizing a multidimensional model would avoid losing information relevant to the connection with other variables and the ability of each subdomain to predict functioning.” Information about the stability and accuracy of the network should be included in the Methods and Results sections. The information would provide useful information for interpreting the strength and instability of the network. We have added the information about the stability and accuracy of the network as follows: Statistical analysis “The accuracy and stability of the centrality indices of the estimated network were examined using edge weight accuracy and case-dropping bootstrap method on the 500 samples. We utilized recommended cut-off for Coefficient Stability (CS-coefficient, correlation = 0.7) at least 0.25 for considering stable network.” 28 Results “Figures 3 and 4 show the stability of central indices and accuracy of the estimated network. In brief, the CS-coefficient indicates that strength had the highest and acceptable stability (CS-coefficient 0.28); whereas closeness (CS-coefficient 0.13) and betweenness (CS-coefficient 0.2) are not stable under sub-setting cases.” Please see Fig 3 (Stability of central indices) and Fig 4 (Accuracy of the edge weight estimates) in the revised manuscript. Limitations “… Second, closeness and betweenness should be interpreted with caution since their estimates showed less stability. Therefore, a larger sample might be needed to ensure a more stable estimated network.” - The sentence in the Discussion section, \"To our knowledge, there are few studies examining this association with network approach.\" does not provide information or citations. I recommend including recent studies using the network approach (as shown in the references below.) The inclusion of the recent papers and discussion on the current and previous findings would enhance the significance and clarify the nature of the research: Hajdúk M, Penn DL, Harvey PD, Pinkham AE. Social cognition, neurocognition, symptomatology, functional competences and outcomes in people with schizophrenia - A network analysis perspective1. Pena-Garijo J, Monfort-Escrig C. The centrality of secure attachment within an interacting network of symptoms, cognition, and attachment dimensions in persons with schizophrenia-spectrum disorders: A preliminary study2. Lui SSY, Zhang RT, Lau WYS, et al. Prospective Memory Influences Social Functioning in People With First-Episode Schizophrenia: A Network Analysis and Longitudinal Study3. I have added some references to this sentence as follows: \"29 Hajdúk M, Penn DL, Harvey PD, et al.: Social cognition, neurocognition, symptomatology, functional competences and outcomes in people with schizophrenia - A network analysis perspective. J. Psychiatr. Res. 2021;144:8-13. 10.1016/j.jpsychires.2021.09.041 30 Lui SSY, Zhang RT, Lau WYS, et al.: Prospective memory influences social functioning in people with first-episode schizophrenia: A network analysis and longitudinal study. J. Clin. Psychiatry. 2022;83(2):21m14114. 10.4088/JCP.21m14114. 31 Galderisi S, Rucci P, Kirkpatrick B, et al.: Interplay among psychopathologic variables, personal resources, context-related factors, and real-life functioning in individuals with schizophrenia: A network analysis. JAMA Psychiat. 2018;75(4):396–404. 29450447 10.1001/jamapsychiatry.2017.4607\" In the Discussion section, the sentence “Our study also supports a multidimensional model of negative symptoms…”, was not supported by the results. In the Results section, the sentence “The negative symptoms and cognitive function variables seem to be as two separate communities…”, suggested that negative symptoms constructed a cluster. Please remove this part. These sentences have been removed as suggested. Regarding the strengths and limitations, please discuss how sample size limits the validity of the study or leads to potential bias, including the results of network stability and accuracy. We have rewritten the limitations. The results of the network stability and sample sizes were also added and discussed as follows: “Our study has some limitations. First, the sample size was relatively modest. Therefore, positive symptoms and neurocognition were constructed as single-measured variables to minimize the number of parameters. Second, closeness and betweenness should be interpreted with caution since their estimates showed less stability. Therefore, a larger sample might be needed to ensure a more stable estimated network. Third, on account of the inclusion of only clinically stable patients with schizophrenia, most had none or very few positive symptoms, therefore, the results might not be able to be generalized to patients with actively psychotic episodes. Lastly, the cross-sectional nature of the data could not allow proper testing of the direction of the causes and effects. Longitudinal study with long term follow-up period would be more appropriate for evaluating the effects of predictors to future functional outcome.” *********************** Minor comments: - Please revise the word “functional outcome” in the title because the outcome implies a longitudinal outcome. “Functional outcome” was substituted with “functioning” in the title and other parts in the manuscript. In the Discussion, the sentence “The finding supports the notion that real-life function should serve as the main target of treatment…”, needs revision because the network analysis only showed the cross-sectional associations between the variables. A more careful statement is preferable. I have edited this statement and softened it as follows: “Our results illustrate that real-life functioning is the most central and key role in the network. It has the highest centrality index in strength, betweenness and closeness. It is also connected to negative symptoms, neurocognition, and social cognition. This finding suggests that real-life functioning should be one of the main targets of schizophrenia treatment and research. Rehabilitation for patients with schizophrenia should be provided and should focus on training of everyday real-life skills.” The sentence “positive symptoms are less important and less influential on functioning in clinically stable…” requires revision. Although this is the reviewer’s opinion, positive symptoms are clinically important targets in schizophrenia. It may have less influence on social functioning in groups. However, it is an essential treatment target in clinical practice. I have revised this statement to make it focuses more on negative and cognitive symptoms as follows: “Conforming to previous studies, our network analysis confirms that negative and cognitive symptoms are the most important and influential on functioning in clinically stable patients with schizophrenia.” 35, 36" } ] } ]	1	https://f1000research.com/articles/10-1258
https://f1000research.com/articles/10-989/v1	30 Sep 21	{ "type": "Software Tool Article", "title": "pubassistant.ch: consolidating publication profiles of researchers", "authors": [ "Reto Gerber", "Mark D. Robinson", "Reto Gerber" ], "abstract": "Online accounts to keep track of scientific publications, such as Open Researcher and Contributor ID (ORCID) or Google Scholar, can be time consuming to maintain and synchronize. Furthermore, the open access status of publications is often not easily accessible, hindering potential opening of closed publications. To lessen the burden of managing personal profiles, we developed a R shiny app that allows publication lists from multiple platforms to be retrieved and consolidated, as well as interactive exploration and comparison of publication profiles. A live version can be found at pubassistant.ch.", "keywords": [ "open access", "publication profiles", "R shiny" ], "content": "Introduction\n\nGiven the increasing number of both researchers and publications as well as publishing modes,1,2 it becomes a challenge to identify and consolidate all publications from a single author. A few of the main issues are the non-uniqueness of names, differently written names (e.g. with or without middle initial) and changing affiliation over time. As a solution to this problem, unique identifiers were created that enable robust linkage of publications to authors, assuming researchers and their collaborators use them consistently. The de facto standard identifier in many fields is the Open Researcher and Contributor ID (ORCID),3 although other identifiers such as Google Scholar ID4 or ResearcherID (Publons)5 are also widely used. Having multiple identifiers on multiple platforms is not unusual and automatic publication detection and syncing between accounts is possible to some degree. However, automatic synchronization of accounts for different identifiers can be hindered by the fact that different document identifiers are used, such as DOI (Digital Object Identifier) or the independent identifier used by Google Scholar.\n\nBecause of this lack of standardized identifiers for both authors and documents, it is often necessary to synchronize publication records on different platforms manually to obtain complete records. For instance, there is no simple one-click solution to synchronize publications between ORCID and Google Scholar. In Google Scholar, publications need to be searched and added manually (if they are not detected automatically) while in ORCID it is possible to input a citation file. A typical workflow to update ORCID based on Google Scholar would therefore be to first search (one by one) in Google Scholar all publications that are listed in ORCID and then add the missing ones. But since it is possible that publications listed in Google Scholar are not in ORCID, the reverse needs to be done to be sure the accounts are up to date. If more accounts need to be synced (e.g. Publons), the complexity and time needed increases accordingly. Although it is possible, and probably advisable, to link accounts for automatic updates (e.g. linking Publons with ORCID), this cannot be done under all circumstances and missing publications are still possible.\n\nWhile some (commercial) services (such as Dimensions6 or Web of Science7) provide extensive data mining to retrieve publication data, they often also rely on unique identifiers (such as ORCID in the case of Dimensions) for correct assignment. Furthermore, on many platforms that combine different sources (e.g. Dimensions), it is not easy to determine where the data originated (e.g. is a publication listed in ORCID or in Publons? or both?), meaning no information about the “completeness” of those sources is given. In addition, data exploration and visualization is often restricted to citations over time (except costly commercial services, such as Dimensions). With the growing awareness, interest and mandates towards Open Science, open access (OA) status of articles can also be of interest. The same is true for preprints, which are often not taken into account despite becoming increasingly important in many research fields.8,9\n\nAnother inconvenience can be the existence of duplicated publications, which can stem either from the association of preprint and peer-reviewed publication or from revisions or different versions. In many cases, it is sensible to treat those closely linked publication as just one publication instead of multiple. Often it is not possible to detect duplicated publications automatically and manual intervention is needed.\n\nTo our knowledge, there does not exist a free tool that allows researchers to interactively explore their publication metadata across multiple platforms, together with the open access status of each publication. Commercial tools exist, such as Elements (from the company Symplectic10) or Dimensions, but they are intended for institutional use. In our case, we took inspiration from the Swiss National Science Foundation’s Open Access Check,11 which allows Swiss researchers to reflect on their publishing practices and encourages various forms of OA, including green OA; importantly, such resources rely on the source databases being up to date in the first place.\n\nFurthermore many of the available tools are not made for individual authors but rather operate on the department, institutions or even country level. A few important tools are: the open science monitoring of the European commission12 (country-level), the German open access monitor (institution-level) and OpenAIRE (Open Access Infrastructure for Research in Europe) provides dashboards (country- or institution-level).\n\nTo facilitate overview and synchronization of publication records, we provide a web-based application that allows publications for an author to be retrieved from different sources, combines entries, checks for duplicates and downloads citations to easily update records across platforms. Furthermore, the open access status of each publication is provided, which can help to select publications that could be “greened” (i.e., depositing documents in institutional repositories). Taken together, this allows researchers to organize their public publication profiles and to interactively explore the accuracy of records across the various entry points.\n\n\nMethods\n\nThe workflow is as follows: The user needs to first specify the unique identifiers of the researcher of interest for at least one of ORCID, Google Scholar and Publons. Additionally, a search query for Pubmed can be generated. Furthermore, the option to search for bibliometrics, obtained from the NIH Open Citation Collection using iCite,13 can be selected. After confirmation, publications are retrieved from the specified sources and combined into a table based on the DOI (see Figure 1) or, in case of publications from Google Scholar, based on (fuzzy) matching of titles and/or metadata retrieval from Zotero (Zotero translator, i.e. web scraping)14 or Crossref (i.e. query the available metadata to obtain a DOI).15 After joining the publications list, the open access status of each publication with a DOI is retrieved using Unpaywall,16 who provide a publicly accessible database containing open access information for publications. The definitions of the different open access status that Unpaywall uses is provided in Table 1. Additionally, preprints are defined as having OA status “green” in Unpaywall with the attribute “version” equal to “submittedVersion”. A database snapshot of Unpaywall can be downloaded https://unpaywall.org/products/snapshot.\n\nThe identifiers given by the user are used to obtain the data from each platform independently. The data is then merged and the open access status (column OA) is obtained using the Digital Object Identifier (DOI). Furthermore duplicates are detected by comparing the titles of the publications.\n\nAfter this step, interactive exploration of the publications is possible. Various options to filter the data according to OA status, year and source (ORCID, Google Scholar, etc.) are available with the possibility to remove or show duplicates (detected using fuzzy matching of titles). Several metrics, tables and plots are available for exploration of the data. Examples include a upset plot that shows how many publications are associated with each identifier, a histogram of the number of publications per year colored by open access status, and a table listing the individual publications. After exploration, specific subsets can be generated using the filtering options, which are then imposed on the visualizations and tables presented. In all cases, relevant snapshots of the citation information can be obtained in the form of a downloadable file.\n\nAnother possible application is the integration of local databases, such as university repositories. For example, the Zurich Open Research Archive (ZORA),17 developed and maintained by the Main Library at the University of Zurich, has been integrated in an alternative version of the app that allows local entries to be compared with public profiles, allowing synchronization of publication profiles with local repositories.\n\nThe application is written in R (Version 4.1.0)18 and shiny (Version 1.6.0),19 see Software availability. As a back-end database, PostgreSQL is used to store a local copy of Unpaywall (and ZORA). Such a local database for Unpaywall is not strictly needed, but a large speedup of the retrieval of the open access status is achieved compared to access over the Unpaywall API. Furthermore, since only a fraction of the data from Unpaywall is used (only the DOI, the open access status and two additional columns for preprint identification) the actual table, containing open access status, is comparably small with a size of about 6 GB (compared to more than 165 GB of the complete version). Unpaywall does daily updates that can be downloaded and are used to update the local database to keep it in sync with the online version. The DOIs for publications listed in Google Scholar are obtained by either matches to publications from other sources, metadata retrieval using the Zotero translator service or a Crossref query.\n\nVarious R packages that facilitate retrieval of publications from a specific resource such as https://docs.ropensci.org/rorcid (ORCID),20 https://github.com/jkeirstead/scholar (Google Scholar)21 or https://docs.ropensci.org/rentrez (Pubmed)22 have been included.\n\nThe app is containerized using Docker (Version 19.03.13, dockerfiles and docker-compose file are provided in Software availability). Multiple, interacting containers are deployed using docker-compose, the two most important are a container running the R shiny application and another running PostgreSQL. Furthermore, the Zotero translator service is run in a separate container. As already stated, the PostgreSQL service is not strictly needed, but substantially increases retrieval speed of the OA status.\n\n\nUse case\n\nFigure 2 shows a use case for an author where the ORCID (0000-0002-3048-5518) and Google Scholar ID (XPfrRQEAAAAJ) were given as an input (collapsed panel in Figure 2A). Panel B provides a summary of the publication list and options to filter by dataset, by year and by OA status. Additionally, the possibility to remove duplicates or only show duplicates is available. The other panels contain visualizations including an upsetplot23 (C), a histogram (D) and a table (E). The table can be further filtered by selecting rows allowing to create specific citation lists that can be created based on the rows in the table. The contents of the table can be copied to the clipboard or downloaded in CSV format.\n\nAfter entering identifiers in panel A, successful retrieval and merging, panels B-E appear. Panel B is the main panel for filtering. Visualizations are in panel C (upsetplot), D (histogram) and E (table).\n\n\nDiscussion\n\nOur method relies on the DOI to retrieve the OA status, which is a limitation in domains where DOIs are not used. The DOI is also used to unambiguously match publications. If no DOI is present, the titles of the publications are used for matching, which can lead to ambiguity. Even if a publication has an assigned DOI, but it is missing in the data, it becomes difficult or time-consuming to retrieve the missing information with services such as the Zotero translator or Crossref.\n\nBecause of the non commercial nature of this application, some additional limits present themselves. Most notably, our application requires freely-available APIs for retrieving the open publication data from their respective sources. While for the two main sources considered (ORCID and Google Scholar) so far no restrictions have been noticed, the APIs of Dimensions or Mendeley are closed and for others, rate limits in the number of requests are quite restrictive (e.g. for Publons).\n\n\nData availability\n\nNo data are associated with this article.\n\n\nSoftware availability\n\nSoftware available from: https://pubassistant.ch/\n\nSource code available from: https://github.com/markrobinsonuzh/os_monitor\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.5509626\n\nLicense: MIT", "appendix": "Acknowledgements\n\nWe thank Izaskun Mallona for help with hosting the application and various helpful suggestions. We thank various members of the Statistical Bioinformatics Group at University of Zurich for feedback.\n\n\nReferences\n\nUNESCO: Science Report.2021. Reference Source\n\nBornmann L, Mutz R: Growth rates of modern science: A bibliometric analysis based on the number of publications and cited references. J. Assoc. Inf. Sci. Technol. 2015; 66(11): 2215–2222. 2330-1643. Publisher Full Text Reference SourceReference Source\n\nHaak LL, Fenner M, Paglione L, et al.: ORCID: a system to uniquely identify researchers. Learned Publishing; 2012; 25(4): 259–264. 1741-4857. Publisher Full Text Reference SourceReference Source\n\nGoogle Scholar. Reference Source\n\nPublons. Reference Source\n\nHook DW, Porter SJ, Herzog C: Dimensions: Building Context for Search and Evaluation. Front. Res. Met. Analy. 23, August 2018; 3: 2504-0537. Publisher Full Text Reference Source\n\nWorld’s largest publisher-neutral citation index and research intelligence platform. Reference Source\n\nVale RD: Accelerating scientific publication in biology. Proc. Natl. Acad. Sci. National Academy of Sciences Section: Perspective; November 2015; 112(44): 13439–13446. 0027-8424, 1091-6490. Publisher Full Text Reference Source\n\nJohansson MA, Reich NG, Meyers LA, et al.: Preprints: An underutilized mechanism to accelerate outbreak science. PLoS Med. Public Library of Science; April 2018; 15(4): e1002549. 1549-1676. Publisher Full Text\n\nOpen Access. Reference Source\n\nSNSF Open Access Check. Reference Source\n\nTrends for open access to publications. Reference Source\n\nICite, B: Ian Hutchins, and George Santangelo. iCite Database Snapshots (NIH Open Citation Collection).The NIH Figshare Archive; 2019. Publisher Full Text Reference Source\n\nZotero Translation Server: July 2021. original-date: 2018-06-11T11:28:53Z. Reference Source\n\nLammey R: CrossRef developments and initiatives: an update on services for the scholarly publishing community from CrossRef.page 6.\n\nPiwowar H, Priem J, Larivière V, et al.: The state of OA: a large-scale analysis of the prevalence and impact of Open Access articles. PeerJ. February 2018; 6: e4375. 2167-8359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWelcome to Zurich Open Repository and Archive - Zurich Open Repository and Archive. Reference Source\n\nR R Development Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; 2011; 3-900051-07-0. 16000706. Publisher Full Text\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R.2020. Reference Source\n\nScott Chamberlain: rorcid: Interface to the ‘Orcid.org’ API.2021.\n\nKeirstead J: scholar: analyse citation data from Google Scholar.2016. Reference Source\n\nWinter DJ: rentrez: an R package for the NCBI eUtils API. The R Journal. 2017; 9(2): 520–526. Publisher Full Text\n\nConway JR, Lex A, Gehlenborg N: UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics. September 2017; 33(18): 2938–2940. 1367-4811. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "96152", "date": "26 Oct 2021", "name": "Daniel W Hook", "expertise": [ "Reviewer Expertise Open Research", "Bibliometrics", "Sociology of Research", "Theoretical Physics (Quantum Statistical Mechanics", "PT-Symmetric Quantum Mechanics)." ], "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 created a free, open source piece of software to bring researcher and publication records together from different data sources. They detail their motivations and methodology in this paper.\nThe authors begin their article by motivating the development of their software based on the difficulty of consolidating all the publications from a single author resulting from: - The non-uniqueness of names of authors - The proliferation of unique identifiers that aim to solve this problem (e.g. ResearcherID/Publons, ORCID et al.)It is reasonable to claim that we, as a community, have not yet realised a comprehensive solution to these problems.\n\nHowever, the authors choose to take a peculiarly western-centric view of this issue. The greatest challenges of name disambiguation are typically found when authors who might not natively use a roman alphabetic system are forced to transliterate their names when publishing in the western-centred publishing system. This fact goes unacknowledged in this paper but would seem to be at the heart of the name disambiguation issue.\nThere are several approaches to name disambiguation but there are broadly two “schools”: attended and unattended. “Attended” is where humans interact with the disambiguation and “unattended” is where humans have no role. Attended disambiguation is the focus for identity management systems like ORCID - incentives need to be aligned for authors and others to participate in this and significant work goes into understanding the motivations and concerns of researchers in order to make these types of system successful. Unattended disambiguation makes use of the data that are available in the ecosystem (including the outputs of the attended disambiguation approach) to create a calculated output. We feel that the authors should give some of this background in their paper for context.\nIn this context, it is important to acknowledge that only one identifier system has been successful in engaging the broad global academic community and that is ORCID. Other solutions are self-acknowledged proprietary solutions that aim to solve this problem in limited contexts (Researcherid.com/Publons to improve the Web of Science data (attended disambiguation); ScopusID - unattended disambiguation approach). Dimensions explicitly leverage ORCID data in an unattended person disambiguation approach.1\n\nThe authors claim that there is no standardization of unique identifiers for authors and documents.\nThis is a very strong claim when viewed at an international ecosystem level. The majority of funders, publishers, institutions, scholarly societies and government agencies involved with research from around the world acknowledge DOIs as the key identifier for a research paper and ORCID as the principal identifier of researchers.\n\nSome complexity lies in the authority that issues and maintains an article DOI (Crossref or DataCite in most cases but also, for example, J-Stage in Japan).\n\nORCID may currently be a “de facto standard” but the authors’ statement to this effect underplays the central role played by ORCID in the scholarly infrastructure community and the extent to which ORCID is the standard with which the majority of the commercial and open infrastructure providers engage. Google Scholar provides no API and makes no claim of persistence of identifiers; authors have limited control of their profile and of privacy. Researcherid.com (the progenitor of Publons/ResearcherID) is a founding member, supporter and participant of ORCID. We suggest that the authors should ensure that acknowledgement be made of ORCID’s suitability and level of adoption in academia. If the authors wish to note that ORCID is not the only standard, it would be appropriate to mention parallel efforts such as researchmap.jp in Japan and note that some countries remain reluctant to adopt ORCID as their principal identifier at this time.\n\nThe authors claim that it is not easy to determine the provenance of data in Dimensions has derived its data from ORCID or Publons. Arguably this is not the case. Dimensions clearly states that algorithmic methods are used with the input of ORCID data.1 Publons/Researchid.com data is a proprietary source that could not be used in Dimensions without explicit acknowledgement of its use. The authors further claim that no information is given about the completeness of these sources (e.g. Dimensions/Web of Science/Scopus), yet significant academic work has been undertaken to understand and benchmark coverage and completeness of these sources (see reference 2 for example).2\n\nWe agree with the authors that Open Science is indeed critical. This is why Dimensions, Web of Science, Scopus and others contain information on Open Access statuses of articles, provided by Unpaywall (https://unpaywall.org/integrations). Dimensions also contains full listings of preprint articles from many preprint servers including ArXiv.org, BioArXiv, the Center for Open Science, PeerJ and so on. As such we find the authors’ comment that these things are “not taken into account” to be misleading.\n\nThe authors then appear to contradict themselves by claiming that preprints and publications cause duplicate entries in these systems. In Dimensions, where the current reviewer has the most experience, publications from preprints are linked to final publications where this information is available, see for example https://app.dimensions.ai/details/publication/pub.1118864658?and_facet_researcher=ur.01123321343.51. We agree that manual intervention is often needed in these cases.\n\nThe authors claim that a free tool does not exist to explore metadata brought together from multiple sources, however, this does not acknowledge the rich lineage of free tools written to serve institutional use cases in author disambiguation and metadata aggregation including: VIVO (https://duraspace.org/vivo/) and Catalyst Profiles (http://profiles.catalyst.harvard.edu), both funded by the NIH; ImpactStory (e.g. https://profiles.impactstory.org/u/0000-0001-6728-7745/publications); and, ReCiter (https://github.com/wcmc-its/ReCiter). The authors should endeavour to situate their work in the context of this prior work.\n\nSymplectic Elements is, as the authors state, a commercial system focused on institutional use cases that meets this need. However, the authors fail to acknowledge that outside STEM subjects, coverage of research outputs becomes more challenging. The strength of commercial tools is often that, as they must meet institutional needs, work is put into diversifying coverage of different output types beyond the journals and conference proceedings that STEM subjects favour. While composing a faithful representation of a STEM academic and their output can be challenging from disparate data sources, doing the same for a non-STEM academic can be an order of magnitude more difficult. We believe that it is also fair to acknowledge that institutional engagement is not unimportant to improving the overall data quality in the bibliographic ecosystem.\n\nThe authors also appear to be unaware that Dimensions does bring records together from multiple sources including author information and that this is transparently documented in scholarly articles written by the Dimensions team,1 as well as in system and API documentation (https://docs.dimensions.ai/dsl/). Open access information and much more (citation information, funding information) and is available in the version of Dimensions that is available for free for personal use.\n\nThe authors note that Dimensions, Mendeley and others do not offer a public open API.\nWhile Dimensions does offer a free end-user tool, it does not offer a full open public API. However, Dimensions does offer a free metrics API for non-commercial purposes (https://www.dimensions.ai/dimensions-apis/) and Mendeley continues to offer a free API (albeit with recent changes to some of the functionality around search functionality - https://dev.mendeley.com/).\nIn addition, given the use of Google Scholar by the authors in their tool, I think that it is important to note explicitly that Google Scholar does not offer an API. Indeed, from the software source code deposited by the authors, it appears that data is scraped from the Google Scholar website contravening the terms of use of Google Scholar. This fact should be explicitly noted in the paper.\n\nThe paper contains no critical analysis on the accuracy or success of the algorithmic approaches taken by the authors as regards fuzzy matching of papers and authors between sources.\n\nThe paper would also be improved by including a flow diagram and description of matching approach taken by the authors.\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? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "7562", "date": "20 Dec 2021", "name": "Reto Gerber", "role": "Author Response", "response": "However, the authors choose to take a peculiarly western-centric view of this issue. The greatest challenges of name disambiguation are typically found when authors who might not natively use a roman alphabetic system are forced to transliterate their names when publishing in the western-centred publishing system. This fact goes unacknowledged in this paper but would seem to be at the heart of the name disambiguation issue. Response: Missing issue of ambiguous transliteration of names into roman alphabetic system has been added to the Introduction. There are several approaches to name disambiguation but there are broadly two “schools”: attended and unattended. “Attended” is where humans interact with the disambiguation and “unattended” is where humans have no role. Attended disambiguation is the focus for identity management systems like ORCID - incentives need to be aligned for authors and others to participate in this and significant work goes into understanding the motivations and concerns of researchers in order to make these types of system successful. Unattended disambiguation makes use of the data that are available in the ecosystem (including the outputs of the attended disambiguation approach) to create a calculated output. We feel that the authors should give some of this background in their paper for context. In this context, it is important to acknowledge that only one identifier system has been successful in engaging the broad global academic community and that is ORCID. Other solutions are self-acknowledged proprietary solutions that aim to solve this problem in limited contexts (Researcherid.com/Publons to improve the Web of Science data (attended disambiguation); ScopusID - unattended disambiguation approach). Dimensions explicitly leverage ORCID data in an unattended person disambiguation approach.1 Response: A short description of the unattended vs attended approaches of name disambiguation were added to the Introduction. The authors claim that there is no standardization of unique identifiers for authors and documents.This is a very strong claim when viewed at an international ecosystem level. The majority of funders, publishers, institutions, scholarly societies and government agencies involved with research from around the world acknowledge DOIs as the key identifier for a research paper and ORCID as the principal identifier of researchers. Response: We rewrote this statement to say that various other identifiers exist beyond the two most important ones, ORCID and DOI. Some complexity lies in the authority that issues and maintains an article DOI (Crossref or DataCite in most cases but also, for example, J-Stage in Japan). ORCID may currently be a “de facto standard” but the authors’ statement to this effect underplays the central role played by ORCID in the scholarly infrastructure community and the extent to which ORCID is the standard with which the majority of the commercial and open infrastructure providers engage. Google Scholar provides no API and makes no claim of persistence of identifiers; authors have limited control of their profile and of privacy. Researcherid.com (the progenitor of Publons/ResearcherID) is a founding member, supporter and participant of ORCID. We suggest that the authors should ensure that acknowledgement be made of ORCID’s suitability and level of adoption in academia. If the authors wish to note that ORCID is not the only standard, it would be appropriate to mention parallel efforts such as researchmap.jp in Japan and note that some countries remain reluctant to adopt ORCID as their principal identifier at this time. Response: We have rephrased parts of the text to highlight the important role of ORCID. The authors claim that it is not easy to determine the provenance of data in Dimensions has derived its data from ORCID or Publons. Arguably this is not the case. Dimensions clearly states that algorithmic methods are used with the input of ORCID data.1 Publons/Researchid.com data is a proprietary source that could not be used in Dimensions without explicit acknowledgement of its use. Response: We have removed this imprecisely-worded statement. The authors further claim that no information is given about the completeness of these sources (e.g. Dimensions/Web of Science/Scopus), yet significant academic work has been undertaken to understand and benchmark coverage and completeness of these sources (see reference 2 for example).2 Response: We have now removed claim about completeness of these sources since in our case, we are more interested in author-level completeness, e.g., are publications listed in Google scholar missing in ORCID for a specific author? We agree with the authors that Open Science is indeed critical. This is why Dimensions, Web of Science, Scopus and others contain information on Open Access statuses of articles, provided by Unpaywall (https://unpaywall.org/integrations). Dimensions also contains full listings of preprint articles from many preprint servers including ArXiv.org, BioArXiv, the Center for Open Science, PeerJ and so on. As such we find the authors’ comment that these things are “not taken into account” to be misleading. Response: We removed the imprecisely-worded claim about Dimensions’ open access status and preprints. The authors then appear to contradict themselves by claiming that preprints and publications cause duplicate entries in these systems. In Dimensions, where the current reviewer has the most experience, publications from preprints are linked to final publications where this information is available, see for example https://app.dimensions.ai/details/publication/pub.1118864658?and_facet_researcher=ur.01123321343.51. We agree that manual intervention is often needed in these cases. Response: This contradiction was removed by acknowledging the listing of preprints on other platforms, such as Dimensions. The authors claim that a free tool does not exist to explore metadata brought together from multiple sources, however, this does not acknowledge the rich lineage of free tools written to serve institutional use cases in author disambiguation and metadata aggregation including: VIVO (https://duraspace.org/vivo/) and Catalyst Profiles (http://profiles.catalyst.harvard.edu), both funded by the NIH; ImpactStory (e.g. https://profiles.impactstory.org/u/0000-0001-6728-7745/publications); and, ReCiter (https://github.com/wcmc-its/ReCiter). The authors should endeavour to situate their work in the context of this prior work. Response: We have added the above mentioned tools to the Introduction text and now describe our application in the context of those tools. Symplectic Elements is, as the authors state, a commercial system focused on institutional use cases that meets this need. However, the authors fail to acknowledge that outside STEM subjects, coverage of research outputs becomes more challenging. The strength of commercial tools is often that, as they must meet institutional needs, work is put into diversifying coverage of different output types beyond the journals and conference proceedings that STEM subjects favour. While composing a faithful representation of a STEM academic and their output can be challenging from disparate data sources, doing the same for a non-STEM academic can be an order of magnitude more difficult. We believe that it is also fair to acknowledge that institutional engagement is not unimportant to improving the overall data quality in the bibliographic ecosystem. Response: We have now added Introduction text, highlighting that commercial systems can make a big impact, especially in institutional cases. The authors also appear to be unaware that Dimensions does bring records together from multiple sources including author information and that this is transparently documented in scholarly articles written by the Dimensions team,1 as well as in system and API documentation (https://docs.dimensions.ai/dsl/). Open access information and much more (citation information, funding information) and is available in the version of Dimensions that is available for free for personal use. Response: We are aware of this functionality within Dimensions, but it is not open for use beyond personal use and most of the data that we are interested in is already in the public domain. The authors note that Dimensions, Mendeley and others do not offer a public open API. While Dimensions does offer a free end-user tool, it does not offer a full open public API. However, Dimensions does offer a free metrics API for non-commercial purposes (https://www.dimensions.ai/dimensions-apis/) and Mendeley continues to offer a free API (albeit with recent changes to some of the functionality around search functionality - https://dev.mendeley.com/). Response: We now removed the imprecisely-worded statement about closed APIs of Dimensions and Mendeley. In addition, given the use of Google Scholar by the authors in their tool, I think that it is important to note explicitly that Google Scholar does not offer an API. Indeed, from the software source code deposited by the authors, it appears that data is scraped from the Google Scholar website contravening the terms of use of Google Scholar. This fact should be explicitly noted in the paper. Response: We have now added a statement that web scraping is done to retrieve information from Google Scholar and that this may contravene the terms of use of Google Scholar. The paper contains no critical analysis on the accuracy or success of the algorithmic approaches taken by the authors as regards fuzzy matching of papers and authors between sources. Response: A description of the fuzzy matching mechanism that was applied was added together with a statement about accuracy of matching. The paper would also be improved by including a flow diagram and description of matching approach taken by the authors. Response: The description of the approach for matching of publications has been expanded." } ] } ]	1	https://f1000research.com/articles/10-989
https://f1000research.com/articles/10-1147/v1	12 Nov 21	{ "type": "Study Protocol", "title": "Palliative care and good death in acute diseases: a scoping review protocol", "authors": [ "Vitri Widyaningsih", "Ratih Puspita Febrinasari", "Adji Suwandono", "Sigid Kirana Lintang Bhima", "Retna Siwi Padmawati", "Ari Probandari", "Vitri Widyaningsih", "Ratih Puspita Febrinasari", "Adji Suwandono", "Sigid Kirana Lintang Bhima", "Retna Siwi Padmawati" ], "abstract": "Increasing cases of emerging and re-emerging infectious diseases, requires healthcare systems to provide essential palliative care for critically ill patients and their families. With the rapid onset and often accelerated deterioration in patients with acute conditions, palliative and supportive care for these patients have different characteristics compared to those for chronic diseases. Furthermore, providing end-of life services for critically ill patients with acute diseases and their families to ensure good death for the patients, will also have its own challenges. This scoping review aims to explore the concept of palliative care and good death for acute diseases. This scoping review will be conducted using the Arksey and O’Malley's framework for scoping reviews: identifying the research question, identifying relevant studies, study selection, charting the data, collating, summarizing, reporting results, and conducting consultation. All original research with a focus on palliative care and good death due to acute diseases will be included. This review will include all original research designs published between the period of 2000–2021 that describe a measure of palliative care management for and good death due to acute diseases. Quantitative, qualitative and mixed-method studies will be included in order to consider different aspects of healthcare services. This review will also include guidelines and gray literature on palliative care and good deaths. The search will be conducted through PubMed, Scopus Database, and ScienceDirect using the key terms related to acute disease palliative care and the concept of good death due to acute diseases. Two authors will screen the titles and abstracts of the studies. Two authors will review the full text of selected studies independently and extract the data. All selected studies will be synthesized qualitatively, and the results will be consulted with experts through discussion and depict the current concept of palliative care and good death in acute diseases.", "keywords": [ "good death", "palliative care", "acute disease", "infectious disease", "health systems" ], "content": "Introduction\n\nPalliative care is provided for a person with an active, progressive and advanced disease who is in serious or life-threatening health conditions.1,2 Palliative care aims to optimize the quality of life and addresses physical suffering of the patient. Furthermore, the palliative care also aims to improve psychological, social and spiritual conditions for patients and their family members.1–3 Palliative care involves a multidisciplinary team beyond healthcare professionals, including spiritual, psychology, and/or social worker team member.4–6 Palliative care is designed as a person-centered and family-centered care, addressing each patient’s and family members specific conditions and needs.7,8 There are two types of palliative care, namely terminal palliative care which focuses on the treatment of immediate disease symptoms, and early palliative care that provides patients and family members support for coping with the diagnosis and helps balance decision-making between values and preferences of the patient and realistic expectations of the outcome of treatment.9 In low to middle income countries, dissimilarity of access to palliative care shows great disparities in global health care.2,10 The concept of palliative care is more commonly discussed and studied in chronic diseases, however, there is still lacking evidence on palliative care on acute diseases.\n\nAcute diseases, such as infections, injuries, acute exacerbations of chronic illnesses, are sudden, rapid and severe diseases that last for a brief time period.11,12 High mortality rate might be found on acute emergency admission, especially amongst the elderly with multiple co-morbid conditions.13 Therefore, palliative care should be an important component in acute disease management, particularly for severe cases.\n\nThe concept of palliative care is also closely related to perceived good death. Good death or successful dying is defined as the person’s preferences for the dying process. These preferences might include how, where, when, pain-free status of dying process.14,15 The concept of good death also include who accompanied the person during the dying process, and the manner of facing death (i.e. awareness and readiness of the dying process, natural or sudden death).14–16 Ironically, studies have shown that patients with contagious disease have been isolated from their family members due to hospital policies and many of them have a deep fear of dying alone.15,17 Majority of people want to be with their loved ones when their life comes to an end.15,18\n\nUp until now, there have been limited summaries on the experience of palliative care and concept of good death in acute diseases. Further, there is a lack of information on the ideal concept of palliative care and good death in acute disease from the perspective of patients, family or caregivers, as well as healthcare providers. This scoping review aims to explore the concept of palliative care and good death for acute diseases from the perspective of patients, families, and providers, hence, depicting the existing palliative care and good death concepts, and providing recommendation on the ideal concept of palliative care and good death in acute diseases.\n\n\nProtocol\n\nTo conduct this scoping review, the framework by Arksey and O’Malley will be used.19 The framework consist of six steps: 1) identification of research questions; 2) identification of relevant studies; 3) selection of relevant studies; 4) data extraction and charting, 5) summary, analyses, and reporting; and 6) consultation with relevant stakeholders.19 The search process, database creation and data extraction will be conducted from September to October 2021. The analysis and reporting will be carried out in December 2021 - April 2022.\n\nIn this scoping review, we aim to assess the concept of palliative care and good death in acute illness. The research questions can be further elaborated as follows:\n\n1. What is the experience of patients, family of patients, and healthcare providers regarding palliative care for acute diseases?\n\n2. What is the experience of patients, family of patients, and healthcare providers regarding good death due to acute illness?”\n\n3. What is the perception of patients, family of patients, and healthcare providers regarding an ideal palliative care for patients with acute diseases?\n\n4. What is the perception of patients, family of patients, and healthcare providers regarding what is considered as good deaths among patients with acute diseases?\n\nThis scoping review includes the relevant studies based on the inclusion and selection criteria shown on Table 1. The format followed the Joanna Briggs Institute (JBI) protocol.20\n\nThe search strategy using keywords and queries can be found in Table 2. The literature search will be conducted in three databases (Pubmed, Scopus, and Science Direct) for articles published between January 2000 to October 2021, hence, providing a review of literature for the past 20 years.\n\nThe search strategy will use Medical Subject Heading (MesH) terms: “Acute Disease”, “Palliative Care”, and “Good Death” on the Pubmed database. An initial search with synonyms of those keywords will be used in the Scopus and ScienceDirect database. Next, analysis of the words contained in the title and abstract will be done. Clinical guidelines database and gray literature will be included and listed in Figure 1.\n\nTwo authors will screen the titles and abstracts of studies according to the selection criteria. In the scoping review, we will include all studies that evaluate the acute disease palliative care and good death. We will exclude studies focusing on chronic disease. Two authors will review the full text of selected studies independently. We will resolve disagreements on study selection and data extraction by discussion with one more reviewer if needed. The details of the study selection are depicted in Figure 2.\n\nThe two reviewers will independently chart the data, discuss the results and continuously update the data-charting form in an iterative process. Data extraction will be carried out following the form that has been prepared in Table 3.\n\nTo summarize the findings, a table will be developed mapping all the studies included in the scoping review. Studies will be categorized by different characteristics, including type of sources, study location, study design, as well as types of integration. EndNote 20 software will be used to store and managed the data obtained from the literature.\n\nAfter data extraction, a table will be developed to summarize and map the findings. Studies will be categorized based on several distinct characteristics. For example: study sample (patients, family or caregiver of patients, and healthcare providers), experience and perception of ideal concept for palliative care and good deaths, and level of healthcare service where the palliative care is provided, or death occurred. Qualitative analyses by using thematic coding will be conducted to present a robust summary of the literature on experience and perception regarding palliative care and good death in acute diseases.\n\nThe results will be consulted to the experts and relevant stakeholders, which include patients, families, and caregivers. As this scoping review will be the basis for recommendation on palliative care and good death for people with acute diseases in Indonesia, relevant stakeholders will be identified from Indonesia. This step is important to ensure that the finding is validated, receive feedback and obtain additional insights into the findings. In the discussion, findings from the scoping review will be presented with feedback by relevant stakeholders provided.\n\n\nEthics and dissemination\n\nWe have obtained ethical permits for this review. The results of this review can be used as a reference for preparing a pilot study on integrated management of acute disease palliative care and good death management as well as policy briefs, presentations in conferences, peer-reviewed journals, and information on related websites.\n\n\nStudy status\n\nThis study is now at early stage of the step two of the scoping review process: identification of relevant studies. We are still searching for literature for relevant studies and abstracting data from the search engines. We estimated the scoping review to be completed by mid-2022.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nReymond L, Parker G, Gilles L, et al.: Home-based palliative care. Aust. J. Gen. Pract. Nov. 2018; 47(11): 747–752. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization (WHO): Integrating palliative care and symptom relief into primary health care: a WHO guide for planners, implementers and managers. Geneva: World Health Organization; 2018.\n\nIreland AW: Access to palliative care services during a terminal hospital episode reduces intervention rates and hospital costs: a database study of 19 707 elderly patients dying in hospital, 2011–2015. Intern. Med. J. May 2017; 47(5): 549–556. PubMed Abstract \| Publisher Full Text\n\nArrieira ICdO, Thofehrn MB, Porto AR, et al.: Spirituality in palliative care: Experiences of an interdisciplinary team. Rev. da Esc. Enferm. 2018; 52: 1–7. Publisher Full Text\n\nGaertner J, et al.: Effect of specialist palliative care services on quality of life in adults with advanced incurable illness in hospital, hospice, or community settings: Systematic review and meta-analysis. BMJ. 2017; 357: j2925. Publisher Full Text\n\nSagha Zadeh R, Eshelman P, Setla J, et al.: Strategies to Improve Quality of Life at the End of Life: Interdisciplinary Team Perspectives. Am. J. Hosp. Palliat. Med. 2018; 35(3): 411–416. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMercadante S, Gregoretti C, Cortegiani A: Palliative care in intensive care units: Why, where, what, who, when, how. BMC Anesthesiol. 2018; 18(1): 106–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiop MS, Rudolph JL, Zimmerman KM, et al.: Palliative Care Interventions for Patients with Heart Failure: A Systematic Review and Meta-Analysis. J. Palliat. Med. 2017; 20(1): 84–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKopecký O, Rusinová K, Kouba M, et al.: The role of palliative medicine in acute conditions. Intern. Med. 2021; 65(schéma 1): 449–455. Publisher Full Text\n\nLuckett T, Phillips J, Agar M, et al.: Elements of effective palliative care models: A rapid review. BMC Health Serv. Res. 2014; 14: 1, 1–22. Publisher Full Text\n\nChamberlain S, et al.: Mortality Related to Acute Illness and Injury in Rural Uganda: Task Shifting to Improve Outcomes. PLoS One. 2015; 10(4): e0122559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChowdhuri PD, Kundu K: Factors determining choice of complementary and alternative medicine in acute and chronic diseases. J. Complement. Integr. Med. 2020; 17(3): 1–10. PubMed Abstract \| Publisher Full Text\n\nKellett J: Acute hospital medicine - A new sub-speciality or internal medicine re-born?. Eur. J. Intern. Med. 2011; 22(4): 334–338. PubMed Abstract \| Publisher Full Text\n\nMeier EA, Gallegos JV, Montross-Thomas LP, et al.: Defining a Good Death (Successful Dying): Literature Review and a Call for Research and Public Dialogue. Am. J. Geriatr. Psychiatry. 2016 Apr.; 24(4): 261–271. Publisher Full Text\n\nCampbell SM: Well-Being and the Good Death. Ethical Theory Moral Pract. Aug. 2020; 23(3–4): 607–623. Publisher Full Text\n\nMiyashita M, Morita T, Sato K, et al.: Good Death Inventory: A Measure for Evaluating Good Death from the Bereaved Family Member’s Perspective. J. Pain Symptom Manage. 2008; 35(5): 486–498. PubMed Abstract \| Publisher Full Text\n\nPowell T, Hulkower A: A Good Death. Hastings Cent. Rep. Jan. 2017; 47(1): 28–29. Publisher Full Text\n\nBorgstrom E: What is a good death? A critical discourse policy analysis. BMJ Support. Palliat. Care. Jul. 2020; bmjspcare-2019-002173. Publisher Full Text\n\nArksey H, O’Malley L: Scoping studies: towards a methodological framework. Int. J. Soc. Resear. 2005; 8(1): 19–32. Publisher Full Text\n\nThe Joanna Briggs Institute: The Joanna Briggs Institute Reviewers’ Manual 2015: Methodology for JBI scoping reviews. Joanne Briggs Inst. 2015." }	[ { "id": "101929", "date": "14 Dec 2021", "name": "Hanan Khalil", "expertise": [ "Reviewer Expertise Health services research and palliative care" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this manuscript. I have the following comments for your consideration:\nI am unclear to which methodology the authors are using, as they quoted both Arksey and Malley and JBI. Please specify.\n\nPlease specify the study types that you are including in the review.\n\nThe research questions are best addressed by a qualitative review rather than a scoping review.\n\nThe questions included are mainly qualitative in nature. Scoping reviews mainly address broader questions addressing; Participants, concept and context. The context is not mentioned in the review.\n\nPlease specify the rationale for conducting the scoping review as opposed to other types of review.\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": [] } ]	1	https://f1000research.com/articles/10-1147
https://f1000research.com/articles/10-1098/v1	29 Oct 21	{ "type": "Research Article", "title": "One Ringgit and five Ringgit Malaysian banknotes reader using backlight mechanism and image processing techniques", "authors": [ "Turki Khaled Salem", "Wai Kit Wong", "Thu Soe Min", "Eng Kiong Wong", "Turki Khaled Salem", "Thu Soe Min", "Eng Kiong Wong" ], "abstract": "Visually impaired persons face challenges in running business activities, especially in handling banknotes. Malaysia researchers had proposed some Ringgit banknotes recognition systems to aid visually impaired persons recognize and classify Ringgit banknotes. However, these electronic banknote readers can only recognize Malaysian Banknotes’ Ringgit value, they have no counterfeit detection features. The purpose of this study is to develop a banknote reader that not only can help visually impaired persons recognize the banknote value, but also to detect the counterfeit of the banknote, safeguarding their losses. This paper proposed a Malaysian banknote reader using backlight mechanism and image processing techniques to read and detect counterfeit for one Ringgit and five Ringgit Malaysian banknotes. The developed handheld banknote reader used visual type sensor to capture banknote image, passed to raspberry pi controller to perform image processing on banknote value and the extracted watermarks features. The developed image processing algorithm will trace out the region of interests: 1)see-thru windows, 2)Crescent and Star, 3)Perfect see though register and detect the watermarks features accordingly. The processed result will be passed back to the handheld banknote reader and broadcast on an attached mini speaker to aid the visually impaired understand the holding banknote, whether it is a real one Ringgit, real five Ringgit or none of them. The experimental result shown by this approach able to accomplish numerous round of banknote reading attempts with successful outcomes. Confusion matrix is further employed to study the performance of the banknote reader, in terms of true positive, true negative, false positive and false negative. Details analysis had been focused on the critical false positive cases (predicted real banknote and actually is fake banknote) and false negative cases (predicted fake banknote and it is actually real banknote).", "keywords": [ "Circuit and System", "Banknote Reader", "Image Processing", "Banknote Counterfeit", "Ringgit Detector" ], "content": "1. Introduction\n\nBanknote readers are machines that are used to check whether the received banknotes are genuine or fake. These devices can be found in a variety of automated equipment, including supermarket self-check-out machines, laundromat washing machines, parking ticket paying machines, automatic fare collecting machines, public transportation ticket selling machines, and vending machines. The operating procedures for these machines’ banknotes reading devices entail inspecting the banknotes that have been entered into the machine and running a series of tests to see if they are counterfeit or not. These currency acceptors must be accurately configured for each item to be accepted since the specifications for each banknote are different.\n\nGenerally, the banknote reader suitable for Malaysian banknotes can be categorized into four processes: FEEL, LOOK, TILT and CHECK.1 Feel is defined by the banknote substrate’s quality. Polymer banknotes featuring raised print effect on the picture of the first SPB Yang di-Pertuan Agong and words made of special plastic. Look involves examining the banknote under the light of a white bulb. A three-dimensional watermark portrait will appear, as well as a perfect see-through registration and a clear window. The security thread will appear in a continuous dark-colored line. Tilt involves tilting the banknote while holding it straight. Examine means examining the security thread and the coloured glossy patch for image and colour changes. Simple equipment may be used to check the banknote, except for certain security features, the Ultraviolet light device will not cause the paper substrate to glow. Micro-letterings will be easily apparent with a magnifying lens. By using the “FEEL, LOOK, TILT, and CHECK” principle, all current Malaysian banknotes counterfeits can be identified clearly without much trouble.\n\nA person who is visually impaired has a vision problem that may not be corrected by wearing glasses. The difference between a blind person and visually impaired person is that the impaired is dim-sighted or visually challenged, not entirely blind, whereas the blind person is entirely blind.2 The challenges experienced by the visually impaired people at conducting daily-life activities, particularly in operating a business, shopping and tasks involving banknotes handling, are similar to those experienced by blind people. A visually impaired person’s banknote transaction is usually handled by their accompanying trusted business vendor or a partner. However, this scenario puts the visually impaired person in danger of being duped in restricting the commercial activities by the accompanying partner or trusted business vendor.\n\nThe Bulgarian Cash Vision team developed the ‘b-note system’,3 a banknote scanner that helps visually handicapped Bulgarians recognize Bulgarian money. They developed a tiny box scanner that employs the camera sensor of a Raspberry Pi controller to record the bill’s middle section of an image using feature extraction algorithm to detect the minimal value (specific stamped marks at one of the banknote’s corners) and the value of the banknote currency. This banknote reader is not suitable to detect Malaysian banknotes because there are no engraved indications on Malaysian banknotes.\n\nNantMobile Money Reader, developed by IPPLEX,4 allows users to aim their iOS device’s camera at a banknote and receive real-time denomination information. It accepts 21 different countries’ currencies, covering the US dollar, Singapore dollar, Australian dollar, etc. The Malaysian ringgit is also disclosed in the reader’s directory. However, this product is just an application software that allowed users to download and install physically on devices such as an iPhone, iPad, or smart tablet to use. The use of a touchscreen is inconvenient for blind and visually impaired people.\n\nTo assess Malaysian banknotes denomination, UTM researchers presented a banknote recognizer with sensor-based modality.5 The system employs an Arduino UNO as the processing component, which has a hefty physical architecture that makes it impractical for holding by consumers. Aside from that, the rule-based technique to identify the worth banknotes is intuitively established, with no classifier intervention or machine learning in the banknote interpretation. In 2018, the same researchers used Arduino Lilypad to improve the recognizer of banknote into a wearable device for identify the Malaysian Ringgit banknote.6 The TCS 34725 colour sensor data was fed into a suggested embedded decision tree classifier, which was then tested using 10-folder cross validation and compared to the k-Nearest Neighbour (k-NN) and Nave Bayesian classifiers.\n\nThe disadvantage of the Malaysian banknote readers proposed above are huge size, and no counterfeit detection. The huge size device makes it difficult to carry by visually impaired person. Therefore, the proposed Malaysian Banknote reader in this paper will relook into the embedded system design to solve the problem of the bulky size reader. Other than that, counterfeit detection will be embedded into the proposed Malaysian Banknote reader to detect the counterfeit of the banknote, safeguarding the users’ losses.\n\nIn this paper, a vision based Malaysian banknote reader has been designed to handle Malaysian banknotes for visually impaired people in order to improve the present Malaysian banknote reader and to meet the needs of visually impaired people when doing their regular business operations.\n\nDifferent values of Malaysian banknotes are having different types of watermarks, for examples RM1 and RM5 required backlight mechanism, Tilting/rotating mechanisms were necessary for the RM10 and RM20, while ultraviolet light shooting mechanisms were necessary for the RM50 and RM100. The current developed banknote reader work is focused on recognized RM1 and RM5, with backlight mechanism and corresponding image recognition techniques.\n\nThe proposed Malaysian banknotes reader’s hardware components include a microprocessor for camera control, a speaker module and illumination. The primary operating idea is that the image of the banknote is captured by a camera, transmitted to the microcontroller for image processing. The developed image processing algorithm will trace out the region of interests: 1)see-thru windows, 2) Crescent and Star, 3) Perfect see though register, from the captured images and detect the watermarks features accordingly to decide the values and counterfeit for the inserted banknote. The detection results are then played as voice message on a mini speaker embedded on the banknote reader. This banknote detection system has a success rate of up to 89% in identifying the proper banknote value and counterfeit.\n\nThe paper is well ordered in following manner. The Malaysian banknote reader system model with backlight mechanism will be briefly detailed in Section II. Section III show the proposed image processing-based RM1 and RM5 Malaysian banknotes detection algorithm. Section IV comments same experimental result and lastly in section V, conclusion is future work are presented.\n\n\n2. RM1 and RM5 banknotes reader system model\n\nThe system model for the RM1 and RM5 banknotes reading system is show in Figure 1. The banknotes detector is consisting of various parts and a slot of banknote insertion. The working principle start with the backlight platform with white light is turned on/off to captured two images of the inserted banknote, one with backlight and one with no backlight images. The two captured images are sent to microcontroller for image processing and check if the inserted banknote is a real RM1, real RM 5 or fake banknote/none of them. The results will be displayed on a speaker to allow the visually impaired person knows the holding paper notes.\n\n• Imaging tool.\n\nAn appropriate imaging tool capable of taking a perfect image of the banknote is selected, allowing the image to be processed accurately. Three types of imaging tool are surveyed. In Type 1, a Raspberry Pi 5MP camera sensor board was surveyed. The sensor itself features a fixed focus lens and a native resolution of 5 megapixels. It can capture static photos with a resolution of 2592 by 1944 pixels. In Type 2, a 5MP OV5647 Fisheye Camera Module for Raspberry Pi was surveyed. This imaging set improves optical performance and provides a clearer, sharper image as well as an integrated IR filter. However, the static photos only have a resolution of 2592 × 1944 pixels. In Type 3, a Raspberry Pi 8MP Camera Module V2 was surveyed. The Raspberry Pi Camera Module V2 is the Raspberry Pi Foundation’s new upgraded official camera board, with an ultra-high-quality 8MP (megapixel) sensor and a fixed focus lens. This V2 camera module can capture static photos at a resolution of 3280 × 2464 pixels. Type 3 imaging tool is selected to be used in this project due to the better resolution and finer focus range.\n\n• Backlight platform.\n\nThe purpose of having a backlight platform is to illuminate the banknotes from the back to aid the imaging tool captured the watermarks (see-thru windows, Crescent and Star, Perfect see though register) hidden in the real RM1 and RM5 banknotes. A custom-made therapy LED white Light with 3 dimming levels and USB powered cable had been fabricated. The maximum light intensity generated is 12000LUX and with the box size of dimension 235 mm (L) × 142 mm (W) × 16 mm (H), fit with the Malaysian RM1 and RM5 banknotes sizes.\n\n• Micro-controller.\n\nThe micro-controller is used to regulate the functionalities of embedded systems in the banknotes detection system. Two types of micro-controllers surveyed. In Type1, an Arduino was surveyed. The CPU, RAM, and ROM are all found on the Arduino board’s Micro-controller. All of the extra hardware on the Arduino Board is used for power, programming, and IO connectivity. In Type2, Raspberry Pi 4 Model B was surveyed. Raspberry Pi 4 Model B is a single-board computer, with CPU, memory, and graphics chip soldered together on a single circuit board. The Arduino clock speed is 16 MHz, while the Raspberry Pi clock speed is roughly 1.2 GHz. Raspberry Pi is ideal for writing Python-based software, but Arduino is ideal for connecting sensors and controlling LEDs and motors. The Raspberry Pi includes Bluetooth and Wi-Fi technology on board, whereas the Arduino does not have wireless connectivity. Raspberry Pi can simply connect to the internet via Wi-Fi, whereas the Arduino requires an extra module to do so. Therefore, taking into consideration of the above advantages, type 2 micro-controller, the Raspberry Pi 4 Model B is selected to be used in this project.\n\n• Speaker.\n\nThe speaker module is applied to output the voice message of the banknote values to the visually impaired person. This is because the visually impaired individual can only “hear” but not “see” the output. As such, the Mini speaker module as shown in Figure 2 is chosen. The module can be controlling using Raspberry Pi. Using a software interface, the Raspberry Pi can convert text to speech and played it on the mini speaker module. The mini speaker module has a very compact size of 5 cm × 3.5 cm (Diameter × Height), which is quite appealing because the system’s hardware should be as tiny as feasible.\n\n• Battery.\n\nThe entire system consumed up to current rating of 1.2 A and voltage rating of 5.0 V. A power bank with a 5 V output can be selected as a power source for this project. The power bank is the power source to Raspberry Pi using Type-C connectors. Raspberry Pi will supply direct power to the speaker module and imaging tools. The purpose of employing a power bank as a power source rather than a power line or socket is to produce a portable gadget that can be carried about. Furthermore, the size of the handheld banknote reader should be as compact as feasible, and cumbersome power sources should be avoided.\n\n\n3. RM1 and RM5 banknotes detection image processing algorithm\n\nThe image processing algorithm for RM1 and RM5 detection can be divided into 6 steps:\n\n• Step 1: Banknote image acquisition\n\nTurn off the back lamp, imaging tool takes image of the slotted in banknote and save it as image “Ba”. Turn on the back lamp, imaging tool take image of the slotted in banknote and save it as image “Bb”. Take the subtraction of image “Bb” and image “Ba” and save it as image “Bc”. A sample set of the RM1 banknote (image “Ba”, “Bb”, and “Bc”) is shown in Figure 3 below.\n\n• Step 2: Image pre-processing\n\nImprove the image quality and reduce image noise by converting image “Bb” from RGB colour to grey scale colour.7\n\nThe two sub-steps below applied for image preprocessing:\n\n1. Resize image\n\nCertain images capture by the imaging tool and pass to the image processing tasks are in different sizes, these images should be standardized in size. Resize all input images (Ba and Bb) to standard size images using the below equation:\n\n2. Remove image noise\n\nUsing Gaussian Blur function image Processing method8 to remove the unwanted noise on images “Ba” and “Bb”. A sample image “Ba” of RM1 is shown in Figure 4, on the original image and the Gaussian Blur converted image.\n\n• Step 3: Songket/Hornbill clear window detection\n\nDetect the clear window of RM1 or RM5 Using Mask detection algorithm.9 HSV colour space is more often used in computer vision owing to its superior performance compared to RGB colour space in varying illumination levels. Thresholding and masking is done in HSV colour space. Figure 5 illustrates Hue, Saturation, Value (HSV) colour model and Figure 6, shows both the original image of RM1 and the converted image in HSV.\n\nSpecify the upper and lower bounds of the pixel’s values in the captured images. Figure 7 shown the track bars in python programming used to detect the features in images “Ba” and “Bb”. The set track bars HSV values will be used for the overall banknote detection later on. Figure 8 shown the original image for RM1 and its corresponding mask image. Figure 9 shown original image for RM5 and its corresponding mask image respectively.\n\nIf neither “Songket” nor “Hornbill” clear window is detected, then “the banknote is neither 1 Ringgit nor 5 Ringgit”.\n\n• Step 4: Three Regions of interest detection\n\nDetect the three regions of interest, namely: Region1 (for transparent see thru window), Region 2 (for crescent and star) and Region 3 (for see-thru register). If clear window (white area in the red box Mask image as shown in Figure 8 for RM1 and Figure 9 for RM5) is detected, in the same area of original image (image “Bb”):\n\ni Detect Regions of interest in RM1\n\nSearch for the biggest and brightest/whitest bounded object, mark it as Region 1 (preparation for “Songket” searching in Step 5). Then in the same clear window area of image “Bb”, search for the second biggest and brightest/whitest bounded object, mark it as Region 2 (preparation for “Crescent and Star” object pair searching in Step 5).\n\nIf Region 2 fall on the left side of the Y-axis symmetrical centreline of Region 1, then locate Region 3 at the right side with respect to the Y-axis symmetrical centreline of Region 1, by an area of ½ Region 1’s horizontal length in square’s dimension. Else if Region 2 fall on the right side of the Y-axis symmetrical centreline of Region 1, then locate Region 3 at the left side with respect to the Y-axis symmetrical centreline of Region 1, by an area of ½ Region 1’s horizontal length in square’s dimension.\n\nDue to the reason that user might slot in banknotes into the banknote reader in different direction, the four possibilities of correct detected 3 Regions of interests for the slot in banknotes are shown in Figure 10 below.\n\nThe reason that Region 2 is not similar size with Region 3 is because in RM1’s banknote design, portion numeric text (“1”) of the see-thru register fall in Region 3 might be clipped, rendering the watermark undetected if similar Region 2’s dimension is used for locating Region 3. Hence Region 3’s area should be assigned slightly bigger than Region 2.\n\nii Detect Regions of interest in RM5:\n\nSearch for the biggest and brightest/whitest bounded object, mark it as Region 1 (preparation for “Hornbill” searching in Step 5). Then in the same clear window area of image “Bb”, search for the second biggest and brightest/whitest bounded object, mark it as Region 2 (preparation for “Crescent and Star” object pair searching in Step 5).\n\nIf Region 2 fall on the left side of the Y-axis symmetrical centreline of Region 1, then locate Region 3 at the right side with respect to the Y-axis symmetrical centreline of Region 1, by an area of ½ Region 1’s horizontal length in square’s dimension. Else if Region 2 fall on the right side of the Y-axis symmetrical centreline of Region 1, then locate Region 3 at the left side with respect to the Y-axis symmetrical centreline of Region 1, by an area of ½ Region 1’s horizontal length in square’s dimension.\n\nDue to the reason that user might slot in banknotes into the banknote reader in different direction, the four possibilities of correct detected 3 Regions of interests for the slot in banknotes are shown in Figure 11 below.\n\nThe reason that Region 2 is not similar size with Region 3 is because in RM5’s banknote design, portion numeric text (“5”) of the see-thru register fall in Region 3 might be clipped, rendering the watermark undetected if similar Region 2’s dimension is used for locating Region 3. Hence Region 3’s area should be assigned slightly bigger than Region 2.\n\niii Synchronize Regions of interest for better watermark detection in Step 5:\n\n– Convert Possibility 2 case into Possibility 1 case\n\nIF Region 1’s Y-coordinates > Region 2’s Y-coordinates\n\nAND Region 2’s X-coordinates > Region 3’s X-coordinates,\n\nTHEN “flipped image “Bb” horizontally, identify Region 1, 2 and 3 again using step (i) or step (ii) above”.\n\n– Convert Possibility 3 case into Possibility 1 case\n\nIF Region 1’s Y-coordinates < Region 2’s Y-coordinates\n\nAND Region 2’s X-coordinates < Region 3’s X-coordinates,\n\nTHEN “flipped the image “Bb” vertically, identify Region 1, 2 and 3 again using step (i) or step (ii) above”.\n\n– Convert Possibility 4 case into Possibility 1 case\n\nIF Region 1’s Y-coordinates < Region 2’s Y-coordinates\n\nAND Region 2’s X-coordinates > Region 3’s X-coordinates,\n\nTHEN “Performs image 180° rotation on the image “Bb”, identify Region 1, 2 and 3 again using step (i) or step (ii) above”.\n\n• Step 5: Watermarks detection\n\nDetect the watermarks characteristics within each of the detected regions of interest.\n\ni Region 1 detection:\n\nNoise object exclusion: Check if the total pixels within the bounded area of the region,\n\nTR = Total Pixels in the Resized Image converted in Step 2.\n\nIF condition in equation (2) is NOT FULFILLED, Region 1 object is a noise object,\n\nTHEN Output: “Region 1 watermark is not detected.”\n\nELSE IF condition in equation (2) is FULFILLED,\n\nTHEN Region 1 object is a possible watermark, proceed to the below Bounding Box measurement.\n\nBounding box measurement:\n\nAssign HR1 as the height of the Region 1 bounding box WR1 as the width of the Region 1 bounding box (as shown in Figure 12 below).\n\nMeasure Region 1 bounding box’s height to width\n\nRegion 1 decision:\n\nIF ThR1,RM1(min) < HR1/WR1 < ThR1,RM1(max), THEN Output: “Region 1’s watermark for RM1 is detected.”\n\nELSE IF ThR1,RM5(min) < HR1/WR1 < ThR1,RM5(max),\n\nTHEN Output: “Region 1’s watermark for RM5 is detected.”\n\nELSE Output: “Region 1’s watermark is not detected.” where\n\n– ThR1,RM1(min) is the minimum threshold of RM1’s “Songket” height to width ratio\n\n– ThR1,RM1(max) is the maximum threshold of RM1’s “Songket” height to width ratio.\n\n– ThR1,RM5(min) is the minimum threshold of RM5’s “Hornbill” height to width ratio.\n\n– ThR1,RM5(max) is the maximum threshold of RM5’s “Hornbill” height to width ratio.\n\nii Region 2 detection:\n\nCompare the colour intensity of the Crescent and Star’s pixels in image “Ba” and image “Bb” (sample of RM1 and RM5 Crescent and Star are shown in Figure 13).\n\nIF “Region 1’s watermark for RM1 is detected” AND ThR2,RM1(min) <\|Blue component for sampled pixel of Crescent and Star in image “Ba” - The same coordinate sampled pixel of Crescent and Star in image “Bb”\|< ThR2,RM1(max),\n\nTHEN Output: “Region 2 watermark for RM1 is detected.”\n\nELSE IF “Region 1’s watermark for RM5 is detected” AND ThR2,RM5(min) <\|Green component for sampled pixel of Crescent and Star in image “Ba” - The same coordinate sampled pixel of Crescent and Star in image “Bb”\|< ThR2,RM5(max),\n\nTHEN Output: “Region 2 watermark for RM5 is detected.”\n\nELSE Output: “Region 2’s watermark is not detected.” where\n\n– ThR2,RM1(min) is the minimum threshold of the acceptable colour intensity change of RM1’s “Crescent and Star” between the banknote image captured with backlight On (“Bb”) and backlight Off (“Ba”).\n\n– ThR2,RM1(max) is the maximum threshold of the acceptable colour intensity change of RM1’s “Crescent and Star” between the banknote image captured with backlight On (“Bb”) and backlight Off (“Ba”).\n\n– ThR2,RM5(min) is the minimum threshold of the acceptable colour intensity change of RM5’s “Crescent and Star” between the banknote image captured with backlight On (“Bb”) and backlight Off (“Ba”).\n\n– ThR2,RM5(max) is the maximum threshold of the acceptable colour intensity change of RM5’s “Crescent and Star” between the banknote image captured with backlight On (“Bb”) and backlight Off (“Ba”).\n\niii Region 3 detection:\n\nConvert Region 3 in image “Ba” to Black and White image, name the new image as image “WBa”.\n\nConvert Region 3 in image “Bb” to Black and White image, name the new image as image “WBb”.\n\nDetect the numerical “1” or “5” in “WBa” and “WBb” using PyTesseract,10,11 an OCR (optical character recognition) tool for python, which is the wrapper for Tesseract,12 a free OCR engine sponsored by Google since 2006.\n\nIF numerical “1” detected in image “WBb” AND not detected in image “WBa” (sample as shown in Figure 14a),\n\nTHEN Output: “Region 3 watermark for RM1 is detected.”\n\nELSE IF numerical “5” detected in image “WBb” AND not detected in image “WBa” (sample as shown in Figure 14b),\n\nTHEN Output: “Region 3 watermark for RM5 is detected.”\n\nELSE Output: “Region 3’s watermark is not detected.”\n\n• Step 6: Decision making\n\nApply fuzzy logic, T norms are used with AND connectors to make decision. The rules are set with at least 2 watermarks detected, only the banknote value is conforming and considered real. The fuzzy rules are set as below.\n\n1. FOR 1 RINGGIT.\n\n– IF “Songket” clear window AND its corresponding Region 1, Region 2 AND Region 3 watermarks are detected, THEN the banknote is a REAL 1 RINGGIT.\n\n– IF “Songket” clear window AND its corresponding Region 1 AND Region 2 watermarks are detected, THEN the banknote is a REAL 1 RINGGIT.\n\n– IF “Songket” clear window AND its corresponding Region 1 AND Region 3 watermarks are detected, THEN the banknote is a REAL 1 RINGGIT.\n\n– IF “Songket” clear window AND its corresponding Region 2 AND Region 3 watermarks are detected, THEN the banknote is a REAL 1 RINGGIT.\n\n2. FOR 5 RINGGITS.\n\n– IF “Hornbill” clear window AND its corresponding Region 1, Region 2 AND Region 3 watermarks are detected, THEN the banknote is a REAL 5 RINGGITS.\n\n– IF “Hornbill” clear window AND its corresponding Region 1 AND Region 2 watermarks are detected, THEN the banknote is a REAL 5 RINGGIT.\n\n– IF “Hornbill” clear window AND its corresponding Region 1 AND Region 3 watermarks are detected, THEN the banknote is a REAL 5 RINGGIT.\n\n– IF “Hornbill” clear window AND its corresponding Region 2 AND Region 3 watermarks are detected, THEN the banknote is a REAL 5 RINGGIT.\n\n3. FOR NOT A REAL BANKNOTE\n\n– IF clear window is NOT detected, THEN the banknote is NOT a REAL BANKNOTE.\n\n– IF Clear window is detected AND Region 1, 2 AND 3 watermarks are NOT detected, THEN the banknote is NOT a REAL BANKNOTE.\n\n– IF Clear window is detected AND ONLY Region 1 watermark is detected, THEN the banknote is NOT a REAL BANKNOTE.\n\n– IF Clear window is detected AND ONLY Region 2 watermark is detected, THEN the banknote is NOT a REAL BANKNOTE.\n\n– IF Clear window is detected AND ONLY Region 3 watermark is detected, THEN the banknote is NOT a REAL BANKNOTE.\n\n\n4. Experiment result\n\nThe prototype of RM1 and RM5 banknote reader is constructed, as shown in Figure 15. The dimension for the banknote reader prototype is 235 mm (Length) × 142 mm (Width) × 135 mm (Height).\n\nSongket area in the real RM1 banknote is measured with dimension of 25 mm × 35 mm = 875 mm2. The whole piece of RM1 banknote is with dimension 120 mm × 65 mm = 7,800 mm2. Therefore, PR1 for RM1 is 11.22% or 0.1122. Hornbill area in the real RM5 banknote is measured with dimension of 25 mm × 40 mm = 1,000 mm2. The whole piece of RM5 banknote is with dimension 135 mm × 65 mm = 8,775 mm2. Therefore, PR1 for RM5 is 11.40% or 0.1140. Since the banknote reader is shared among RM1 and RM5 detection, hence the minimum PR1 among the two is selected, and rounded to 0.11.\n\nTR is Total Pixels in the Resized Image converted in Step 2, TR = 250 × 450 = 112,500 pixels. Hence, in step 5 Noise Object Exclusion part, any object with bounding box region smaller than 0.11 × 112,500 = 12,375 pixels will not be considered as Region 1.\n\nThe measured height of Songket’s pattern in RM1 banknote is 35 mm and the width of Songket’s pattern in RM1 banknote is 20 mm. Therefore, the height to width ratio of Songket’s pattern in RM1 banknote is 1.75. The measured height of Hornbill’s pattern in RM5 banknote is 43 mm and the width of Hornbill’s pattern in RM1 banknote is 23 mm. Therefore, the height to width ratio of Hornbill’s pattern in RM5 banknote is 1.87. To better classify RM1 and RM5 from one another, for RM1, ThR1,RM1(min) is set to 1.69 and ThR1,RM1(max) is set to 1.81; whereas for RM5, ThR1,RM5(min) is set to 1.82 and ThR1,RM5(max) is set to 1.93. Such setting is with the best tolerance gap to classify the two types of banknotes effectively. To get ThR2,RM1, 100 different real banknotes of RM1s’ images were captured for 100 pairs of image “Bb” (backlight On) and image “Ba” (backlight Off). The Blue colour intensity value on the Crescent and Star’s sampled pixels were recorded and the difference between image “Bb” and image “Ba” were calculated and tabulated in the plots of no. of attempts vs. \|Blue colour intensity difference between image “Bb” and image “Ba” \|as shown in Figure 16. From Figure 16, it is shown that most occurrence happened in between Blue colour intensity value of 112 to 131. Hence ThR2,RM1(min) is set to 112 and ThR2,RM1(max) is set to 131.\n\nDifference between Image “Bb” and Image “Ba”\|) for RM1\n\nTo get ThR2,RM5, 100 different real banknotes of RM5s’ images were captured for 100 pairs of image “Bb” (backlight On) and image “Ba” (backlight Off). The Green colour intensity value on the Crescent and Star’s sampled pixels were recorded and the difference between image “Bb” and image “Ba” were calculated and tabulated in the plots of no. of attempts vs. \|Green colour intensity difference between image “Bb” and image “Ba”\| as shown in Figure 17. From Figure 17, it is shown that most occurrence happened in between Green colour intensity value of 114 to 135. Hence ThR2,RM5(min) is set to 114 and ThR2,RM5(max) is set to 135.\n\nIn step 3 of the image processing algorithm, if the clear window of a songket (for 1 Ringgit) or a hornbill (for 5 Ringgit) can be detected, the banknote is genuine; otherwise, it is counterfeit. In mask, the HSV values of the colour that are filtered out. Figures 18–21 illustrate the test run for some real and fake Malaysian banknotes. Experimental test was carried out with 100 pieces of real RM1, 100 pieces of real RM5 banknotes, 100 pieces of fake RM1 and 100 pieces of fake RM5 banknotes respectively revealed that the proposed banknote reader achieved around 99% accuracy for RM1 detection and around 78% accuracy for RM5 detection. The success rate of this system is up to 89% in recognizing the correct banknote value. From experimental test the threshold value of the acceptable colour intensity changes between the banknote image captured with and without backlight for RM1 (THB) from 41 to 57 and for RM5 (THG) from 60 to 78.\n\nThe total time for the banknote reader to complete 100 pieces of real RM1 banknotes detection is 1,148 seconds. Therefore, on average, the time required for one cycle of the banknote reader to capture in related banknote images, send to microcontroller to perform image processing and output the results on a speaker is 11.48 seconds.\n\nAmong the tested banknotes, for RM1, all the 100 pieces of the real banknotes and the 98 pieces of fake banknotes detected correctly. For RM5, 56 pieces of the real banknotes and all the 100 pieces of the fake banknotes detected correctly. To probe deep in to the failed banknote detection cases, confusion matrix is adopted.13 The four possible outcomes for the banknote’s detection scenario are diagnosed as list in Table 1 and Table 2 for RM1 and RM5 respectively.\n\nNoteworthy attentions are placed on False Positive and False Negative cases, because these two cases may cause the visually impaired person losing credits in their business. For RM 1 detection, 2 banknotes detection cases, related to False Positive class and none cases related to False Negative class. Further analysed on these 2 False Positive cases, it is found that the fake RM1 banknotes were not placed properly into the Malaysian banknote reader (center of the banknote slot) and the Malaysian banknotes reader had mistreated some other areas on the corresponding fake banknote as the three Region of interest area (as shown in Figure 22), and this further identified the fake RM1 as the real RM1. To overcome this problem, normalized sizes were assigned on RM1 and RM5 at the Step 2 Algorithm (resizing image portion) to better locking the three Region of interest area.\n\nFor RM5 detection, 44 cases related to False Negative class and none of the case relate to False Positive class. Further probed on these 44 False Negative Class cases, it is found out that majority of the captured “Bb” images were not fully covered, as shown in Figure 23. The slotted RM5 banknotes cannot fully picture by the imaging tool, causing some of the regions of interest on the inserted banknotes (especially Region 2 and Region 3) cannot be detected. This is due to the size of the RM5 is much bigger compare to RM1. To overcome this problem, imaging area for the inserted banknote should be increased to cover the full banknote’s image. However, with existing imaging tool, this might need to be tolerance with a longer focal length with bigger size of banknote reader. Another alternative is to search for a wide view imaging tool to replace the current imaging tool for optimizing the current Malaysian Banknotes Reader’s size.\n\n\n5. Conclusions\n\nA Malaysian banknote reader employing image processing techniques was developed for visually impaired person to read and identify counterfeit on one Ringgit and five Ringgit Malaysian banknotes. The proposed portable banknote reader employed a visual type sensor to capture the inserted banknote image, sent to a Raspberry Pi controller for extracting the banknote’s watermarks and identify the banknote’s value. The detection result will be broadcasted on a mini speaker mounted on the banknote reader to help the visually impaired comprehend if it is a real one Ringgit, real five Ringgit, or none of them. The experimental results had proven that the proposed banknote reader is capable of completing several rounds of successful tries. In future, tilting/rotating mechanism and Ultraviolet light shooting mechanism can be embedded on the banknote reader to allow the visually impaired persons to cover the full series of Malaysian banknotes reading capabilities. The Malaysian banknote reader can also be expanded to support additional foreign currencies reading in the future. Aside from that, the size of the banknote reader can be improved, as well as the classifier intervention in the banknote interpretation. These issues will be resolved 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.", "appendix": "References\n\nBank Negara Malaysia: Current Banknote Series.2021.Reference Source\n\nTan CJ, Wong WK, Min TS: Malaysian Banknote Reader For Visually Impaired Person. 2020 18th IEEE Student Conference on Research and Development (SCOReD), 27-29 September, 2020, Universiti Tun Hussein Onn Malaysia (UTHM), Malaysia, paper ID 1570650473. 2020; pp. 74–79.\n\nStavri N: Avaliable Now: Banknote Reader (b-Reader) for Visually Impaired People. Imagga Technologies Blog. 2017. Reference Source\n\nIPPLEX: NantMobile Money Reader. AppAdvice LLC. 2020. Reference Source\n\nMohamed A, Ishak MI, Buniyamin N: Development of a Malaysian Currency Note Recognizer for the Vision Impaired. 2012 Spring Congress on Engineering and Technology, Xian. 2012; pp. 1–4.\n\nNurul Fathiah G, Muhammad A, Mohd Najeb J, et al.: Wearable Device for Malaysian Ringgit Banknotes Recognition Based on Embedded Decision Tree Classifier. J. Telecommunication, Electronic and Computer Eng. 2018; 10(1): 129–137.\n\nAlnowaini G, Alabsi A, Ali H: Yemeni Paper Currency Detection System. 2019 First International Conference of Intelligent Computing and Engineering (ICOICE), 2019. 2019.\n\nTowards Data Science: Image Preprocessing.2020.Reference Source\n\nCicolani J: Beginning Robotics with Raspberry Pi and Arduino. Springer Science and Business MediaLLC. 2018; 2018. Publisher Full Text\n\nDario R: Read Text from Image with One Line of Python Code. Towards Data Sci. 2019.Reference Source\n\nImtiaz H: A Beginners Guide to Tesseract OCR Using Pytesseract. Gitconnected. 2020.Reference Source\n\nVincent L: Announcing Tesseract OCR. The Official Google Code Blog. 2006.Reference Source\n\nPehrsson A, Gunnar T, Engblom C, et al.: Roadside oral fluid testing: Comparison of the results of Drugwipe 5 and Drugwipe Benzodiazepines on-site tests with laboratory confirmation results of oral fluid and whole blood. Forensic Science International. 2008; 175: 140–148. Publisher Full Text" }	[ { "id": "120762", "date": "25 Jan 2022", "name": "Neeraj Dhanraj Bokde", "expertise": [ "Reviewer Expertise Data science", "deep learning", "time series analysis", "computer vision" ], "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 presented an image processing technique for two Malaysian banknotes detection using a microcontroller-based mechanism. The research can have a good impact on society and is worth investigating, however, the research component of the manuscript is minimal. The authors may consider the following comments to revise the manuscript:\nThe research contribution and novelty in terms of image processing are not well discussed in the manuscript. It is advised to discuss the methodology proposed by the authors to solve the problem statement.\n\nBesides, it is very crucial to compare the performance of the proposed methodology with the state-of-the-art methods and evaluate its performance in terms of different error metrics.\n\nThe author tried different IF-ELSE situations to detect the currency, however, the presentation of the same is very poor in the manuscript. It is advised to discuss these things in the form of block diagrams and Psuedo codes with proper formatting.\n\nThe quality of figures in terms of resolution and aesthetics are very poor. It is advised to revise all figures with improved qualities.\n\nThe manuscript in the present form is like a project report, and not suitable for a research article. It is recommended to revise the manuscript with an improved case study that will discuss the research contributions in more detail than the hardware-software interface systems.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "8065", "date": "13 Apr 2022", "name": "Wai Kit Wong", "role": "Author Response", "response": "1. Reviewer: The research contribution and novelty in terms of image processing are not well discussed in the manuscript. It is advised to discuss the methodology proposed by the authors to solve the problem statement. Author: The research contribution and novelty for this work is a new model of Malaysian banknotes counterfeit detection using watermarks image processing analysis and classifier with fuzzy logic. In particular, the three watermarks features: 1) see-thru windows, 2) Crescent and Star 3) Perfect see though register will be extracted from the one Ringgit and five Ringgit banknotes to determine the real/fake in a dynamic environment with ambiguous, distorted or imprecise banknotes images. Added in Second last paragraph of Section 1. A new Figure 4 is added with methodology proposed by the authors to solve the problem statement of banknote counterfeit detection. The detail of the algorithm is explained in Section 3. 2. Reviewer : Besides, it is very crucial to compare the performance of the proposed methodology with the state-of-the-art methods and evaluate its performance in terms of different error metrics. Author: Comparison of the proposed banknote reader detection accuracy and processing speed is done with three state-of-the-art methods: 1) VGG16 model using 2D Convolution Layer (32 neural) at TensorFlow's Keras API [14], 2) MobileNet model using RMSprop Loss Function (learning_rate=0.0001) at TensorFlow's Keras API [15] and 3) Fuzzy Logic Based Perceptual Image Hashing Algorithm [16]. Experimental setup for method 1: Total of one hundred RM1 banknotes and one hundred RM5 banknotes are captured as the dataset for training the model. VGG16 model using 2D Convolution Layer (32 neural) at TensorFlow's Keras API being trained and tested with 100 real RM1, 100 real RM5, 100 fake RM1 and 100 fake RM5. The average time to load the model and build up the interpreter objects (Training time) was 60 seconds with batch size=32 and epochs=20 and the average inference time while modeling detecting banknote (Testing time) was 1 second. The test Accuracy was 60%. Experimental setup for method 2: It is understood that the model MobileNet with Loss Function RMSProp was Selected as best accuracy of about 96.80% in paper [15]. Convolutional Neural Networks using MobileNet model with Loss Function RMSProp (0.0001) optimization technique being trained with one hundred RM1 banknotes and one hundred RM5 banknotes and tested with 100 real RM1, 100 real RM5, 100 fake RM1 and 100 fake RM5. The average time to load the model and build up the interpreter objects (Training time) was 81 seconds with batch size=32 and epochs=20 and the average inference time while modeling detecting banknote (Testing time) was 1 second. The test Accuracy was 50%. Experimental setup for method 3: following paper [16] algorithm. Fuzzy Logic Based Perceptual Image Hashing Algorithm first sorting Database using Perceptual Hashing with one hundred RM1 banknotes and one hundred RM5 banknotes and tested with 100 real RM1, 100 real RM5, 100 fake RM1 and 100 fake RM5. The average time to load the model and build up the interpreter objects (test 100 banknotes) was 130 seconds and the average inference time while detecting banknote (Per banknote) was 1.30 seconds. The test Accuracy was 42%. The accuracy and required processing time for the experimented methods were summarized in Table 3. By comparing the above works on different Ringgit recognizers, it is observed that Fuzzy logic based light intensity variation watermark detection algorithm required longest processing time (both training and detection times for details watermark features extraction), however it has the best accuracy in detecting fake banknotes (minimum false positive and false negative cases) among the compared state-of-the-art methods. The VGG16 model, MobileNet model and Fuzzy Logic Based Perceptual Image Hashing Algorithm managed to be trained and detected the banknotes currency faster but with limitation of unable to accurately detecting fake banknotes (high false positive and false negative cases recorded) due to no watermarks detection consideration. This write-up is added in Section 4 Experimental Session. 3. Reviewer : The author tried different IF-ELSE situations to detect the currency, however, the presentation of the same is very poor in the manuscript. It is advised to discuss these things in the form of block diagrams and Psuedo codes with proper formatting. Author: The algorithm had been revised according to reviewers’ comments. IF-ELSE statement pseudocodes are well occupied in Section 3’s algorithm Step 4 onwards. General block diagram for the image processing algorithm well defined in Figure 4. Here in Section 3, the algorithm steps further details up the operation sequence of the banknote watermarks counterfeit detection. 4. Reviewer: The quality of figures in terms of resolution and aesthetics are very poor. It is advised to revise all figures with improved qualities. Author: All figures revised with improved qualities. 5. Reviewer : The manuscript in the present form is like a project report, and not suitable for a research article. It is recommended to revise the manuscript with an improved case study that will discuss the research contributions in more detail than the hardware-software interface systems. Author: More case studies were reviewed as per Reviewer 2 comment 20 as well. Whole Section 1 Introduction revised accordingly." } ] }, { "id": "122605", "date": "21 Feb 2022", "name": "Haidi Ibrahim", "expertise": [ "Reviewer Expertise Digital image processing and analysis. Digital signal processing and analysis." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere are some suggestions for the improvements of the manuscript (page numbers refer to the pdf version of the article):\nTo highlight the contribution of the work, it would be better to add terms “counterfeit detection” and “visually impaired person” to the title. For example, “One- and five-Ringgit Malaysia banknotes reader with counterfeit detection for visually impaired person using backlight mechanism and image processing techniques”.\n\nAbbreviations should be defined properly. For examples, what are SPB, IPPLEX and UTM on page 3?\n\nOn page 3, 2nd last paragraph, the authors did mention that the available Malaysian banknote readers are huge in size. However, the system developed, as shown in Figure 15, also seems bulky. (It is also better if the labels and dimensions of Figure 15 are provided). Besides, if the authors want to show that the proposed system has advantage in terms of size, it would be nicer if there is a table to compare the size of the proposed system with the available systems.\n\nIn Section 1, it would be better if the authors could provide an introduction to RM1 and RM5 banknotes. Better to provide figure(s) with labels (e.g., songket, hornbill) for this purpose.\n\nBetter to treat divisions in Section 2 as subsections, and given number, such as Section 2.1, Section 2.2, etc.\n\nIn Section 2, it would be better to discuss about the micro-controller first before the other components. Thus, it would be clearer, for example, why the authors are considering the use of Raspberry Pi cameras.\n\nSection 3 is mostly in point form. A better presentation is needed. The authors could describe the methods in paragraphs, and explain with the help of figures, flowchart, or pseudocodes.\n\nThe method in Section 3 is not clear. For example, on page 6, in Figure 3, it is shown image “Bc”, but when image “Bc” is used for banknotes detection it is not mentioned clearly.\n\nPage 7, 2nd line. How can converting the RGB to grayscale image help in improving image quality and reduce image noise?\n\nIn equation (1), why are the input images (Ba and Bb) located on the left side of the equation, and not on the right side? Usually, the left side is for the output.\n\nIn equation (1), better to mention the values of “width”, “height”, and “no. RGBchannels” used in this work.\n\nFor the Gaussian Blur function, what is the filter size, or the standard deviation used?\n\nFigure 4 does not show the Gaussian Blur converted image, but the image after grayscale conversion.\n\nIf the image is already converted to grayscale image, why should we convert it to HSV space? Or is the conversion from the RGB image? If this is from the RGB image, then why do we need to convert the image into grayscale? Besides, why we do not use “Bc” for this purpose?\n\nIf Figure 5 is taken somewhere, a proper permission should be asked to re-publish this figure. Citation should be given in the figure’s caption.\n\nFigure 7 shows how the thresholding process is done by using track bars. The question is, are these threshold values fixed for all input images, or need to be changed, depending to the input image? If it is not fixed, then the method is not automated, and the user needs to set it every time a banknote is input to the system. Besides, is this process suitable for a visually impaired person?\n\nPage 9, descriptions for part (i) and part (ii) are similar to each other.\n\nFigure 15 should also label where the slot to input the banknote to the system, and where the banknote will exit from the system.\n\nThe system has a speaker. What is the sound/notification given to the user? Some description on how to set up this sound/notification should be given.\n\nIn Section 1, more review on the related works should be done. For example:\n\nhttp://eprints.utar.edu.my/4289/1/17ACB01383_FYP.pdf https://www.hindawi.com/journals/js/2022/4505089/ https://iopscience.iop.org/article/10.1088/1742-6596/2089/1/012008/meta\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? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8066", "date": "13 Apr 2022", "name": "Wai Kit Wong", "role": "Author Response", "response": "1. Reviewer : To highlight the contribution of the work, it would be better to add terms “counterfeit detection” and “visually impaired person” to the title. For example, “One- and five-Ringgit Malaysia banknotes reader with counterfeit detection for visually impaired person using backlight mechanism and image processing techniques”. Author: The title of the article is revised to “One- and Five-Ringgit Malaysia banknotes reader with counterfeit detection for visually impaired person using backlight mechanism and image processing techniques”. 2. Reviewer : Abbreviations should be defined properly. For examples, what are SPB, IPPLEX and UTM on page 3? Author : The abbreviations are well defined: SPB in Section 1 INTRODUCTION Paragraph 2 is replaced with full form “Seri Paduka Baginda”. IPPLEX is a company develops application services enabled by fixed and wireless IP networks and the convergence of media communications. UTM in Section 1 INTRODUCTION Paragraph 6 is added with full form “Universiti Teknologi Malaysia”. 3. Reviewer 2: On page 3, 2nd last paragraph, the authors did mention that the available Malaysian banknote readers are huge in size. However, the system developed, as shown in Figure 15, also seems bulky. (It is also better if the labels and dimensions of Figure 15 are provided). Besides, if the authors want to show that the proposed system has advantage in terms of size, it would be nicer if there is a table to compare the size of the proposed system with the available systems. Author: The proposed system is not having advantage in terms of size compared to some available Malaysian banknote reader in the market. Therefore, the issue of bulky size reader will not be discussed in the paper. Labels and dimensions of Figure 17 are provided 235 mm (Length) x142 mm (Width) x135 mm (Height), for readers to justify the banknote reader size themselves. 4. Reviewer : In Section 1, it would be better if the authors could provide an introduction to RM1 and RM5 banknotes. Better to provide figure(s) with labels (e.g., songket, hornbill) for this purpose. Author: An introductory paragraph of RM1 and RM5 banknotes is added in Section 1 INTRODUCTION Paragraph 3 (after \"FEEL, LOOK, TILT, and CHECK\" principle), together with Figure 1 with labels (songket, hornbill): The RM1 and RM5 Malaysian Banknotes are shown in Figure 1a and Figure 1b respectively. These banknotes are made from polymer substrate and with security features/watermarks with label 1-8. In sequence: 1) Intaglo, 2) Clear Window, 3) Shadow Image, 4) Crescent & Star Non-transparent window, 5) Perfect see thru Register, 6) Micro-Lettering, 7) Two color fluorescent element for Perfect see-thru, 8) UV BNM Text and Logo. Among the eight types of watermarks, there are three types of watermarks that related to the use of front-backlight mechanism ( 1)see-thru windows, 2)Crescent and Star non transparent window, 3)Perfect see though register). These three types of watermarks will be selected for the proposed prototype to run test. Added Figure 1 (a) RM1 (b) RM5 and their Corresponding watermarks. 5. Reviewer : Better to treat divisions in Section 2 as subsections, and given number, such as Section 2.1, Section 2.2, etc. Author: Divisions in Section 2 are treated as subsections e.g: Section 2.1 Micro-controller, Section 2.2 Imaging Tools, Section 2.3 Backlight Platform, Section 2.4 Speaker and Section 2.5 Battery. 6. Reviewer : In Section 2, it would be better to discuss about the micro-controller first before the other components. Thus, it would be clearer, for example, why the authors are considering the use of Raspberry Pi cameras. Author: Revised Section 2, micro-controller is discussed first (moved to Section 2.1) before the other components. 7. Reviewer : Section 3 is mostly in point form. A better presentation is needed. The authors could describe the methods in paragraphs, and explain with the help of figures, flowchart, or pseudocodes. Author: Revised as per in Reviewer 1’s Comment 3. 8. Reviewer : The method in Section 3 is not clear. For example, on page 6, in Figure 3, it is shown image “Bc”, but when image “Bc” is used for banknotes detection it is not mentioned clearly. Author: Image “Bc” and its related definition is removed from the text to avoid confusion. 9. Reviewer : Page 7, 2nd line. How can converting the RGB to grayscale image help in improving image quality and reduce image noise? Author: The numeral printed on the banknote can be easily traced out by optical character recognition software in grayscale format. 10. Reviewer : In equation (1), why are the input images (Ba and Bb) located on the left side of the equation, and not on the right side? Usually, the left side is for the output. Author: The equation actually carried the meaning of the new Ba and Bb images will be resized to the desired width; height and number of RGBchannel after execution. 11. Reviewer : In equation (1), better to mention the values of “width”, “height”, and “no. RGBchannels” used in this work. Author: The values of “width”, “height”, and “no. RGBchannels” used in this work are selected in Section 4 Paragraph 4 (TR = 250(width) x 450(height) = 112,500 pixels). The no. RGBchannels is 3. Added in Section 4 Paragraph 4. 12. Reviewer : For the Gaussian Blur function, what is the filter size, or the standard deviation used? Author: The filter size, or the standard deviation used for the Gaussian Blur function is 5x5 pixels. Such filter removed outlier 5x5 pixels that may be noise elements in the image. Added in Section 4 Paragraph 1. 13. Reviewer : Figure 4 does not show the Gaussian Blur converted image, but the image after grayscale conversion. Author: Figure 6 added a Gaussian Blur converted image. 14. Reviewer : If the image is already converted to grayscale image, why should we convert it to HSV space? Or is the conversion from the RGB image? If this is from the RGB image, then why do we need to convert the image into grayscale? Besides, why we do not use “Bc” for this purpose? Author: To extract region 1 the RGB image “Bb” needs to convert to a Gaussian Blur image and further convert to an HSV space image. However, to extract region 3 the RGB image “Ba” and “Bb” need to convert to grayscale images. 15. Reviewer : If Figure 5 is taken somewhere, a proper permission should be asked to re-publish this figure. Citation should be given in the figure’s caption. Author: Figure 7 is taken from this paper: Vazquez Saraullo, Federico Alejandro & Larosa, Facundo & Ghignone, Ramiro & Lanzillotta, Lucas. (2019) “Diseño, implementación y ensayo de un lector de colores de bajo costo para personas ciegas y disminuidas visuals”, X Congreso de Microelectrónica Aplicada (μEA2019)At: San Martín, Buenos Aires, Argentina. Citation for Figure 7 is done and listed in the paper with citation [18]. 16. Reviewer : Figure 7 shows how the thresholding process is done by using track bars. The question is, are these threshold values fixed for all input images, or need to be changed, depending to the input image? If it is not fixed, then the method is not automated, and the user needs to set it every time a banknote is input to the system. Besides, is this process suitable for a visually impaired person? Author: Yes, the threshold values are fixed according to the banknote reader box internal environment and the front-backlight intensity. There are two set of threshold values set, one set for RM1 and another set for RM5. For RM1 the HSV value for the raspberry pi processor is fixed at Hue Min = 0 , Hue Max = 179, Sat Min= 0, Sat Max=255, Val Min=170,Val Max=255 . For RM5 the HSV value for the raspberry pi processor is fixed at Hue Min = 0 , Hue Max = 179, Sat Min=0, Sat Max=255, Val Min=205,Val Max=255 . Figure 9 (a) will show Track bars detect features in RM1 images. Figure 9 (b) will show Track bars detect features in RM5 images. This detail explanation is added in Section 3 Step 3 Second paragraph. 17. Reviewer : Page 9, descriptions for part (i) and part (ii) are similar to each other. Author: In Section 3, Step 4 description for part (i) and part (ii) are similar to each other. Part (i) is detecting Region of Interest for RM1 whereas Part (ii) is detecting Region of Interest for RM5. Hence the two parts are combined to become one part. 18. Reviewer : Figure 15 should also label where the slot to input the banknote to the system, and where the banknote will exit from the system. Author: Figure 17 revised to label where the slot to input the banknote to the system, and where the banknote will exit from the system. 19. Reviewer : The system has a speaker. What is the sound/notification given to the user? Some description on how to set up this sound/notification should be given. Author: The notification messages given to the users include: “Real one Ringgit”, “Real five Ringgit” and “Not a Malaysian banknote.” Description : Import pyttsx3 library in Python. It is a a text-to-speech conversion library in Python. The results in step 6 Decision making part will be sent to activate the text (eg. Real one Ringgit, Real five Ringgit or Not a Malaysian banknote.” The pyttsx3 command will transfer the text to speech and display at the speaker. Below is the sample of codings: engine = pyttsx3.init() engine.say(\"Real five ringgit\") engine.runAndWait() engine.stop() Added in the Section 2.4 Speaker 20. Reviewer : In Section 1, more review on the related works should be done. For example: http://eprints.utar.edu.my/4289/1/17ACB01383_FYP.pdf 1. Lee KL: Malaysia currency recognizer mobile application for visual impairment. Universiti Tunku Abdul Rahman, Malaysia. 2022. Reference Source https://www.hindawi.com/journals/js/2022/4505089/ 2. Aseffa D, Kalla H, Mishra S: Ethiopian Banknote Recognition Using Convolutional Neural Network and Its Prototype Development Using Embedded Platform. Journal of Sensors. 2022; 2022: 1-18 Publisher Full Text https://iopscience.iop.org/article/10.1088/1742-6596/2089/1/012008/meta 3. Padmaja B, Naga Shyam Bhargav P, Ganga Sagar H, Diwakar Nayak B, et al.: Indian Currency Denomination Recognition and Fake Currency Identification. Journal of Physics: Conference Series. 2021; 2089 (1). Publisher Full Text Author: The review of the THREE (3) works above are considered and added in Section 1 Paragraph 8 (After NantMobile): Convolutional Neural Networks using MobileNet model was selected by [15] in detecting Ethiopian banknotes. Convolutional Neural Networks using Canny Edge detection and multiscale template matching methods were selected by [17] in detecting Indian banknotes. Both these two models are detecting banknotes denomination and counterfeit. However, their counterfeit detection only focus on banknotes’ surface security features, like micro-lettering and only can detect single - sided of banknotes. Unlike other hidden type of watermarks, micro-lettering is easy to be printed by current high-resolution printers." } ] } ]	1	https://f1000research.com/articles/10-1098
https://f1000research.com/articles/10-223/v1	18 Mar 21	{ "type": "Study Protocol", "title": "Cross-sectional study to assess etiology and associated factors for anaemia during first trimester of pregnancy in Anuradhapura District, Sri Lanka: a protocol", "authors": [ "Gayani Amarasinghe", "Vasana Mendis", "Thilini Agampodi", "Vasana Mendis", "Thilini Agampodi" ], "abstract": "Background: Anaemia in pregnancy, which can lead to adverse maternal and fetal outcomes, is a significant global health problem. Despite Sri Lanka’s strong public health system and commitment towards prevention, maternal anaemia remains a major problem in the country. While prevention is focused on iron deficiency, detailed etiological studies on this topic are scarce. Moreover, estimates of socio demographic and economic factors associated with anaemia in pregnancy, which can provide important clues for anaemia control, are also lacking. This study aims to evaluate the hemoglobin distribution, spatial distribution, etiology and associated factors for anaemia in pregnant women in Anuradhapura, Sri Lanka. Methods: This is a cross sectional study of pregnant women in their first trimester registered for antenatal care from July to September 2019 in the Anuradhapura district. The minimal sample size was calculated to be 1866. Initial data collection has already been carried out in special field clinics for pregnant women between June to October 2019. An interviewer-administered questionnaire, a self-completed dietary questionnaire and an examination checklist were used for data collection. In addition, all participants underwent complete blood count testing. Further investigations are being conducted for predicting the etiology of anaemia based on a developed algorithm (such as high-performance liquid chromatography [HPLC] and peripheral blood film analysis). Discussion: Being the largest study on anaemia during pregnancy in a single geographical area in Sri Lanka, this study will provide important clues about geographical clustering of anaemia cases with similar etiology, associated factors and etiologies which would help to develop interventions to improve the health of pregnant women in the area. The possibility of selection bias is a potential limitation associated with the study design.", "keywords": [ "Anemia", "Pregnancy", "Sri Lanka", "Anuradhapura" ], "content": "Introduction\n\nAnaemia in pregnancy is associated with many adverse outcomes for the mothers (such as loss of productivity, heart failure and even death) as well as her offspring (such as low birth weight, anaemia and developmental problems)1–4. Despite exceptional maternal care standards and numerous nutritional interventions, anaemia in pregnancy remains a major problem in Sri Lanka5. Assuming iron deficiency is the major underlying cause of anaemia6, anaemia screening during preconception and antenatal clinics (at their initial appointment and 28 weeks), universal iron supplementation, provision of double dose iron (elemental iron 120 mg/day) for anaemic mothers, nutritional education, prophylaxis for malaria, and treatment for worm infestations have long been incorporated to Sri Lanka’s pregnancy care package7. Interventions with a lifecycle approach such as iron supplementation at schools are also in place8. Despite all these measures, maternal anaemia remains a major public health problem in certain districts of the country9–21.\n\nIn 2018, 22.8% of pregnant women in the Anuradhapura district were identified as anemic during their initial visit to an antenatal clinic and despite routine interventions for iron deficiency anaemia, the prevalence was almost triple among the second trimester women22; amounting to a severe public health problem23. This raises the question of whether etiologies other than iron deficiency are significantly responsible for anaemia in this population. Previous reports on the presence of hemoglobinopathies, heterogeneous nutrient deficiencies and hereditary disorders among Sri Lankans supports this assumption24–26.\n\nIdentifying dietary, socio economic11,17, demographic12,14,16,20, biological and behavioral factors that lead to anaemia in pregnancy will shed light on this unsolved problem as published literature exploring these factors is scarce. Even though a significant variation in prevalence between districts has been reported14,16,20 intra-district (micro-geographical) variations have not yet been studied thoroughly.\n\nThis study will explore the prevalence, distribution and etiologies of anaemia among first trimester pregnant women in the Anuradhapura district, Sri Lanka.\n\n\nMethods\n\nThis study was carried out in Anuradhapura, the largest of 25 districts in Sri Lanka. The Anuradhapura district is divided into 275 public health midwife (PHM) divisions within 22 medical officers of health (MOH) areas (Figure 1) which conduct village level antenatal clinics. Early registration for antenatal care and clinic attendance is very high (96%) in this district14.\n\nThis map depicts the position of Anuradhapura district (yellow) in the map of Sri Lanka (blue) and the area is enlarged to depict the 22 medical officer of health areas in the district. The figure has been reproduced with permission from Agampodi TC, Wickramasinghe ND, Prasanna RIR, Irangani MKL, Banda JMS, Jayathilake PMB, et al. The Rajarata Pregnancy Cohort (RaPCo): study protocol. BMC Pregnancy Childbirth [Internet]. 2020 Jun 26 [cited 2020 Nov 30];20(1):374. Available from: https://bmcpregnancychildbirth.biomedcentral.com/articles/10.1186/s12884-020-03056-x.\n\nThis cross sectional study was conducted as the baseline assessment of the Rajarata Pregnancy Cohort (RaPCo)27. This study includes the baseline assessment of etiology of anaemia in early pregnancy, supported by an evidence-based algorithm adopted and tested in a sub sample (n=200) of women selected consecutively from the same study sample. The study includes two components; the basic investigations conducted at the first special antenatal clinic held in early pregnancy (component 1) and a second assessment if further investigations were needed to identify the etiology of anaemia (component 2). Although the cohort of women were followed up until delivery, this particular study protocol is focused on identification of etiology of anaemia of first trimester pregnant women.\n\nAll pregnant women in their first trimester (less than 13 weeks pregnant) and registered in the pregnancy care programme from July to September 2019 were invited to take part in the study. There were no exclusion criteria.\n\nWe hypothesized that causes of anaemia affecting at least 10% of anemic pregnant women would be significant from a public health point of view. Therefore, the minimum sample size needed to detect causes in at least 10 % of the anemic pregnant women was calculated using the following formula28:\n\n\n\n\n\nwhere n = minimum sample size, z = level of confidence 95% – 1.96, d = precision - 2%, p = expected proportion of participants with a selected cause for anaemia in the population – 10%.\n\nWhen a 10 % nonresponse and loss to follow-up rate was applied for the minimum sample size needed for the second component the required sample size was determined as 425. Anaemia prevalence of first trimester pregnant women in the Anuradhapura district in 2018 was 22.8%29. Therefore, the number of participants needed for the first component in order to get 425 anemic pregnant women was 1866.\n\nAn invitation to participate in the study was sent to all eligible participants through their PHM. From July to October 2019, a special clinic was conducted fortnightly at each MOH area where the eligible participants were recruited consecutively. The special clinic was continued till October 2019 so that eligible participants registering with the PHM towards the end of August could also join.\n\nBaseline data collection was carried out by four teams, each led by a fully qualified MBBS doctor. Each team included several medical graduates, a nursing officer and four to six trained data collectors. All team members received three days of training and all procedures were carried out according to a manual.\n\nThree study instruments were used; an interviewer administered questionnaire on socio demographic, economic, behavioral and medical factors (filled in by data collectors), a self-report questionnaire on diet (filled in by the participants at home and returned through the public health midwives, and a form to record findings of a clinical examination conducted by a medical graduate30. All the tools were developed specifically for this study. They were validated by a panel of multidisciplinary experts including a consultant community physician, social epidemiologist, hematologist and physicians and public health midwives (PHMs) working in the area where study is conducted. All tools were pretested among 20 pregnant women with the same eligibility criteria but registered in the maternal care program prior to the establishment of the cohort. The questions were comprehendible according to pretest results with the consensus of the expert panel.\n\nThe short clinical interview was conducted to identify presence or absence of symptoms of anaemia (shortness of breath, fatigue on exertion or at rest, faintness and chest pain). Anthropometric measures (height, weight, waist circumference and hip circumference), pallor and murmurs were recorded after a clinical examination. The dietary assessment included a food frequency questionnaire, assessment of consumption frequency of selected iron rich food items and the type of usual main meal\n\nCollection of blood samples. Blood sample collection, handling, storage and analysis were conducted according to a protocol30. For all the participants, a complete blood count was performed at the time of baseline data collection during the special clinic. A slide was prepared for peripheral blood film analysis for each participant at the time of blood collection by trained laboratory technicians.\n\nA working algorithm was developed using available research evidence31–38 and hematological expert opinion to identify the etiology of anaemia (Figure 2). The algorithm was developed as performing each investigation on all anemic women was not technically and financially feasible. Considering the feasibility, the different pathways of the algorithm were tested in 200 consecutively taken blood samples representing the district in the ongoing cohort. Serum ferritin level was assessed to determine iron status in all 200 of this sub-sample in addition to analysis of peripheral blood film In anaemic pregnant women of this sample, further investigations (HPLC, serum B12, Folate levels and serum homocysteine levels) were conducted to test the algorithm.\n\nThis figure shows the evidence-based algorithm developed to guide the laboratory investigations to determine etiology of anaemia among first trimester pregnant women in the Anuradhapura district. Anaemia is determined by haemoglobin level less than 11 g/dl. Mean corpuscular volume is used to identify microcytic (MCV less than 80 fl), normocytic (MCV 80 to 95.9 fl) and macrocytic anaemia (MCV more than 96). Those who have microcytic anaemia with high red cell counts (five or more106/µl) will be tested for minor haemoglobinopathies using HPLC testing. Those who have microcytic anaemia with normal red cell counts or have normocytic anaemia with mean corpuscular haemoglobin (MCH) 27 pg or less will undergo serum ferritin testing to determine iron deficiency. A peripheral blood film of participants with normocytic anaemia with MCH more than 27 will be examined to see if there are membrane disorders. Then serum Homocysteine and ferritin will be estimated in them to determine mixed deficiency. If this etiology is not confirmed with these investigations, thyroid stimulating hormone (TSH) and liver enzymes will be tested along with ferritin assessment. A peripheral blood film will be examined in macrocytic anaemic participants. If macrocytes are round, serum Homocysteine will be estimated. If it is raised, serum folate and B12 levels will be conducted to determine which nutrient is deficient. If oval macrocytes are present, TSH, liver enzymes and serum ferritin will be assessed.\n\nAccording to the algorithm high red cell count (RCC ≥ 5106) is used to differentiate minor hemoglobinopathies from iron deficiency among microcytic anemic cases (MCV <80fl)31,32. Early iron deficiency is suspected in normocytic hypochromic anaemia (MCV 80 – 95.9 fl). Suspected etiology is confirmed through serum ferritin and High Performance Liquid Chromatography (HPLC)33. A peripheral blood film will be analyzed in those with normochromic normocytic anaemia and macrocytic anaemia (MCV ≥96 fl). Serum Homocysteine, serum B12 and folate levels will be used for confirming mixed deficiency, B12 deficiency and folate deficiency respectively34–38.\n\nWhen vitamin B12 or folate level assessment or performing HPLC was necessary according to the algorithm, participants were invited for a special hematology clinic. This clinic was conducted at the teaching hospital in Anuradhapura, the main center providing tertiary care for pregnant women in the Anuradhapura district. The invitation to participate was sent within a week following initial assessment and the appointment was booked within four weeks for serum vitamin levels assessment and according to their convenience for the thalassemia assessment. A telephone reminder was provided for all invited participants on the day prior to the scheduled appointment.\n\nSerum ferritin, Homocysteine, B12 and Folate assessments were performed in a commercial laboratory with external quality control methods. HPLC is performed at the thalassemia unit of teaching hospital, Anuradhapura. Other investigations (complete blood count, peripheral blood film analysis and liver functions) are performed in a public health research laboratory with internal and external quality control.\n\nThis study will have several important outcomes. The prevalence of anaemia in the first trimester will be assessed. This will be a valid statistic for the district as the current indicators calculated using routine data from the reproductive health management information system (RHMIS) need to be validated. Evidence on etiology of anaemia is not available for the province. This study will be valuable at the national level as large community-based studies on etiology of anaemia are scarce in Sri Lanka. Based on the outcomes the study will be able to influence maternal health policies at both national and regional level.\n\nThe study will also depict the important associations of anaemia in pregnancy, namely socio-demographic (age, ethnicity, religion, education level and economic details of the family); obstetric (period of gestation, consumption of folic acid, parity, age at each conception, outcomes and complications); medical and biological (medical conditions, regularity and length of menstrual cycle and approximate estimation of menstrual blood loss using a pictogram); contraceptive history (methods that has been used, duration of use and side effects); and pre pregnancy and inter pregnancy care (breast feeding duration, intake of iron and folate supplements during postpartum period and/or pre-pregnancy). These assessments will help to develop a bio-psychosocial model for anaemia in early pregnancy which could be applicable to other LMICs as well.\n\nThe results of the tested algorithm will guide policies and strategies for screening for anaemia which will be a benefit for future pregnant women and their families.\n\nData from the interviewer administered questionnaire will be entered directly electronically. The rest of the data including laboratory investigations will be entered by single entry technique by research assistants and 10% of data will be manually verified by the investigator (GA).\n\nAll the hard copies of questionnaires and copies of investigation reports will be stored in an access restricted place, separate from the consent forms and identification details. Only investigators will have access to these copies. The database will be password protected on a separate computer. Access is limited to the investigators.\n\nData analysis will be performed using SPSS version 22. Mean haemoglobin distribution and anaemia prevalence will be reported with confidence intervals. Distribution of anaemia in MOH divisions will be mapped using GeoDa software. Causes for anaemia will be presented as percentages with 95% confidence levels. To determine factors associated with anaemia, chi squared tests for binary outcomes (having a history of anaemia, consumption of folic acid and current breast feeding status) and t tests for continuous outcomes (mothers’ ethnicity, religion, parity, age category, education level, severity of menstrual blood loss, BMI category) will be used. For all tests, 2-sided p value with alpha ≤0.05 level of significance will be used. A logistic regression model will be attempted to predict the anaemia among first trimester pregnant women.\n\nEthical approval for the ‘Rajarata Pregnancy Cohort’ of which this study is a part of, has been obtained from the ethics review committee of Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka (ERC 2009/07).\n\nTrained medical graduates introduced the study to participants and distributed information leaflets in native languages (Sinhala or Tamil)30. Written informed consent was obtained from all participants30. Participation is voluntary and refusal to take part or withdrawal from the study will not lead to any disadvantages during routine care provision. Already collected data will be retained if participation is withdrawn. Participants will receive the originals of all investigations and will be referred for further management as necessary if any abnormality is detected.\n\nTo maintain confidentiality participants will be allocated an ID number which will link their data. Consent forms will be stored separately in a locked filing cabinet at the Maternal and Child Health Research Unit (MCHRU) of our institution. All questionnaires and lab reports will be stored in one center to which only the investigators will have access.\n\nFindings will be reported to the scientific community as peer reviewed research articles, abstracts and presentations. They will be conveyed to the district level stakeholders at monthly MOH conferences, special dissemination meetings and as policy briefs.\n\nFrom July to October 2019, 3018 pregnant women were recruited in this study. Baseline assessments have all been done (interviewer administered questionnaire; clinical examination; dietary assessment; complete blood count). Blood films have been prepared and serum samples have been obtained for further assessments. Confirmatory investigations for minor hemoglobinopathies such as Thalassemia is still being conducted due to difficulties in following up participants due to the coronavirus disease 2019 (COVID-19) pandemic. Peripheral blood film analysis is also yet to be completed due to lockdown related to COVID-19. Batch processing and testing of stored samples and data analysis is also ongoing.\n\n\nDiscussion\n\nThis is the largest study on anaemia in pregnancy in a single district of Sri Lanka. Therefore, more specific spatial data on etiological distribution and pocketing may emerge. As studies investigating several causes of anaemia at the same time are scarce, this study will provide a wider understanding about etiology and distribution of anaemia in Sri Lankans. This will enable policy change to go beyond routine prevention methods focused on iron deficiency anaemia. Data on associated factors will also be helpful for planning further preventive interventions.\n\nSince the participants are pregnant women who registered for routine antenatal care, a selection bias could occur. However, since the clinic registration rate is very high this bias would be minimal.\n\n\nData availability\n\nNo data are associated with this article.\n\nOpen Science Framework: Anaemia Tools. https://doi.org/10.17605/OSF.IO/KSX2F30.\n\nThis project includes the following extended data:\n\n1. Anaemia questionnaire - interviewer administered (in Sinhala and an English translation)\n\n2. Diet questionnaire (in Sinhala and an English translation)\n\n3. Examination reporting form.docx\n\n4. Protocol for Sample Collection.docx\n\n5. Consent form\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAlwan N, Hamamy H: Maternal Iron Status in Pregnancy and Long-Term Health Outcomes in the Offspring. J Pediatr Genet. 2015; 04(02): 111–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrannon PM, Taylor CL: Iron Supplementation during Pregnancy and Infancy: Uncertainties and Implications for Research and Policy. Nutrients. 2017; 9(12): 1327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJwa SC, Fujiwara T, Yamanobe Y, et al.: Changes in maternal hemoglobin during pregnancy and birth outcomes. BMC Pregnancy Childbirth. 2015; 15(1): 80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhoigani MG, Goli S, Hasanzadeh A: The relationship of hemoglobin and hematocrit in the first and second half of pregnancy with pregnancy outcome. 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PubMed Abstract\n\nAgampodi TC, Wickramasinghe ND, Prasanna RIR, et al.: The Rajarata Pregnancy Cohort (RaPCo): study protocol. BMC Pregnancy Childbirth. 2020; 20(1): 374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLemeshow S, Hosmer Jr DW, Klar J, et al.: Adequacy of Sample Size in Health Studies. 1st ed. Chchester: John Wiley and Sons Ltd. 1990; 1–5. Reference Source\n\nDepartment of Health North Central Province: Department of Health- North Central Province - pregnant Mother Care. [cited 2020 Nov 26]. Reference Source\n\nAgampodi SB, Amarasinghe G: Anemia Tools. http://www.doi.org/10.17605/OSF.IO/KSX2F\n\nEngland JM, Fraser PM: Differentiation of Iron Deficiency from Thalassaemia Trait by Routine Blood-Count. Lancet. 1973; 1(7801): 449–52. PubMed Abstract \| Publisher Full Text\n\nHoffmann JJML, Urrechaga E, Aguirre U: Discriminant indices for distinguishing thalassemia and iron deficiency in patients with microcytic anemia: a meta-analysis. Clin Chem Lab Med. 2015; 53(12): 1883–94. PubMed Abstract \| Publisher Full Text\n\nThomas DW, Hinchliffe RF, Briggs C, et al.: Guideline for the laboratory diagnosis of functional iron deficiency. Br J Haematol. 2013; 161(5): 639–48. PubMed Abstract \| Publisher Full Text\n\nGreen R, Dwyre DM: Evaluation of Macrocytic Anemias. Semin Hematol. W.B. Saunders; 2015; 52(4): 279–86. PubMed Abstract \| Publisher Full Text\n\nButtarello M: Laboratory diagnosis of anemia: are the old and new red cell parameters useful in classification and treatment, how? Int J Lab Hematol. 2016; 38 Suppl 1: 123–32. PubMed Abstract \| Publisher Full Text\n\nSobczyńska-Malefora A, Harrington DJ: Laboratory assessment of folate (vitamin B 9) status. J Clin Pathol. 2018; 71(11): 949–56. PubMed Abstract \| Publisher Full Text\n\nChatthanawaree W: Biomarkers of cobalamin (vitamin B12) deficiency and its application. J Nutr Health Aging. 2011; 15(3): 227–31. PubMed Abstract \| Publisher Full Text\n\nOosterhuis WP, Niessen RW, Bossuyt PM, et al.: Diagnostic value of the mean corpuscular volume in the detection of vitamin B12 deficiency. Scand J Clin Lab Invest. 2000; 60(1): 9–18. PubMed Abstract \| Publisher Full Text" }	[ { "id": "81701", "date": "15 Apr 2021", "name": "Manjula Danansuriya", "expertise": [ "Reviewer Expertise Community 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\nAbstract body should be homogenous. Objectives are stated differently at different places. Propose to improve the title as -assessing/exploring prevalence of known causes of anaemia- rather than \"assessing etiology\".\n\nJustification could have been backed by more stronger reference from 2015 National nutrition survey which revealed - prevalence of anaemia among preg. women as 31.8% ; of them only 9.9% were identified as having iron def. anaemia (IDA) as per serum ferritin levels, highlighting other causes for anaemia is more prevalent among SRL pregnant women compared to IDA.\n\nSample size should be 865 not 383. Equation should be:\n\nIdeally sample size should have been calculated to different entities of anaemia (as per available literature) i.e.: thalassemia, mixed deficiency, etc and sample size for the study should be the largest N of those (see here).\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6610", "date": "13 May 2021", "name": "Gayani Amarasinghe", "role": "Author Response", "response": "We would like to express our gratitude for the valuable comments given by the reviewer. Please find a point by point reference for the comments below. Comment 1: Abstract body should be homogeneous. Objectives are stated differently at different places. Propose to improve the title as -assessing/exploring prevalence of known causes of anemia- rather than \"assessing etiology\". Response: Thank you for pointing out the discrepancy in stated objectives at different places. We have corrected the objectives in the abstract and the main text to improve the clarity and homogeneity. We would like to adhere to the original title of the article rather than updating to ‘exploring prevalence of known etiologies’ as our original aim is to actually identify etiologies that may matter in the study population. That’s why the algorithm includes examination of peripheral blood film assessments (in hope of identifying clues to alternate etiologies) when the suspected etiology proves unlikely according to the algorithm based testing. Comment 2: Justification could have been backed by stronger reference from 2015 National nutrition survey which revealed - prevalence of anaemia among preg. women as 31.8% ; of them only 9.9% were identified as having iron def. anaemia (IDA) as per serum ferritin levels, highlighting other causes for anaemia is more prevalent among SRL pregnant women compared to IDA. Response: Thank you for this valuable suggestion. We updated the text accordingly. Comment 3: Sample size should be 865 not 383. Response: Thank you very much for pointing this out. We have used the same formula for calculation. However, we have mentioned that we used 2% precision for the calculation whereas we had actually used 3% precision. This has been a mistake from us and has been corrected in the new version. Comment 4: Ideally sample size should have been calculated to different entities of anaemia (as per available literature) i.e.: thalassemia, mixed deficiency, etc. and sample size for the study should be the largest N of those (see here). Response: We agree that this would have been the ideal method. However, when the known population prevalence of etiologies (iron deficiency and thalassemia) among pregnant women are applied for the sample size calculation, obtained minimum sample sizes are smaller than the sample size we have used (n-1866). Hence we employed this method also taking into consideration the feasibility." } ] } ]	1	https://f1000research.com/articles/10-223
https://f1000research.com/articles/10-427/v1	28 May 21	{ "type": "Systematic Review", "title": "The efficacy of adjunctive alpha-blockers on ureteroscopy procedure for ureteral stones: a systematic review and meta-analysis", "authors": [ "Saras Serani Sesari", "Widi Atmoko", "Ponco Birowo", "Nur Rasyid", "Saras Serani Sesari", "Widi Atmoko", "Ponco Birowo" ], "abstract": "Background: Urolithiasis cases are a common condition, and the number is still growing today. The prevalence of urinary tract stones globally currently ranges from 2-20% with a recurrence rate of around 50%. The present study aims to investigate the efficacy of adjunctive alpha-blockers in improving the success rate of ureteroscopy (URS) procedure for urolithiasis. Methods: We reviewed articles obtained from MEDLINE, CENTRAL, CINAHL, and Elsevier from 14 August to 9 September 2020, comparing alpha-blockers as adjunctive therapy, versus either a placebo or no drug at all, in post-URS urolithiasis patients. There were no restrictions on the type of URS and alpha-blockers given to patients. The quality of studies included was assessed using Cochrane’s Risk of Bias Assessment for Randomized-Controlled Trials. Results: Forest plot analysis emphasizes the statistically significant difference among the group, where the adjunctive alpha-blocker group had pooled relative risk (RR) of being stone-free, readmitted due to initial URS failure, having an overall complication, having haematuria, getting their ureteral mucous injured, and suffering a colic episode was 1.71 (95% CI, 1.11–1.24), 0.50 (95% CI, 0.25–1.01), 0.41 (95% CI, 0.27–0.61), 0.42 (95% CI, 0.22–0.79), 0.31 (95% CI, 0.13–0.73), and 0.21 (95% CI, 0.06–0.69), respectively. Conclusions: Alpha blockers minimize the frequency and duration of ureteral contractions, allowing smooth stone expulsion. With this knowledge, it is expected to help clinicians decide the importance of adjunctive alpha-blocker administration.", "keywords": [ "adjunctive alpha-blocker", "ureteral stone", "ureteroscopy" ], "content": "Introduction\n\nIn the last decade, urolithiasis has become a common condition, and the number continues to increase. The prevalence of urinary tract stones globally currently ranges from 2-20% with a recurrence rate of around 50%. The increase in urinary tract stones incidence was also followed by the rise in the frequency of urinary tract endoscopy, one of which was retrograde or antegrade ureterorenoscopy, which was indicated to treat ureteral stones and kidney stones.1 Compared to the extra-corporeal shock-wave lithotripsy (ESWL) procedure, (URS) is more preferred, as it has been proven to achieve higher success rates in a single operation.2,3\n\nIn recent literature, the adjunctive alpha-blocker is recommended to facilitate ureteric stone expulsion, decrease postoperative complications, improve stents tolerability, and reduce colic episodes to reduce the necessity for secondary procedure retreatment.1\n\nThe present study aims to investigate the efficacy of adjunctive alpha-blockers for improving the success rate of the URS procedure for urolithiasis. By conducting this review and analysis, a definite conclusion regarding the effectiveness can be achieved. Thus, clinicians can decide the necessity of adjunctive alpha-blockers.\n\n\nMethods\n\nThis review was done according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statements.4 This study attempted to improve alpha-blocker therapy effectiveness in post-URS urolithiasis patients, with success rate parameters. Thus, this meta-analysis included studies which compared alpha-blockers as adjunctive therapy, versus either a placebo or no drug at all, in post-URS patients. No restrictions on the type of URS were performed in patients. There were also no restrictions on the kind of alpha-blocker given to patients. The success rate was then defined as the stone-free rate and overall postoperative complication rate.\n\nWe performed an article search on four electronic databases (MEDLINE/Pubmed, CENTRAL/Cochrane, CINAHL/EBSCOHost, and EMBASE/Elsevier). The investigation was carried out from 14 August to 9 September 2020. PICOS were used to trace studies and identify the suitability of any we found.5 We used specific keywords, adjusted to each search engine specification, in the form of (postoperative OR adjunctive) AND (alpha-blocker OR tamsulosin OR alpha-adrenergic antagonists OR Alpha-adrenoreceptor antagonists OR doxazosin OR terazosin OR alfuzosin OR prazosin) AND (ureteroscopy OR URS OR ureterorenoscopy OR retrograde intrarenal surgery) AND ureteral stone. We also looked at a reference list of several reviews to expand the search coverage of the study.\n\nStudy selection was carried out independently and duplicated by each author, referring to inclusion and exclusion criteria. The inclusion criteria in this study included: 1) RCT or quasi-RCT studies that were compatible with PICOS; 2) English/Indonesian written articles; 3) Full-text articles available; 4) The output assessed were, at least, one of postoperative stone-free rate or overall complication rate; and 5) Published between 1 January 2000 and 31 December 2020.\n\nThere were no restrictions on the type of URS and alpha-blockers given to patients. The exclusion criteria for this study included review articles, case reports, case series, editorial letters, studies on animals, and/or studies in the process of peer review (has not been published yet).\n\nThe decision to study eligibility was determined by each author independently. Any disagreement was resolved by discussion.\n\nData extraction was carried out by each author independently and in duplication. We extracted the study's primary characteristics, including the first author, location, sample size, and publication year.\n\nFollowing the dependent variables in this meta-analysis, we also extracted patient baseline data and postoperative data, including a stone-free rate and overall complication rate. We also noted the type of alpha-blocker and the duration of alpha-blocker administration.\n\nThis study explored the efficacy of adjunctive alpha-blockers in increasing URS's success rate in urolithiasis patients, divided into the stone-free rate and overall complication rate, in the form of relative risk (RR). We used a 2×2 contingency table to obtain each study's RRs and pooled the overall RRs using the Review Manager 5.3 application.6 Analysis using the DerSimonian and Laird random-effects model was performed when high heterogeneity was found.\n\nTo see the precision and examine publication bias, this meta-analysis utilized funnel plots, which were also produced via Review Manager 5.3.6\n\n\nResults\n\nWe searched five electronic databases, using specific keywords tailored to each database, to improve search sensitivity and specificity. The total records retrieved were 520 studies, and 131 studies were then excluded as there were duplications. From 389 studies screened, 376 studies were further excluded because of unrelated topics and objectives, resulting in 13 studies to be assessed for eligibility. Another five studies were later excluded due to unsuitable study design (n = 2) and review articles (n = 3). We obtained eight studies that were included in the qualitative and quantitative synthesis. The summary of study identification and selection according to the PRISMA Statement flow diagram are shown in Figure 1 and Table 1.\n\nInitial database searching yielded 520 items, 389 of which were left after duplicate screening. Abstract screening excluded 376 more items. Eight items survived full-text assessment and were included in both the qualitative and quantitative synthesis.\n\nThree of the eight studies we included in this study were multicenter, prospective, randomized trials.7,8,9 While the other five were a single-center, prospective, randomized trial.1,3,10,11 However, the numbers of patients enrolled in the pilot studies did not differ significantly between studies. Overall, the total number of patients included in this meta-analysis was 913 patients.\n\nSeven of the eight studies gave alpha-blockers before URS, three of those gave Tamsulosin 0.4 mg once daily for seven days before surgery.1,8,10 Two studies administered alpha-blockers only once, the day before surgery.3,9 One study gave Silodosin 8 mg once daily for ten days before surgery,10 and one other study gave Tamsulosin 0.4 mg once daily for 14 days postoperatively.11 Bhattar et al.12 also looked at Tamsulosin independently and in combinations. Data on the characteristics of the included studies are shown in Table 2.\n\n* Intervention/Control; mm: millimeters; qd: drug was given once daily; HFU: Hounsfield unit; NR: not reported\n\n* I/C: Intervention/Control; comp: complication; †: recorded at 4 weeks postoperatively; ††: recorded at the end of study; NR: not reported.\n\nOf the eight studies included in this meta-analysis, five had a high risk of bias because assessor outcome blinding was not performed. One study clearly stated no blinding in patients who were given intervention or control.11 Selection bias in some studies was also considered high, because no allocation concealment was performed. In general, the quality of the studies included in this meta-analysis varied from low to high.\n\nThe bias assessment was carried out independently by each author and in duplication, in which all authors assessed all articles. Disagreements between the authors were resolved by discussion or consensus. Figure 2 visualizes the summary of bias risk.\n\nSelection bias from allocation concealment is present in three studies and not enough information was given to assess random sequence generation. Detection bias was detected in five studies. Only one study presented with performance bias. No attrition or reporting bias were detected.\n\nThe stone-free rate was found to be higher in patients given adjunctive alpha-blockers in most of the studies. A study by Aydin et al. 2017,9 found no difference at all in the stone-free rates of patients with and without adjunctive alpha-blockers one day before URS surgery. However, this study also compared alpha-blocker administration three days before surgery with a placebo and found a significant difference between the placebo and alpha-blocker groups.\n\nBased on the results of the meta-analysis in Figure 3, it could be seen that the pooled RR favors the experimental group (adjunctive alpha-blocker), with a value of 1.17 (95% CI, 1.11–1.24). This indicates that a significantly higher stone-free rate was found in the adjunctive alpha-blocker patient.\n\nThe lower risk of readmissions due to initial URS failure also supported a higher stone-free rate in the alpha-blocker group. The risk of readmission to URS in the alpha-blocker group tends to be lower than the placebo group, with a pooled RR of 0.50 (95% CI, 0.25–1.01).\n\nIn all studies, the complication rate was higher in the control group, either general complication or overall complication, hematuria, or mucosal injury. The alpha-blockers group had a significantly lower risk of complications than the placebo group, with a pooled RR of 0.41 (95% CI, 0.27–0.61). In this meta-analysis, the heterogeneity was recorded at only 0%; thus, we used a fixed-effect model to pool the effect estimate.\n\nSeveral studies break down their patients' complications into several types of complications, such as hematuria, mucosal injury, and colic episode. For each of these complications, the alpha-blocker group was shown to have a lower risk of developing these complications. For all three types of complications, the alpha-blocker group had a significantly lower risk, with the pooled RR for hematuria, mucosal injury, and a colic episode of 0.42 (95% CI, 0.22–0.79), 0.31 (95% CI, 0.13–0.73), and 0.21 (95% CI, 0.06–0.69), respectively. These are summarized in Figure 4.\n\n\nDiscussion\n\nAccording to this systematic review and meta-analysis, postoperative alpha-blocker is associated with URS procedure's success rate for urolithiasis.2,13 From the forest plot, it was discovered that patients having postoperative alpha-blockers are more likely to be stone-free with the RR=1.17 (95% CI, 1.11–1.24). This means that patients consuming postoperative alpha-blockers are 1.17 times more likely to be stone-free. The risk ratio ranges from 1.11–1.24, which are both greater than 1. Thus, it can be concluded that postoperative alpha-blocker is effective in increasing the stone-free rate after ureteroscopy. The study with the highest weight is by Ahmed et al. 2017,2 with a weight of 72.2%, followed by a study by Bayar et al. 2019,8 with a weight of 27.8%. This result is similar to a study done by Alsaikhan et al. in 2020,14 albeit using different parameters. They did a systematic review and meta-analysis about preoperative alpha-blockers usage for ureteroscopy for ureteric stones with parameters including risk reduction in need for intraoperative ureteral dilatation, stone-free status at four weeks post-operatively, and at final follow-up, the likeliness of urologists to reach the stone with the ureteroscope, operative time, and length of hospital stay. They study the preoperative use of alpha-blockers, meanwhile this study analyzed the usage of alpha-blockers as an adjunctive both preoperatively and postoperatively. The study result showed that at four weeks post-operatively and at final follow-up, patients have increased stone-free status with RR 1.17 (95% CI: 1.08 to 1.26), p < 0.0001 and 1.18 (95% CI: 1.11 to 1.24), p < 0.00001 respectively. It is important to note that some studies that were assessed by this study and Alsaikhan et al. 2020 are different.14\n\nTamsulosin, an alpha-1A blocker, has been shown to improve the stone expulsion rate and minimize the probability of colic episodes in patients during watchful waiting.15,16 It relaxes the muscle of the ureteral wall, aiding gravel clearance after URS or ESWL procedure. Furthermore, a relaxed ureter allows the instrument forwarding to become easier. In patients with Tamsulosin, the ureteral orifices were dilated, easily identified, and provided a more accessible entrance for the ureteroscope.8,17 Tamsulosin also lessens the amplitude of ureteral contractions and shortens the duration between contractions.15,18\n\nPostoperative complications outcome is also affected by the administration of postoperative alpha-blockers.19,20 It was found that endoscopic treatments without the administration of adjunctive alpha-blockers are associated with a higher probability of complications.2,21 This was shown by the forest plot, where the adjunctive alpha-blocker gives a protective effect from postoperative complications with RR = 0.41 (95% CI, 0.27–0.61). Therefore, postoperative alpha-blocker administration reduces the odds of postoperative complications. For this outcome, a study by Mohey et al. 2018,10 weighs 19.3%, while a study by Ahmed et al. 20172 weighs 47.8%. Extra benefits, such as shorter hospital stay, lesser hospital bills, milder postoperative complications, and better symptomatic improvement were obtained by simultaneous administration of Tamsulosin.18,20\n\nPostoperative alpha-blocker improves the incidence of colic episodes. This was shown by RR=0.21 (95% CI, 0.06–0.69). Numerous reports have demonstrated alpha-adrenoreceptors on the ureteral wall, with the distal ureter's highest density.1,2,15 Variable distribution of alpha receptor subtypes were found in the proximal, middle, and distal ureter. Alpha-blockers are now commonly prescribed for ureteral colic in various hospitals.15,22\n\nThis systematic review and meta-analysis have several strengths. Firstly, this systematic review and meta-analysis included a relatively broad scope of population. This review then assesses the primary outcome and considers other additional outcomes, which is also essential in clinical practices, albeit not widely studied. Low risk of bias in included studies, utilization of guidelines, no heterogeneity between studies, symmetrical funnel plots as shown in Figure 5, and high specificity also strengthen this study.\n\nAside from the strengths, this systematic review and meta-analysis has a limitation. This study's limitation is that there were only eight studies eligible for review, and there were only two studies for each outcome, which makes its representability somewhat questionable.\n\n\nConclusions\n\nIn conclusion, our study shows that the administration of adjunctive alpha-blockers improves the URS procedure's success rate for ureteral calculi in terms of increasing stone-free rate, reducing postoperative complications, and minimizing colic episodes. This is because the alpha-blocker relaxes and reduces the ureteral wall's contractions, allowing easier stone clearance.\n\n\nData availability\n\nOpen Science Framework: PRISMA checklist and flow chart for 'The efficacy of adjunctive alpha-blocker on ureteroscopy procedure for ureteral stones: a systematic review/meta-analysis'\n\nDOI 10.17605/OSF.IO/RM4AG4\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors acknowledge that no contributions to this review originated outside the listed authors.\n\n\nReferences\n\nAbdelaziz AS, Kidder AM: Tamsulosin therapy improved the outcome of ureterorenoscopy for lower ureteral stones: A prospective, randomised, controlled, clinical trial. Afr J Urol. 2017; 23(2): 148–53. Publisher Full Text\n\nAhmed AF, Maarouf A, Shalaby E, et al.: Semi-rigid ureteroscopy for proximal ureteral stones: Does adjunctive tamsulosin therapy increase the chance of success? Urol Int. 2017; 98(4): 411–7. PubMed Abstract \| Publisher Full Text\n\nRashahmadi N, Sofimajidpour H, Saedi A: The effect of Tamsulosin on the quality and clinical trial. Discovery. 2018; 22(91).\n\nSerani S: PRISMA checklist and flow chart for ‘The efficacy of adjunctive alpha-blocker on ureteroscopy procedure for ureteral stones: a systematic review/meta-analysis’. OSF. 2021; Publisher Full Text\n\nEriksen MB, Frandsen TF: The impact of patient, intervention, comparison, outcome (PICO) as a search strategy tool on literature search quality: a systematic review. J Med Libr Assoc. 2018 Oct; 106(4): 420–431. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReview Manager (RevMan) [Computer program]: Version 5. 2014; vol. 3. . Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration.\n\nKetabchi AA, Mehrabi S: The effect of tamsulosin, an alpha-1 receptor antagonist as a medical expelling agent in success rate of ureteroscopic lithotripsy. Nephrourol Mon. 2014 Jan; 6(1): e12836. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBayar G, Kilinc MF, Yavuz A, et al.: Adjunction of tamsulosin or mirabegron before semi-rigid ureterolithotripsy improves outcomes: prospective, randomized single-blind study. Int Urol Nephrol. 2019 Jun; 51(6): 931–6. PubMed Abstract \| Publisher Full Text\n\nAydın M, Kılınç MF, Yavuz A, et al.: Do alpha-1 antagonist medications affect the success of semi-rigid ureteroscopy? A prospective, randomised, single-blind, multicentric study. Urolithiasis. 2018 Nov; 46(6): 567–72. PubMed Abstract \| Publisher Full Text\n\nMohey A, Gharib TM, Alazaby H, et al.: Efficacy of silodosin on the outcome of semi-rigid ureteroscopy for the management of large distal ureteric stones: blinded randomised trial. Arab J Urol. 2018 Dec; 16(4): 422–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohn TT, Razdan S: Adjunctive tamsulosin improves stone free rate after ureteroscopic lithotripsy of large renal and ureteric calculi: a prospective randomized study. Urology. 2010 May 1; 75(5): 1040–2. PubMed Abstract \| Publisher Full Text\n\nBhattar R, Tomar V, Yadav SS, et al.: Comparison of safety and efficacy of tamsulosin, tadalafil, combinations and deflazacort in lower ureteric orifice negotiation by large size ureteroscope (8/9.8Fr) prior to intracorporeal lithotripsy. Afr J Urol. 2018 Jun 1; 24(2): 139–45. Publisher Full Text\n\nRamesh A, Karthick P, Kumar R: Medical expulsion therapy for ureteric calculus - possible! F Int Surg J. 2016; 3(1): 113–8. Publisher Full Text\n\nAlsaikhan B, Koziarz A, Lee JY, et al.: Preoperative alpha-blockers for ureteroscopy for ureteral stones: a systematic review and meta-analysis of randomized controlled trials. J Endourol. 2020 Jan 1; 34(1): 33–41. PubMed Abstract \| Publisher Full Text\n\nJohn TT, Razdan S: Adjunctive tamsulosin improves stone free rate after ureteroscopic lithotripsy of large renal and ureteric calculi: a prospective randomized study. Urology. 2010; 75(5): 1040–2. PubMed Abstract \| Publisher Full Text\n\nLi S, Penniston K: Combination versus monotherapy with alpha-blockers and anticholinergics for the relief of urinary stent symptoms – a randomized control trial.2017: 1–15.\n\nLim KT, Kim YT, Lee TY, et al.: Effects of tamsulosin, solifenacin, and combination therapy for the treatment of ureteral stent related discomforts. Korean J Urol. 2011; 52(7): 485–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAydın M, Kılınç MF, Yavuz A, et al.: Do alpha-1 antagonist medications affect the success of semi-rigid ureteroscopy? A prospective, randomised, single-blind, multicentric study. Urolithiasis. 2018; 46(6): 567–72. PubMed Abstract \| Publisher Full Text\n\nAta ur Rehman DrR, Muzammil Tahir DrM, Seerwan DrM: Effect of Tamsulosin on Stentrelated Symptoms; a Prospective Study. Profession Med J. 2016; 23(01): 114–118.\n\nZhu J, Liang Y, Chen W, et al.: Effect of alpha1-blockers on stentless ureteroscopic lithotripsy. Int Braz J Urol. 2016; 42(1): 101–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNavanimitkul N, Lojanapiwat B: Efficacy of tamsulosin 0.4 mg/day in relieving double-J stent-related symptoms: A randomized controlled study. J Int Med Res. 2010; 38(4): 1436–41. PubMed Abstract \| Publisher Full Text\n\nFerrandino MN, Monga M, Preminger GM: Adjuvant therapy after surgical stone management. Adv Chronic Kidney Dis. 2009; 16(1): 52–59. PubMed Abstract \| Publisher Full Text" }	[ { "id": "88750", "date": "28 Jul 2021", "name": "Ferry Safriadi", "expertise": [ "Reviewer Expertise Endourology and oncology urology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is interesting. Some limitations I see in this study are:\nOnly 2 studies with multicenter and each outcome parameter.\n\nIn this review, it is not mentioned about stone location in the ureter, which is in a proximal, mid ureter, or distal ureter. As we know, an alpha-blocker only works in the distal part of ureter.\n\nBased on that, the author should say this study is only good for distal ureter stones.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "8008", "date": "06 Apr 2022", "name": "Saras Serani Sesari", "role": "Author Response", "response": "I already added the clarification of which alpha-blockers exert their effects on the distal region of the ureter muscle. Because kidney and urinary stones can occur in a variety of locations, it was vital to state this explicitly. The author hopes that by identifying the place in which alpha-blockers are effective, which was distal ureter, this study will be easier to implement in the future." } ] }, { "id": "125918", "date": "14 Mar 2022", "name": "Evangelos Liatsikos", "expertise": [ "Reviewer Expertise Urology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have several comments for the authors.\nThe authors should clearly indicate the aim of the study in the Introduction.\n\nThe authors mention in the study selection that studies were selected if full-text articles were available. So they might have missed many important articles which full text was not available.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-427
https://f1000research.com/articles/11-412/v1	12 Apr 22	{ "type": "Research Article", "title": "Role of metalinguistic awareness intraining for reading: a quasi-experimental study with Saudi EFL learners", "authors": [ "Raniyah Mohammad Almarshedi" ], "abstract": "Background Metalinguistic awareness plays an important role in developing foreign learners' repertoire in the underlying system of the target language. Therefore, this study addresses the research gap in the Saudi context regarding the role of metalinguistic awareness in teaching reading comprehension. Moreover, it also verifies the level of application ofmetalinguistic strategies in the English as a first language (EFL) classroom.\nMethods The study employs a quasi-experimental research design with 70 EFL Saudi learners at Hail University as the participants. The duration of the experiment was twelve weeks and results are compared between control and experimental groups who were tested for homogeneity by administering the TOEFL reading comprehension test. The impact of metalinguistic awareness in developing Saudi learners' reading comprehension skills was verified by administering a questionnaire to the participants.\nResults The findings of the study show that the reading abilities of both groups of learners developed, but the development in the experimental group was significant. Planning was not enhanced in either the experimental or control group, monitoring and assessment were enhanced only in the experimental group.\nConclusions Consequent to the findings the study recommends that EFL instructors directly train their students on metacognitive strategies to improve their reading comprehension.", "keywords": [ "Language Learning", "Metalinguistic Awareness", "Saudi EFL Learners", "Reading Comprehension", "Training" ], "content": "Introduction\n\nReading ability is the capacity to comprehend and make sense of a written piece or text. It provides a link between the passive and active readers, as well as a critical relationship between them and successful reading, which is vital for a fulfilling academic, professional, and personal life. It is the process of obtaining and constructing meaning from written language via interaction and participation with the text (Pourhosein Gilakjani & Sabouri, 2016). Because knowledge is provided in text form across the globe, learners must be able to read fluently in order to succeed in institutionalized education. In addition to providing photographs for visual reference, print media such as websites and books as well as magazines and newspapers are used to communicate information to the reader (Cimmiyotti, 2013). Reading ability is a complicated cognitive task, and different components of metalinguistic awareness are beneficial for successful reading in different situations.\n\nMany previous studies have presented evidence that metalinguistic awareness and reading fluency may be used to predict reading comprehension in a variety of situations (Akbulut, 2019; Dong et al., 2020; Li & Wu, 2015). Metalinguistic awareness is defined as the understanding that a person has necessary to transform language in a variety of ways. In other words, it is a person’s ability to control language. As described by the Oxford Dictionary, metalinguistic awareness is the ability to separate oneself from the content of communication to reflect on and modify the structure of language. According to Altman et al. (2018), metalinguistic awareness, which demands the speaker to pay attention to the structure and form of the language, emerges in the later stages of language acquisition, around the age of 5–6 years, and builds on linguistic information acquired earlier in life.\n\nHaving a strong sense of metalinguistic awareness is essential for multilingual competency, and it distinguishes speakers of numerous languages from those who only know one or two languages. Language awareness refers to a speaker’s ability to approach and see language in abstract terms, as well as to analyze and comprehend language as a system or object that can be worked with and controlled (Al-Ahdal, 2020; Alfallaj, 2020; Hofer & Jessner, 2019; Kitishat et al., 2020). According to the author’s knowledge, however,though there are many studies that focused on metalinguistic awareness and reading comprehension, none have examined the use impact of metalinguistic awareness (Akbulut, 2019; Dalona & Dalona, 2019; Dong et al., 2020; Li & Wu, 2015), in developing EFL Saudi learners’ reading comprehension (Al-Ahdal, 2020; Amin, 2019) in addition to the enhancement in the sub-skills of planning, monitoring and assessment in quasi-experimental study design. This study is inspired by Habibian (2015) who checked whether explicit teaching of metacognitive strategies urges learners to use them in text comprehension in a Malaysian context. The current study verifies the use of these strategies in a Saudi context and also checks which ones of the three sub-elements are developed and which one is enhanced as a result of the intervention.\n\n\nLiterature review\n\nAccording to Sinar (2018), metalinguistic awareness can be compared to a large, benevolent giant. It entails a greater awareness of competence achievement at phonology level in rhymes, syllables and phonemes, in addition to acquiring meaning bearing components whether morphological, words leading to syntax-like phrases, and sentences and semantics as denotation, or connotation, ambiguities and at the level of figurative language as metaphor, idioms and the like. All of these factors contribute to ensuring understanding during reading and give the reader alternatives for repairing comprehension or forming predictions and inferences when comprehension fails or is not completely realized. Similarly, Zhang et al. (2017) believed that the acquisition of reading skills is basically metalinguistic in nature. Considering print as a representation of the spoken language, the capacity to reflect on and control diverse linguistic components, referred to as metalinguistic awareness, is essential in the process of learning to read. Moreover, the findings of Yang et al. (2019) are particularly significant because they provide further evidence that, even though metalinguistic skills such as phonological awareness and orthographic knowledge are still in the early stages of development in early childhood, they play bridging roles in reading among beginning readers. Tighe et al. (2019) in the findings of their recent study on children has demonstrated that metalinguistic abilities have a direct and/or indirect influence (by word reading and/or vocabulary skills) on reading comprehension across various grades and across a range of diverse language backgrounds. As stated by Dong et al. (2020), when it comes to cognitive characteristics that predict reading comprehension ability through decoding and word recognition on the single-word or single-character cognition process, metalinguistic awareness has been identified as a major component. Zhang et al. (2017) conducted a longitudinal study to investigate themorphological and phonological awareness of young Singapore learners in reading bilingual words. Findings showed that all elements of metalinguistic awareness were boosted significantly and considered as predictors of word reading concurrently in the two languages.\n\nGenerally, this study assesses the role of metalinguistic awareness in training for reading through an experimental study with EFLstudents. Specifically, it aims to answer the following queries:\n\n1. Is there any significant difference in the students’ reading comprehension test achievement attributable to the intervention between the control and the experimental groups?\n\n2. Is there any significant difference between the two groups in terms of the use of metacognitive strategies at the end of the intervention?\n\n\nMethods\n\nThis study verifies the efficacy of the metacognitive approach in reading comprehension using a quasi-experimental design with a pre- and post-test. It was inspired by Habibian’s (2015) study in verifying the role of metacognitive strategies in developing EFL learners’ reading comprehension. It used both a TOEFL reading test as well as a questionnaire to check the participants’ attitudes at the end of the intervention on the impact of metalinguistic awareness in developing Saudi EFL learners’ reading comprehension. It specially tries to identify which one of the four elements of metalinguistic awareness was applied more according to the participants’ attitudes. Ethical procedures were highly taken into the researcher’ consideration. Initially, the researcher got permission to conduct the research at hand from the ethical committee affiliated to the deanship of higher studies from Hail University, and consent letter was submitted to the department of English where the study took place. Moreover, the researcher explained the purpose of the study to the students who also agreed to take role in the study by appearing in the pre- and post-TOEFL exams. Furthermore, students were assured that all their information will remain confidential and just be used for the purpose of the study at hand, and if they want to know about the findings of the study, the researcher will support them with the study outcome as soon as she finishes.\n\nThe number of participants in this study is 70, with 35 each constituting the experimental and control groups. All participants are enrolled at the English department, Hail University. This is a purposefully chosen sample, i.e. the sample was chosen with the specific research objective in mind (Creswell, 2014). Purposeful sampling may be implemented in a variety of ways. In this study, criterion-based sampling was applied to ensure the quality of the results. Accordingly, the first-level students at the English Department were chosen as subjects for this study. Students at this level are assumed to be unfamiliar with metacognitive techniques. Using criterion-based sampling was deemed the most suitable to conduct this inquiry.\n\nInitially, both experimental and control group were administered a TOEFL test of reading comprehension skills to check their homogeneity. Only the experimental group received instruction in metacognitive methods, while the control group was taught the reading course traditionally. The former received instructions on how to read in order to encourage active engagement of students. Both the teacher and participants considered the pragmatic aspects of the situation in order to provide reliable results. Metacognitive strategies were taught three times a week, for an hour each time, to the experimental group. Students were given three categories of information: declarative knowledge (learning what strategies are), situational knowledge (learning where strategies might be used), and procedural knowledge (learning how to apply strategies). The data were collected after a 12-week period of metacognitive instruction through a questionnaire.\n\nThe following is a short description of the instruments employed in this study: A TOEFL test extracted from the book “Building Skills for the TOEFL iBT, Beginning Reading” by Edmunds et al. (2009). It was clearly cited in the list of references. All the questions used for the pre-test were multiple choice type and the aim was to ascertain whether or not the students’ reading abilities were similar. The same TOEFL test was also used in the post-test to check the enhancement, if any, in the students’ reading comprehension skills. Each test contained 10 questions and no modifications were made in the original test. The test checks some reading strategies in the students’ comprehension including the meaning of certain vocabulary, the topic of a paragraph, detailed information, referent of a pronoun, comprehending the inference of a paragraph, the synonyms of a word, summary of a paragraph, inclusion of a sentence inside a paragraph, and summarizing the whole text in a few sentences. All the participants had to take reading exams at the beginning of the term. The assessments were designed to test students’ reading comprehension and ensure that the two groups were taught at the same level of understanding. Moreover, Beyer’s (1987) metacognitive strategy questionnaire was employed in this study to assess metacognitive methods by employing the four-pointLikert Scale (Never, Rarely, Often, and Always numerically converted to 1, 2, 3, 4 to quantify the results) for responses. The questionnaire is designed to obtain data on how students plan, monitor, and analyze their own progress while they learn a new language. As part of the pilot study, the questionnaire was administered to 12 students who shared demographics with the sample but were not included in the final survey. The reliability of the questionnaire used to assess metacognitive strategy was determined to be 0.85%. The questionnaire responses were interpreted using the proposed guide to determine their awareness of metacognitive.\n\n\nResults and discussion\n\nAs stated earlier, all the participants took a TOEFL reading comprehension test before the program started to make sure they shared reading comprehension level. Both the experimental and control groups had almost the same mean scores. The categorization of the two groups is appropriate since both groups had the same reading comprehension level. Table 1 shows that the experimental group scored an average of 10.28 in the pretest while the control group scored marginally lower at 10.12. However, the difference is not significance, since the Sig. value is 0.34 which is greater than 0.05.\n\nPrior to and during the course, participants were tested on their reading comprehension. Table 2 shows no significant difference between the two tests at the significance level of.001 according to the t-test.\n\nFor the experimental group, Table 3 shows that participants’ mean score of planning strategy use before and after training did not increase. As a result, the total mean score of monitoring (5.45, 11.35) and assessment (2.65, 7.01) may be interpreted to mean that the reading strategy use increased due to metacognitive strategy instruction. Monitoring and assessment strategies included the following: keeping the goal in mind, finding errors and determining when a sub-goal is met, identifying how to become free from errors, keeping one’s spacein sequence, choosing the next appropriate operations, and determining when to proceed. There are no significant changes in the control group when it comes to planning, monitoring, and assessment. Preparation strategies included the following steps: defining the goal, selecting an operation, predicting desirable results, identifying likely mistakes, and planning how to recover from errors. Metacognitive approaches were studied before and after the test to see whether explicit instruction had an effect.\n\nStudents’ performance in the experimental group improved when they are closely observed and evaluated, according to Table 4. Consequent to the training, the participants also utilize a larger variety of strategies. Monitoring and assessment are shown in Table 4 to have a significant impact on performance in the experimental group when compared to the control group. After training sessions, the former employs more strategies than they previously did. According to Table 4, there is no significant difference between the experimental group and control groups for planning in the pre- and post-tests: the Sig. values are (0.286, & 0.194), i.e., Sig. values >0.05. For the second element of metalinguistic awareness, i.e., monitoring, Table 4 shows that the difference is significant between the levels of application of this strategy only in the experimental groups. Sig. values are (0.000). Finally, for assessment the Sig values are (0.000 & 0.147). It is only significant in the experimental group.\n\nTeaching metacognitive strategies proved effective in promoting the use of these tactics, according to the findings. Explicit instruction in these strategies is likely to enhance students’ comprehension scores on reading comprehension tests, based on the results of this study. As is reflected in the data, following training, the experimental group outperformed the control group and employed more monitoring and assessment processes than the control group. This finding is confirmed by Habibian’s (2015) who found that Singapore young learners began to use monitoring and assessment as a result of explicit training in metacognitive awareness. A person’s metacognitive talents allow him or her to choose and employ acceptable methods. Metacognitive training seems to have enhanced students’ reading comprehension. According to the findings of this study, metacognitive strategies may help students become more self-aware and better comprehend the language world around them. Using these approaches more often shows their value and usefulness once they are taught as in the present case scenario. Providing students with regular teaching on this method may help them build a habit of using these strategies when reading. Metacognitive awarenessshows that students who are familiar with these approaches expect them to be effective in the classroom. As a consequence, it is impossible to exaggerate the significance of a positive reading attitude in the classroom.\n\nAs stated by Amin (2019), it is reasonable to regard reading as the ultimate collaborative skill to be employed in school and throughout life. To seek information, acquire knowledge, and read books, English is required and is frequently employed as the medium of teaching in higher education. Reading is a critical life skill that contributes to a child’s success in school and later in life. In elementary sections, children must develop a variety of reading skills. According to Good et al. (2019), the major problem facing general education instructors and students in grades one through three is the learning of fundamental reading abilities. Nothing in education is more widely used as a measure to judge the effectiveness of schooling than literacy, which is founded on a solid foundation of fundamental reading abilities. Cirino et al. (2019) confirmed that for reading comprehension to be successful, a significant amount of time and effort must be put in, particularly when the text content or the words themselves are challenging. Students are expected to read more independently and to utilize reading to grasp topics in content courses beyond the early elementary grades, which is frequently the case during the first few years of school.\n\nIzadi and Yarahmadzehi (2018) found that Baluch respondents in English detected and corrected a larger proportion of grammatical mistakes than Persian respondents in a study on metalinguistic awareness of students studying English. Furthermore, Baluch participants fixed errors in a more grammar-focused manner than Persian participants, who repaired errors in a more content-oriented manner. Although the differences were not statistically significant in this example, Baluch participants supplied a plentynumber of errors explanations and a more grammar-centredmethodology than Iranian participants in terms of error explanation. These differences were discovered on factors that were both comparable and different across the three languages. Dalona and Dalona (2019) investigated the metalinguistic awareness of multilingual first graders, and the findings showed that the children’s metalinguistic awareness is average. Grade 1 multilingual learners still need to improve their linguistic skill in English and Filipino, particularly in identifying syntactic errors, because they will be using these languages in their future academic pursuits. The study also asserts that early school-age children’s Cebuano metalinguistic knowledge helps them complete their linguistic tasks in Filipino and English. Similarly, Woll (2019) investigated how French people perceive their mother tongue. In the meta-semantic half of the exam, greater than one third of participants achieved the toplevel of metalinguistic analysis, compared to only 5% in the meta-grammatical half. This tendency may be associated with the fact that direct reference to grammatical elements was necessary to get maximum scores in the meta-grammatical segment, but not in the meta-semantic section, according to a study of coding processes.\n\nThe purpose of the study of Akbulut (2019) was to determine the effect of morphological therapy on the morphological awareness and reading comprehension skills of students enrolled in a foreign language class. The study employed an experimental approach and comprised 74 freshmen at the Translation & Interpreting Department. The experimental group outperformed the control group in MCT and RCT, as determined by the findings. In other words, the experimental group’s morphological awareness improved significantly; also, their reading comprehension abilities improved significantly. Additionally, it can be stated that the participants profited efficiently and consciously from the explicit morphological awareness training session, which aided in their metalinguistic capacity development. The strengthof using video-based in training students on reading comprehensionstrategiesand metalinguistic awareness in learning a target language was investigated by Núñez-Vázquez and Crismán-Pérez (2017) in a non-parametric research. The test was completed by 30 pupils. The cloze test is part of the TAELIS test (Test for the Assessment of the English Language in the School). The results showed that there are statistical differences between school 2 (single videos) and school 3 (many videos or video-based). This presupposes that pupils have more reading practice, that words become more familiar, and that their orthographic vocabulary expands. As a result, pupils learn to play with language, which has an impact on metalinguistic awareness. The goal of the study of Hung and Loh (2020) was to see how cognitive flexibility affects metalinguistic abilities and reading comprehension in elementary school. The cognitive flexibility, metalinguistic skills such as syntactic awareness (word order knowledge), morphosyntactic competence, and discourse awareness (sentence order knowledge), and reading comprehension tests were completed by 49 third-grade primary school students. Reading comprehension is substantially predicted by syntactic awareness, morphosyntactic competence, discourse awareness, and cognitive flexibility, according to the results of hierarchical regression analysis.\n\n\nConclusion\n\nThis study addressed the research gap in the Saudi context regarding the role of metalinguistic awareness in teaching reading. The study confirmed and validated previous finding of Habibian (2015) in whose study the experimental group outperformed their counterparts in reading comprehension. However, this study found both the control and experimental groups got enhancement in the reading comprehension skills. The difference between the mean score of the control and experimental groups was statistically significant. The study, through the application of a questionnaire found that both monitoring and assessment procedures of the experimental group improved as a result of metacognitive strategy training that they were exposed to. However, the improvement in planning was not significant. In the control group, students got enhancement in the monitoring procedures while improvement was not significant in planning and assessment. This finding matched with Habibian’s (2015). Accordingly, as the experimental group showed enhanced ability in two of the three strategies tested, it can be claimed that this metalinguistic awareness is beneficial, and training learners on these strategies may lead to better reading comprehension, to put it in other words, the findings from this study indicate that teaching metacognitive strategies explicitly may help students improve their reading comprehension, and we have data to back up this claim.\n\nAs a result of this study, the EFL classroom stands to benefit in several ways. It is essential for students to understand and practice metacognitive procedures, as well as be conscious of their own thoughts and behaviors while implementing them. Teachers can assist their students achieve higher success through training them on metacognitive strategies. The outcomes of this and other previous research (e.g., Akbulut, 2019; Dalona & Dalona, 2019; Núñez-Vázquez & Crismán-Pérez, 2017) may help teachers to achieve their objectives of encouraging students to use these methods to their utmost extent. Students may be taught metacognitive skills to help them become better readers and speakers of their target language. It is hoped that students would be able to take care of their education and grow into self-directed, resourceful learners when they gain these abilities. Practitioners’ ability to learn may be improved by providing teachers with the training, mentoring, and instruction they require to upgrade their skills in this direction. Educators who want to use metacognitive approaches must be aware of the factors that might influence their lesson preparations.\n\nEven with its pronounced success, the study had certain limitations in its conclusions, as well as in its methodology. Due to the fact that only Saudi EFL students were studied, the findings cannot be applied to ESL or other EFL situations. Furthermore, the improvement in students’ final score was modest but not drastic (10.28 into 14.35). Statistically, though, it was significant. It may be argued that the improvement was not exclusively due to the use of metacognitive skills. These may be attributed to the different instructors and other uncontrolled variables. Future studies in this filed need to rule out these obstacles to make the findings more widely usable.\n\n\nData availability\n\nFigshare: Underlying data for ‘Role of metalinguistic awareness intraining for reading: a quasi-experimental study with Saudi EFL learners’\n\nAssessment Questionnaire: https://doi.org/10.6084/m9.figshare.18925811.v1\n\nDescriptive Analysis: https://doi.org/10.6084/m9.figshare.18925223.v1\n\nThree Way ANOVA Test: https://doi.org/10.6084/m9.figshare.18923801.v1\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nEthical approval\n\nThe study has been reviewed and approved by the Research Ethics committee (REC) at University of Hail (Letter no. H2021-248, Dated 03/01/2022). This study is inspired by Habibian (2015) who checked whether explicit teaching of metacognitive strategies urges learners to use them in text comprehension in a Malaysian context. The current study verifies the use of these strategies in a Saudi context and also checks which ones of the three sub-elements are developed and which one is enhanced as a result of the intervention.", "appendix": "References\n\nAl-Ahdal AAMH: EBook interaction logs as a tool in predicting learner performance in reading. Asiatic: IIUM. J. Engl. Lang. Lit. 2020; 14(1): 174–188.\n\nAlfallaj FSS: Technology in Saudi EFL undergraduate classrooms: Learning tool or weapon of distraction?. Asian ESP J. 2020; 16(4): 97–115.\n\nAkbulut DF: Role of morphological and metalinguistic awareness on reading among Turkish EFL learners. International Journal of Contemporary Educational Research. 2019; 6: 261–277.\n\nAltman C, Goldstein T, Armon-Lotem S: Vocabulary, metalinguistic awareness and language dominance among bilingual preschool children. Front. Psychol. 2018; 9: 1953. PubMed Abstract \| Publisher Full Text\n\nAmin M: Developing reading skills through effective reading approaches. International Journal of Social Science and Humanities. 2019; 4(1): 35–40. Publisher Full Text\n\nBeyer BK: Practical strategies for the teaching of thinking. 7 Wells Avenue, Newton, MA 02159: Allyn and Bacon, Longwood Division; 1987.\n\nCimmiyotti CB: Impact of reading ability on academic performance at the primary level. (Master’s theses and Capstone). projects.2013.\n\nCirino PT, Miciak J, Ahmed Y, et al.: Executive function: association with multiple reading skills. Read. Writ. 2019; 32: 1819–1846. Publisher Full Text\n\nCreswell JW: Research design: Qualitative, quantitative, and mixed methods approaches. USA: Sage Publications; 2014.\n\nDalona IM, Dalona AD: Metalinguistic awareness of multilingual first graders: An exploratory study. English Lang. Teach. Educ. J. 2019; 2(3): 102–111.\n\nDong Y, Peng SN, Sun YK, et al.: Reading comprehension and metalinguistic knowledge in Chinese readers: A meta-analysis. Front. Psychol. 2020; 10: 3037. PubMed Abstract \| Publisher Full Text\n\nEdmunds P, McKinnon N, Zeter J: Building skills for the TOEFL iBT, beginning reading. 2nd ed.USA: Compass Publishing; 2009.\n\nGood RH, Simmons DC, Smith SB: Effective academic interventions in the United States: Evaluating and enhancing the acquisition of early reading skills. Sch. Psychol. Rev. 2019; 27: 45–56. Publisher Full Text\n\nHabibian M: The impact of training metacognitive strategies on reading comprehension among ESL learner’s. J. Educ. Pract. 2015; 6(28): 61–69.\n\nHofer B, Jessner U: Multilingualism at the primary level in South Tyrol: how does multilingual education affect young learners’ metalinguistic awareness and proficiency in L1, L2 and L3?. Lang. Learn. J. 2019; 47(1): 76–87. Publisher Full Text\n\nHung CO, Loh EK: Examining the contribution of cognitive flexibility to metalinguistic skills and reading comprehension. Educ. Psychol. 2020; 41: 712–729. Publisher Full Text\n\nIzadi M, Yarahmadzehi N: The metalinguistic awareness of bilingual (Persian-Baluchi) and monolingual (Persian) learners of English language. Linguist. Approaches Biling. 2018.\n\nKitishat AR, Al Omar KH, Al Momani MAK: The Covid-19 crisis and distance learning: E-teaching of language between reality and challenges. Asian ESP J. 2020; 16(5.1): 316–326.\n\nLi L, Wu X: Effects of metalinguistic awareness on reading comprehension and the mediator role of reading fluency from grades 2 to 4. PLoS One. 2015; 10(3): e0114417. PubMed Abstract \| Publisher Full Text\n\nNúñez-Vázquez I, Crismán-Pérez R: Development of metalinguistic awareness in reading comprehension from video-based instruction. Proceedings of the 5th International Conference on Technological Ecosystems for Enhancing Multiculturality. 2017.\n\nPourhoseinGilakjani A, Sabouri NB: How can students improve their reading comprehension skill?. J. Stud. Educ. 2016; 6(2): 229–240. Publisher Full Text\n\nSandiford C, Macken-Horarik M: Changing stories: Linguistically-informed assessment of development in narrative writing. Assess. Writ. 2020; 45: 100471. Publisher Full Text\n\nSinar B: Promoting metalinguistic awareness in a classroom to improve reading comprehension: Examples from Roald Dahl’s novel The BFG. Acta Didactica Norge. 2018; 12(2): 11–22. Publisher Full Text\n\nTighe EL, Little CW, Arrastia-Chisholm MC, et al.: Assessing the direct and indirect effects of metalinguistic awareness to the reading comprehension skills of struggling adult readers. Read. Writ. 2019; 32(3): 787–818. Publisher Full Text\n\nWoll N: How French speakers reflect on their language: a critical look at the concept of metalinguistic awareness. Lang. Aware. 2019; 28: 49–73. Publisher Full Text\n\nYang X, Peng P, Meng X: How do metalinguistic awareness, working memory, reasoning, and inhibition contribute to Chinese character reading of kindergarten children?. Infant Child Dev. 2019; 28(3): e2122. Publisher Full Text\n\nZhang D, Chin CF, Li L: Metalinguistic awareness in bilingual children’s word reading: A cross-lagged panel study on cross-linguistic transfer facilitation. Appl. Psycholinguist. 2017; 38(2): 395–426. Publisher Full Text" }	[ { "id": "134720", "date": "19 Apr 2022", "name": "Wagdi Rashad Ali Bin-Hady", "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 well-written and organized. Both the abstract and the introduction contain the main required moves and gaps. On the other hand, the literature review needs more development by including the studies added to the discussion section. The methodology is good and clear and the conclusion reported the main findings. A few problems need fixing for the improvement of the paper. I will mention them below.\n\nIs the work clearly and accurately presented and does it cite the current literature?\nThe paper seems interesting and shows good and smooth flow in its parts. The abstract is well-written; it includes all the necessary moves. There are, however, simple points that need to be fixed. Initially, the two words in training have been clubbed in the title; these need to be separated. The introduction is well-presented; it showed the research gap in the Saudi context very satisfactorily.\nA few mistakes need to be fixed, though: the definition of “Metalinguistic awareness\": \"the understanding that a person has necessary to transform language in a variety of ways”, it seems to me that the word necessary is out of context. Still in the introduction, “According to the author’s knowledge, however, though “, I think the use of two subordinate conjunctions at the same time violates the rule of grammar. One of them should be deleted.\nTo go back to the above question, the paper cited some of the up-to date literature. Yet, the second sentence in the literature review is very long. It needs division to free the ambiguity. “It entails a greater awareness of competence achievement at phonology level in rhymes, syllables and phonemes, in addition to acquiring meaning bearing components whether morphological, words leading to syntax-like phrases, and sentences and semantics as denotation, or connotation, ambiguities and at the level of figurative language as metaphor, idioms and the like”.\nIs the study design appropriate and does the work have academic merit?\nThe study would be of significance to scholars interested in metalinguistic studies. The design is also clear, but there seems to be a problem in the instruments “four-point Likert Scale\" has been written in place of frequency Scale. A small paragraph on the analysis of data and the type of analysis used needs to be added.\nAre all the source data underlying the results available to ensure full reproducibility?\nIn the discussion, previous studies used to justify the findings should preferably be brought from the literature review. What is noticed is that some studies appeared in the discussion for the first time for example Dalona and Dalona (2019); Woll (2019) and others. Furthermore, the mentioning of previous studies in the discussion should be in brief, not in details. The elaborate explanations are required in the literature not in the discussion. But that is not a big deal. It could go this way, too. Another point could be added to the limitations section: the the sampling mechanism used it was not random\nAre the conclusions drawn adequately supported by the results?\nYes, the conclusion reported the findings of the study. It also it compares and contrasts with the study of Habibian’s (2015), another great feather in the author's cap.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "134719", "date": "18 May 2022", "name": "Jamal Kaid Mohammed Ali", "expertise": [ "Reviewer Expertise Applied Linguistics · Second Language Acquisition · Language Learning Strategies" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 is well-chosen as it deals with an issue that is not over-researched in Applied Linguistics. It is of pedagogic orientation. It addresses \"reading\" which is considered one of the four English language skills that any learner of English adheres to acquire the language successfully. This type of topic is required as it helps the syllabus makers/designers make the required amendments suggested/recommended for the English language acquisition. However, there seems to be a typo in \"Intraining.\" It should be written as \"in Training.\"\nThe manuscript is well-presented and well-organized as it follows the standard style of a research paper. The author's clear grasp of academic research is shown throughout the manuscript. Moreover, the study to be conducted followed a quasi-experiment. This research design can be perfect for determining what is best for the population. It gave the researcher power over the variables by being able to control them. The way used by the researcher in writing the introduction is perfect and precise. The introduction seems gradual, smooth, and coherent in employing the main concepts and keywords of the title. It shows the significance of such study to the Saudi context.\nThe literature review covers the topic appropriately. It demonstrates the most important recent previous studies related to the topic. There is a need, however, for citing more recent studies.\nThe research questions are clearly stated and fully answered by the instruments employed and the statistical results reached. The methodology section is well-built and framed. It demonstrates all the steps required, such as determining the instrument, participants, and procedures applied in the study. The number of participants in this study being 70 is considered to be a small number for such a study. The references are varied, well-organized and up to date.\n\nIn the \"results and discussion\" section, the manuscript displays the results and findings reached at supported with tables containing all the statistical data. Yet, the problem is with the placement of the tables. It is better to place Table 1 just after the introductory sentence related. Demonstrating the previous studies in this section is perfect as they aim to justify the findings.\n\nThe conclusion is well-stated as it restates the purpose of the study and how it is achieved. The conclusion reports the findings of the study. Moreover, it compares and contrasts with Habibian’s (2015) study that this research was inspired by. The conclusions drawn are adequately supported by the results. One last point here. For the paper to be more valuable and inspiring, the author needs to add a section on “Limitations” so that future researchers can work on the gaps pointed out by the author and try to bridge them.\nIn conclusion, the paper appears to be interesting and flows well. The topic researched is great and would certainly be relevant to many researchers interested in teaching in general and teaching reading in particular.\n\nIs the work 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/11-412
https://f1000research.com/articles/11-411/v1	12 Apr 22	{ "type": "Research Article", "title": "Efficacy and safety of CT-guided percutaneous fine needle aspiration and biopsy for malignant pulmonary lesions", "authors": [ "Santosh Rai", "Vinay BS", "Vishak Acharya", "Jyoti R Kini", "Madhav Kamath M", "Basavaprabhu Achappa", "Jane Mendonca", "Santosh Rai", "Vinay BS", "Vishak Acharya", "Jyoti R Kini", "Madhav Kamath M", "Basavaprabhu Achappa" ], "abstract": "Background: CT-guided percutaneous transthoracic fine needle aspiration (FNA) and core biopsy (CB) are commonly used to characterise lung lesions. There is conflicting information on which method is superior and wide variation in reported complication rates. Our objectives were to establish the efficacy and safety of percutaneous CT-guided FNA and CB in the diagnosis of malignant lung lesions. Methods: This retrospective study included patients who underwent CT-guided percutaneous FNA and/or CB for lung parenchymal lesions at Kasturba Medical College Mangalore, from January 2013 to December 2020. Ethical clearance was obtained from the Institutional Ethics Committee. Efficacy was determined by the adequacy of samples, sensitivity, specificity and diagnostic accuracy. Safety was assessed using the incidence of complications. Results: A total of 326 patients underwent both FNA and CB, and 49 underwent FNA alone. Adequate samples were obtained in 82.9% of FNA cases and 95.7% of CB cases. Considering biopsy as the gold standard, the sensitivity, specificity and diagnostic accuracy of FNA for malignancy were 95.19%, 80% and 91.27%, respectively. Kappa agreement between the two methods was substantial (0.767). Pneumothorax was the only complication observed, and was seen in 31 patients (8.2%), of which only one required chest tube drainage. The incidence of pneumothorax was significantly higher in patients with pre-existing lung disease such as COPD/emphysema (p value 0.000), patients with smaller lesions (p = 0.009), and deeper lesions from the pleura (p <0.0001). Conclusions: FNA and CB are both safe and effective procedures. In the absence of an onsite cytopathologist, we recommend a combination of both techniques.", "keywords": [ "fine needle aspiration", "biopsy", "lung", "CT-guided", "pulmonary lesions", "malignancy", "pneumothorax", "safety" ], "content": "Introduction\n\nComputed tomography (CT) imaging of the thorax has become the cornerstone of initial imaging in lung cancer and can provide accurate anatomic localization of the tumour, along with valuable diagnostic clues about tumour histology.1 However, tissue sampling remains the gold standard for the diagnosis and subtyping of malignant lung pathologies and in the era of personalised medicine, obtaining adequate tumour tissue is essential for molecular profiling and targeted therapy.2 In this regard, minimally invasive procedures such as fine needle aspiration (FNA) and core biopsy (CB) performed under CT guidance have become increasingly common. There is controversy regarding which technique is ideal.3 Recent surveys have shown that in practice the core needle biopsy alone is preferred over FNA alone as a sampling technique in the United States (42% vs. 15%) and the United Kingdom (88.7% vs 1.3%).4,5 Forty three percent of respondents in the United States and only ten percent in the United Kingdom reported using both FNA and CB, despite evidence that a combination of the two techniques is superior to either alone in terms of yield and diagnostic accuracy.6–8 This study was conducted to evaluate the efficacy and safety of CT guided FNA and core needle biopsy for lung pathologies. This study will add to the existing literature on the safety and efficacy of these procedures, and also serve as an audit of our institutional practice.\n\n\nMethods\n\nThis retrospective study was conducted in the Department of Radiodiagnosis of Kasturba Medical College Mangalore, a tertiary care hospital, from January 2013 to December 2020 in patients with intrathoracic lesions suspicious of lung malignancies. Ethical clearance was obtained from the Institutional Ethics Committee, Kasturba Medical College Mangalore, Manipal Academy of Higher Education (approval number: IEC KMC MLR 08-16/181), prior to the start of the study and written informed consent was obtained from all the subjects. Relevant investigations (bleeding time, clotting time, prothrombin time/INR and platelet count) were done to exclude coagulopathy. Anticoagulants were withheld five days before the procedure if the patient was receiving these medications.\n\nThe inclusion criteria were patients of all ages undergoing percutaneous transthoracic lung biopsy and/or fine needle aspiration under computed tomography guidance. Exclusion criteria included uncooperative patients, patients with contraindications to the procedure (haemodynamic instability, coagulopathy, etc), those with significant paraseptal emphysema surrounding the lesion and patients who did not consent to the procedure and/or study.\n\nDemographic and relevant clinical data were collected from the records of eligible patients. Contrast enhanced computed tomography of the chest was performed using a multidetector 16-slice CT scanner (G E Brivo and G E Bright Speed Elite General Electric, Milwaukee, United States). Contrast enhancement of the lesions was undertaken using 80 ml of non-ionic contrast given intravenously. Venous phase images were acquired 50–60 seconds after administration of contrast. After localization of the lesion, FNA and/or CB were performed. Only FNA was performed in lesions less than 10 mm in size and lesions within 2 cm of the proximal bronchial tree, heart, great vessels, trachea, or other mediastinal structures. The final decision was made by the radiologist performing the procedure.\n\nAfter a detailed explanation of the procedure, the patient was appropriately positioned in the CT gantry based on pre-procedure imaging. A plain CT scan of the suspected lesion was performed (Figure 1A). A radiopaque grid was placed on the skin at the expected needle entry site (Figure 1B). A CT of the targeted area was performed to determine the skin site for needle entry and needle trajectory. Subsequently, the grid was removed. For the FNAC, a 22-gauge spinal needle (BD Spinal Needle Quincke Type Point, Becton Dickinson India Pvt Ltd, 22Gx3.50IN) was used. The needle was advanced in a stepwise manner while obtaining interval short-segment CT images to guide placement (Figure 1C). When the needle was confirmed to be inside the target tissue by CT, the stylet was removed. A 10 ml suction syringe was attached to the hub of the needle and the plunger was gently withdrawn 6–8 times. The suction pressure was then released, and the needle was withdrawn. Smears were prepared from the aspirated material on glass slides, half of which were inserted into a jar of methanol fixative (for Papanicolaou staining) and the other half were air dried.\n\nFor the core biopsy, an 18-gauge spring loaded cutting needle automatic biopsy gun (Max Core Disposable Core Biopsy Instrument, CR Bard Inc) with a 22 mm throw side cutting needle was used. For deeper intraparenchymal lesions the needle was advanced till the pleura in a stepwise manner and inserted into the lesion by assessing the depth from the skin (Figure 1D). In cases of multiple lesions, the largest lesion with the safest access in proximity to the pleura was chosen. Pleural separation with saline was attempted to obtain a wider window for passage of the biopsy gun. The specimen obtained was transferred to a container with formalin for histopathological examination. A single pass was performed routinely. Repeat passes were performed only if the first pass was unsuccessful or the specimen was fragmented on visual inspection. A cytopathologist was not available on site, and specimens were transported to the laboratory after the procedure.\n\nAdequate compression and sterile dressing were applied immediately after the procedure. A check CT was performed within 30 minutes of the procedure to evaluate for complications including pneumothorax, haemorrhage, and soft tissue hematoma.\n\nData were analysed using SPSS version 25 (IBM SPSS Statistics, RRID:SCR_019096). Categorical data were represented in the form of frequencies and proportions. Continuous data were represented as mean and standard deviation. The Chi-square test was used as test of significance for qualitative data. The Independent t test was used as test of significance to identify the mean difference between two quantitative variables.\n\n\nResults\n\nA total of 375 patients met the inclusion criteria and were included in the study, of which 326 underwent concurrent FNA and CB and 49 underwent FNA alone. The mean age of the study population was 60.19 ± 12.09 years. The demographic characteristics of the study population are presented in Table 1.\n\nThe mean size of histologically benign lesions on CT was 4.3 cm ± 2 cm, and the mean size of histologically malignant lesions was 4.6 cm ± 1.7 cm. There was no significant difference between the size of benign and malignant lesions (p value 0.116). Out of 375 sampled lesions, 301 (80.3%) were abutting the pleura, and 74 (19.7%) were located at some distance from the pleura. The mean distance from the pleura in these lesions was 9.67 cm ± 7.686 cm.\n\nThe FNA smears and biopsy samples obtained were categorised as benign, malignant, atypical/suspicious or inadequate. Those reported to be benign included 20.3% (76/375) of FNA samples and 26.1% (84/326) of core biopsy samples. Malignancy was detected in 58.9% (221/375) of FNA and 69% (225/326) of biopsy samples. Atypical or suspicious for malignancy were reported in 3.7% (14/375) and 0.6% (2/326) of FNA and biopsy samples respectively. Only 4.6% (15/326) of biopsy samples were inadequate, as were 17% (64/375) of FNA smears.\n\nIn cases where both FNA and CB were performed, the result of the CB was considered to be the final diagnosis. Of the CB samples 309 out of 326 were classified as either benign or malignant (15 cores were inadequate and two were atypical). Forty-nine patients underwent FNA alone, of which 37 smears were classified as either benign or malignant (12 smears were inadequate). The histologic diagnoses reported from these specimens are presented in Table 2. Non-small cell lung cancer (NSCLC) was the commonest diagnosis (n=220). The benign specific diagnoses included tuberculosis (n=10), cryptogenic organising pneumonia (n=9), interstitial pneumonia (n=5), neurofibroma (n=1), actinomycosis (n=1), aspergillosis (n=2), lipoid pneumonia (n=1) and lipoma (n=1). A total of seven cases were reported as positive for malignancy without further classification of tumour type.\n\nConsidering biopsy as the gold standard, the sensitivity and specificity of FNA for malignancy was found to be 95.19% and 80% respectively. The positive predictive value and negative predictive value were 93.19 and 85.25, respectively. Overall, FNA had a diagnostic accuracy of 91.27%. Kappa agreement between the two methods was found to be substantial (0.767).\n\nThirty one out of 375 patients (8.3%) developed pneumothorax following the procedure, of which 30 were small pneumothoraces (less than 2 cm) and only one was a large pneumothorax that required chest tube drainage. No other complications apart from pneumothorax were observed.\n\nPatients who developed pneumothorax had significantly smaller lesions and significantly larger mean distance of the lesion from the pleura. Significantly higher occurrence of pneumothorax was also observed in patients who had pre-existing lung disease versus those who did not (Tables 3 and 4).\n\n\nDiscussion\n\nBronchogenic cancer is the leading cause of cancer related death worldwide, causing more deaths than colorectal, breast, brain, and prostate cancer put together.9,10 The treatment of primary lung cancer hinges on distinguishing non-small cell lung cancer from small cell lung cancer, and recent advances have made it possible to identify genetic mutations in tumour tissue which can guide targeted therapy.11 The lung is also one of the commonest sites of metastasis from other primary tumours and adequate tissue sampling of these lesions is essential for identification of the primary malignancy and treatment.12 All of this necessitates finding safe and efficient techniques to obtain adequate amounts of tissue from lung lesions. Percutaneous CT-guided fine needle aspiration (FNA) and core biopsy (CB) are minimally invasive procedures that are commonly performed for the diagnosis of intrathoracic lesions, including lung lesions. Both techniques have their advantages and disadvantages and controversy exists over which technique can independently provide samples that are adequate for diagnosis and ancillary studies.\n\nFNA is an easy to perform and cost-effective method of sampling, with established accuracy for diagnosing malignancy. Several studies have reported that the diagnostic accuracy of FNA for malignant lesions ranges from 80–95%, and sensitivities > 90% have been reported.6,13–17 The sensitivity and diagnostic accuracy of percutaneous CT guided FNA for malignant lesions in our study were comparable to the existing literature (95.19% and 92.17%, respectively). However, the adequacy of FNA sampling in our study was lower than that of CB (82.9% versus 95.7%, respectively). This is in contrast to the findings of Poulou et al, who reported that performing CB alone decreased the diagnostic yield, especially with lesions ≤ 4 cm in size.18 The authors have hypothesized that this may be due to the inability of CB needles to obtain samples within the margin of smaller lesions, a drawback that may be overcome with FNA. Guidelines from the British Thoracic Society (BTS) regarding percutaneous transthoracic FNA and CB of the lung recommend that an adequacy of >90% be achieved for samples.19 The adequacy and diagnostic accuracy of FNA samples in our study could have been improved by Rapid On-Site Evaluation (ROSE) of the aspirate by a cytopathologist, which is not available at our institution. Previous studies have shown that with ROSE, the diagnostic accuracy of FNA was similar to or even higher than CB for malignancy.20,21 Coley et al. reported that performing FNA alone with ROSE provided adequate samples even for immunohistochemical (IHC) and molecular analysis. The authors compared three modalities – FNA, CB and FNA plus CB – with regard to providing sufficient tissue for subtyping malignancy (with IHC if necessary) and for molecular analysis when required. They reported that no significant statistical difference was found between the three for adequacy of samples.2 These studies might suggest that FNA with ROSE could obviate the need for performing a more invasive core biopsy. However, performing both procedures is beneficial, especially in patients who may have benign lesions or non-epithelial malignancies, where FNA has been shown to be inferior to CB.6,16 Combined FNA and CB could also aid in risk stratification of patients who have atypical cytology – the highest risk of developing malignancy being in patients who had atypical cells on both FNA and CB – followed by those with atypical cytology on CB but negative FNA, and the lowest risk in those with atypical FNA/negative CB.7\n\nThe incidence of complications in our study was 8.3%. Apart from pneumothorax, no other complications such as haemoptysis or pulmonary haemorrhage were observed. Pneumothorax is the commonest complication following percutaneous lung biopsy and has a reported incidence of up to 61% for FNA and 26–54% for cutting needle biopsies.19 Of these, up to 18% of patients in the former group and 3.3–15% of patients in the latter group may require drainage.19 Our study demonstrated an acceptable complication rate and only one patient required chest tube drainage for pneumothorax. Increased depth of the lesion from the pleura, smaller size of the lesion and the presence of pre-existing parenchymal disease such as emphysema were found to be risk factors for the development of pneumothorax in our study. This agrees with the findings of Dennie et al, who reported that deeper lesions, smaller lesions and the presence of chronic obstructive pulmonary disease (COPD) were more likely to be associated with development of a pneumothorax.22 Between 40–70 % of patients with lung cancer may also have underlying COPD, which could be attributed to the presence of common risk factors such as smoking.23 COPD has also been found to be an independent risk factor for the development of lung cancer, irrespective of smoking status.24 The high prevalence of COPD in lung cancer can create challenges with regard to selecting the modality for tissue diagnosis when the risk of pneumothorax is considered. A study evaluating the use of ultra-thin 25 G needles for FNA lung in patients with functional lung impairment reported an overall pneumothorax rate of 20.6%, with chest tube insertion frequency of 8.7%, in a study population where nearly half the subjects had moderate to severe functional lung impairment.17\n\nThis study had certain limitations. Firstly, due to its retrospective nature, we could not randomize patients to undergo either one or both of these procedures, which could have provided a better comparison of the two techniques. We also considered the biopsy diagnosis as the final diagnosis due to a lack of follow-up data. Lastly, since we do not routinely request cell blocks for fine needle aspirates, we could not compare the adequacy of the two techniques for IHC and/or molecular studies.\n\n\nConclusions\n\nIn conclusion, we report that CT guided FNA is a safe and reliable method for diagnosis of malignant lung lesions. The complication rate in the present study was minimal. In the absence of an on-site cytopathologist, we recommend a combined approach of FNA and core biopsy in all cases.\n\n\nData availability\n\nDryad: Underlying data for ‘Efficacy and safety of CT-guided percutaneous fine needle aspiration and biopsy for malignant pulmonary lesions’. https://doi.org/10.5061/dryad.5mkkwh76w\n\nThis project contains the following underlying data:\n\n• Data file: Study data.xlsx\n\n• ReadMe.txt\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 and their images was obtained from the patients.", "appendix": "References\n\nHollings N, Shaw P: Diagnostic imaging of lung cancer. Eur. Respir. J. 2002 Apr 1; 19(4): 722–742. Publisher Full Text\n\nColey SM, Crapanzano JP, Saqi A: FNA, core biopsy, or both for the diagnosis of lung carcinoma: obtaining sufficient tissue for a specific diagnosis and molecular testing. Cancer Cytopathol. 2015 May; 123(5): 318–326. Publisher Full Text\n\nYao X, Gomes MM, Tsao MS, et al.: Fine-Needle Sspiration Biopsy versus Core-Needle Biopsy in Diagnosing Lung Cancer: A Systematic Review. Curr. Oncol. 2012 Feb; 19(1): 16–27. PubMed Abstract \| Publisher Full Text\n\nTavare AN, Hare SS, Miller FN, et al.: A survey of UK percutaneous lung biopsy practice: current practices in the era of early detection, oncogenetic profiling, and targeted treatments. Clin. Radiol. 2018 Sep 1; 73(9): 800–809. PubMed Abstract \| Publisher Full Text\n\nLee C, Guichet PL, Abtin F: Percutaneous lung biopsy in the molecular profiling era. J. Thorac. Imaging. 2017 Jan 1; 32(1): 63–67. PubMed Abstract \| Publisher Full Text\n\nGong Y, Sneige N, Guo M, et al.: Transthoracic fine-needle aspiration vs concurrent core needle biopsy in diagnosis of intrathoracic lesions: a retrospective comparison of diagnostic accuracy. Am. J. Clin. Pathol. 2006 Mar 1; 125(3): 438–444. PubMed Abstract \| Publisher Full Text\n\nChen L, Jing H, Gong Y, et al.: Diagnostic efficacy and molecular testing by combined fine-needle aspiration and core needle biopsy in patients with a lung nodule. Cancer Cytopathol. 2020 Mar; 128(3): 201–206. PubMed Abstract \| Publisher Full Text\n\nAviram G, Greif J, Man A, et al.: Diagnosis of intrathoracic lesions: are sequential fine-needle aspiration (FNA) and core needle biopsy (CNB) combined better than either investigation alone?. Clin. Radiol. 2007 Mar 1; 62(3): 221–226. PubMed Abstract \| Publisher Full Text\n\nTorre LA, Bray F, Siegel RL, et al.: Global cancer statistics, 2012. CA Cancer J. Clin. 2015 Mar; 65(2): 87–108. PubMed Abstract \| Publisher Full Text\n\nSiegel RL, Miller KD, Fuchs HE, et al.: Cancer statistics, 2021. CA Cancer J. Clin. 2021 Jan 12; 71(1): 7–33. PubMed Abstract \| Publisher Full Text\n\nMayekar MK, Bivona TG: Current landscape of targeted therapy in lung cancer. Clin. Pharmacol. Ther. 2017 Nov; 102(5): 757–764. Publisher Full Text\n\nHerold CJ, Bankier AA, Fleischmann D: Lung metastases. Eur. Radiol. 1996 Oct; 6(5): 596–606. PubMed Abstract \| Publisher Full Text\n\nUzun Ç, Akkaya Z, Atman ED, et al.: Diagnostic accuracy and safety of CT-guided fine needle aspiration biopsy of pulmonary lesions with non-coaxial technique: a single center experience with 442 biopsies. Diagn. Interv. Radiol. 2017; 23(2): 137–143. PubMed Abstract \| Publisher Full Text\n\nKlein JS, Salomon G, Stewart EA: Transthoracic needle biopsy with a coaxially placed 20-gauge automated cutting needle: results in 122 patients. Radiology. 1996 Mar; 198(3): 715–720. PubMed Abstract \| Publisher Full Text\n\nKim HK, Shin BK, Cho SJ, et al.: Transthoracic fine needle aspiration and core biopsy of pulmonary lesions. A study of 296 patients. Acta Cytol. 2002 Nov 1; 46(6): 1061–1068. Publisher Full Text\n\nEftekhar-Javadi A, Kumar PV, Mirzaie AZ, et al.: Diagnostic accuracy of fine needle aspiration cytology versus concurrent core needle biopsy in evaluation of intrathoracic lesions: a retrospective comparative study. Asian Pac. J. Cancer Prev. 2015; 16(16): 7385–7390. PubMed Abstract \| Publisher Full Text\n\nOikonomou A, Matzinger FR, Seely JM, et al.: Ultrathin needle (25 G) aspiration lung biopsy: diagnostic accuracy and complication rates. Eur. Radiol. 2004 Mar; 14(3): 375–382. PubMed Abstract \| Publisher Full Text\n\nPoulou LS, Tsagouli P, Ziakas PD, et al.: Computed tomography-guided needle aspiration and biopsy of pulmonary lesions: a single-center experience in 1000 patients. Acta Radiol. 2013 Jul; 54(6): 640–645. PubMed Abstract \| Publisher Full Text\n\nManhire A, Charig M, Clelland C, et al.: Guidelines for radiologically guided lung biopsy. Thorax. 2003 Nov 1; 58(11): 920–936. Publisher Full Text\n\nCharig MJ, Phillips AJ: CT-guided cutting needle biopsy of lung lesions—safety and efficacy of an out-patient service. Clin. Radiol. 2000 Dec 1; 55(12): 964–969. PubMed Abstract \| Publisher Full Text\n\nCapalbo E, Peli M, Lovisatti M, et al.: Trans-thoracic biopsy of lung lesions: FNAB or CNB? Our experience and review of the literature. Radiol. Med. 2014 Aug 1; 119(8): 572–594. PubMed Abstract \| Publisher Full Text\n\nDennie CJ, Matzinger FR, Marriner JR, et al.: Transthoracic needle biopsy of the lung: results of early discharge in 506 outpatients. Radiology. 2001 Apr; 219(1): 247–251. PubMed Abstract \| Publisher Full Text\n\nYoung RP, Hopkins RJ, Christmas T, et al.: COPD prevalence is increased in lung cancer, independent of age, sex and smoking history. Eur. Respir. J. 2009 Aug 1; 34(2): 380–386. Publisher Full Text\n\nPark HY, Kang D, Shin SH, et al.: Chronic obstructive pulmonary disease and lung cancer incidence in never smokers: a cohort study. Thorax. 2020 Jun 1; 75(6): 506–509. PubMed Abstract \| Publisher Full Text" }	[ { "id": "134795", "date": "29 Apr 2022", "name": "Ravikanth Balaji", "expertise": [ "Reviewer Expertise Oncology Imaging" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPertinent study to daily clinical practice on use of FNA and biopsies for lung malignancies. .\n\nExcellent comparison of use of these techniques and yield of pathology to differentiate benign and malignant pathologies. The pathology support is essential for evaluating samples for cytology.\n\nGood statistical correlations.\n\nIncludes commonly encountered complications.\n\nIs the work 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/11-411
https://f1000research.com/articles/11-409/v1	12 Apr 22	{ "type": "Research Article", "title": "Effects of bariatric surgery on renal function and associated factors with bivariate analysis: a cohort study", "authors": [ "Juliana Amaro Borborema Bezerra", "Eduardo Pachu Raia dos Santos", "Carlos Teixeira Brandt", "Eduardo Pachu Raia dos Santos", "Carlos Teixeira Brandt" ], "abstract": "Background: Obesity is a global pandemic, caused by genetic, biological and social factors, increasing the risk of many chronic diseases, including kidney disease. Bariatric surgery can control associated comorbidities and also improve renal function. Thus, the aim of this study was to evaluate the effect of bariatric surgery on renal function and associated factors, through bivariate analysis. Methods: A cohort, prospective, analytical study was carried out in the Department of surgery and obesity, in Campina Grande - Paraíba, Brazil. Thirty-five obese patients were evaluated in the preoperative period of bariatric surgery and after one year of bariatric surgery. Sociodemographic and anthropometric data were collected, body mass index was calculated, and renal function was estimated through the glomerular filtration rate. Results: Predominantly female (71.4%) and white (77.1%) patients were observed. Regarding the type of bariatric surgery, most patients (65.7%) underwent the sleeve technique. There was no significant difference between the mean levels of cystatin C pre and post bariatric surgery. There was a significant improvement in the mean glomerular filtration rates in the postoperative period (p=0.09). In further analysis of the association between the variables: type of bariatric surgery, diabetes or hypertension outcomes, no association was observed with the outcome improvement in glomerular filtration rate, without statistical significance, respectively (p = 0.312; p =0.217; p = 0.476). Similarly, there was no statistically significant difference between the variable loss of body mass index, under the effect of bariatric surgery, in relation to the outcome of the glomerular filtration rate (p = 0.904). Conclusion: After analyzing the association between outcome of glomerular filtration rate and the studied variables, no association was observed between these variables, under the effect of bariatric surgery, with improvement in glomerular filtration rate. Thus, bariatric surgery is associated with improvement of renal function independently.", "keywords": [ "Obesity", "Bariatric surgery", "Glomerular filtration rate", "Cystatin C", "Renal function" ], "content": "Introduction\n\nObesity is a global pandemic related to excessive adiposity and mediated by inflammation, with long-term damage that impairs the quality of life of obese people.1,2 It may affect all age groups and ethnicities in many countries.3–5\n\nAdopted life-style and genetic factors are related to the genesis of obesity and associated with an increase in cancer, cardiovascular diseases, blood hypertension, type 2 diabetes mellitus (DM) and chronic kidney disease (CKD).6–10\n\nThe causes of obesity-related renal impairment are multifactorial, ranging from the deleterious mechanisms of obesity-associated comorbidities in the kidneys, as well as the production of adiponectin by adipose tissue. In addition to weight gain itself with deleterious renal action leading to the onset or worsening of CKD.11,12\n\nOther mechanisms for the onset of kidney injury in obesity involve oxidative stress, activation of the renin-angiotensin-aldosterone system and insulin resistance.13–17\n\nBariatric surgery has become the main method for controlling morbidities associated with obesity, with adequate glycemic and blood pressure control, in addition to promoting sustained weight loss.18–22\n\nThe effect of bariatric surgery on the renal function is a subject of intensive research. In most studies, one can observe, after bariatric surgery, an improvement of glomerular filtration rate (GFR) and in preventing the onset of CKD and its progression. But in other papers, the improvement of kidney function is not so clear.23–25\n\nThe aim of this study was to evaluate the impact of bariatric surgery on patients' renal function and associated factors through a bivariate analysis, corroborating the findings of improvement in GFR in most studies.\n\n\nMethods\n\nA cohort, prospective, analytical study was carried out in a Department of surgery and obesity, in Campina Grande - Paraíba, Brazil, between February 2019 and August 2020. The study was approved by the Ethics and Research Committee of the Faculty of Medical Sciences - UNIFACISA - Campina Grande - Paraíba, Brazil, registration number - 79501417.0.0000.5175. All patients gave written informed consent before inclusion.\n\nThe sampling process took place in a non-probabilistic way. To calculate the sample size, the free software GPower version 3.1.9.7 (RRID:SCR_013726, http://www.gpower.hhu.de/); was used, with a significance level of 0.05 and a test power commonly used in the literature equal to 0.80. The existence of an average effect size equal to 0.5 was also considered. The minimum number of patients estimated for the research were 34. Sixty-five patients were recruited to participate in this research.\n\nObese individuals undergoing bariatric surgery were included in this study. Those with microalbuminuria ≥ 30 mg/g were excluded, to avoid previous kidney disease, and patients with thyroid disease were excluded too. Hyperthyroidism has been shown to increase, while hypothyroidism is shown to decrease cystatin C serum concentrations, the reason why participants with thyroid disease were excluded in this study.26\n\nAll the patients were evaluated using a form regarding sociodemographic characteristics (age, gender, ethnicity, schooling and health insurance), comorbidities (pre-existing diseases) and types of surgery they would undergo. At the same time, blood pressure, weight and height were measured. The body mass index (BMI) was calculated by weight (kilograms), divided by height (by meter squared).27\n\nLater, blood samples were collected to measure serum creatinine and cystatin C. The cystatin C was measured by nephelometry and calibrated to recent cystatin C standardization, with a result expressed in mg/L. This is an endogenous marker of glomerular filtration rate (GFR) for renal function due to its high sensitivity and specificity and it is not influenced by the weight loss.28,29 The GFR was estimated using Nefrocalc version 2.0 (http://www.nefrocalc.net/filtracao-7.html) through the Chronic Kidney Disease Epidemiology (CKD-Epi) collaboration equation creatinine-cystatin C and corrected for the corresponding body surface.30 Hypofiltration was defined by GFR < 90 mL/min/1.73 m2, normal GFR ranged between 90 and 120 mL/min/1.73 m2 and hyperfiltration was defined by GFR > 120mL/min/1.73 m2.31\n\nOnly 35 patients could be re-evaluated one year after the bariatric surgery. The others refused to undergo any type of re-evaluation because of the Covid-19 pandemic. Therefore, only 35 patients were able to continue in this cohort study out of a total of 65 obese patients were included initially.\n\nQuantitative variables were expressed as means ± standard deviation. Qualitative variables were expressed by their absolute and relative frequencies. p ≤ 0.05 was established for rejection of the null hypothesis.\n\nIn order to investigate the effect of bariatric surgery on means (pre-surgery vs post-surgery), Student's t test for paired samples was applied. In the case of variables that did not meet the normality assumption, the bootstrapping procedure (1000 re-samplings; 95% CI) was performed to obtain greater reliability of the results. Similarly, one-way ANOVA with bootstrapping correction was applied in order to verify a possible influence of the GFR outcome (remained, improved or worsened GFR between pre-surgery and post-surgery) on the mean effects of loss of BMI (loss of BMI = BMI (pre-surgery) – BMI (post-surgery)). In the case of dichotomous variables, McNemar's test was performed. Fisher's exact test was used to investigate the association between variables created in order to express the effect of bariatric surgery. Such variables were: GFR outcome, type of diabetes outcome (DM: remained non-diabetic, improved or remained diabetic) and type of hypertension outcome (Hypertension: remained non-hypertensive, improved or remained hypertensive).\n\n\nResults\n\nPredominantly, female (71.4%) and white (77.1%) patients were observed in this study. All had high school education and health insurance. The mean time of follow-up was 16.2 ± 2.6 months. Ages ranged from 24.0 to 57.0 years. Regarding the type of bariatric surgery, most underwent the sleeve type (65.7%).\n\nIn the preoperative bariatric surgery, it was observed that 31.4% were diabetic patients and 40.0% were hypertensive patients. In the postoperative surgery, among the patients with hypertension and diabetes, the majority of obese patients obtained control. Of the total of 11 pre-surgery diabetic patients, seven were no longer diabetic, and of the total of 14 pre-surgery hypertensive patients, 12 became non-hypertensive post-surgery (Table 1).\n\n McNemar's test. Binomial distribution used.\n\nIn the post bariatric surgery there was a significant reduction in the mean abdominal circumference (p < 0.0001) and in the mean BMI in postoperative period of bariatric surgery (p < 0.0001) (Table 2). No significant difference was observed between the mean levels of cystatin C pre and post bariatric surgery (p = 0.094) (Table 2). There was a significant improvement in glomerular filtration rates in obese patients undergoing bariatric surgery (Table 2) (Figure 1).\n\n* BMI: body mass index kg/m2 (kilograms per square meter);\n\n paired t test with bootstrapping procedure;\n\n* GFR: Glomerular filtration rate mL/min/1.73 m2 (milliliters per minute per1.73 square meters);\n\n** Abdominal circumference cm (centimeters);\n\n* paired t test;\n\n**** Cystatin C mg/L (milligrams per liter); SD: Standard Deviation.\n\nOn analyzing the association between the outcome of the variables: type of bariatric surgery, diabetes outcome and hypertension outcome, no association was observed with the outcome improvement in GFR, under the effect of bariatric surgery, without statistical significance, respectively (p = 0.312; p = 0.217; p = 0.476) (Table 3).\n\n* Fisher's exact test.\n\nThere is an indication that bypass surgery tends to lead a greater number of patients to an improvement in the GFR (91.7%), although this result is not statistically significant (p = 0.312) (Table 3).\n\nOne-Way ANOVA results demonstrated that there was no difference between the mean BMI losses associated with the GFR outcome groups (p = 0.904). The type of GFR outcome under the effect of surgery did not affect the result of BMI expressed by the loss between pre and postoperative periods (Figure 2).\n\nANOVA One-Way with bootstrapping procedure.\n\n\nDiscussion\n\nAccording to the literature, in this cohort study one can observe a prevalence of white, females with an average age of 41 years, having high school education and health insurance, as women tend to take more care of themselves. They had a good socioeconomic level, given the difficulties of access to bariatric surgery in public services.32\n\nBariatric surgery has shown encouraging results in the control of comorbidities in obese individuals, proving to be effective in sustained weight loss, corroborating the findings of the present study.33,34\n\nWith regard to comorbidities (hypertension and diabetes), a significant decrease in the prevalence of these diseases can be observed in rated obese individuals after bariatric surgery, similar to most studies.35\n\nSome studies have pointed out the improvement of renal function after bariatric surgery, but not in other studies.36–38 In the present result, an improvement in renal function was observed after bariatric surgery.\n\nIn this study there was trend towards an improvement in the GFR in the bypass technique in relation to the sleeve as opposed to the one observed in the literature.39 The explanation for this would be the evidence of the relationship of the bypass technique with urinary calculus formation, as well as malabsorption syndrome.39\n\nKidney disease can be caused by hypertension, diabetes as well as obesity.10,40 However, obesity is also associated with the emergence of diabetes, hypertension and kidney disease. In this sense, bariatric surgery promotes a reduction in BMI, glycemic control and blood pressure, and is even associated with improved renal function.6–12 Therefore, it is hard to know what came first in this cascade of improvements.\n\nAfter performing a bivariate analysis between outcome of glomerular filtration rate and factors such as outcome of hypertension, outcome of diabetes and surgical techniques, no association of these factors with improved renal function was found. Furthermore, the type of GFR outcome under the effect of bariatric surgery does not affect the result of BMI which reinforces evidence suggesting that there is a strong and independent association between obesity and development of kidney disease.24\n\n\nConclusion\n\nAfter bariatric surgery, there was a significant improvement in the GFR, abdominal circumference and BMI, in addition to the control of associated comorbidities in obese patients with diabetes and hypertension. After bivariate analysis, no association was found between the improvement in GFR in the post-operative period of bariatric surgery and the analyzed variables. However, other prospective and more robust studies, as well as those with a larger sample size are needed to answer this challenging question: “Does bariatric surgery have an effect on improving renal function as an independent factor?”\n\nThe sample size that followed in this longitudinal study was too small because of the Covid-19 pandemic. The short follow-up time for assessing renal function after bariatric surgery stems from the fact that this manuscript is part of an ongoing PhD thesis research in surgery, from the Federal University of Pernambuco.\n\n\nData availability\n\nHarvard Data verse: Effects of bariatric surgery on renal function and associated factors with bivariate analysis: a cohort study. https://doi.org/10.7910/DVN/5C4F0I41\n\nThe project contains the following underlying data:\n\n- 01_bariatric_surgery_nephrological_study_database.tab (raw 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\n\nAuthor contribution\n\nBezerra JAB: collected the data, contributed in interpretation of data and prepared the manuscript.\n\nBrandt CT: contributed in interpretation of data and made the final revision.\n\nSantos EPR: contributed in interpretation of data.", "appendix": "Acknowledgement\n\n\n\n• Gilberto S. Matos: PhD. Associate Professor of Statistics at The Federal University of Campina Grande (UFCG), Brazil- contributed to the statistical analysis.\n\n• Edmilson de Albuquerque Borborema Filho: PhD. Associate Professor of English and Linguistics at The Federal University of Paraiba (UFPB), Brazil- contributed in english language review.\n\n\nReferences\n\nWharton S, Lau D, Vallis M, et al.: Obesity in adults: a clinical practice guideline. CMAJ: Canadian Medical Association Journal = Journal de l'Associationmedicalecanadienne. 2020; 192: E875–E891. PubMed Abstract \| Publisher Full Text\n\nLee BY, Bartsch SM, Mui Y, et al.: A systems approach to obesity. Nutr. Rev. 2017; 75: 94–106. PubMed Abstract \| Publisher Full Text\n\nRangel-Huerta OD, Villaescusa BP, Gil A: Are we close to defining a metabolomic signature of human obesity? A systematic review of metabolomics studies. Metabolomics. 2019; 15: 31–93. 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PubMed Abstract \| Publisher Full Text\n\nMemarian E, Carrasco D, Thulesius H, et al.: Primary care physicians' knowledge, attitudes and concerns about bariatric surgery and the association with referral patterns: a Swedish survey study. BMC Endocr. Disord. 2021; 21: 1–10. PubMed Abstract \| Publisher Full Text\n\nDe Paris FGC, Padoin AV, Mottin CC, et al.: Assessment of changes in body composition during the first postoperative year after bariatric surgery. Obes. Surg. 2019; 29: 3054–3061. PubMed Abstract \| Publisher Full Text\n\nJi Y, Lee H, Kaura S, et al.: Effect of Bariatric Surgery on Metabolic Diseases and Underlying Mechanisms. Biomolecules. 2021; 11: 1582. PubMed Abstract \| Publisher Full Text\n\nHolcomb CN, Goss LE, Almehmi A, et al.: Bariatric surgery is associated with renal function improvement. Surg Endoscopy. 2018; 32: 276–281. PubMed Abstract \| Publisher Full Text\n\nDewantoro D, Fultang J, Lowe K, et al.: Impact of Bariatric Surgery on Renal Function. Cureus. 2021; 13: e18534. PubMed Abstract \| Publisher Full Text\n\nLi K, Zou J, Ye Z, et al.: Effects of bariatric surgery on renal function in obese patients: A systematic review and metaanalysis. PLoS One. 2016; 11: e0163907. PubMed Abstract \| Publisher Full Text\n\nProchaska M, Worcester E: Risk Factors for Kidney Stone Formation following Bariatric Surgery. Kidney360. 2020; 1: 1456–1461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuyckx VA, Tonelli M, Stanifer JW: The global burden of kidney disease and the sustainable development goals. Bull. World Health Organ. 2018; 96: 414–422D. PubMed Abstract \| Publisher Full Text\n\nBorborema J, Santos E, Brandt C: Replication Data for: Nephrological study of people undergoing bariatric surgery.2022. Harvard DataverseV1, UNF:6:E+vW60wi/RTS6z09UCAywQ== [fileUNF].Publisher Full Text" }	[ { "id": "218469", "date": "29 May 2024", "name": "Tarek Arabi", "expertise": [ "Reviewer Expertise Kidney transplantation", "obesity", "chronic kidney disease", "molecular mechanisms", "immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study by Bezerra et al. is interesting and is still a topic of controversy and discussion. However, there are a few comments that must be addressed:\nThe language of the manuscript can be better refined.\n\nI believe the statistical methods used are weak. The authors relied heavily on comparing categorical variables instead of continuous ones. Instead of categorizing patients into 'improved', 'worsened', and 'remained', it might be more adequate to compare the value of the GFR change itself. For example, the change of GFR in bypass vs. sleeve patients. I think this would help strengthen the statistical value and would give the readers an accurate representation of how much the change was.\n\nThe authors stated their method of calculating sample size. I do not think they mentioned what the population size they based off their calculations off was.\n\nI believe it is important to provide a mean and SD of the age itself, instead of simply a range.\n\nThe tables are difficult to follow, in my opinion. I think the tables would need to restructured after adding the GFR change instead of just categories. Furthermore, a table describing the general demographics and relevant parameters of the study sample is needed.\n\nThe discussion is too simple. Authors should delve deeper into the role of obesity in renal function and better compare with studies in the literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-409
https://f1000research.com/articles/11-407/v1	11 Apr 22	{ "type": "Research Article", "title": "Minimization of Total Variable Cost through Determination of Batch Transfer Size at m Serial CNC-Machining Centers", "authors": [ "Chaznin R Muhammad", "A Harits Nu'man", "A Harits Nu'man" ], "abstract": "Background: In a batch production series consisting of several process stages for discrete products, the determination of the transfer batch size greatly affects the batch waiting time. For production using a Computer Numerical Control (CNC)-machining center, several process stages can be carried out on one machine with quick setup. The use of a single machine for all stages of the process certainly does not require transfer to the next machine. Thus, there are no material handling costs. However, the use of a single machine will result in longer batch waiting times as the production batch size increases. An increase in the batch waiting time will result in an increase in work-in-process (WIP) inventory cost. This research was conducted to reduce the total variable cost consisting of WIP inventory cost and material handling cost. Methods: This research was conducted in two steps. The first step is to divide the process stages, I into m serial CNC machining centers, where in this study I=m. The second step is to determine the same size of transfer batch, Qij for a certain number of transfers, J that produces the minimum total variable cost. Results: The results showed that J=5 resulted in a total variable cost of Rp. 146,800.00, while J=10 resulted in a total variable cost of Rp. 178,900.00. Conclusion: In conclusion, the total variable cost decreases if the size of transfer batch is reduced to a certain amount and will increase again along with the reduction in the size of transfer batch.", "keywords": [ "CNC-Machining Center", "batch production", "production batch", "WIP inventory", "transfer batch", "total variable cost" ], "content": "1. Introduction\n\nIn batch production, a machine is assigned to complete a job in a large batch size before moving on to complete the next job. As the batch size increases, the batch waiting time will increase. If a job requires multiple processes, the batch waiting time will be longer because switching from one process to the next requires setup. The switch from one process to the next is done on one machine or it can be switched to the next machine. A Computer Numerical Control (CNC)-machining center can perform multiple processes on one machine.\n\nThe advantage of a CNC-machining center is that it can perform several processes singly with quick setup. The ability to perform multiple process stages in a CNC-machining center is not found on conventional machines. In general, the use of conventional machines in a series of batch production requires a transfer process to another machine because one machine cannot perform all stages of the process. Advantages of the CNC-machining center resulted in the tendency to use a single machine to carry out multiple processes of a product. This is understandable because all stages of the process can be completed on one machine so there is no need for a transfer process, setup can be done quickly. Single use result in the batch waiting time to be long which has implications for increasing work in process (WIP) inventory. If there is more than one CNC-machining center, single use can be avoided by dividing the process stages into available machines in series (sequentially). Using m machines to complete a batch, requires transfer between machines. The use of m machines allows two or more process stages to be carried out concurrently. The selection of the transfer batch size is important because it affects the batch waiting time and the number of transfers. On one side, small batch size can reduce batch waiting time. On the other hand, small batch size can increase the transfer frequency which of course increases material handling costs. The use of serial machines will make it possible to determine the size of the transfer batch that can minimize the total variable cost that consisting of WIP inventory costs and material handling costs.\n\nResearch on batch production has been carried out by several authors. Sukoyo et al.1 conducted research on batch scheduling with the performance goal is the actual flow time. Research on batch scheduling to minimize makespan was conducted by Ozturk et al.2 Research with the aim of minimizing total weighted work delays in real-world single batch processing machine (SBPM) scheduling with fuzzy due dates, was conducted by Niroom et al.3 Research aimed at minimizing makespan on a single processing machine was carried out by Li and Wang.4 Research with the aim of minimizing the number of setups required by independent job orders grouped into several classes based on similarity in style was carried out by Yimer and Demirli.5 Mathematical modeling of batch scheduling problems to minimize start and delay was carried out by Ogun and Uslu.6 Muhammad, et al.7 conducted research about batch production at m serial CNC-machining center to minimizing WIP inventory cost. This study is a development of a study that has been done by Muhammad, et al.7 This study proposes to schedule I stage to m machines and then determine the optimal Qij, which minimizes the total variable cost.\n\n\n2. Methods\n\nIn a series of stages of batch production, number of stages I can be assigned to m serial machines. For example, I=3 can be assigned to m=3. Initially, batch of 50 units (Q =Qij= 50) and requires I=3 carried out on m=1. If t1 is 48 minutes/unit, (t2) is 25 minutes/unit, t3 is 23 minutes/unit, S1 is 35 minutes/batch, S2 is 5 minutes/batch, S3 is 15 minutes/batch, then the results of scheduling are illustrated in Figure 1.\n\nNext, J = 5, I=3 is scheduled on m=3 where one machine for one stage. This scheduling requires 5 transfers each from Machine 1 to Machine 2 and from Machine 2 to Machine 3. For example, using Qij=10 units, then results of scheduling for J=5, m=3 is illustrated in Figure 2.\n\nIn Figure 1, the waiting time for each unit in the batch in Stage 1 is obtained from the size of the production batch multiplied by the processing time per unit then added with the setup time minus the processing time per unit, which is 50 units × 48 minutes/unit + 35 minutes – 48 minutes = 2,387 minutes. Therefore, the batch waiting time in Stage 1 is 50 units × 2,387 minutes per unit = 119,350 minutes. In Figure 2, the waiting time for each unit in the batch in Stage 1 is 10 units × 48 minutes/unit + 35 minutes – 48 minutes = 467 minutes. Therefore, the batch waiting time in Stage 1 is 10 units × 457 minutes per unit = 4,570 minutes. However, the number of transfers from the schedule in Figure 2 is 10 times, more than 0 times from the schedule in Figure 1.\n\nThe following notation is used for discussion of determining the transfer batch size that minimizes the total variable cost:\n\nThe number of transfers is determined by the size of the transfer batch. The number of transfers will be more if the size of the transfer batch is smaller. On the other hand, the number of transfers will be less if the size of the transfer batch is larger. Waiting time is also influenced by the size of the number of transfers. Waiting time increases if the number of transfers is reduced and vice versa. That is, WIP inventory costs are caused by waiting time, while material handling costs are caused by the number of transfers. The sum of these two costs is the total variable cost.\n\n\n3. Results and discussion\n\nIn this section, we begin by scheduling a production batch size of 50 units (Q=50) which are produced through three process stages (I=3) on one machine. Next, I=3 is assigned to three machines (m-3) serially, where each machine performs one process stage in sequence. The use of three machines serially require transfer from Machine 1 to Machine 2 and continued from Machine 2 to Machine 3. In this scheduling, a batch transfer size of 10 units is selected so that the number of transfers is 5 times (J=5) from Machine 1 to Machine 2. and from Machine 2 to Machine 3. In comparison, a transfer batch size of 5 units (J=10) was also selected. Finally, the total variable cost is calculated for I=3 performed on one machine, I=3 and J=5 on 3 machines, I=3 and J=10 on three machines.\n\nTable 1 represents the scheduling results of I = 3, m = 1. Next, Table 2 to Table 4 represent the scheduling results of J=5, I=3, m=3.\n\nScheduling in Table 1 shows the results W1 is 544,200 minutes. Thus, the results WIP inventory cost, CWIP=544,200minutesxRp1.00perminute=Rp544,200.00, material handling cost,CMH=0xRp5,000.00=Rp0.00,TVC=Rp544,200.00+Rp0.00=Rp544,200.00.\n\nScheduling in Table 2 shows the result of total waiting time until Stage 1, W1 is 71,350 minutes. Total waiting time, W1 is obtained from the sum of W11, W12, W13, W14, and W15.\n\nScheduling in Table 3 shows the result of total waiting time until Stage 2, W2 is 85,050 minutes. Total waiting time, W2 is obtained from the sum of W21, W22, W23, W24, and W25.\n\nTable 4 shows the results of Stage 3 scheduling (i = 3). For example, for Batch 5, from Eq. (1) obtained F35 = 10x23 +0 + max [2,685; 2,435] = 2,915 minutes. From Eq. (2) obtained W35 = 2,915-23 = 2,892 minutes. The waiting time of batch 5, Q35xW35, is 28,920 minutes. From Eq. (3) obtained total waiting time until Stage 3, W3 is 96,800 minutes. Thus, the results CWIP=96,800minutesxRp1.00perminut=Rp96,800.00,CMH=5+5xRp5,000.00=Rp50,000.00,TVC=Rp96,800.00+Rp50,000.00=Rp146,800.00.\n\nIf the transfer batch size is 5 units (J=10), then W3 is 78,900 minutes. Thus, the results CWIP=78,900minutesxRp1.00perminut=Rp78,900.00, CMH=10+10xRp5,000.00=Rp100,000.00,TVC=Rp78,900.00+Rp100,000.00=Rp178,900.00.\n\nFrom this case, it is found that the reduction in the transfer batch size causes the total variable cost to decrease and will increase again as the transfer batch size decreases. For example, when the transfer batch size is 10 units (J=5), the total variable cost is Rp 146,800.00, but when the batch transfer size is 5 units (J=10), the total variable cost is Rp 178,900.00.\n\n\n4. Conclusion\n\nAssignment of several stages of the process, I to m CNC-machining centers will reduce WIP inventory costs compared to using one machine for I. Using m machines requires transfer to the next machine which results in material handling costs. There is a tradeoff between WIP inventory costs and material handling costs. Therefore, in this study, the minimum total variable cost is used as a criterion in determining the size of the transfer batch, Qij which results in the number of transfers, J. From the results and discussion, it is obtained that J=5 produces a total variable cost of Rp. 146,800.00, smaller compared to J=10 which results in a total variable cost of Rp. 178.900.00.\n\nHowever, this research is still limited to the case of the same number of process stages as the number of machines (I=m), not yet discussing the I≠m case. If the research is extended to the I≠m case, then what must be considered as a decision variable is not only the number of transfers, J but also the number of process stages, I to be assigned to a particular machine. Furthermore, there is a situation where the size of transfer batch, Qij is not always constant for the same J. If there is a situation like this, then the determination of Qij will affect J which of course has implications for the total variable cost.\n\n\nData availability\n\nFigshare. Data Artikel Minimization of Total.xlsb. DOI: https://doi.org/10.6084/m9.figshare.194306188\n\nThis project contains the following underlying data:\n\n- The data used to calculate the waiting time and the number of transfers at the minimum m CNC-Machining Centers that minimize the Total Varable Cost.\n\n- The data consists of: Production batch size, setup time, processing time, transfer batch size.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nUniversitas Islam Bandung.", "appendix": "References\n\nSukoyo, Samadhi TMAA, Iskandar BP, et al.: Model penjadwalan batch multi item dengan dependent processing time. J. Teknik Industri. 2010; 12(2): 69–80.\n\nOzturk O, Espinouse ML, Mascolo MD, et al.: Makespan minimization on parallel batch processing machines with non-identical job sizes and release dates. Int. J. Prod. Res. 2012; 50(20): 6022–6035. (Taylor & Francis).\n\nNiroomand S, Mahmoodirad A, Zavardehi SMA: Single batch-processing machine scheduling problem with fuzzy due-date: mathematical model and metaheuristic approach. Handbook of Research on Modern Optimization Algorithms and Applications in Engineering and Economics. USA: Engineering Science Reference; 2016; pp 751–769 chapter 28.\n\nLi XL, Wang Y: Scheduling batch processing machine using max-min ant system algorithm improved by a local search method. Math. Probl. Eng. 2016; 2018: 1–10. Publisher Full Text\n\nYimer AD, Demirli K: Fuzzy scheduling of job orders in a two-stage flowshop with batch processing machines. Int. J. Approx. Reason. 2009; 50: 117–137. Publisher Full Text\n\nOgun B, Uslu CA: Mathematical models for a batch scheduling problem to minimize earliness and tardiness. J. Ind. Eng. Manag. 2018; 11(3): 390–405. Publisher Full Text\n\nMuhammad CR, Nu'man AH, Shofia N: Minimization of WIP inventory cost at CNC-machining centers through assignment of m serial machines anf transfer batch size reduction. IOP Conf. Series: Materials Science and Engineering. 2019; 830: 032096. Publisher Full Text\n\nMuhammad CR, Nu'man AH: Data Artikel Minimization of Total.xlsb. figshare. Dataset. 2022. Publisher Full Text" }	[ { "id": "173062", "date": "26 May 2023", "name": "Agathoklis A. Krimpenis", "expertise": [ "Reviewer Expertise CNC machine tools", "Subtractive Manufacturing", "Additive Manufacturing", "Artificial Intelligence", "Machine design" ], "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 proposes a method to minimize costs of production series using multiple processes on multiple CNC machine tools. There is an actual need for cost minimization in manufacturing environments, and the authors have presented their work in very good English. In the reviewer's opinion there are some major points to be taken into account before the manuscript is fit for indexing.\nThe state-of-the-art could be improved with an more extended presentation of previous work and the inclusion of a bigger number of references. This can help substantiate the present work better and provide a solid background to it.\n\nAlthough scheduling methods have been explored by various researchers, the present work can bring a valid method for its objective, if the minimization process (as it is an optimization problem) is more clearly described and results do not come from simple calculations.\n\nThe reviewer does not see a clear optimization algorithm and its application to solve the scheduling problem.\n\nAlthough the method is scientifically valid, there does not appear (or it is not clearly described) any innovation in the method or the obtained results.\n\nThe presented analysis should be improved to allow for replication.\n\nThe drawn conclusions are expected and should be better presented, so as to stress out the importance of the work.\n\nIn the reviewer's opinion, the manuscript title does not reflect the presented study adequately and should be improved.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "169795", "date": "22 May 2024", "name": "Nicla Frigerio", "expertise": [ "Reviewer Expertise Production system design", "modelling and ooptimization", "discrete eventi simulation", "energy efficiency", "optimization", "manufacturing" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper focuses on a multi-stage serial system composed by machining centers and intermediate buffers. The paper investigates how to allocate tasks (or operations) to each machine to minimize costs and proposes a method to perform the allocation. Results on a numerical.case are reported.\nMajor:\nThe addressed problem is actually a system design problem including the selection of the number of stages and the task allocation among such stages. This problem is well known in literature and not new. Therefore, it is hard to support the claim of providing an innovative contribution. The authors should investigate deeply the literature, analyse the lacks in literature and provide a significant contribution with repeat to the state of the art. Currently, the paper appears not bringing any significant contribution with respect to the state of the art.\nThe paper does not include any comparison and it is not possible to support paper finding with numerical evidence. Many statements of the authors are not clearly supported by evidence, neither numerical and theoretical. Numerical results are not sufficient for publication. Extensive numerical analysis should support the paper and show the actual impact of the proposed approach.\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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-407
https://f1000research.com/articles/11-338/v1	21 Mar 22	{ "type": "Research Article", "title": "Thyroid cancer incidences in the United Arab Emirates: a retrospective study on association with age and gender", "authors": [ "Asma Almansoori", "Hauke Busch", "Riyad Bendardaf", "Rifat Hamoudi", "Hauke Busch", "Riyad Bendardaf" ], "abstract": "Background: Thyroid cancer is the ninth most common malignancy worldwide, but the third most common malignancy in the United Arab Emirates (UAE). To our knowledge, this is the first UAE nationwide study aimed at presenting incidence rates of thyroid cancer at the national level of UAE based upon data from the national cancer registry and GLOBOCAN. Methods: Between 2011 and 2017, a total of 2036 thyroid cancer cases from UAE patients were registered, of which 75.3% were female and 24.7% male patients. Results: The results showed 6.6% increase in thyroid cancer cases in the UAE from 2011 to 2017 (p < 0.001) with a rise of approximately 400 cases per year from 2011 to 2040. Age standardized rate calculations showed increase in prevalence from 1.18 in 2011 to 4.32 in 2017 but decreases in incidence from 1.05 in 2011 to 0.15 in 2017. This trend is confirmed by the predictive model showing increase in incidence from 0.15 in 2017 to 0.64 by 2040. Gender was shown to be significantly associated with thyroid cancer. The female to male ratio was significantly higher in Emirati patients (4.86:1) (p < 0.001) than expat patients (2.47:1) (p < 0.01). Interestingly, expat patients contributed to the majority of thyroid cancer cases despite having lower female to male ratio. The age at diagnosis was significantly associated with thyroid cancer (p = 0.03) with the highest frequency diagnosed at 35-39 years of age. Globally, data from the predictive model showed that Asia had the highest rate of increase per year and UAE the lowest. Conclusions: The slight increase in thyroid cancer prevalence and incidence, together with the different female to male ratio and diagnosis at younger age warrants further investigation at the molecular level from UAE thyroid cancer patients to elucidate the molecular basis of thyroid cancer.", "keywords": [ "Thyroid carcinoma", "Epidemiology", "Cancer incidence", "United Arab Emirates", "Age Standardized Rate" ], "content": "Introduction\n\nOver the last decades, the overall worldwide incidence of thyroid cancer has increased significantly.1 In the United States, thyroid carcinoma has become the most rapidly increasing cancer amongst both genders. The malignancy has been on the rise by an average of 1.9% over the ten last years with rising mortality rate of 0.7% annually between 2007-2016. According to the National Cancer Institute statistics, it constitutes 3.0% of all cancer types and the newly diagnosed cases were 15.8 per 100,000 among both genders per year.2 In 2016, there was an estimate of 822,242 persons living with thyroid carcinoma in the United States. Ultrasound is most commonly used in detecting papillary thyroid cancer, however the diagnosis of papillary thyroid carcinoma should not only use ultrasound as it is not accurate predictor of papillary thyroid carcinoma and should be complimented with other diagnostic tests such as fine needle aspirate. The reasons for the increase in thyroid cancer are unclear and have been attributed to improved diagnosis of papillary thyroid cancer through the use of fine needle aspirate. It was also estimated that 52,070 new cases will be diagnosed in 2019 and approximately 2,170 deaths among both genders in the USA.3 The highest incidence rate was between 45-54 years of age with a median age of 51 at the time of diagnosis. Patients aged between (75-84), have the highest mortality rate of 27.4% with a median age of 73 at death.3 In the UAE, the incidence of thyroid cancer has increased at a rate that exceeds the incidence in neighboring countries and Western populations.4\n\nThyroid cancer is the top ranked endocrine malignancy and the second most common female malignancy in the United Arab Emirates.5 According to the Ministry of Health and Prevention (MOHAP), thyroid cancer is among the top ranked cancer cases in both genders besides Breast and Colorectal cancer. Between 2011 and 2017, thyroid cancer incidence has an overall peak from 4.37% to 9.99% among all new cancer cases.6\n\nAccording to the Global Cancer Observatory (GLOBOCAN)7 collected in 2018, a total of 360 thyroid cancer cases were diagnosed in the UAE, 279 (77.5%) among females and 81 (22.5%) among males. The age-standardized incidence rate (ASR) per gender was 13.6/100,000 in females and 1.9/100,000 in males 17-20.7 In 2020, a total of 405 (8.4%) cases were reported. The age-standardized incidence rate (ASR) per gender was 12.5/100,000 in females and 2.0/100,000 in males.7\n\nAt the regional level in Gulf Cooperation Council Countries (GCC),8 thyroid carcinoma is the fifth most common malignancy in both genders and again the second most common malignancy in females. During 1998–2007, a total of 5587 (5.9%) were recorded with an overall age-standardized incidence rate (ASR) of 5.9/100,000 in females and 1.8/100,000 in males. Thyroid cancer incidence in GCC was projected to continue increasing by 24% in males and 63% in females over the 10 years period.5,7,9 Thus, investigating existing estimates for thyroid cancer in the UAE and worldwide is useful for future studies of the disease.\n\nWith the absence of recent national studies of UAE cancer epidemiology, and in compliance with the World’s Health Organization recommendations to establish cancer baselines and monitoring trends to address tumor related to thyroid gland,10 therefore, this study’s main aim is to compile and analyze the estimates of thyroid malignancies across the UAE and compares them to worldwide incidence of thyroid cancer based on multiple attributes including ethnicity, gender and age groups.\n\n\nMethods\n\nGlobal data was collected using the GLOBOCAN repository.7 Estimates of global future cancer incidence was carried out using tools available from the Cancer Tomorrow website, which is part of the Global Caner Observatory software suite.11,12\n\nLocal UAE data in this study were collected from the UAE National Cancer Registry (UAE-NCR) between 2011-2017. The inclusion criterion is for primary thyroid carcinoma. The UAE-NCR is systematically collecting, storing, summarizing and analyzing cancer estimates of UAE populations on annual basis. It provides population-based incidence rates in accordance to demographics, cancer staging and treatment information and in compliance with international coding and registration protocols (ICD-10 and ICD-0). The UAE-NCR’s abstractors used active and passive data collection methods. The active method involves data abstraction through regular visits to MOHAP departments. The passive method involves notifications by healthcare providers on submitted forms containing patient’s data obtained from: Health information management system (HIM), pathology reports, Abu Dhabi Health Authority (HAAD) central cancer registry, Dubai Health Authority (DHA) central cancer registry. Crude incidence and crude mortality rates were computed and expressed as an annual mean per 100,000 residents; using the total UAE population estimated by the Department of Economic and Affairs, population division of the United Nations.13 In addition, predictive model for total malignancies in the UAE was constructed by applying linear regression to UAE data collected from 2011 to 2017 obtained from the (UAE-NCR). In order to determine the rate of change of thyroid cancer per year, gradient analysis derived from the linear regression equation was applied to the data derived from the model. For the ASR calculations, we used population data obtained from US Census Bureau and UAE Federal Competitiveness and Statistics Centre.\n\nIn this study, categorical variables were reported as count and percentages. Continuous variables were presented as mean ± standard deviation. Group comparison was carried out using Student t-test. Correlation was carried out using Pearson and Rank correlations where appropriate. Unsupervised hierarchical clustering was carried out using Euclidean distance measure and average linkage. Statistical analysis was carried out using a mixture of R and SPSS. P < 0.05 was taken to be statistically significant.\n\n\nResults\n\nThe data from Figure 1 show that the number of thyroid cancer cases rises globally, with the exception of Europe, at a steady rate from 2020 to 2040. The largest number of thyroid cancer cases is from the Asian continent as shown in Figure 1A. Figure 1C show that the UAE follows the global trend of steady but small increase in thyroid cancer cases.\n\nTable 1 data is generated using linear regression models for each population and calculating the rate of change of thyroid cancer cases per year from the gradient of the linear regression equation for each population. The data indicates that overall, the rate of increase of thyroid cancer cases is small compared to other cancers. The unsupervised hierarchical clustering in Figure 2 is based on the predictive model and shows that there is a steady increase in the number of thyroid cancer cases in all regions except in Europe where the cases increase at a faster rate than the other region until 2030 but then from 2030 to 2040 the cases will start to decrease.\n\nFigure 3 shows that thyroid cancer has a high ratio of female to male globally across various continents suggesting that gender might be a risk factor globally. Interestingly, the highest thyroid cancer incidence was found in Polynesia with the lowest in Africa. The average world-wide incidence of thyroid cancer is 11.6 and 3.5 for females and males, indicating an approximately 3 times higher incidence rate in female.\n\nTaken together, Table 1 confirms the data in Figures 2 and 3 in that the largest increase of thyroid cancer cases is in Asia followed by Africa and Latin America. UAE shows the smallest rate of change at 15.2 and Europe has a negative rate of change due to the predicted decrease in cases from 2030 to 2040.\n\nIn order to predict the total malignancies, linear regression model was used to calculate all cancer incidence rates from 2011 to 2017. The data from the linear regression model was compared with that from the predicted model as shown in Table 2.\n\n* Represents predicted data.\n\nTable 2 shows that the linear regression model prediction matches the actual data as seen by the significant correlation between predicted and actual data between 2011 and 2017 (r = 92.5, p = 0.003). In addition, Table 2 shows an increase of 6.6% between 2011 and 2017 which is similar to that in Table 1, however it drops to 3.3% of total thyroid cancer in UAE between 2011 and 2040, which is a slight increase.\n\nTable 3 shows slight but steady increase in the incidence and prevalence in UAE from 2011 to 2040 with the average increase for prevalence is 4.02 and incidence is 0.52.\n\n* Represents predicted data.\n\nTable 4 shows that between 2011 and 2017, a total of 2036 newly diagnosed thyroid cancer cases were recorded by UAE-NCR. Out of those (n = 634, 31%) are Emirati and (n = 1402, 69%) are expats.\n\nTable 5 shows that thyroid cancer patients between 2011 and 2017 comprised of 75.2% females and 24.7% males. Age standardized rate calculations showed slight increase in prevalence from 1.18 in 2011 to 4.32 in 2017 but a decrease in incidence from 1.05 in 2012 to 0.15 in 2017. This trend is confirmed by the predictive model showing the incidence to be 0.64 by 2040.\n\nFigure 4 show the rise of thyroid cancer is in line with the rise of total malignancies within the UAE with the percentage rise of 5.7% for total cases comprising of 1.3% for Emirati and 4.4% for Expat patients between 2011 and 2017. In addition, Figure 4 show steady rise in both Emirati and expats patients with expats providing more of the contribution to the total number of cases of thyroid cancer.\n\nFigure 5 shows that the female to male ratio in 2011 was high but leveled from 2012 to 2017 with the Emirati ratio being highest than from expats throughout that period. The trend in Figure 5 is confirmed by data shown in Table 6, which indicates that overall, thyroid cancer incidences are higher in females than males. This is shown to be the case from 2011 to 2017 with 2011 having the highest ratio of 12 for Emirati and 3.92 for expat patients. Generally, the Emirati patients have higher ratio than expats. Using independent Student’s t-test, the female to male ratio is shown to be significant for both Emirati (p < 0.0001) and expats (p < 0.001), respectively.\n\nTaken together, the data show that in all populations thyroid cancer have high female to male ratio, however within the UAE the Emirati patients have higher ratio than the expats group, but the expats contribute more towards the thyroid cancer cases within the UAE.\n\nTable 7 and the cluster in Figure 6A show that in UAE the peak thyroid cancer diagnosis tends to be within the young age group of 35-39 (p = 0.03) with 17.5% of all thyroid cancer patients diagnosed between 2011 and 2017, followed by the 30-34 age group comprising of 15.9% of all thyroid cancer cases. Figure 6B confirms the findings with the highest number of patients of thyroid cancer are diagnosed with the age of 30-34 and 35-39. Also, Table 7 and Figure 6B show that the age range 30-44 account for 47.8% of all thyroid cancer patients.\n\n\nDiscussion\n\nTo our knowledge, this is the first study focused on thyroid cancer incidence in the UAE. The data showed that thyroid cancer constitutes the highest percentage of endocrine malignancies.13 The incidence rate of thyroid cancer has increased from 4.37% in 2011 to 9.99% in 2017, indicating a slow but steady increase compared to worldwide incidence. This is supported by the recent article showing incidence of thyroid cancer in the UAE between 2011 and 2017 showed an increase.13\n\nThe results showed that both globally and locally within the UAE, thyroid cancer is associated with gender, in that the disease occurs in more female patients. Since thyroid cancer is an endocrine disease, the higher female incidence might be partly attributed to female hormone fluctuations as it was shown that some of the female hormones such as estrogen are associated with thyroid cancer.14 The higher incidence in UAE females5 may be associated with a mixture of endocrine genetic factors together with other environmental factors such as obesity, which is prevalent in UAE, smoking and a sedentary lifestyle.15,16\n\nThis increase in incidence of thyroid cancer in the UAE female population is the main reason why the results showed that although thyroid cancer incidence in UAE is low, the prevalence in the UAE female population between 2011 and 2017 is higher than the rest of the world.\n\nInterestingly, the data showed that female to male ratio is generally high in Emirati than expats, but with expats contributing to more cases of thyroid cancer. Also, the data show that male thyroid cancer incidence is low, leading to high female to male incidence ratio with the Emirati having higher ratio than expats. However, the ratios stabilizing after 2012, probably due to the UAE adopting more sensitive thyroid cancer diagnostic testing including genetic testing.17,18 The differences between Emirati and expats ratio may be due to a mixture of the population pyramid of expats arriving to the UAE for economic purposes and possibly medical tourism.19\n\nIn addition, the analysis showed that another risk factor for thyroid cancer in the UAE is age, where the highest frequency (17.5%) of thyroid cancer patients tends to be between 35-39 years of age and around 47.8% of the patients tend to be between 30-44 years of age. This might be explained by the fact that the younger generation in UAE adopted changes in lifestyle, including poor diet, which may be deficient in iodine,8 however further studies are needed to confirm such association. Taken together, the results showed that the peak incidence of thyroid cancer was during approximately the third and early fourth decades of life amongst the UAE patients, which is a decade behind neighboring Arabian Gulf countries,8 where thyroid cancer patients are diagnosed in their fourth decade of life. Taken together, the strengths of the study included data related to the thyroid cancer cases diagnosed in UAE from 2011 to 2017 and made predictive observations for the years 2020 to 2050. In addition, this is the first of the kind to report the thyroid cancer incidence in UAE population. The major limitations include the lack of availability of patient characteristics such as BMI, family history as well as other clinical characteristics. Overall, the incidence of thyroid cancer in UAE showed a small but steady increase, probably due to better diagnostic tests, as the data showed that the rate of increase is similar to other malignancies in the UAE. In addition, the methodologies used in this study may be applied to investigate other chronic complex diseases in the UAE from data stored in disparate sources.\n\nIn conclusion, this study showed that the incidence of thyroid cancer in the UAE increases at a slow but steady rate per year in line with worldwide data. The study identified that gender is associated with increase in risk of thyroid cancer probably due to a combination of genetic and environmental factors. The study also showed that although the female to male ratio of thyroid cancer is higher in Emirati, the contribution of the majority of thyroid cancer cases in UAE come from the expats. In addition, the study showed that more than a third of UAE thyroid cancer patients are diagnosed below the age of 40. In addition, the predictive models used in the study showed that future trend of thyroid cancer incidence in Europe may be different than the rest of the rest of the world.\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: Thyroid cancer incidences in the UAE\n\nhttps://doi.org/10.6084/m9.figshare.19232841.v120\n\nThis project contains the following extended data:\n\n• Supplementary data for figures.xlsx\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\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.", "appendix": "References\n\nRossouw JE, et al.: Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial. JAMA. 2002; 288(3): 321–333. PubMed Abstract \| Publisher Full Text\n\nVaccarella S, et al.: Worldwide Thyroid-Cancer Epidemic? The Increasing Impact of Overdiagnosis. N. Engl. J. Med. 2016; 375(7): 614–617. PubMed Abstract \| Publisher Full Text\n\nCarcangiu ML, Zampi G, Rosai J: Papillary thyroid carcinoma: a study of its many morphologic expressions and clinical correlates. Pathol. Annu. 1985; 20(Pt 1): 1–44.\n\nAbubaker J, et al.: Clinicopathological analysis of papillary thyroid cancer with PIK3CA alterations in a Middle Eastern population. J. Clin. Endocrinol. Metab. 2008; 93(2): 611–618. Publisher Full Text\n\nAl-Zaher N, Al-Salam S, El Teraifi H: Thyroid carcinoma in the United Arab Emirates: perspectives and experience of a tertiary care hospital. Hematol. Oncol. Stem Cell Ther. 2008; 1(1): 14–21. PubMed Abstract \| Publisher Full Text\n\nAl-Nuaim AR, et al.: Papillary thyroid cancer in Saudi Arabia. Clinical, pathologic, and management characteristics. Clin. Nucl. Med. 1996; 21(4): 307–311. PubMed Abstract \| Publisher Full Text\n\nBray F, et al.: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018; 68(6): 394–424. PubMed Abstract \| Publisher Full Text\n\nHussain F, et al.: Incidence of thyroid cancer in the Kingdom of Saudi Arabia, 2000-2010. Hematol. Oncol. Stem Cell Ther. 2013; 6(2): 58–64. PubMed Abstract \| Publisher Full Text\n\nBray F, et al.: Improving the quality and coverage of cancer registries globally. Lancet. 2015; 386(9998): 1035–1036. PubMed Abstract \| Publisher Full Text\n\nRyerson AB, Massetti GM: CDC's Public Health Surveillance of Cancer. Prev. Chronic Dis. 2017; 14: E39. Publisher Full Text\n\nBray F, Møller B: Predicting the future burden of cancer. Nat. Rev. Cancer. 2006; 6(1): 63–74. Publisher Full Text\n\nFerlay J, Ervik M, Lam F, et al.: Global Cancer Observatory: Cancer Tomorrow.2020. Reference Source\n\nNCR-UAE, Cancer Incidence In United Arab Emirates Annual Report 2011-2017, in The Statistics & Research Dept. - National Disease Registry Section-Ministry of Health&Prevention2011-2017.\n\nDerwahl M, Nicula D: Estrogen and its role in thyroid cancer. Endocr. Relat. Cancer. 2014; 21(5): T273–T283. Publisher Full Text\n\nCarcangiu ML, et al.: Papillary carcinoma of the thyroid. A clinicopathologic study of 241 cases treated at the University of Florence, Italy. Cancer. 1985; 55(4): 805–828. Publisher Full Text\n\nFrauenhoffer CM, Patchefsky AS, Cobanoglu A: Thyroid carcinoma: a clinical and pathologic study of 125 cases. Cancer. 1979; 43(6): 2414–2421. PubMed Abstract \| Publisher Full Text\n\nAl-Salam S, et al.: BRAF and KRAS mutations in papillary thyroid carcinoma in the United Arab Emirates. PLoS One. 2020; 15(4): e0231341. PubMed Abstract \| Publisher Full Text\n\nAl-Salam S, et al.: Ultrasound-guided fine needle aspiration cytology and ultrasound examination of thyroid nodules in the UAE: A comparison. PLoS One. 2021; 16(4): e0247807. PubMed Abstract \| Publisher Full Text\n\nBulatovic I, Iankova K: Barriers to Medical Tourism Development in the United Arab Emirates (UAE). Int. J. Environ. Res. Public Health. 2021; 18(3). PubMed Abstract \| Publisher Full Text\n\nAlmansoori A, Busch H, Bendardaf R, et al.: 2022. Thyroid cancer incidences in the UAE. figshare. Online resource. Publisher Full Text" }	[ { "id": "128255", "date": "05 Apr 2022", "name": "Yuanping Zhou", "expertise": [ "Reviewer Expertise Clinical cancer and cancer epidemiology related investigation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlmansoori et al. presented an interesting and useful article on thyroid cancer incidence in the United Arab Emirates. The article is concise and well written and the experimental part is well designed and some of the methods presented in the article can be applied to study the epidemiology of other rare tumours.\nThere are two minor typos that needs correcting:\nFirstly, I think it is better for the title should be 'Thyroid Cancer incidence' (not the plural incidences).\n\nSecondly it will be more complete to include the equation for linear regression that the authors used, which I believe is y = mx+c. This will clarify to the reader which of the equations was used in the study and allow for reproducibility of the results generated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8072", "date": "11 Apr 2022", "name": "Rifat Hamoudi", "role": "Author Response", "response": "Dear Dr Zhou, Thank you for taking the time to review this article. We hope that the corrections below implemented in the revised will be to your satisfaction. Comments Almansoori et al. presented an interesting and useful article on thyroid cancer incidence in the United Arab Emirates. The article is concise and well written and the experimental part is well designed and some of the methods presented in the article can be applied to study the epidemiology of other rare tumours. > We thank you for the constructive comments regarding the study. There are two minor typos that needs correcting: Firstly, I think it is better for the title should be 'Thyroid Cancer incidence' (not the plural incidences) > In the revised version we have replaced the word \"incidences\" with the word \"incidence\" Secondly it will be more complete to include the equation for linear regression that the authors used, which I believe is y = mx+c. This will clarify to the reader which of the equations was used in the study and allow for reproducibility of the results generated. > We have added the line equation, y = mx + c to the methods section" } ] } ]	1	https://f1000research.com/articles/11-338
https://f1000research.com/articles/10-853/v1	26 Aug 21	{ "type": "Research Article", "title": "Seroprevalence and risk factors of SARS-CoV-2 infection in an urban informal settlement in Nairobi, Kenya, December 2020", "authors": [ "Patrick K Munywoki", "Caroline Nasimiyu", "Moshe Dayan Alando", "Nancy Otieno", "Cynthia Ombok", "Ruth Njoroge", "Gilbert Kikwai", "Dennis Odhiambo,", "Mike Powel Osita", "Alice Ouma", "Clifford Odour", "Bonventure Juma", "Caroline A Ochieng", "Immaculate Mutisya", "Isaac Ngere", "Jeanette Dawa", "Eric Osoro", "M Kariuki Njenga", "Godfrey Bigogo", "Peninah Munyua", "Terrence Q Lo", "Elizabeth Hunsperger", "Amy Herman-Roloff", "Caroline Nasimiyu", "Moshe Dayan Alando", "Nancy Otieno", "Cynthia Ombok", "Ruth Njoroge", "Gilbert Kikwai", "Dennis Odhiambo,", "Mike Powel Osita", "Alice Ouma", "Clifford Odour", "Bonventure Juma", "Caroline A Ochieng", "Immaculate Mutisya", "Isaac Ngere", "Jeanette Dawa", "Eric Osoro", "M Kariuki Njenga", "Godfrey Bigogo", "Peninah Munyua", "Terrence Q Lo", "Elizabeth Hunsperger", "Amy Herman-Roloff" ], "abstract": "Introduction: Urban informal settlements may be disproportionately affected by the COVID-19 pandemic due to overcrowding and other socioeconomic challenges that make adoption and implementation of public health mitigation measures difficult. We conducted a seroprevalence survey in the Kibera informal settlement, Nairobi, Kenya, to determine the extent of SARS-CoV-2 infection.\nMethods: Members of randomly selected households from an existing population-based infectious disease surveillance (PBIDS) provided blood specimens between 27th November and 5th December 2020. The specimens were tested for antibodies to the SARS-CoV-2 spike protein. Seroprevalence estimates were weighted by age and sex distribution of the PBIDS population and accounted for household clustering. Multivariable logistic regression was used to identify risk factors for individual seropositivity.\n\nResults: Consent was obtained from 523 individuals in 175 households, yielding 511 serum specimens that were tested. The overall weighted seroprevalence was 43.3% (95% CI, 37.4 – 49.5%) and did not vary by sex. Of the sampled households, 122(69.7%) had at least one seropositive individual. The individual seroprevalence increased by age from 7.6% (95% CI, 2.4 – 21.3%) among children (<5 years), 32.7% (95% CI, 22.9 – 44.4%) among children 5 – 9 years, 41.8% (95% CI, 33.0 – 51.1%) for those 10-19 years, and 54.9%(46.2 – 63.3%) for adults (≥20 years). Relative to those from medium-sized households (3 and 4 individuals), participants from large (≥5 persons) households had significantly increased odds of being seropositive, aOR, 1.98(95% CI, 1.17 – 1.58), while those from small-sized households (≤2 individuals) had increased odds but not statistically significant, aOR, 2.31 (95% CI, 0.93 – 5.74).\n\nConclusion: In densely populated urban settings, close to half of the individuals had an infection to SARS-CoV-2 after eight months of the COVID-19 pandemic in Kenya. This highlights the importance to prioritize mitigation measures, including COVID-19 vaccine distribution, in the crowded, low socioeconomic settings.", "keywords": [ "Population-based", "Households", "Serosurvey", "Serology", "IgM and IgG", "SARS-CoV-2", "COVID-19", "urban informal settlement", "Kibera", "Kenya" ], "content": "Introduction\n\nRecent discovery and spread of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and the resulting disease associated with this virus, Coronavirus Disease 2019 (COVID-19), has brought unprecedented morbidity and mortality worldwide.1-3 Tracking the extent of the virus spread and disease severity in various populations is important in informing the local, national, and global public health response. Real time reverse transcription–polymerase chain reaction (rRT-PCR) testing has been the mainstay diagnostic test for COVID-19 surveillance. rRT-PCR is expensive and requires specialised infrastructure, equipment, and skills. These laboratory challenges compounded by global shortages of supplies and restrictions in shipping has resulted in sub-optimal implementation of rRT-PCR in low resource settings. Serologic tests that are cheaper than rRT-PCR are important in determining population level prevalence of SARS-CoV-2 infections. Infected individuals, including those with asymptomatic and mild disease, develop an immune response with detectable antibodies within two weeks of exposure4,5 and for months afterwards6 allowing inferences to be made on the true extent of exposure in the population.\n\nIn Kenya, the first case of SARS-CoV-2 infection was detected on 12th March 2020, and as of 30th November 2020, a total of 83,316 rRT-PCR confirmed cases and 1,452 deaths (case fatality rate, 1.7%) were reported by the Ministry of Health (MoH).7,8 The national MoH data shows two major waves of increased transmission in Kenya observed prior to this serosurvey; the first wave happened between June and August 2020 and the second wave between October and November 2020.9 Nevertheless, with limited testing resources, Kenya implemented a strategy to prioritize testing only symptomatic persons who presented at health facilities and met the suspect case definitions.10 Along with the suboptimal contact tracing, the MoH's counts likely underreports cases by excluding individuals with asymptomatic and mild cases of COVID-19 who are less likely to seek healthcare. Serologic testing may offer additional surveillance insights. Previous findings from SARS-CoV-2 antibody testing of serum from Kenya's National Blood Transfusion Services by Kenya Medical Research Institute (KEMRI)-Wellcome Trust investigators correlated well with the observed increase in community transmissions. The investigators reported a marked increase in crude prevalence of SARS-CoV-2 antibodies from 5.6% in May to 13.3% by August 2020.11,12 In Nairobi County, the increase in seroprevalence was more than double in the same period; from a baseline of 8.9% in May to 21.5% in August 2020.\n\nThe distribution of SARS-CoV-2 infections is unlikely to be homogeneous across all communities and regions, and informal settlement environments such as Kibera in Nairobi may be disproportionately affected due to overcrowding, water, sanitation, and hygiene (WASH) infrastructure constraints, and socio-economic challenges that make adoption and implementation of COVID-19 public health mitigation measures difficult. A serosurvey in July 2020 in Mumbai, India found the seroprevalence among slum residents to be nearly 3.6 times that of non-slum residents.13 There have been very few serosurvey data in informal settlements in Kenya.14 This article provides findings on seroprevalence and risk factors associated with history of SARS-CoV-2 infection from a population-based seroprevalence survey in Kibera, the largest urban, densely populated, informal settlement in Nairobi, Kenya.\n\n\nMethods\n\nKibera is a densely populated informal settlement within Nairobi, Kenya, characterised by overcrowding, poor sanitation, and poor infrastructure. This seroprevalence survey was embedded in an ongoing population-based infectious disease surveillance (PBIDS) within the informal settlement.15,16 The Kibera PBIDS covers an area < 0.5 km2 with an estimated population of about 23,103 individuals in 5,265 households (as of December 2020) living in two villages, Soweto and Gatwekera, that are under active surveillance. The PBIDS area is divided into 10 zones, referred to as residential areas (Figure 1). The platform is maintained by KEMRI-Centre for Global Health Research (CGHR) and Washington State University-Global Health in Kenya (WSU-GH) with technical and financial support from U.S. Centers for Disease Control and Prevention (CDC). The seroprevalence survey in Kibera was implemented at the same time as a wider seroprevalence survey across Nairobi County.17\n\nThe subdivisions show the areas of residence i.e. PBIDS zones 1-10.\n\nWe selected a sample size of 684 persons from 171 households (assuming each household had an average of 4 individuals). This was based on an expected seroprevalence of 25% with a precision of 5.0%, a design effect of 2, and 20% attrition should a repeat seroprevalence survey be possible in the future. More households (n = 181) were eventually included to boost the number of participants enrolled.\n\nWe conducted a cross-sectional household-based survey aligned with World Health Organisation's (WHO) UNITY seroepidemiological protocol.18 The study households were randomly selected from the most recent PBIDS dataset and household members were consented before enrolment. Efforts were made to recruit all household members, both adults and children, regardless of current or prior COVID-19 infection status. Individuals residing in the selected households who were not yet registered in the PBIDS platform were also approached for consenting if they were residents for a minimum of four months. We conducted a minimum of three study visits to a household before replacing it due to unavailability of household members. When a household was enrolled in the study, we conducted a minimum of three return visits for household members not available at the time of the initial study visit. The household enrolment and data collection were conducted from 27th November to 5th December 2020 by five trained field teams, each consisting of a field worker and a phlebotomist.\n\nAll participants were interviewed for sociodemographic data such as age, sex, education level, and occupation. Data on current occupation and highest education level were collected from adult participants (≥18 years) only. Data were collected and managed using REDCap (Research Electronic Data Capture) electronic data tools hosted at Washington State University.19,20 Venous blood samples (approximately 5 ml for persons aged >12; 2-3 ml for children 2-12 years and 1.5 ml for children <2 years) were collected from each participant and transported in a cool box at 2-8°C to the KEMRI-Diagnostics and Laboratory Systems Program (DLSP) laboratory in Nairobi the same day. Sera were extracted from the whole blood specimen and stored at −80°C before testing.\n\nWe tested for total immunoglobulins (IgM and IgG) antibodies to the SARS-CoV-2 spike protein using the Wantai SARS-CoV-2 two-step antigen sandwich enzyme immunoassay kit (Catalogue number, WS-1096; Wantai Biological Pharmacy Enterprise Ltd, Beijing, China). The assay was optimised at KEMRI-DLSP serology lab by including 10 washes instead of five washes recommended by the manufacturer to reduce background cross-reactivity, as described elsewhere.17 The test results were considered positive when the ratio of antibody titer in the sample to a negative control exceeded 1.5 according to manufacturer's instructions. All lab tests were performed in an ISO15189 certified and GCP-accredited KEMRI-DLSP laboratory in Nairobi, Kenya.\n\nIndividual seroprevalence of SARS-CoV-2 antibodies was expressed as a percentage of the seropositive among the individuals tested. The disaggregated individual seropositivity estimates accounted for household clustering and weighted by the age and sex structure of the PBIDS population (Figure 2). The standard errors for generating the 95% confidence intervals were computed using the Taylor linearized variance estimation method.21 Pearson's chi-square test was used to assess the association of categorical variables with individual seropositivity. Household seroprevalence (defined as the percentage of households with at least one seropositive member) was estimated and stratified by household size (usual number of persons living in the household), number of persons enrolled in the serosurvey per household and location in the PBIDS area. Age, sex, area of residence, relationship to head of household, household size, and underlying medical conditions (known hypertensive, asthmatic or diabetic) were considered in the univariable logistic regression model for determining the factors associated with individual seropositivity. Age and sex were considered a priori for inclusion in the multivariable logistic regression. The final multivariable logistic regression model included statistically significant variables, accounting for sampling weights and clustering by household using the clustered sandwich estimator.22,23 Adjusted odds ratio (aOR) and 95% confidence intervals (CI) were presented and two-sided p-values <0.05 were considered significant.\n\nThe red dashed line shows the overall expected probability of selection with bars above the red line indicating overrepresentation while those below the line denoting underrepresentation of the age-sex groups in the sero serosurvey.\n\nStata 15.1 software [STATA Corp, Texas, USA] was used for random selection of households to be enrolled in the study, data cleaning, management, and analyses.\n\nIndividual written informed consent/assent was obtained from all the study participants and/or their parents/guardian. Ethical approval for the study was provided by the KEMRI Scientific and Ethical Review Committee in Kenya (#4098) and reliance approval provided by the Washington State University. This activity was also reviewed by CDC and was conducted consistent with applicable federal law and CDC policy as provided for in the Code of Federal Regulations (45 C.F.R part 46 and 21 C.F.R. part 56). The PBIDS platform is approved by KEMRI Scientifical and Ethical Review Committee in Kenya (#2761), Washington State University reliance agreement and CDC reliance approval (#6775).\n\n\nResults\n\nOf the 252 randomly selected households, 175 (69.4%) agreed to participate in the survey (Figure 3). Of the 77 households that did not participate, 38 (49.4%) did not have a household head available for consenting, 26 (33.8%) had moved, and 13 (16.9%) declined participation. Consent was obtained from 523 individuals yielding 511 blood samples; field teams were unable to get a blood specimen from 12 participants. The median number of individuals with a specimen collected per household was 3 (interquartile range, IQR, 2-4; range, 1 – 10).\n\nOf the 511 sampled individuals, 58.5% (299) were female, 23.5% (120) were below the age of 10 and 1.4% (7) were 60 years or older (Table 1). Males aged 5-9 years and females aged 30-59 years were overrepresented, while both sexes below 5 years and males aged 30 years and above were underrepresented in the surveyed participants relative to the PBIDS general population (Figure 2).\n\nOf the 511 tested individuals, 222 (43.4%) were seropositive. The overall weighted-seroprevalence was 43.3% (95% CI, 37.4 – 49.5%), with no difference detected between females and males (Table 1, Figure 4). Seroprevalence increased with age from 7.6% (95% CI, 2.4 – 21.3%) among young children (<5 years), 32.7% (95% CI, 22.9 – 44.4%) among children 5 – 9 years, 41.8% (95% CI, 33.0 – 51.1%) for those 10-19 years, and 54.9% (46.2 – 63.3%) for adults 20 years and above. The elderly (60 years and above) had a seroprevalence of 52.6% (95% CI, 13.2 – 89.0%). The age effect was also observed for seroprevalence estimates by relationships to household head with grandchildren (30.2%; 95% CI, 8.4 – 67.2%) and children (37.3%; 95% CI, 30.4 – 44.8%) registering lower estimates compared to the household head (53.1%; 95% CI, 41.6 – 64.2%) and other adults including spouses (48.4%; 95% CI, 35.4 – 61.7%) and other relatives (63.1%; 95% CI, 35.1 – 84.5%).\n\nThe prevalence of SARS-CoV-2 antibodies by area of residence ranged from 25.8% in zone 7 to 69.7% in zone 4. However, the differences in prevalence by area of residence were not statistically significant [Pearson's design-based F statistic = 1.5421, p-value = 0.144]. Participants (≥18 years) with primary, secondary, and post-secondary level of education had similar seroprevalence of 54.6%, 54.5% and 67.2%, respectively. Those with no formal education were few (n = 7) and had a seroprevalence of 21.9% (95% CI, 4.3 – 63.3%). All occupation groups (Table 1) had a seroprevalence of between 43.3% to 65.0%. Only six health care workers were included in the survey and their seroprevalence was 42.9% (95% CI, 16.3 – 74.3%).\n\nParticipants with any underlying medical condition (n = 38) had a seroprevalence of 48.8% (95% CI, 29.5 – 68.5%) which was not statistically different compared with those without, 42.9% (95% CI, 36.7 – 49.3%); (Pearson design-based F statistic = 0.3100, p-value = 0.5784).\n\nOf the households enrolled, 122 (69.7%) had at least one individual with detectable SARS-CoV-2 antibodies (Table 2). The proportion of households with at least one seropositive individual varied by area of residence ranging from 50.0% in zone 3 and 9 to 82.1% in zone 10, but the differences were not statistically significant (Figure 5, Table 2). For 132 households with two or more members enrolled, 98 (74.2%) had at least one person seropositive. The median seropositivity within these households with at least one seropositive person (‘exposed’) and two or more participants enrolled was 50.0% (range, 16.7% to 100%). The vast majority (81/98, 82.7%) of these ‘exposed’ households also included one or more seronegative individual(s). The largest proportion of the seronegative household contacts (n = 72) were children of the household head (55, 76.4%), followed by grandchildren of household head (6, 8%), household head (6, 8.3%), spouse (3, 4.2%) and other relatives (2, 2.8%) (Figure 6).\n\nHousehold (HH) seropositivity (at least one person testing positive in the household) by household size, number of enrolled persons per household and location in the population-based infectious disease surveillance (PBIDS) area as of December 2020. PBIDS zones are areas of residence numbered 1-10.\n\nDistribution of 72 seronegative individuals by relationship to household (HH) head, age groups in years and sex from the 98 households with least one seropositive person in Kibera urban informal settlement, Nairobi, Kenya.\n\nSex, area of residence, relationship to head of household, and underlying medical conditions were not significantly associated with individual seropositivity (Tables 3 and 4). Individual's age and household size were the independent predictors of seroconversion. Relative to adults aged 20-29 years, young age groups (<20 years) had reduced odds of being seropositive. The odds of being seropositive were similar for older adults (age groups ≥30 years) compared to the reference group, 20-29 years. The odds for being seropositive among the elderly groups (≥60 years) were not different from the referent 20-29 years age group (adjusted odds ratio, aOR, 0.83 (95% CI, 0.19 – 3.64).\n\nRisk factors for individual seropositivity from univariable logistic regression model in Kibera urban informal settlement, Nairobi, Kenya.\n\nRisk factors for individual seropositivity from multivariable logistic regression model in Kibera urban informal settlement, Nairobi, Kenya.\n\nRelative to those from medium-sized households (of three and four individuals), participants from large (≥5 persons) households had significantly increased odds of being seropositive, aOR, 1.98 (95% CI, 1.17 – 3.34), while those from small-sized households (≤2 individuals) had increased odds but not statistically significant, aOR 2.31 (95% CI, 0.93 – 5.74).\n\n\nDiscussion\n\nWe report findings from a population-based seroprevalence survey in an urban informal settlement setting in Kenya aligned with WHO's UNITY seroepidemiological protocol.18 An overall seroprevalence of 43.3% in Kibera, the largest urban, densely populated informal settlement in Nairobi, Kenya was observed. A Nairobi-wide serosurvey conducted at the same time utilizing similar methods (specimen collection and testing) reported a lower overall seroprevalence of 32.7% in the County.17 However, the authors noted the seroprevalence in the Nairobi county-wide survey varied across populations with densely populated areas having the highest seroprevalence. The larger Kibera area (known as Kibra subcounty) had a seroprevalence of 42.8%, which corresponds with our finding of 43.3%. Though there are no other published population-based serosurveys in Kenya, estimates from convenient samples of mothers attending antenatal services at Kenyatta National Hospital located in the same administrative area and various cadres of healthcare workers from the same hospital had comparable seroprevalences of 41% and 44%, respectively.12,24 There are limited serosurveys from informal settlements beyond Kenya but one such study conducted in July 2020 in Mumbai, India found the seroprevalence among slum dwellers to be nearly 3.6 times that of non-slum residents.13 Taken together, the associated challenges for residents of informal settlement to implement mitigation measures such as social distancing, wearing of face masks, and optimal hygiene practices could explain the increased transmission in these populations.\n\nA high level of SARS-CoV-2 exposure in households was recorded with more than two-thirds (69%) of the study households having at least one seropositive member. The seropositivity within households with at least one seropositive person and two or more participants enrolled ranged from 17% to 100% with a median of 50% compared to the overall household seropositivity of 69%. The lower seroprevalence among younger household members suggests transmission outside the household may have played an important role in infection among adults. This finding aligns with observations from the Nairobi county-wide serosurvey and strengthens the argument of increased risk of infection from outside the household, especially among the working populations.17 However, a serosurvey in Singapore showed higher seroprevalence among household contacts compared to work and other social contacts.25 Further, household age structure appeared to play a role with majority of seronegative persons in the exposed households being less than 20 years old. These findings conform to the lower incidence and prevalence of COVID-19 infection found among children in Kenya and other countries.26 Consistent with epidemiological findings of rRT-PCR confirmed SARS-CoV-2 infections, children had a lower cumulative risk of infection than adults. Lower expression of angiotensin converting enzyme 2 in children relative to adults has been considered as one hypothesis for the observed reduced risk.27 Higher prevalence among adolescents 10-19 years compared to younger children below 5 years, also conform with earlier documentation of increasing risk of infection with increasing age among children and adolescents28 which could partly be attributed to increased interaction outside households. Adherence to COVID-19 mitigation measures within the households such as hand hygiene was not assessed in this study but highly unlikely to have reduced the infections rates among younger children given the WASH challenges reported in Kibera. Schools in Kenya had been closed since the confirmation of the first case in March 2020 to the time of this serosurvey, potentially reducing young children's contact to persons living beyond their immediate neighbourhood hence exposure to infectious individuals. The schools have since reopened and a follow up survey would delineate any changes in transmission in the school going children as well as the rest of the population.\n\nOur data show that most of the older persons were in their own business or employment potentially increasing their risk of exposure while on public transport and/or at workplaces. Adherence to mitigation measures may be suboptimal in these settings in Kenya. Although children were not going to school, in the informal settlement with limited indoor space, the anecdotal evidence point to considerable peer interactions as children played outdoors. This is a paradox to disentangle with further investigations when schools open.\n\nIndividuals from small (≤2 members) and large (≥5 persons) households had increased odds of being seropositive compared to those from medium-sized (3-4 persons) households. While this observation appears counter intuitive with respect to the role of crowding, most medium-sized households had parents and their children which dovetail well with the lower risk of infection among the young children as discussed earlier. Small-sized households consisted of mainly adults (spouses) who needed to go out for work, while for the larger households, there were more adults, suggesting overcrowding and more adults who needed to go out for work. Sharing of bed space overnight by couples could also partly explain the relatively high risk of infection in the small sized households.\n\nThis study had some limitations. First, not all members of the selected households were enrolled, and as shown in Figure 2, the probability of inclusion varied by age and sex. For instance, adult males were underrepresented as they were frequently working at the time of household visits and sampling. This would most likely lead to underestimation of the true population SARS-CoV-2 exposure given the working populations seemed to have higher seroprevalence. However, we have weighted the reported estimates by probabilities of inclusion to generate population level estimates. Second, sample size was limited and possibly inadequate for some of the stratified and regression analyses. Third, the reported seroprevalence was not adjusted for assay performance. Although the Wantai kit was verified in a CDC-supported laboratory,17 sensitivity and specificity estimates from local or similar populations were lacking. The manufacturer's estimates are from a different population to provide meaningful interpretation. The kinetics of antibodies are not fully elucidated, and we may have missed those who were infected several months prior due to waning of detectable antibodies. It would be informative to have serial serosurveys in the seropositive participants to assess the longevity of detectable SARS-CoV-2 antibodies. Finally, seropositivity was not confirmed by a neutralisation or a secondary assay.\n\nIn densely populated urban settings where the implementation of mitigation measures – such as case identification and isolation, contact tracing and quarantine, and social distancing – remained very challenging, close to half of the individuals have had aSARS-CoV-2 infection eight months into the COVID-19 pandemic in Kenya. This highlights the importance to prioritize additional mitigation measures, including COVID-19 vaccines, in these crowded, low socioeconomic settings.\n\n\nData availability\n\nThe dataset and analyses code are available at Harvard Dataverse. DOI: https://doi.org/10.7910/DVN/LWJH9N.29\n\nThis project contains the following underlying data:\n\n- pbids_age_gender_weights.tab\n\n- PBIDS_December_Serosurvey_Dataset_Version_13Aug2021.tab\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nwAccess to the dataset is restricted as it contains sensitive participant identifying information. Accompanying documentation is available under open access. For more detailed information beyond the metadata and documentation provided, there is a process of managed access requiring submission of a request, detailing the intended use, for consideration by our Data Governance Committee. Please contact the Data Governance Committee via this email address - gbigogo@kemricdc.org.\n\nThe findings and conclusions in this study are those of the authors and do not necessarily represent the official position of the US National Institutes of Health, KEMRI, or U.S. Centers for Disease Control and Prevention.", "appendix": "Acknowledgements\n\nWe thank the Kenya Ministry of Health (MOH) and Nairobi Metropolitan Services for granting permission and actively participating in public sensitization for the study. We acknowledge the Kenya Medical Research Institute (KEMRI) who provided ethical approval and oversight, field staff that carried out household visit, and the Washington State University Global Health Kenya administration staff that supported the project. This paper is published with the permission of the Director of KEMRI.\n\n\nReferences\n\nCucinotta D, Vanelli M: WHO Declares COVID-19 a Pandemic. Acta Biomed. 2020; 91(1): 157–160. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDan JM, Mateus J, Kato Y, et al.: Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science. 2021, 371(6529): eabf4063. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Health - Republic of Kenya: COVID-19 Operations Dashboard on 30th November 2020. Accessed on 15th April 2021. Reference Source\n\nWorldometer: Coronaviruses in Kenya on 30th November 2020. 30th November 2020 edn: Accessed on 15th April 2021; 2021. Reference Source\n\nMinistry of Health - Republic of Kenya: COVID-19 Outbreak in Kenya: Daily Situation Reports.\n\nMinistry of Health - Republic of Kenya: Targeted testing trategy for COVID-19 in Kenya. Accessed on 15th April 2021. Reference Source\n\nUyoga S, Adetifa IMO, Karanja HK, et al.: Seroprevalence of anti-SARS-CoV-2 IgG antibodies in Kenyan blood donors. Science. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKEMRI-Wellcome Trust Research Programme - Kilifi Kenya: Status of the COVID-19 pandemic in Kenya: Evidence from national case-based surveillance, serosurveillance and hospital-based clinical surveillance. Published, October 2020.\n\nMalani A, Shah D, Kang G, et al.: Seroprevalence of SARS-CoV-2 in slums and non-slums of Mumbai, India, during June 29-July 19, 2020.Preprint 2020.\n\nLai CC, Wang JH, Hsueh PR: Population-based seroprevalence surveys of anti-SARS-CoV-2 antibody: An up-to-date review. Int J Infect Dis: IJID: official publication of the International Society for Infectious Diseases. 2020; 101: 314–322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeikin DR, Olack B, Bigogo GM, et al.: The burden of common infectious disease syndromes at the clinic and household level from population-based surveillance in rural and urban Kenya. PloS one. 2011; 6(1): e16085. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreiman RF, Cosmas L, Audi A, et al.: Use of population-based surveillance to determine the incidence of rotavirus gastroenteritis in an urban slum and a rural setting in Kenya. Pediatr Infect Dis J. 2014; 33(Suppl 1): S54–S61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNgere I, Dawa J, Hunsperger E, et al.: High seroprevalence of SARS-CoV-2 eight months after introduction in Nairobi, Kenya.2021; Reference Source\n\nWorld Health Organisation: Population-based age-stratified seroepidemiological investigation protocol for coronavirus 2019 (COVID-19) infection.2020; World Health Organisation; 2020.\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\n\nHarris PA, Taylor R, Minor BL, et al.: The REDCap consortium: Building an international community of software platform partners. J Biomed Inform. 2019, 95: 103208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaylor DJ, Muller KE: Computing Confidence Bounds for Power and Sample Size of the General Linear Univariate Model. Am Stat. 1995; 49(1): 43–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhite H: A Heteroskedasticity-Consistent Covariance Matrix Estimator and a Direct Test for Heteroskedasticity. Econometrica. 1980; 48(4): 817–838. Publisher Full Text\n\nHuber PJ: The behavior of maximum likelihood estimates under nonstandard condition. Unversity of California Press; 1967.\n\nEtyang AO, Lucinde R, Karanja H, et al.: Seroprevalence of Antibodies to SARS-CoV-2 among Health Care Workers in Kenya. Clin Infect Dis: an official publication of the Infectious Diseases Society of America. 2021. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNg OT, Marimuthu K, Koh V, et al.: SARS-CoV-2 seroprevalence and transmission risk factors among high-risk close contacts: a retrospective cohort study. Lancet Infect Dis. 2021; 21(3): 333–343. Publisher Full Text\n\nDong Y, Mo X, Hu Y, et al.: Epidemiology of COVID-19 Among Children in China. Pediatrics. 2020, 145(6). PubMed Abstract \| Publisher Full Text\n\nBunyavanich S, Do A, Vicencio A: Nasal Gene Expression of Angiotensin-Converting Enzyme 2 in Children and Adults. JAMA. 2020; 323(23): 2427–2429. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCenters for Disease Control and Prevention C-R: COVID-19 Case Surveilance Public Use Data Access, Summary, and Limitations. 1st September 2020 edn; 2020. Reference Source\n\nMunywoki PK, Nasimiyu C, Alando MD, et al.: Replication data for: Seroprevalence and risk factors of SARS-CoV-2 infection in an urban informal settlement in Nairobi, Kenya, December 2020. In: Bigogo G: V1 edn: Harvard Dataverse. 2021." }	[ { "id": "92857", "date": "01 Sep 2021", "name": "Sarah R. Haile", "expertise": [ "Reviewer Expertise I am biostatistician with experience in both clinical trials and epidemiological (incl. observational) data. I work in a variety of different subject areas", "including a study of covid seroprevalence in Swiss children." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this well-written manuscript. I've seen similar work on seroprevalence in other populations recently, and such studies are certainly complicated to perform and analyse.\nMy main question is what are the test performance characteristics of the antibody test used (sensitivity, specificity)? If I have missed them in a citation, I apologize but would ask that they be restated here for completeness.\nThe statistical analysis took care of many factors of the study design (household clustering and population weights, for example). Would it nevertheless be possible to explicitly account for test sensitivity and specificity, as performed for example in this study1 (see also the supplementary material for full model write-up and link to GitHub repository)?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8067", "date": "11 Apr 2022", "name": "Patrick K Munywoki", "role": "Author Response", "response": "Thank you for the review and the very useful comments. We tested for total immunoglobulins (IgM and IgG) antibodies to the SARS-CoV-2 spike protein using the Wantai SARS-CoV-2 two-step antigen sandwich enzyme immunoassay kit (Catalogue number, WS-1096; Wantai Biological Pharmacy Enterprise Ltd, Beijing, China) whose published sensitivity and specificity is 94.4% and 100% respectively from populations in China (https://www.ystwt.cn/wp-content/uploads/2020/05/Brochure-Wantai-SARS-CoV-2-Ab-ELISA.pdf). However, local or data from similar populations is lacking on the test performance hence the reported seroprevalence did not account for the sensitivity and specificity. We have made this decision explicit in the revised manuscript including in the discussion as a limitation." } ] }, { "id": "126232", "date": "28 Mar 2022", "name": "Tatjana Vilibic-Cavlek", "expertise": [ "Reviewer Expertise I am a Medical Microbiology specialist. I work at the Department of Virology", "and my areas of research include emerging and re-emerging viral zoonoses", "hepatitis viruses and TORCH infections. I have participated in seroepidemiological studies of COVID-19 in different population groups in Croatia." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have read with great interest the manuscript entitled \"Seroprevalence and risk factors of SARS-CoV-2 infection in an urban informal settlement in Nairobi, Kenya, December 2020\". The manuscript is generally well written and the results are clearly presented. Please see some minor comments regarding the methodology below.\nMethods:\nData and specimen collection Page 5, line 6: please correct \"Serum samples were extracted from the whole blood ...\"\n\nSerological testing Page 5, lines 1-3: please add characteristics of the ELISA test used for detection of SARS-CoV-2 antibodies (sensitivity, specificity).\n\nIn my opinion, one of the main limitation of the study is that confirmatory virus neutralization test has not been performed. However, the authors stated this limitation in the discussion section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "8068", "date": "11 Apr 2022", "name": "Patrick K Munywoki", "role": "Author Response", "response": "Thank you for the review and the minor comments raised. A detailed response to the review comments is provided below: 1. Data and specimen collection: Page 5, line 6: please correct \"Serum samples were extracted from the whole blood ...\" We have corrected the statement as advised. Thank you 2. Serological testing, Page 5, lines 1-3: please add characteristics of the ELISA test used for detection of SARS-CoV-2 antibodies (sensitivity, specificity) We have added the reported sensitivity and specificity of the serological assay used (https://www.ystwt.cn/wp-content/uploads/2020/05/Brochure-Wantai-SARS-CoV-2-Ab-ELISA.pdf). 3. In my opinion, one of the main limitation of the study is that confirmatory virus neutralization test has not been performed. However, the authors stated this limitation in the discussion section. We acknowledge the limitation on lack of confirmatory virus neutralization test and point that out in the study limitation section. Thank you" } ] } ]	1	https://f1000research.com/articles/10-853
https://f1000research.com/articles/11-406/v1	11 Apr 22	{ "type": "Research Article", "title": "Machine learning methods to predict particulate matter PM2.5", "authors": [ "Naveen Palanichamy", "Su-Cheng Haw", "Subramanian S", "Rishanti Murugan", "Kuhaneswaran Govindasamy", "Su-Cheng Haw", "Subramanian S", "Rishanti Murugan", "Kuhaneswaran Govindasamy" ], "abstract": "Introduction Pollution of air in urban cities across the world has been steadily increasing in recent years. An increasing trend in particulate matter, PM2.5, is a threat because it can lead to uncontrollable consequences like worsening of asthma and cardiovascular disease. The metric used to measure air quality is the air pollutant index (API). In Malaysia, machine learning (ML) techniques for PM2.5 have received less attention as the concentration is on predicting other air pollutants. To fill the research gap, this study focuses on correctly predicting PM2.5 concentrations in the smart cities of Malaysia by comparing supervised ML techniques, which helps to mitigate its adverse effects. Methods In this paper, ML models for forecasting PM2.5 concentrations were investigated on Malaysian air quality data sets from 2017 to 2018. The dataset was preprocessed by data cleaning and a normalization process. Next, it was reduced into an informative dataset with location and time factors in the feature extraction process. The dataset was fed into three supervised ML classifiers, which include random forest (RF), artificial neural network (ANN) and long short-term memory (LSTM). Finally, their output was evaluated using the confusion matrix and compared to identify the best model for the accurate prediction of PM2.5. Results Overall, the experimental result shows an accuracy of 97.7% was obtained by the RF model in comparison with the accuracy of ANN (61.14%) and LSTM (61.77%) in predicting PM2.5. Discussion RF performed well when compared with ANN and LSTM for the given data with minimum features. RF was able to reach good accuracy as the model learns from the random samples by using decision tree with the maximum vote on the predictions.", "keywords": [ "Air Pollution", "Particulate Matter (PM2.5)", "Artificial Neural Network", "Random Forest", "Long Short-Term Memory" ], "content": "Introduction\n\nAir pollution has become a major issue around the world, particularly in smart cities. Pollution of air is defined as any substance from any atmospheric source that continues to exist in any state and causes damage or the ability to alter the atmosphere’s typical characteristics, putting living things’ health at risk, or causing ecological disruption.1\n\nThe concentration of air pollutants, particularly PM2.5, a very small particle with a diameter of 2.5 micrometers in the air, during the haze season in Malaysia determines the API readings [http://apims.doe.gov.my/public_v2/aboutapi.html]. PM2.5, most dangerous among air pollutants, is a microscopic particle that can enter into lungs and have a negative consequence on the human respiratory system [https://www.epa.gov/pm-pollution/particulate-matter-pm-basics].\n\nTraffic pollution and industrialization are the primary sources of PM2.5 emissions. This stage of the PM2.5 problem can be seen in smart cities. In recent times, the size of population in the urban area has significantly risen because of industrialization and rural to urban area migration. The increase in population has increased in the modes of transportation and consumption of energy, which contributes to the growth of vehicles and industry in the urban city.2\n\nSeveral scientific investigations have shown that air quality is a major concern for smart cities and ML has started to emerge as a truly outstanding solution for predicting PM2.5 that takes the required steps to mitigate its negative effects, as the prediction must be accurate. However, in comparison with other countries, Malaysian PM2.5 prediction in the context of ML is not well established.\n\nThere has been significant progress in the prediction of PM2.5 concentrations in the air in smart cities around the world over the last decade, thus, using ML techniques to predict air PM2.5 concentrations in Malaysian smart cities could be beneficial. The goal of this research is to use ML to predict PM2.5 concentrations in the Air Pollutant Index (API) of six Malaysian smart cities. The sections that follow will be discussed and organized in the following order: Related works: this topic’s related works were discussed; Methods: to discuss the research methodology used to conduct this study; and Results and discussion: to discuss the study’s findings.\n\n\nRelated works\n\nMost cities in the world witnessed the levels of pollution have surpassed all the global standard guidelines in the past few decades of global modernization, resulting in existing issues. Because of the potentially fatal effects of PM2.5, research teams are working on developing a dependable method for analyzing PM2.5 concentrations in the polluted air. First, PM2.5 prediction approaches will be discussed, followed by a summary of related works in the second section.\n\nA research paper on air pollution forecasting using an ML model in Delhi’s smart cities was conducted.3 ANN and support vector machine (SVM) algorithms were used as ML methods. Among the six SVM functions used to predict accuracy, medium Gaussian SVM had the highest accuracy.\n\nAnother study4 predicted roadside PM10 and PM2.5 concentrations using ANN, Boosted Regression Trees (BRT), and SVM. The ML models were applied to pollutant, meteorological, and traffic data collected at nineteen London Air Quality Monitoring (AQM) sites between 2007 and 2012. The ANN and BRT models performed well. Shahriar et al.5 evaluated the effectiveness of Autoregressive Integrated Moving Average (ARIMA)-SVM and ARIMA-ANN, for daily PM2.5 prediction in Bangladesh. The ARIMA-ANN and CatBoost models performed well when the hybrid models were compared with DT, Catboost.\n\nThe daily PM2.5 levels in the Greater London area was predicted using an ensemble ML approach.6 Random forest (RF) was one of the ML models of the ensemble. RF outperformed the k-nearest neighbor (KNN) and gradient boosting machine (GBM). With a ten-fold cross-validated R2 of 0.828, the model performed exceptionally well. A comparison study of ML methods for predicting PM2.5 concentrations in smart cities was successfully undertaken in Malaysia.7 Multi-layer perceptron (MLP) and RF were the techniques used. RF was effective, according to the findings of this study.\n\nDeep learning (DL) models, which are algorithms inspired by the function and structure of the brain, have also been studied for PM2.5 predictions. In the study,8 hybrid model long short-term memory (LSTM)-convolutional neural network (CNN) hybrid model performed well compared to the hybrid gated recurrent Unit (GRU)-CNN. In another research,9 the proposed deep neural network (DNN)-LSTM hybrid model, was effective when compared with multiple additive regression trees (MART) and deep feedforward neural network (DFNN). Zhang et al.10 suggested a DL model, which included bidirectional LSTM (Bi-LSTM) and an auto-encoder (AE) for the PM2.5 prediction.\n\nFollowing the review of papers, it is evident that supervised ML models are considered for PM2.5 predictions. Next, ANN, LSTM and RF are the repeatedly used models for the prediction of PM2.5 but were not compared. Nonetheless, in comparison to the rest of the world, Malaysia has limited studies. As a result, the authors proposed using ML to predict air quality (PM2.5).\n\n\nMethods\n\nThe methods for determining the research’s outcome are discussed in this section.\n\nIn the world of smart cities, haggling with pollution of air is one of the most pressing issues in the environment. In Malaysia, smart cities such as Kuala Lumpur, Johor Bharu, Penang, Putrajaya, Kota Kinabalu, and Kuching have experienced severe pollution of air. Air pollution forecasting has become a significant strategy for Malaysia’s Department of Environment in preventing air pollution. As a result, ML must be utilized to determine the PM2.5 prediction’s accuracy.\n\nThe data was obtained from the Malaysia’s Department of Environment.2 The data was collected between 2017 and 2018. PM2.5 readings have been taken since July 2017, according to the Department of Environment. The data were gathered from the air quality monitoring stations, including: Batu Muda and Cheras (Wilayah Persekutuan Kuala Lumpur); Batu Pahat, Kota Tinggi, Kluang, Larkin, Muar, Pasir Gudang, Pengerang, and Segamat (Johar Bharu); Seberang Jaya, Seberang Prai, Minden, and Balik Pulau (Penang); Putrajaya; Kota Kinabalu (Sabah); and Kuching (Sarawak).\n\nThe data span the months of July 2017 to December 2018. Beginning in July 2017, the DOE began taking readings of PM2.5 concentrations in the air. The features included in the dataset were station id, location, ate and PM2.5.\n\nDOE provided clean datasets with no noisy data, such as missing data or outliers. As a result, data transformation was carried out. The data types were transformed to integers during data transformation so that the classification algorithm could operate on them. To convert the categorical variables station id and location to integers, they were encoded.\n\nIn addition, the data is in the form of time series. The date was thus divided into the following sections: day, day of the year, week of the year, month, quarter, and weekdays. In order to improve ML accuracy, this procedure is taken to measure various points.\n\nFollowing a review of the PM2.5 values, it was decided to introduce a new column called Label. The air quality status indication is defined by the label column is relies on the Malaysia’s Air Quality Status Indication rating. The target column for ML to forecast values will be the label column.\n\nThe data factors were converted to numerical data. The data is formatted as integer as it has to be fed into the supervised training classifiers.\n\nA key stage that must be accomplished is the identification of relevant characteristics that are connected to the target variable. The accuracy of the classifier will increase as a result of picking the best feature. On this dataset, the SelectKBest technique was employed. The characteristics are picked based on the k number, which decides which features receive the greatest ratings. In this work, chi-squared statistics for classification tasks are evaluated using the chi2 function.\n\nFollowing the implementation of the approaches, five characteristics are chosen: PM2.5, Station Id, location, day of the year, and day of the year. These five characteristics, according to the chi-square scores, show a significant relationship with the target feature, which is the \"Label.\" These characteristics will be utilised to train the model, resulting in greater accuracy when assessing the real PM2.5 label values.\n\nFor this experiment, the train-test split is 60-40. The data with labels is used to train and test the classifiers. Supervised ML algorithms ANN, LSTM and RF were used to predict PM2.5 accurately.\n\nFirst, Artificial Neural Network (ANN): The interconnection of assembly of nodes with their structure using a directed link [https://www.mathworks.com/discovery/neural-network.html]. It has three layers: an input layer, a concealed layer, and an output layer. To begin, 10 neurons will be in the input layer, 200 neurons in the hidden layer and their output layer will have ten neurons. To determine the number of epochs to update the network weights during training, the maximum number of iterations was set to 100. Adam, which works well on large datasets, was the solver employed. Finally, for this dataset, inactivation parameters, ReLU, were used. ReLU allows the model to run faster, especially in backpropagation. After the model has been set up, the predicted values are obtained by running it against test data.\n\nNext, LSTM: recurrent neural networks (RNNs) have a long-term dependency problem that LSTM networks were created to solve due to the vanishing gradient problem. LSTMs differ from more traditional feedforward neural networks by having feedback connections. This trait allows LSTMs to process whole data sequences without having to treat each point in the sequence separately, instead storing important information from prior data in the sequence to aid in the processing of new data points [https://towardsdatascience.com/lstm-networks-a-detailed-explanation-8fae6aefc7f9].\n\nThis model’s LSTM has three layers, each of which takes the output of the previous LSTM and outputs a prediction for the next time step. The three LSTMCell objects that were created were encapsulated using MultiRNNCell in TensorFlow. The model is run against test data after it has been set up.\n\nFinally, random forest: a model comprised of hundreds or thousands of decision trees [https://builtin.com/data-science/random-forest-algorithm#how]. Each one is trained with a random dataset and splits the nodes in each tree by few features. The n estimator parameter is set to 100, implying that 100 decision trees will be constructed. The model trains each tree before determining the best predicted values, which are determined by the number of votes. Next, to acquire the expected values, the model is run on the test data.\n\nThe performance of a ML model is assessed with the confusion matrix. The confusion matrix will display the comparison between the actual and predicted values of the ML models [https://www.analyticsvidhya.com/blog/2020/04/confusion-matrix-machine-learning/]. The evaluation’s goal is to see how well our model performed in terms of ML. The result of the ANN, LSTM and RF are analyzed with confusion matrix. There are four parts to the confusion matrix:\n\n1. True positive (TP) indicates that the actual and predicted values are same. Both are positive values.\n\n2. True negative (TN): The real values are predictable. Even if the real value is positive, negative is predicted. The expected value is improperly forecasted.\n\n3. False positive (FP): Although the actual value is negative, the predicted value is positive. The expected value is improperly forecasted.\n\n4. False negative (FN): Incorrect forecasting of a value. The real value is positive, however, but is expected to be negative.\n\nThese equations are derived from the confusion matrix in order to determine ML performance.\n\nRandom forest, LSTM, and ANN are compared in this study because these ML algorithms have shown to be more accurate in many studies, as discussed in the related work. Since limited research on pollution of air (PM2.5) in Malaysia has been done, in this study, ML methods will be analysed, and the best ML method will be chosen. This study will be conducted entirely in Python. Anaconda Jupyter Notebook is used to stimulate the module.\n\n\nResults and discussion\n\nThe results of each classifier will be discussed and justified in this section.\n\nThe ML algorithms were employed on the DOE data, which has few features for the data over the period 2017 to 2018. The ANN and LSTM predict PM2.5 with an accuracy of 61.14% and 61.77% respectively and cannot improve further after 150 epochs as they require more data to get a significant conclusion. Though ANN, LSTM and RF have 100 layers, the accuracy of RF (97.7%) for PM2.5 predictions was higher as the model learns from the random samples by using decision tree with the maximum vote on the predictions, which improves the interpretation unlike ANN and LSTM, that decimate the interpretability of the features. Figure 1, shows the accuracy of the comparison algorithms. Overall, the research results reveal that the RF model outperformed ANN and LSTM.\n\n\nConclusion and future work\n\nIn this paper, air pollution prediction using ML models were tested in Smart Cities of Malaysia because of the severity of air pollution in urban areas. Only supervised ML techniques were used for comparison in this paper to predict PM2.5. Previous research has used supervised ML techniques to predict PM2.5 levels. However, comparative analysis of them is often required for the identification of the best model for accurately forecasting the concentration of PM2.5. When compared, RF outperformed ANN and LSTM in predicting PM2.5 in Malaysian Smart Cities with an accuracy of 97.7% for a dataset with less data and features.\n\n\nData availability\n\nDANS: Underlying data for ‘Machine learning methods to predict particulate matter PM2.5’. https://doi.org/10.17026/dans-2zd-rgue\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor contributions\n\nRishanti and Kuhaneswaran did the conception of the work, data collection, data analysis and interpretation, drafting the article, and revision to the final version under the guidance of S Subramanian and their supervisors Su-Cheng Haw and Palanichamy Naveen. Palanichamy Naveen is the corresponding author for this paper.", "appendix": "Acknowledgements\n\nWe would like to thank Department of Environment for providing us the dataset to complete this research successfully.\n\n\nReferences\n\nSentian J, Herman F, Yin CY, et al.: Long-term air pollution trend analysis in Malaysia. International Journal of Environmental Impacts 2019; 2(4): 309–324. Publisher Full Text\n\nAmeer S, Ali Shah M, Khan A, et al.: Comparative Analysis of Machine Learning Techniques For Predicting Air Quality in Smart Cities. Urban Computing and Intelligence 2017; 7: 128325.\n\nMahalingam U, Elangovan K, Dobhal H, et al.: A Machine Learning Model to Air Quality Prediction for Smart Cities. International conference on wireless communications signal processing and networking (WiSPNET). IEEE 2019, March 2019; vol. 19: p. 452.\n\nSuleiman A, Tight MR, Quinn AD: Applying machine learning methods in managing urban concentrations of traffic-related particulate matter (PM10 and PM2. 5). Atmos. Pollut. Res. 2019; 10(1): 134–144. Publisher Full Text\n\nShahriar SA, Kayes I, Hasan K, et al.: Potential of ARIMA-ANN, ARIMA-SVM, DT and CatBoost for Atmospheric PM2. 5 Forecasting in Bangladesh. Atmos. 2021; 12(1): 100. Publisher Full Text\n\nDanesh Yazdi M, Kuang Z, Dimakopoulou K, et al.: Predicting Fine Particulate Matter (PM2.5) in the Greater London Area: An Ensemble Approach using Machine Learning Methods. Remote Sens. 2020; 12(6). Publisher Full Text\n\nMurugan R, Palanichamy N: Smart City Air Quality Prediction using Machine Learning. 2021 5th International Conference on Intelligent Computing and Control Systems (ICICCS). IEEE 2021, May; (pp.1048–1054).\n\nYang G, Lee H, Lee G: A hybrid deep learning model to forecast particulate matter concentration levels in Seoul, South Korea. Atmos. 2020; 11(4): 348. Publisher Full Text\n\nKarimian H, Li Q, Wu C, et al.: Evaluation of different machine learning approaches to forecasting PM2. 5 mass concentrations. Aerosol Air Qual. Res. 2019; 19(6): 1400–1410. Publisher Full Text\n\nZhang B, Zhang H, Zhao G, et al.: Constructing a PM2. 5 concentration prediction model by combining auto-encoder with Bi-LSTM neural networks. Environ. Model Softw. 2020; 124: 104600. Publisher Full Text" }	[ { "id": "134550", "date": "12 May 2022", "name": "Stella Morris", "expertise": [ "Reviewer Expertise Power system control", "stability", "and optimization" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 focuses on the prediction of PM2.5 concentrations in Malaysia which would help to combat air pollution.\nAn increase in particulate matter, or PM2.5, is a hazard since it can have unpredictable implications such as asthma and cardiovascular disease worsening. Machine learning (ML) techniques have gotten less attention in Malaysia because the focus is on forecasting other air contaminants. Smart cities are concerned about air pollution. PM2.5 emissions are mostly caused by traffic pollution and industrialization.\nThis study has applied machine learning to estimate the Air Pollution Index (API) of six Malaysian smart cities, among other things. An ensemble ML technique was used to forecast daily PM2.5 levels in the Greater London area. Random forest (RF) was one of the ensemble's machine learning models. The RF machine outperforms the KNN and gradient boosting machines (GBM).\nSmart cities in Malaysia have suffered from significant air pollution. Therefore, this proposed machine learning approach would help to improve the accuracy of the PM2.5 prediction and hence mitigate air pollution.\nThe investigation has been conducted using data sets from 2017 to 2018. To validate their effectiveness, those predicted outputs could be compared to the most recent available data sets.\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?\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": "156478", "date": "05 Dec 2022", "name": "Charles Raja S.", "expertise": [ "Reviewer Expertise Machine learning", "Deep learning and Smart grids." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 discussed the topic of Machine learning methods to predict particulate matter PM2.5.\nThe authors have done an extensive literature survey and they have collected real-time data to train the ML program.\nThe authors are requested to clarify some minor clarifications:\n•\n\nKindly explain how you performed pre-processing for your real-time data set.\n•\n\nWhat are the features available in the considered data set?\n•\n\nMention the reason for labelling the data.\n•\n\nWrite the accuracy of the methods in terms of RMSE values if possible.\n\nIs the work 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/11-406
https://f1000research.com/articles/10-1031/v1	11 Oct 21	{ "type": "Research Article", "title": "Status of self-esteem in medical students of a college in Kathmandu: A descriptive cross sectional study", "authors": [ "Bikal Shrestha", "Stuti Yadav", "Subodh Dhakal", "Pooja Ghimire", "Yubika Shrestha", "Ela Singh Rathaure", "Bikal Shrestha", "Subodh Dhakal", "Pooja Ghimire", "Yubika Shrestha", "Ela Singh Rathaure" ], "abstract": "Background: Self-esteem is vital to living a happy, confident and content life. Medical students experience various forms of stress due to academic, financial and social pressures which could affect their levels of self-esteem. This study aims to study the status of self-esteem among undergraduates of a medical college at Tribhuvan University, Nepal. Methods: After receiving the ethical approval from the Institutional Review Committee (IRC) of NAIHS; we conducted a descriptive cross-sectional study among the first- to fifth-year medical students from December 2020 to April 2021. 190 were selected for the study using a stratified random sampling technique. This study used the Rosenberg self-esteem scale to measure self–esteem of the participants. A Google Forms questionnaire was sent to the participants via email. Then, the data obtained were entered in the Google sheet and later analyzed using SPSS 27. A Chi-square test was used to identify potential differences in self-esteem scores among different variables. A p-value of < 0.05 was considered statistically significant. Results: This study included a total of 180 participants, among which, 18.9% (13.19% to 24.61%; at 95% CI) students showed low self -esteem. 74.4% (68.02% to 80.78%; at 95% CI) students had normal self-esteem and 6.7% (3.05% to 10.35%; at 95% CI) students had high self-esteem. The mean self-esteem score was 19.19 (15.01 to 23.37; at 95% CI). Female participants suffered more than males from low self-esteem, and third-year students had the highest percentage of low self-esteem (30.77%). Conclusion: The majority (74.4%) of medical students had normal self-esteem. However, 18.9% students had low self-esteem, among which, third-year students suffered the most (30.77%). Likewise, females exhibited higher prevalence of low self-esteem compared to males. Interventions to boost the level of self-esteem should be carried out to help medical students become confident and efficient doctors.", "keywords": [ "self-esteem", "college", "medical students", "Kathmandu", "Nepal", "undergraduates", "doctors", "self-perception" ], "content": "Introduction\n\nSelf-esteem is one’s sense of self-worth, acceptance, and confidence that individuals feel toward themselves.1 Mentally and physically active people are the foundation of a healthy society and serve the community. Doctors and medical students in particular must have a healthy mental state. A doctor suffering from anxiety, dissatisfaction, and low self-esteem will not be able to fulfil his/her duty properly,2 and a medical student with low self-esteem will never grow to become a healthy, efficient doctor.\n\nHaving balanced self-esteem is highly important for healthcare professionals, including medical students.3 Medical students undergo major stress in the form of academic workload,4 family expectations, peer pressure, financial issues5,6 and excessive working hours.5-7 Studies show that a large proportion of medical students are unhappy and have low self-esteem,8 and that students with low self-esteem are likely to suffer from anxiety and mental breakdowns, doubt themselves and feel a sense of inadequacy.9 These students tend to appear less competent among their peers10 which contributes to depression and unhappiness. Self-development and well-being is also negatively affected by inadequate self-esteem.8\n\nSelf-esteem influences a student's performance, as well as their physical and psychological health and therefore is important to be maintained. A person who has high self-esteem can cope more effectively with stress and anxiety-inducing events in life without any negativity or impact on their mental health.11 It helps one to foster confidence, appear competent among their colleagues, tackle challenges with optimism and take on criticism without it affecting their self-image.8\n\nIn Nepal, studies evaluating the self-esteem of medical students are lacking, but existing literature studying nursing students found that nearly 78% of the students had low self-esteem.12 Producing healthy doctors is a crucial part of human resource planning.13 Hence, assessing their self-esteem and designing necessary interventions for students with inadequate self-esteem can help us produce confident and optimistic doctors. Since confidence is regarded as a subjective marker of competence,14 we can expect to produce highly competent doctors from the timely intervention. Having this competency in student life will help them acquire good knowledge and later, as a doctor, enable them to effectively manage patients. This study aims to report the status of self-esteem among medical students at a college in Kathmandu.\n\n\nMethods\n\nA descriptive cross-sectional study was conducted from December 2020 to April 2021. Data collection took place from 30th of December, 2020 to 25th of January, 2021 among medical students at the Nepalese Army Institute of Health Sciences-College of Medicine (NAIHS-COM) situated in Kathmandu. Medical undergraduates from all years of study were included in the study. The study was conducted after receiving ethical approval from the Institutional Review Committee (IRC), NAIHS; on December 2020 (Reg. No. 373).\n\nWe calculated the adequate sample size using the formula for infinite population and then adjusted it according to our population size. Based on Syed et al’s study, we estimated the proportion of students with low self-esteem to be 18%.15 Using this estimation, we calculated the minimum sample size that could represent our population.\n\n[where, N = sample size for infinite population, z = factor to achieve (1 − a) % level of confidence (z = 1.96 at 95% C.I) p = estimated rate of population proportion i.e. 18% here.15 d = margin of error to be tolerated (5% here)]\n\nFor our study population, adjusted sample size is:\n\n[where, n = adjusted sample size for our finite population, P = our study population i.e. 550 students.]\n\nHence, our sample size (n) is 161.\n\nAdjusting the sample size by taking non-response rate at 15%, the final sample size becomes 190.\n\nStudents 18 years or older who gave consent were eligible to participate in the study. For the purpose of sampling, students were divided into two strata: pre-clinical (first, second) and clinical year (third, fourth, fifth). This was done because students of pre-clinical years shared some common characteristics – such as studying basic medical subjects without clinical exposure, while students of clinical years had clinical exposure. Students were selected through a Proportionate Stratified Random sampling technique so that every student from the first to fifth year had an equal probability of being selected in the study. This would ensure that the sample within each stratum properly represented our study population. The sampling frame was prepared by collecting the list of students from administrative section of the institute, who were numbered from 1 to 550. Within that limit, 190 random numbers were generated with the help of Google Random Number Generator as per the proportion of students from each year and the proportion of males and females in each year. The random numbers that would represent the student researchers involved in this study were excluded.\n\nTo measure self-esteem, we used a questionnaire based on the Rosenberg Self Esteem Scale (RSES).16,17 The questionnaire had two parts; demographic details and the Rosenberg Self Esteem Scale. Demographic details covered general information including: age, sex, province of permanent address, year of study and type of institution they had attended for primary level education. A copy of the questionnaire can be found in the extended data.18\n\nThe RSES was developed by sociologist Morris Rosenberg.16 It is a 10-item Likert-type scale with items answered on a four-point scale from strongly agree to strongly disagree. For items 1, 3, 4, 7, 10: Strongly Agree = 3, Agree = 2, Disagree = 1, and Strongly Disagree = 0. For items 2, 5, 6, 8, 9 (which are negative statements; hence reversed in score): Strongly Agree = 0, Agree = 1, Disagree = 2, and Strongly Disagree = 3.16,18 A total is obtained by adding these markings which ranges from 0–30. A score less than 16 indicates low self-esteem, more than 25 indicated high self-esteem and scores from 16 to 25 shows normal self-esteem in the respondents.17\n\nThe RSES has high reliability: test-retest correlations are in the range of .82 to .88 and Cronbach's alpha for various samples are in the range of .77 to .88.19,20 We also calculated Cronbach’s alpha for our study sample and the value for it was 0.811 which showed good internal consistency and reliability of the instrument for our population as well.\n\nThe questionnaire was sent to 190 students through Google forms via email. Out of 190 students, 182 responded among which 2 refused to give consent. Hence, we obtained complete data from 180 students which was then entered into a Google spreadsheet18 and later analyzed using Microsoft Excel and SPSS version 27. Chi Square Test of Association was used to compare between and among different categorical variables by taking the median values of RSES score. A p-value of < 0.05 was considered statistically significant.\n\nThe participant responses could have been subjected to response bias and subjective bias. Response bias includes responding to questionnaire items without understanding them and also giving socially desirable answers. Subjective bias could have occurred as the questionnaire is based on the subjective feelings of the respondents. We could not control subjective bias as it is inevitable when measuring abstract parameters such as self-esteem. But for response bias, we employed certain techniques to minimize it as far as possible. The questionnaire was self-administered, without individual interpretation by the researcher, but with explanation for some possible ambiguities in it. We checked the clarity of the items by translating the items to Nepali and retranslating it back to English with the help of two individuals who were not involved in the study. Moreover, we assured participants regarding concealed identity and confidentiality issues at the beginning and the Likert scale used was a 4-point Likert without the neutral responding.\n\nEthical approval was obtained from the Institutional Review Committee (IRC), NAIHS in December 2020 (Reg. No. 373). There was no patient or public involvement in the design, conduct or reporting of our research. The first section of our Google forms survey had an information sheet with information about our study and an informed consent section where the participants could choose to agree or disagree to participate in the study via a tick-box. Participants who chose to agree could proceed to the questionnaire section. They were informed that their identity would be concealed and their de-identified data would be accessed only by the researchers. In this way, only after obtaining informed consent, the randomly selected medical students were involved as participants for data collection.\n\n\nResults\n\n180 responses were received from 190 study participants (94.73% response rate). Out of the 180 participants, 123 (68.3%) were male and 57 (31.7%) were female. Majority of the respondents were from clinical years (third, fourth and fifth year) making up 63.5%, whereas only 36.1% respondents were from Pre-Clinical years (first and second year). The average age of the students was 22.17 ± 1.78 years. The mean self-esteem score of the respondents was 19.19 ± 4.18 (15.01 to 23.37 at 95% CI) which lies in the normal range of RSES score.\n\nAmong 180 participants, 18.9% (13.19 to 24.61 at 95% CI) were found to have low self-esteem. 74.4% (68.02% to 80.78% at 95% CI) students had normal self-esteem and 6.7% (3.05% to 10.35% at 95% CI) students had high self-esteem.\n\nFemale participants were found to have suffered lower self-esteem than males. The mean self-esteem score of males was 19.63 ± 4.29 and that of females was 18.26 ± 3.81. There is an association between sex and median values of RSES scores (i.e., whether students had higher or lower self-esteem than the median value). Significant difference in self-esteem was found between males and females at 5% level of significance (χ 2 = 4.33, p = 0.037). Not enough evidence was found to suggest a significant difference in RSES scores between and among any other variables. Out of 65 pre-clinical year students, 11 (16.92%) and out of 115 clinical year students 23 (20%) were found to have low self-esteem. Third year medical students had the highest percentage of low self-esteem i.e., 12 (30.77%) as shown in Table 1.\n\nIt was found that 20% (14.16% to 25.84% at 95% CI) of the students who had attended private school for their primary level of schooling had low self-esteem whereas its prevalence among government school students was reported to be 10% (5.62% to 14.38% at 95% CI). Further details of the findings have been presented in Table 1.\n\n\nDiscussion\n\nSelf-esteem refers to an individual’s overall sense of value or worth.19 Various factors including age,21 sex,22,23 socio-economic status,23,24 level of study,25 academic stress12 and family environment26 influence self-esteem. A reasonably high degree of self-esteem is considered an attribute of good mental health,27 and students with high self-esteem are found to have better academic achievements compared to those with low self-esteem.28 So, in order to improve the academic performance of medical students, healthy self-esteem is imperative. Moreover, healthcare professionals with high self-esteem show higher tendency to influence and induce positive well-being not only in patients but also in the healthcare team.29 Empathy is an essential element of the physician-patient relationship and is also an essential quality for medical students.30 A medical student with low self-esteem is likely to grow less empathetic since self-esteem and empathy are positively correlated.30 Low self-esteem has also been allied with unhappiness,31 suicidal ideation,32 isolation and avoidance of social settings33 and psychological disorders.34\n\nNumerous studies have been conducted on self-esteem among the general population but there have been limited studies conducted among medical students. According to studies carried out by Syed, et al. and Nair, et al. among medical students, the prevalence of low self-esteem was found to be 18%15 and 19.4%35 respectively. According to our study, the prevalence of low self-esteem among the medical students of NAIHS-COM was found to be 18.9% which was similar to the above mentioned studies. This prevalence of low self-esteem may be due to high academic stress,12 low perceived family support12 and learning environment.24 Since not many studies have been conducted on self-esteem among medical students in Nepal, other contributing factors are yet to be explored. 74.4% of the students had normal levels of self-esteem. Having high but realistic self-esteem is important for sound mental health. But considering the potential for subjective bias in this study, it cannot be implied that the high scores (>25) attained by the respondents are an accurate reflection of their actual self-esteem status, and not a subjective, narcissistic trait. The prevalence of high self-esteem was reported to be 6.7% in our study. Studies among Arab participants suggest that self-esteem positively and significantly linked with attitude towards life, mental health, and general satisfaction.36-41 People with high self-esteem are expected to persist in the face of challenges than those with low self-esteem.42 But it is also suggested that people with high self-esteem are more likely to be conceited, arrogant and sometimes narcissistic.43\n\nThe mean RSES score of males was found to be significantly higher at 19.63 ± 4.29, whereas females had a score of 18.26 ± 3.8. Studies conducted among Malaysian and Indian students showed no significant difference between males and females.44,45 A study done among four Arab counties showed Kuwaiti men had a significantly higher mean score than their female peers.40 A study conducted in Loni, India also showed higher prevalence of low self-esteem in males than females,15 whereas our study indicated that low self-esteem was more prevalent among females.\n\nIn our study, significant differences between self-esteem were not found among clinical and pre-clinical year students. However, third year medical students had the highest prevalence of low self-esteem. This could be due to lack of adaptation to academic burden while transitioning from pre-clinical phase to clinical phase. A study conducted in India by Syed et al had a dissimilar finding that the cohort of first year males had the highest prevalence of low self-esteem.15 In a study conducted by Naz, et al, the highest prevalence of low self-esteem was found in fourth year medical students.46 As the third year medical students had the highest prevalence of low self-esteem, proper orientation and pre-exposure sessions should be conducted before entering the clinical phase. Psychological counselling should be frequently provided to all medical students, focusing more on the students with low self-esteem. Medical students should also be encouraged to participate in extracurricular activities which may help in boosting their self-esteem. This is because involvement in extracurricular activities can help students achieve personal identity development and a sense of self-worth47 which will in turn, add to their self-esteem.\n\nA Nigerian study showed that self-esteem is positively and significantly correlated with the primary school attended.24 The self-esteem of medical students who attended private schools was significantly higher than those of public school students.24,48 In our study, we tried to figure out whether there was any difference in self-esteem between students who had attended government schools for primary level of schooling and those who had attended private schools. Although we found that a proportionately higher number of students who had acquired primary levels of education through private schools had low self-esteem, the difference was not significant. We propose that this could be because the majority of the students (88.9%) were from private schools and a comparatively higher percentage of low self-esteem was seen among them. But the statistical significance of this difference was not obtained which could have been due to the very low number of students from government schools for the comparison.\n\nWe were also interested to know if the permanent address of students created an impact on their self-esteem since not all parts of Nepal are equally developed and students from some areas may be deprived of opportunities. But we did not find any significant difference in the self-esteem of students from different provinces.\n\nSelf-esteem can be classified as explicit and implicit self-esteem.49 Explicit self-esteem is presented in the reflective and deliberative evaluations that one makes about oneself, while implicit self-esteem results from responses to self-relevant stimuli and is the function of automatic process.50 It is found that people with high explicit self-esteem and low implicit self-esteem showed higher levels of narcissism49 and the self-esteem level measured with RSES is usually the explicit one.51 So, a self-esteem score of more than 25 cannot always be guaranteed to be healthy since high self-esteem can be relatively secure or defensive.49 Our study showed that 6.7% students had high self-esteem. The normal range of self-esteem falls within the 16-25 score range which 74.4% of the students come under.\n\nHealthy self-esteem in healthcare professionals is a personal quality which, when combined with professionalism and accountability, can greatly reinforce patient satisfaction.52 This is the reason why strengthening medical students’ self-esteem warrants special consideration from the beginning of their medical studies as early intervention could yield better results.\n\nThe findings of this study contribute evidence to the limited literature on the status of self-esteem in medical undergraduates at a college in Nepal. Our findings provide estimations of our future doctors’ levels of self-esteem. This would help the policy makers and medical institution to develop interventions that could support those with low self-esteem. The study used well-known standard screening tools i.e. RSES to assess self-esteem and measured its difference among several variables. The RSES has high reliability.19,20 We also calculated Cronbach’s alpha for our study sample and the value for it was 0.811 which showed good internal consistency of the instrument for our population as well.\n\nAs the study was conducted among medical students at a single medical college, the results cannot be generalized to the larger populations of the students of other medical colleges of Nepal as well as the general population. Therefore, multi-centric studies on self-esteem should be conducted in order to expand the generalizability of the findings. Likewise, Rosenberg’s self-esteem scale only measures general self-esteem neglecting self-esteem in relation to peers, parents and schooling.53 This leads to partial assessment of levels of self-esteem. The participants may have felt pressure to give answers that were socially desirable which could have contributed to response bias. Response bias includes responding to questionnaire items without understanding them and also, providing what they consider to be socially desirable answers. There might also be subjective bias associated with our study as the questionnaire is based on the subjective feelings of the respondents.\n\n\nConclusion\n\nThis study found that the majority of medical students had normal self-esteem. Several factors including age, gender, schooling, and year of study could have an impact upon self-esteem. Further studies on self-esteem should be conducted on larger populations to determine the contributing factors of low self-esteem in medical students. In our study, low self-esteem was most prevalent in third year students which may be due to the transition from pre-clinical to clinical study. Proper counseling and clinical exposure in pre-clinical years may help students to manage this transition more easily. Considering the conducted study and the related studies, we can conclude that self-esteem along with medical knowledge and skills play a great role in shaping future doctors. Hence, it is recommended that essential measures such as clinical exposure in pre-clinical years and proper counseling should be used to enhance the levels of self-esteem and mental health among medical students.\n\n\nConflict of interest\n\nNo competing interests were disclosed.\n\n\nFunding\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nData availability\n\nFigshare: ‘Status of self-esteem in medical students of a college in Kathmandu: A descriptive cross sectional study’. https://doi.org/10.6084/m9.figshare.15149589.18\n\nFigshare: ‘Status of self-esteem in medical students of a college in Kathmandu: A descriptive cross sectional study’. https://doi.org/10.6084/m9.figshare.15149589.18\n\nThis project contains the following extended data:\n\n- Questionnaire (includes consent form)\n\nSTROBE checklist for ‘Status of self-esteem in medical students of a college in Kathmandu: A descriptive cross sectional study’, https://doi.org/10.6084/m9.figshare.15149589.18\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to thank the students of NAIHS for their participation and the Community Medicine Department, NAIHS for allowing and helping us to conduct this study.\n\n\nReferences\n\nVan Vugt M, Howard C, Moss S: Being better than some but not better than average: Self‐enhancing comparisons in aerobics. 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Publisher Full Text\n\nBaumeister RF, Campbell JD, Krueger JI, et al.: Does high self-esteem cause better performance, interpersonal success, happiness, or healthier lifestyles?. Psychol. Sci. Public Interest. 2003 May; 4(1): 1–44. PubMed Abstract \| Publisher Full Text\n\nBaumeister RF, Smart L, Boden JM: Relation of threatened egotism to violence and aggression: The dark side of high self-esteem. Psychol. Rev. 1996 Jan; 103(1): 5–33. PubMed Abstract \| Publisher Full Text\n\nAhmat SN, Muda MR, Neoh CF: Self-esteem level and its relationship to academic performance among undergraduate pharmacy students in a Malaysian public university. Indian J Pharm Educ Res. 2018 Apr 1; 52(2): 197–201.\n\nChaudhari B: The Relationship of Eating Disorders Risk with Body Mass Index, Body Image and Self-Esteem among Medical Students. Ann. Med. Health Sci. Res. 2017 [cited 2021 Apr 15]; Vol. 7. Annals of Medical and Health Sciences Research.Reference Source\n\nNaz F, Aijaz S, Ullah Khan S: Level of self esteem in medical students according to their educational year, gender and socio economic status.[cited 2021 Apr 15]. Reference Source\n\nExtracurricular Activity Involvement and Adolescent Self-Esteem:[cited 2021 Sep 14].Reference Source\n\nEremie MD, Chikweru AE: Self Esteem among Private and Public Secondary Schools Students in Rivers State: Implications for Counselling. Kuwait Chapter of the Arabian J. Busi. Manage. Rev. 2015 Jul 1; 4(11): 1–6. Publisher Full Text\n\nZeigler-Hill V, Jordan CH:Two faces of self-esteem: implicit and explicit forms of self-esteem.Gawronski B, Payne BK, editors. Handbook of Implicit Social Cognition: Measurement, Theory, and Applications. New York:Guilford Press;2010; p. 392–407.\n\nJordan CH, Whitfield M, Zeigler-Hill V: Intuition and the Correspondence Between Implicit and Explicit Self-Esteem. J. Pers. Soc. Psychol. 2007 Dec; 93(6): 1067–1079. PubMed Abstract \| Publisher Full Text\n\nDi Pierro R, Mattavelli S, Gallucci M: Narcissistic Traits and Explicit Self-Esteem: The Moderating Role of Implicit Self-View. Front. Psychol. 2016 November 22; 7: 1815. Publisher Full Text\n\nDecker PJ: The hidden competencies of healthcare: Why self-esteem, accountability, and professionalism may affect hospital customer satisfaction scores. Hosp. Top. 1999; 77(1): 14–26. PubMed Abstract \| Publisher Full Text\n\nPotard C:Self-Esteem Inventory (Coopersmith). Encyclopedia of Personality and Individual Differences. Springer International Publishing;2017; p. 1–3. Publisher Full Text" }	[ { "id": "100126", "date": "22 Nov 2021", "name": "Dr. Richard Mottershead", "expertise": [ "Reviewer Expertise I am a qualified researcher and mental health specialist with experience as an educator within higher education in undergraduate and postgraduate programmes across nursing", "medical and health and social care provision." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 would like to say thank you for asking me to review this research. I have read the article and have the following comments to make:\nThis is a generally well-composed paper, which will be of interest to healthcare educators who wish to be aware of issues which may hamper student performance and well-being. The authors appear to have a creditable background and research experience which gives the article credibility and allows for an interesting insight into student mental well-being within Nepal. The paper proposes that female students are are at greater risk of reduced self-esteem and the causality needs now to be ascertained in order reduce further stigma and shame. Comparisons have been made with relevant contemporary research and this allows for a global overview of the topic and how this article fits within the body of evidence.\nI think that the article is correct not to generalize and to highlight the subjective nature of assessing se-esteem but no doubt the data will highlight the need to support and the participants may have developed a collective consciousness on the necessity to self-care in order to be effective healthcare practitioners.\n\nI wish the authors well with their future efforts to support this cohort and in progressing future research and support networks which I am sure will provide long-term benefits to the health and well-being of medical students with results that can be referred to the wider student healthcare 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": "98205", "date": "03 Feb 2022", "name": "Christian Sunday Ugwuanyi", "expertise": [ "Reviewer Expertise Educational research", "measurement and 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 study explored the state of self-esteem among undergraduates at Tribhuvan University in Nepal's medical college. The majority of medical students reported normal self-esteem, according to the findings of a descriptive cross-sectional survey conducted among first- to fifth-year medical students from December 2020 to April 2021. The findings, however, revealed that 18.9% of students showed low self-esteem, with third-year students being the most affected (30.77 percent). The researchers recommended that to help medical students become confident and effective doctors, interventions to improve self-esteem should be implemented. This study was properly conceptualised and documented as an academic paper, but more efforts should be made by the authors to use recent empirical studies to support their line of argument. The methodology section of the manuscript was properly handled to allow for its replication. In other words, sufficient details of methods and analysis were provided to allow replication by others. The results were also adequately presented.\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": "123050", "date": "09 Mar 2022", "name": "Sunil Kumar Joshi", "expertise": [ "Reviewer Expertise Public health", "Occupational health and safety", "injury and violence prevention." ], "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\nI appreciate the efforts put forward by a group of young students to participate in such a novel study in Nepal. This study on self-esteem has been conducted in a single medical college in Kathmandu.\nHere are a few comments from me: There is no explanation on the classification of self-esteem and its relation to the RSES scoring in the Introduction section, it comes for the first time in the Methods section. Few sentences need to be rephrased or restructured as they are not very clear to me. The manuscript needs to be checked by an English language expert. The authors should elaborate on the technical words mentioned in the text, e.g., narcissism. In the pdf format of the manuscript \"Table 1. Socio-demographic characteristics and RSES of the students along with p-value\" comes in the Methods section, it should go to the Results section.\nI have some reservations about the sample size calculation. An estimated rate of population proportion from a study in a rural Indian medical college i.e.18% has been cited to calculate the sample size (ref 15). NAIHS is not a rural medical college, and as it is the first of its kind study in Nepal, I suggest using the population proportion of 50%. It would bring a sample size of approximately 384. I do not support the adjusted sample size calculation for the finite population in this study. As the total number of students in this medical college is 550, the researchers would be easily able to get the required study respondents. So, I suggest the students do recalculate the sample size as I suggested, extend the data collection and reanalyse data and resubmit for publication. This will definitely make the findings more robust. I suggest being considerate in citing the published literature. I think a total of 53 references is a little high for this article.\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? Yes", "responses": [ { "c_id": "8053", "date": "07 Apr 2022", "name": "Stuti Yadav", "role": "Author Response", "response": "Thank you for the comments, sir. The manuscript has been changed as per the comments and the table has also been placed in the results section." } ] } ]	1	https://f1000research.com/articles/10-1031
https://f1000research.com/articles/10-758/v1	05 Aug 21	{ "type": "Research Article", "title": "Philympics 2021: Prophage Predictions Perplex Programs", "authors": [ "Michael J. Roach", "Katelyn McNair", "Sarah K Giles", "Laura K Inglis", "Evan Pargin", "Simon Roux", "Przemysław Decewicz", "Robert A. Edwards", "Katelyn McNair", "Sarah K Giles", "Laura K Inglis", "Evan Pargin", "Simon Roux", "Przemysław Decewicz", "Robert A. Edwards" ], "abstract": "Background Most bacterial genomes contain integrated bacteriophages—prophages—in various states of decay. Many are active and able to excise from the genome and replicate, while others are cryptic prophages, remnants of their former selves. Over the last two decades, many computational tools have been developed to identify the prophage components of bacterial genomes, and it is a particularly active area for the application of machine learning approaches. However, progress is hindered and comparisons thwarted because there are no manually curated bacterial genomes that can be used to test new prophage prediction algorithms. Methods We present a library of gold-standard bacterial genome annotations that include manually curated prophage annotations, and a computational framework to compare the predictions from different algorithms. We use this suite to compare all extant stand-alone prophage prediction algorithms to identify their strengths and weaknesses. We provide a FAIR dataset for prophage identification, and demonstrate the accuracy, precision, recall, and f1 score from the analysis of seven different algorithms for the prediction of prophages. Results We identified different strengths and weaknesses between the prophage prediction tools. Several tools exhibit exceptional f1 scores, while others have better recall at the expense of more false positives. The tools vary greatly in runtime performance with few exhibiting all desirable qualities for large-scale analyses. Conclusions Our library of gold-standard prophage annotations and benchmarking framework provide a valuable resource for exploring strengths and weaknesses of current and future prophage annotation tools. We discuss caveats and concerns in this analysis, how those concerns may be mitigated, and avenues for future improvements. This framework will help developers identify opportunities for improvement and test updates. It will also help users in determining the tools that are best suited for their analysis.", "keywords": [ "software comparison", "bioinformatics tool", "lysogen genome", "temperate phage", "prokaryotic virus" ], "content": "Introduction\n\nBacteriophages (phages), viruses that infect bacteria, can be either temperate or virulent. Temperate phages may integrate into their bacterial host genome and the host-integrated phage genome is referred to as a prophage. Prophages are ubiquitous and may constitute as much as 20 percent of bacterial genomes (Casjens, 2003). Prophages replicate as part of the host bacterial genomes until external conditions trigger a transition into the virulent lytic cycle, resulting in replication and packaging of phages and typically the death of the host bacteria. Prophages generally contain a set of core genes with a conserved gene order that facilitate integration into the host genome, assembly of phage structural components, replication, and lysis of the host cell (Kang et al., 2017; Canchaya et al., 2003). As well as these core genes, phages can contain an array of accessory metabolic genes that can effect significant phenotypic changes in the host bacteria (Breitbart, 2012). For instance, many prophages encode virulence factors such as toxins, or they can encode fitness factors such as nutrient uptake systems (Brüssow et al., 2004). Lastly, most prophages encode a variety of super-infection exclusion mechanisms to prevent concurrent phage infections, including restriction/modification systems, toxin/antitoxin genes, repressors, etc. (Calendar, 1988). The function of most prophage accessory genes remains unknown.\n\nCore (pro) phage genes have long been used for identifying prophage regions. However, there are other unique characteristics that can distinguish prophages from their host genomes: bacterial genomes have a GC skew that correlates with direction of replication, and the insertion of prophages will generally disrupt this GC bias (Grigoriev, 1998). Transcript direction (Campbell, 2002) and length of prophage proteins have also proven to be useful metrics in predicting prophages (Akhter et al., 2012; Song et al., 2019), where phage genes are generally smaller and are oriented in the same direction (Dutilh et al., 2014). Likewise, gene density tends to be higher in phage genomes and intergenic space shorter (Amgarten et al., 2018; McNair et al., 2019).\n\nOver the last two decades many prophage prediction tools have been developed, and they fall into two broad classes: (1) web-based tools where users upload a bacterial genome and retrieve annotations including PHASTER (Arndt et al., 2016), Prophage Hunter (Song et al., 2019), Prophinder (Lima-Mendez et al., 2008), PhageWeb (Sousa et al., 2018), and RAST (Aziz et al., 2008); and (2) command-line tools where users download a program and database to run the predictions locally (although some of these also provide a web interface for remote execution). In this work we focus on this latter set of tools (Table 1) because web-based tools typically do not handle the large numbers of simultaneous requests required to run comparisons across many genomes.\n\nDespite the abundance of prophage prediction algorithms, there has never been either a set of reference genomes against which all tools can be compared, nor a unified framework for comparing those tools to identify their relative strengths and weaknesses or to identify opportunities for improvement. We generated a set of manually annotated bacterial genomes released under the FAIR principles (Findable, Accessible, Interoperable, and Reusable), and developed an openly available and accessible framework to compare prophage prediction tools.\n\n\nMethods\n\nTo assess the accuracy of the different prophage prediction tools, a set of 49 gold-standard publicly available bacterial genomes with manually curated prophage annotations was generated. The genomes and prophage annotations currently included are available in Tables S1 and S2. The genomes are in GenBank format and file conversion scripts are included in the framework to convert those files to formats used by the different software. The tools that are currently included in the framework are outlined in Table 1. Snakemake (Köster & Rahmann, 2012) pipelines utilising conda (Anaconda Software Distribution. Conda. v4.10.1, April 2021 (Conda, RRID:SCR_018317)) package manager environments were created for each tool to handle the installation of the tool and its dependencies, running of the analyses, output file conversion to a standardized format, and benchmarking of the run stage. Where possible, annotations from the GenBank files were used in the analysis to promote consistency between comparisons. Additional pipelines were created for running PhiSpy using the included training sets for the appropriate genera, and for running PhiSpy with pVOG (Grazziotin et al., 2017) HMMs and these are also available in the repository. DBSCAN-SWA was not able to consistently finish when using GenBank files as input, and instead the genome files in fasta format were used. Another pipeline was created to pool the results from each tool and some comparisons are illustrated in the included Jupyter notebook. Testing and development of the pipelines were conducted on Flinders University’s DeepThought HPC infrastructure. The final benchmarking analysis was performed on a stand-alone node consisting of dual Intel® Xeon® Gold 6242R processors (40 cores, 80 threads), 768 GB of RAM, and 58 TB of disk space. Each tool was executed on all genomes in parallel (one thread per job), with no other jobs running.\n\n\n\nThere are many potential ways to compare prophage predictions: For instance, is it more important to capture all prophage regions or minimise false positives? Is it more important to identify all the phage-encoded genes, or the exact locations of the attachment site core duplications (attL and attR)? The runtime and CPU time in seconds, peak memory usage and file write operations were captured by Snakemake (Snakemake, RRID:SCR_003475) for the steps running the prophage tools only (not for any file conversion steps before or after running each tool). The predictions were then compared to the gold standard annotations and the number of true positive (TP), true negative (TN), false positive (FP) and false negative (FN) gene labels were used to calculate the performance metrics. Each application marks prophages slightly differently, and therefore we used the designation of coding sequence (CDS) features as phage or not to assess prophage predictions.\n\nWe developed the framework to simplify the addition of new genomes to the benchmarks. Each genome is provided in the standard GenBank format, and the prophages are marked by the inclusion of a non-standard flag for each genomic feature that indicates that it is part of a prophage. We use the qualifier/is_phage=“1” to indicate prophage regions.\n\n\nResults and discussion\n\nWe compared the availability, installation, and results from ten different prophage prediction algorithms (Table 1). Two—ProphET (Reis-Cunha et al., 2019) and LysoPhD (Niu et al., 2019)—could not be successfully installed and were not included in the current framework (see below). The remaining eight PhiSpy (Akhter et al., 2012), Phage Finder (Fouts, 2006), VIBRANT (Kieft et al., 2020), VirSorter (Roux et al., 2015), Virsorter2 (Guo et al., 2021), Phigaro (Starikova et al., 2020), PhageBoost (Sirén et al., 2021), and DBSCAN-SWA (Gan et al., 2020) were each used to predict the prophages in 49 different manually curated microbial genomes.\n\nMost of these programs utilize protein sequence similarity and HMM searches of core prophage genes to identify prophage regions. PhageBoost leverages a large range of protein features (such as dipeptide and tripeptide combinations) with a trained prediction model. PhiSpy was originally designed to identify prophage regions based upon seven distinct characteristics: protein length, transcript directionality, AT and GC skew, unique phage words, phage insertion points, optionally phage protein similarity and sequence similarity. DBSCAN-SWA likewise uses a range of gene metrics and trained prediction models to identify prophages.\n\nRegardless of whether annotations are available, Virsorter2, Phigaro, and PhageBoost all perform de novo gene prediction with Prodigal (Hyatt et al., 2010) and VirSorter uses MetaGeneAnnotator (Noguchi et al., 2008) for the same purpose. VIBRANT can take proteins if they have ‘Prodigal format definition lines’ but otherwise performs predictions with Prodigal. PhageBoost can take existing annotations but this requires additional coding by the user. DBSCAN-SWA can take annotations or can perform gene predictions with Prokka (Seemann, 2014). PhiSpy takes an annotated genome in GenBank format and uses the annotations provided.\n\nThe prophage prediction packages Phigaro, PhiSpy, VIBRANT, VirSorter, and VirSorter2 are all able to be installed with conda from the Bioconda channel (Grüning et al., 2018), while Phispy, Phigaro, and PhageBoost can be installed with pip—the Python package installer. Phigaro, VIBRANT, VirSorter, and VirSorter2 require a manual one-time setup to download their respective databases. Phigaro uses hard-coded file paths for its database installation, either to the user’s home directory or to a system directory requiring root permissions. Neither option is ideal as it is impossible to have isolated versions or installations of the program, and it prevents updating the installation paths of its dependencies. For PhageBoost to be able to take existing annotations, a custom script was created to skip the gene prediction stage and run the program. Basic PhiSpy functionality is provided without requiring third-party databases. However, if the HMM search option is invoked, a database of phage-like proteins—e.g. pVOG (Grazziotin et al., 2017), VOGdb (https://vogdb.org), or PHROGS (Terzian P et al., 2021)—must be manually downloaded before it can be included in PhiSpy predictions. DBSCAN-SWA is not currently available on any package manager and must be pulled from GitHub, however all its dependencies are available via conda and it could easily be added in the future. All the above “manual” installation and setup steps are uncomplicated and are automatically executed by the Snakemake pipelines provided in the framework.\n\nPhage Finder was last updated in 2006 and is not available on any package manager that we are aware of. The installation process is dated with the package scripts liberally utilising hard-coded file paths. The Snakemake pipeline for this package resolves this with soft links between the framework’s directory to the user’s home directory (where the package expects to be installed). The dependencies are available via conda allowing the complete installation and setup to be handled automatically by Snakemake.\n\nLysoPhD does not appear to be available to download anywhere and was dropped from the comparison. ProphET requires the unsupported BLAST legacy and EMBOSS packages. It is not available on any package manager and instructions for a clean installation are incomplete and not compatible with conda. The codebase was last updated in 2019. Numerous issues were encountered installing dependencies and despite significant effort we were not able to create a working installation. ProphET’s installation script reported many errors during setup, but alarmingly finished with an exit code zero to indicate a successful installation. Preparing the necessary GFF files in a format that the program could use was non-trivial. The program reported errors during runtime that we believe are related to the errors encountered during installation; ProphET terminated with incomplete output but again returned an exit code zero to indicate a successful run. ProphET was dropped from the comparison.\n\nThere was minimal difference in the performance metrics for the different methods of running PhiSpy, and we have recently shown (Roach et al in preparation) that including HMM searches with PhiSpy results in less than one additional prophage being identified. Therefore, only PhiSpy using default settings will be discussed in comparison to the other tools. PhiSpy, VIBRANT, and Phigaro performed best for mean accuracy (Figure 1a; Table S3) while DBSCAN-SWA performed the worst. PhiSpy, Phigaro, and Phage Finder performed best for mean precision (Figure 1b; Table S3). DBSCAN-SWA, PhageBoost, VirSorter, and VirSorter2 all performed poorly for mean precision. This was mostly driven by a high false-positive rate compared to the other tools (Figure S1). PhiSpy, VirSorter, VirSorter2, VIBRANT, DBSCAN-SWA, and PhageBoost all had high mean recall scores.\n\nViolin plots for each tool are shown with individual points for each genome indicated. The graphs show: ‘Accuracy’ (a) as the ratio of correctly labelled genes to all genes, ‘Precision’ (b) as the ratio of correctly labelled phage genes to all predicted phage genes, ‘Recall’ (c) as the ratio of correctly labelled phage genes to all known phage genes, and ‘f1 Score’ (d) as defined in the methods. For all graphs, more is generally better.\n\nEach tool balances between recall and precision. For example, the more conservative Phage Finder performed relatively well in terms of precision, making very confident predictions, but had one of the lower mean recall ratios and was not predicting prophages based on limited information. In contrast, the more speculative DBSCAN-SWA and PhageBoost both exhibited the opposite trend.\n\nThe f1 Score is a more nuanced metric, as it requires high performance in both precision and recall. PhiSpy, VIBRANT, Phigaro, VirSorter, and VirSorter2 all averaged above 0.5, while the remaining tools suffered from too many false predictions (FP or FN) (Figure 1d).\n\nMany users will not be too concerned about runtime performance, for instance if they are performing a one-off analysis on a genome of interest all the tools will finish in a reasonable time. However, efficient resource utilization is an important consideration for large-scale analyses. Provisioning computing resources costs money and a well optimised tool that runs fast translates to real-world savings. The runtime distributions across the genomes are shown for each tool in Figure 2a. The slowest prophage predictors were generally VirSorter and VirSorter2 with mean runtimes of 1,316 and 2,118 seconds respectively, except for a single DBSCAN-SWA run taking 4,697 seconds. PhiSpy using the trained datasets was by far the fastest performing tool (8.4 seconds mean runtime), although if an appropriate training set is not available for the genus of interest it would first need to be generated to benefit from these reduced runtimes. PhageBoost was the next fastest (37.8 seconds mean runtime) and Phage Finder, Phigaro, and PhiSpy with default parameters all performed similarly well in terms of runtime.\n\nViolin plots for each tool are shown with individual points for each genome indicated. The graphs show total runtime in seconds (a), peak memory usage in MB (b), total file writes in MB (c) and the final total disk usage (all genomes) in MB (d). For all graphs, less is better.\n\nMemory requirements also remain an important consideration for provisioning resources for large-scale analyses. For instance, inefficiency is encountered where the memory required by single-threaded processes exceeds the available memory per CPU. Peak memory usage for each tool is shown in Figure 2b. Memory requirements were lowest for VirSorter and trained PhiSpy with 210 and 450 MB mean peak memory respectively. There was a single notable exception for trained PhiSpy (predicting prophages in E. coli O157:57 EDL933) with a peak memory usage of 6.13 GB. DBSCAN-SWA had the highest mean peak memory of 6.0 GB with one run requiring 35 GB at its peak. Apart from the DBSCAN-SWA outlier, there were no situations where the peak memory usage would prevent the analysis from completing on a modest personal computer, but at larger-scales, Phigaro, PhiSpy, VirSorter, and VirSorter2 have an advantage in terms of peak memory usage.\n\nAnother important consideration for large-scale analyses are the file sizes that are generated by the different tools. Large output file sizes can place considerable strain on storage capacities, and large numbers of read and write operations can severely impact the performance of a system or HPC cluster for all users. Total file writes for the default files (in MB, including temporary files) are shown in Figure 2c and the final disk usage for all genomes for each tool is shown in Figure 2d. VirSorter, DBSCAN-SWA, and VirSorter2 performed the most write operations with mean file writes of 2.063, 0.262, and 0.034 GB respectively. The other tools performed similarly well and have a clear advantage at scale as they perform far fewer disk writes. VirSorter and DBSCAN-SWA removed most of their generated files, however, the final disk usage for these tools were still the highest at 5.36 and 2.96 GB respectively. Disk usage for PhageBoost and PhiSpy was by far the lowest at 0.14 and 15 MB respectively.\n\nEvery bioinformatics comparison involves many biases. In this comparison, PhiSpy performs well, but we developed PhiSpy and many of the gold-standard genomes were extensively used during its development to optimize the algorithm. VirSorter and VirSorter2 were primarily developed to identify viral regions in metagenomes rather than prophages in bacterial genomes—although they have been used for that e.g. in Glickman et al. (2020)—and filtering VirSorter and VirSorter2 hits with CheckV (Nayfach et al., 2021) is recommended. By openly providing the Prophage Prediction Comparison framework, creating a framework to install and test different software, and defining a straightforward approach to labelling prophages in GenBank files, we hope to expand our gold-standard set of genomes and mitigate many of our biases. We welcome the addition of other genomes (especially from beyond the Proteobacteria/Bacteroidetes/Firmicutes that are overrepresented in our gold-standard database).\n\nRecent developments in alternative approaches to predict prophages, including mining phage-like genes from metagenomes and then mapping them to complete genomes (Nayfach et al., 2021) and using short-read mapping to predict prophage regions from complete bacterial genomes (Kieft & Anantharaman, 2021) have the potential to generate many more ground-truth prophage observations. However, both approaches are limited as they will identify prophages that are active, but are unable to identify quiescent prophage regions, and thus for prophage prediction algorithms they will provide useful true positive datasets but may not provide accurate true negative datasets.\n\n\nConclusions\n\nIn this comparison, PhiSpy, VIBRANT, and Phigaro were the best performing prophage prediction tools for f1 score. PhiSpy and Phigaro were also among the best in terms of runtime performance metrics. Phage Finder performs well in terms of precision at the expense of false-negatives, whereas VirSorter, VirSorter2, DBSCAN-SWA and PhageBoost perform well for recall at the expense of false-positives. Currently, DBSCAN-SWA, VirSorter, and VirSorter2 are not as well suited for large-scale identification of prophages from complete bacterial genomes when compared to the other tools. More genomes with manually curated prophage annotations are needed, and we anticipate that these benchmarks will change with the addition of new genomes, the addition of new tools, and as the tools are updated over time. Developers are strongly encouraged to contribute by adding or updating their tool and adding their manually curated genomes to be included in the benchmarking. Users are strongly encouraged to check the GitHub repository for the latest results before making any decisions on which prophage prediction tool would best suit their needs.\n\n\nAuthor contributions\n\nRAE conceived of the study; KM and PD generated the initial gold-standard set and SKG, LI, and EP contributed to the gold-standard set; RAE and MJR created the framework; RAE, MJR, and SR performed the analysis. All authors contributed to the manuscript writing.\n\n\nData availability\n\nZenodo: linsalrob/ProphagePredictionComparisons: Review release. https://doi.org/10.5281/zenodo.4739878. (Roach and Edwards, 2021b).\n\nThis project contains the following underlying data;\n\n• genbank/\n\nº The gold-standard prophage-annotated genomes in genbank format\n\n• snakefiles/\n\nº Snakemake pipeline files for running each of the prophage prediction tools against the gold-standard prophage-annotated genomes\n\n• rules/\n\nº Snakemake files with generic rules used by one or more of the Snakemake pipelines\n\n• conda_environments/\n\nº Configuration files for creating conda environments for use in the Snakemake pipelines\n\n• data/\n\nº Any custom small datasets required by the prophage prediction tools\n\n• scripts/\n\nº Perl and Python scripts that are used in the Snakemake pipelines for performing various tasks\n\n• ProphagePredictionsLib/\n\nº Library files required by the Perl and Python scripts\n\n• jupyter_notebooks/\n\nº Summary metric tables for all of the tools, and example Jupyter notebook for producing the comparison figures\n\n• img/\n\nº Example figures generated by the Jupyter notebook\n\n• LICENCE\n\nº Licence file for the github repository\n\n• Supplementary/\n\nº SupplementaryTables.xlsx\n\n▪ (Sheet 1) Table S1. Genomes provided in the gold-standard library with manually curated prophages\n\n▪ (Sheet 2) Table S2. Prophages identified in the genomes\n\n▪ (Sheet 3) Table S3. Mean metrics for each tool as measured from our gold-standard set of genomes\n\nº FigureS1.tif\n\n▪ False positive comparison.\n\nUnderlying data is also available at:\n\nGithub: Comparisons of multiple different prophage predictions https://github.com/linsalrob/ProphagePredictionComparisons/tree/v0.1-beta (Roach and Edwards, 2021a).\n\nZenodo: Extended data for ‘Philympics 2021: Prophage Predictions Perplex Programs’: https://doi.org/10.5281/zenodo.4739878.\n\nThis project contains the following extended data:\n\nSupplementaryTables.xlsx:\n\n\n\n• Table S1. Genomes provided in the gold-standard library with manually curated prophages\n\n• Table S2. Prophages identified in the genomes\n\n• Table S3. Mean metrics for each tool as measured from our gold-standard set of genomes\n\nFigureS1.tif:\n\n\n\n• Figure S1. False positive comparison. Violin plots for each tool show ‘False Positives’ as the number of genes incorrectly labelled prophage genes in each genome. Less is better.", "appendix": "References\n\nAkhter S, Aziz RK, Edwards RA: PhiSpy: a novel algorithm for finding prophages in bacterial genomes that combines similarity- and composition-based strategies. Nucleic Acids Res. 2012; 40: e126–e126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmgarten D, Braga LPP, Da Silva AM, et al.: MARVEL, a Tool for Prediction of Bacteriophage Sequences in Metagenomic Bins. Front Genet. 2018; 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArndt D, Grant JR, Marcu A, et al.: PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res. 2016; 44: W16–W21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAziz RK, Bartels D, Best AA, et al.: The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008; 9: 75. 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Publisher Full Text\n\nNoguchi H, Taniguchi T, Itoh T: MetaGeneAnnotator: detecting species-specific patterns of ribosomal binding site for precise gene prediction in anonymous prokaryotic and phage genomes. DNA res. 2008; 15: 387–396. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReis-Cunha JL, Bartholomeu DC, Manson AL, et al.: ProphET, prophage estimation tool: A stand-alone prophage sequence prediction tool with self-updating reference database. PLOS ONE. 2019; 14: e0223364. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoach MJ, Edwards RA: linsalrob/ProphagePredictionComparisons [Online]. GitHub . 2021a. [Accessed].Reference Source\n\nRoach MJ, Edwards RA: linsalrob/ProphagePredictionComparisons: Review release (Version v0.1). Zenodo. 2021b.\n\nRoux S, Enault F, Hurwitz BL, et al.: VirSorter: mining viral signal from microbial genomic data. PeerJ. 2015; 3: e985. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeemann T: Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014; 30: 2068–2069. PubMed Abstract \| Publisher Full Text\n\nSirén K, Millard A, Petersen B, et al.: Rapid discovery of novel prophages using biological feature engineering and machine learning. NAR Genom Bioinform. 2021; 3. Publisher Full Text\n\nSong W, Sun H-X, Zhang C, et al.: Prophage Hunter: an integrative hunting tool for active prophages. Nucleic Acids Res. 2019; 47: W74–W80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSousa ALD, Maués D, Lobato A, et al.: PhageWeb – Web Interface for Rapid Identification and Characterization of Prophages in Bacterial Genomes. Fron Genet. 2018; 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStarikova EV, Tikhonova PO, Prianichnikov NA, et al.: Phigaro: high-throughput prophage sequence annotation. Bioinformatics. 2020; 36: 3882–3884. PubMed Abstract \| Publisher Full Text\n\nTerzian P, Olo Ndela E, Galiez C, et al.: PHROG: families of prokaryotic virus proteins clustered using remote homology. [Online].2021. [Accessed June 2021]. Reference Source" }	[ { "id": "91324", "date": "23 Aug 2021", "name": "Franklin Nobrega", "expertise": [ "Reviewer Expertise Phage biology", "phage-host interactions" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Roach and co-authors focuses on a crucial question on how to accurately determine prophage regions. With the increasing accessibility of HPC infrastructure by scholars, and the increasing number of open datasets available for mining, there is an urgent need to establish FAIR datasets to set a ground-truth for data analysis. This work sets the tone so the community can move away from using a favourite tool, to using the most accurate and reliable to address the question at hand. In particular, the tool performance part is excellently achieved, providing details for both the novice and the advanced user when considering a new tool for their pipeline. In sum, it was a pleasure to read this well-structured and well-balanced work, which I fully endorse. I would like to leave the authors with a few recommendations and suggestions.\nRecommendations:\nI believe the authors could use a more recent reference for Calendar, 1988 (Introduction).\n\nPlease provide the methods used for manual curation of prophage annotations, as this will contribute to increase the gold-standard genomes available.\n\nWhat was the rationale for running PhiSpy using all modes, and not doing the same for other tools that also have different run modes? This would make for a more fair comparison.\n\nCould the authors clarify which prophage prediction categories were considered for VirSorter and Virsorter2, as these will certainly affect the accuracy and recall results obtained. Similar comment for VIBRANT.\n\nData would benefit from statistical analysis.\nSuggestions:\nThe first three lines of “Benchmark metrics” (Methods) seem to be more adequate to the Results section.\n\nI would suggest that the authors summarize the information provided in “Software compared” (Results and discussion) as a supplementary table, since this will certainly be useful to the community.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8049", "date": "08 Apr 2022", "name": "Michael Roach", "role": "Author Response", "response": "Thank you so much for reviewing our manuscript. We hope that version 2 addresses the concerns with the manuscript which we outline below: I believe the authors could use a more recent reference for Calendar, 1988 (Introduction). Response: We have updated this section with more recent references. Please provide the methods used for manual curation of prophage annotations, as this will contribute to increase the gold-standard genomes available. Response: Guidelines are now included in additional data. What was the rationale for running PhiSpy using all modes, and not doing the same for other tools that also have different run modes? This would make for a more fair comparison. Response: We developed PhiSpy and are well familiar with the different ways of running the pipeline. To keep the comparison fair, we now only present running PhiSpy with default parameters. Could the authors clarify which prophage prediction categories were considered for VirSorter and Virsorter2, as these will certainly affect the accuracy and recall results obtained. Similar comment for VIBRANT. Response: Following valuable contributions and input from SR—now a coauthor on this paper—the Virsorter categories 1-5 are taken as prophage predictions. Virsorter2 is run with --high-confidence-only --exclude-lt2gene and we accept both predicted whole phages and integrated phage genomes as prophage predictions. VIBRANT is run with default parameters and we use all predictions from the integrated_prophage_coordinates output file. Data would benefit from statistical analysis. Response: We now include some statistical analysis as part of out database bias evaluation. Suggestions: The first three lines of “Benchmark metrics” (Methods) seem to be more adequate to the Results section. Response: We agree, the sentence has been moved to the results section. I would suggest that the authors summarize the information provided in “Software compared” (Results and discussion) as a supplementary table, since this will certainly be useful to the community. Response: We considered adding a more simplified summary table, such as ticks and crosses or 5-start ratings for various features, however these can be very subjective and would simply be our interpretation complete with our personal biases of the results." } ] }, { "id": "91781", "date": "31 Aug 2021", "name": "Karthik Anantharaman", "expertise": [ "Reviewer Expertise Metagenomics", "Microbial and Phage ecology" ], "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 by Roach et al. describes the creation and benchmarking of a gold standard bacterial dataset that can be used for prediction of prophages from bacterial genomes. Additionally, they also benchmark seven currently available software for prediction of prophages. Overall, this manuscript and the datasets provided represent a valuable resource to the community that can be used widely. We do have some comments that in our opinion can improve the manuscript, the benchmarking and the utility of the gold standard dataset that the authors aim to to provide to the community.\n\nIntroduction:\n\nLast paragraph: the reasoning for this study is sound and accurate. Though the usage of the term “annotated” is slightly ambiguous here, and throughout the paper, because it is meant to designate either prophage locations or protein/gene annotations.\n\nMethods:\nFirst sentence: the actual method of “manually curated prophage annotations was generated” is never explained.\nWhat method was used? How were prophages manually identified, annotated, and validated? To me, gold standard would imply each prophage has been experimentally validated.\n\nWas there is specific method by which certain hosts were selected? I can see that there is “Escherichia_coli_O157-H7” and “Escherichia_coli_O157-H7_EDL933”. Why were so similar hosts chosen? Do they have significantly different prophages? There are multiple examples of this.\n\nMany also appear to be common model organisms. How was diversity ensured? Is there a possibility to use organisms more widely distributed across the tree of life?\n\nIf this component is at all incorrect then the performance metrics, especially false positives if the prophage boundaries are wrong, will be biased.\n\nHow was Supplemental Table 2 generated?\nOverall, there is no indication as to how the gold standard dataset, the centerpiece of the paper, was actually generated. This does not significantly affect the study’s results, but this information needs to be included before publication. For example, were the host sequences chosen at random from a database? Represent variable phylogenetic backgrounds? Source environments? Was prophage phylogeny taken into account? There are certainly questions left unanswered.\n\nResults:\nProphage prediction performance: rather than only providing subjective “best” and “worst” designations, the numerical results should be provided too (e.g., accuracy of 0.9 +/- 0.1). Furthermore, what statistical metrics were used to designate “best” and “worst”?\n\nCaveats: The VIBRANT manuscript says that it also was developed to identify both temperate and virulent viral regions in metagenomes. FYI - we were not able to access the Supplementary datasets through the paper (but were able to retrieve them from bioRxiv).\n\nSuggestions:\nOverall, this comparison framework would benefit from simple, minor additions.\nDoes the specific host (e.g., taxonomy) affect the results of prophage prediction for some tools? For tools that utilize HMM searches, the HMM databases may be biased towards certain groups (e.g., E. coli phages). We suggest that a comparison of precision/recall be compared to the source host.\n\nDo the performance comparisons only take into account total prophage CDS predictions or also the completeness of predicted prophages? For example, identifying all prophages but only 50% of each of those prophages is different than identifying half of all prophages but 100% of each of those.\n\nIs there any effect on hosts with multiple prophages? Are some tools affected by this?\n\nSome tools will have predicted prophages that were not in the gold standard set (false positives). What measures were taken to ensure that none of these are real prophages that were missed within the manual curation?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8050", "date": "08 Apr 2022", "name": "Michael Roach", "role": "Author Response", "response": "Thank you so much for reviewing our manuscript. We hope that version 2 address the concerns with the manuscript which we outline below: Last paragraph: the reasoning for this study is sound and accurate. Though the usage of the term “annotated” is slightly ambiguous here, and throughout the paper, because it is meant to designate either prophage locations or protein/gene annotations. Response: We agree that this could be clearer. We have updated all instances of ‘annotations’ in the manuscript to explicitly refer to either prophage or gene annotations. Methods: First sentence: the actual method of “manually curated prophage annotations was generated” is never explained. What method was used? How were prophages manually identified, annotated, and validated? To me, gold standard would imply each prophage has been experimentally validated. Response: Experimentally validating all prohages by inducing them is not possible currently, as we do not know the signal for triggering the lytic cycles for all the prophages we know about. It is also not possible for elements such as cryptic phages, which may still offer a strong phrophage signal and represent true phage genome sequence, as they are not able to be induced. For cryptic prophages we believe it is still important to include these predictions as prophage annotations. The next best approach to a gold-standard library is to manually inspect and annotate the genomes. We have included the guidelines we use as supplementary material. Was there is specific method by which certain hosts were selected? I can see that there is “Escherichia_coli_O157-H7” and “Escherichia_coli_O157-H7_EDL933”. Why were so similar hosts chosen? Do they have significantly different prophages? There are multiple examples of this. Many also appear to be common model organisms. How was diversity ensured? Is there a possibility to use organisms more widely distributed across the tree of life? Response: For PhiSpy’s original development it was important to include multiple similar genomes with dissimilar numbers and positions of phages for training the algorithm. This selection bias is something that we have more recently been trying to address. We have earmarked many new diverse genomes for manual curation, and this update marks the inclusion of 10 new prophage annotated genomes from under-represented phyla. There is still a long way to go. Annotating prophages remains an extremely challenging task for underrepresented bacterial phyla, and it will remain so until our knowledgebase of known phages and phage proteins for these phyla improves. If this component is at all incorrect then the performance metrics, especially false positives if the prophage boundaries are wrong, will be biased. Response: The current state is not perfect, rather, it is the best we can do right now. We agree with the sentiment, and it is why we have designed the repository around making it easy to add and refine tools, genomes and prophage annotations over time. However, we don’t believe there are enough errors to significantly affect the outcome of the evaluation. How was Supplemental Table 2 generated? Response: It was originally compiled during manual curation. We now include a script for generating this table from the prophage-annotated GenBank files, as well as an updated table to include the new genomes. Overall, there is no indication as to how the gold standard dataset, the centerpiece of the paper, was actually generated. This does not significantly affect the study’s results, but this information needs to be included before publication. For example, were the host sequences chosen at random from a database? Represent variable phylogenetic backgrounds? Source environments? Was prophage phylogeny taken into account? There are certainly questions left unanswered. Response: We hope the inclusion of our guidelines for manual curation and our recent progress has alleviated these concerns. There is still a long way to go to achieve a more diverse representation of prophages and this framework is intended to support this journey. Results: Prophage prediction performance: rather than only providing subjective “best” and “worst” designations, the numerical results should be provided too (e.g., accuracy of 0.9 +/- 0.1). Furthermore, what statistical metrics were used to designate “best” and “worst”? Response: This was partially available in Supp table 3, but given its importance, we have move this to a new table (Table 2) and include a new table (Table 3) for the benchmarking results. We have also calculated and report standard deviations where applicable. “Best” and “Worst” are simply designated based on the rankings of each prophage caller based on their scores for a given metric. Caveats: The VIBRANT manuscript says that it also was developed to identify both temperate and virulent viral regions in metagenomes. FYI - we were not able to access the Supplementary datasets through the paper (but were able to retrieve them from bioRxiv). Response: Our apologies. The Genbank files are stored and can be retrieved using git-lfs. The Zenodo repo is a verbatim copy of the GitHub repo and does not resolve the git-lfs links to the GenBank files. We have added a section in underlying data to clarify this. We have also made sure there are instructions in the GitHub readme and genbank subfolder readme for syncing the GenBank files with git-lfs. Suggestions: Overall, this comparison framework would benefit from simple, minor additions. Does the specific host (e.g., taxonomy) affect the results of prophage prediction for some tools? For tools that utilize HMM searches, the HMM databases may be biased towards certain groups (e.g., E. coli phages). We suggest that a comparison of precision/recall be compared to the source host. Response: This is absolutely a consideration for every program and comes back to the problem of selection bias in the database. It’s fundamentally not something we can solve in this update of the framework. We compared f1 scores for each tool against genus and the f1 scores of each genus across the population averages (see results and Figure S2a/b). Do the performance comparisons only take into account total prophage CDS predictions or also the completeness of predicted prophages? For example, identifying all prophages but only 50% of each of those prophages is different than identifying half of all prophages but 100% of each of those. Response: The performance comparisons currently only compare the numbers of correctly labelled genes. If a user were manually curating their predictions, then it could be argued that partially capturing all prophages would be better than completely capturing half of the prophages. This is something we might be able to add in the future if there is enough interest. Is there any effect on hosts with multiple prophages? Are some tools affected by this? Response: The number of prophages shouldn’t impact the performance of any of the tools but this is not something we’ve looked at specifically. Most genomes only have 1 or a few prophages and it would be difficult to draw any conclusions without more examples at the fringes (0 prophages or say more than 6 or so). It is certainly something to consider as the dataset grows. Some tools will have predicted prophages that were not in the gold standard set (false positives). What measures were taken to ensure that none of these are real prophages that were missed within the manual curation? Response: The genomes are small enough that the entire genomes are thoroughly examined during manual curation. We don’t anticipate enough errors to significantly affect the outcome of the evaluation. Nevertheless, we do anticipate that some corrections will need to be made over time, be that from missed prophages or incorrect prophage boundaries and we welcome feedback from the community about the prophage annotations." } ] } ]	1	https://f1000research.com/articles/10-758
https://f1000research.com/articles/11-400/v1	07 Apr 22	{ "type": "Research Article", "title": "Interaction of surface glycoprotein of SARS-CoV-2 variants of concern with potential drug candidates: A molecular docking study", "authors": [ "Anuj Mavlankar", "Afzal Ansari", "Mukul Sharma", "Purna Dwivedi", "Pushpendra Singh", "Anuj Mavlankar", "Afzal Ansari", "Mukul Sharma", "Purna Dwivedi" ], "abstract": "Background: COVID-19 has become a global threat. Since its first outbreak from Wuhan, China in December 2019, the SARS-CoV-2 virus has gone through structural changes arising due to mutations in its surface glycoprotein. These mutations have led to the emergence of different genetic variants threatening public health due to increased transmission and virulence. As new drug development is a long process, repurposing existing antiviral drugs with potential activity against SARS-CoV-2 might be a possible solution to mitigate the current situation. Methods: This study focused on utilizing molecular docking to determine the effect of potential drugs on several variants of concern (VOCs). The effect of various drugs such as baricitinib, favipiravir, lopinavir, remdesivir and dexamethasone, which might have the potential to treat SARS-CoV-2 infections as evident from previous studies, was investigated for different VOCs. Results: Remdesivir showed promising results for B.1.351 variant (binding energy: -7.3 kcal/mol) with residues Gln319 and Val503 facilitating strong binding. Favipiravir showed favorable results against B.1.1.7 (binding energy: -5.6 kcal/mol), B.1.351 (binding energy: -5.1 kcal/mol) and B.1.617.2 (binding energy: -5 kcal/mol). Molecular dynamics simulation for favipiravir/B.1.1.7 was conducted and showed significant results in agreement with our findings. Conclusions: From structural modeling and molecular docking experiments, it is evident that mutations outside the receptor binding domain of surface glycoprotein do not have a sharp impact on drug binding affinity. Thus, the potential use of these drugs should be explored further for their antiviral effect against SARS-CoV-2 VOCs.", "keywords": [ "Molecular docking", "SARS-CoV-2", "VOCs", "Drug repurposing" ], "content": "Abbreviations\n\nCOVID-19: Coronavirus disease 2019\n\nFDA: Food and Drug Administration\n\nGISAID: Global initiative on sharing all influenza data\n\nhACE2: Human angiotensin converting enzyme\n\nMD: Molecular dynamics\n\nRBD: Receptor binding domain\n\nRMSD: Root mean square deviation\n\nSARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus-2\n\nVOCs: Variant of Concern\n\n\nIntroduction\n\nThe spread of human coronavirus SARS-CoV-2 has been increasing since it was first detected in the Chinese city of Wuhan in December 2019.1 Several efforts have been taken to prevent its spread after the World Health Organization (WHO) declared it a public health emergency on January 31, 2020. However, its continual spread across the world compelled the WHO to declare it a pandemic.2 Different genetic variants of this novel coronavirus have appeared and been transmitted across the world amidst the pandemic.3 WHO classified some of these genetic variants into three categories: variant of interest (VOI), variant of concern (VOC) and variants of high consequence (VOHC).4 Five different genetic variants have been placed in the VOC category, due to their increased transmission rates, more severe disease, or significant reduction in antibodies generated due to previous infection or vaccination namely by B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), AY.1 (Delta plus) and P.1 (Gamma). Certain anti-retroviral drugs have been used owing to their promising results for the emergency treatment of COVID-19 patients.5 Remdesivir was the first drug to be approved by the United States Food and Drug Administration (USFDA) for the treatment of COVID-19 patients.6 However, emerging mutations in drug targets (such as the receptor binding domain [RBD] of the surface glycoprotein) are likely to affect the binding affinity through altered drug-receptor interaction.7 Considering the emergence of several VOCs in different parts of the world, it is important to ascertain the effect of their signature genomic variants such as single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). To the best of our knowledge, there is a paucity of such information, especially regarding the newly emerged Delta variant.8 Docking studies are very helpful and serve as the first starting point for such investigations.9 Therefore, in this study, surface glycoprotein sequences of different VOCs were modeled in silico and their interactions with the drugs baricitinib, dexamethasone, favipiravir, lopinavir and remdesivir were studied using molecular docking. These drugs have shown promising results in various clinical studies and thus have been considered to determine their binding affinity on SARS-CoV-2 VOCs.10–12 The main aim of this study was to utilize an in silico docking approach to estimate relative changes in the binding affinity of these potential drugs against the characteristic mutational profile of the spike protein sequence in different VOCs, to predict their potential therapeutic efficacy against VOCs.\n\nThe surface glycoprotein (Spike) allows the virus to bind to hACE2 receptors and thereby promotes the virus’s entry into the host cell.13,14 It is divided into two subunits, S1 and S2. The S1 subunit consists of the RBD which directly binds to hACE2. It is also the target of neutralizing antibodies. Thus, it is the region with most mutations with clinical significance in terms of viral transmissibility and virulence.15,16 Major mutations reported in different VOCs are shown in Table 1.\n\n* AY.1 is commonly referred as Delta plus,17 although this is not as per WHO classification which considers it as one of the types within Delta lineage.\n\n\nMethods\n\nA total of 24 full-length sequences of SARS-CoV-2 genomes categorized into five VOCs from different geographical regions were selected and retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) database.18,19 The first sequence of SARS-CoV-2 originating from Wuhan was retrieved from the National Center for Biotechnology Information (NCBI) nucleotide database as a reference (NCBI reference sequence: NC_045512.2). Mutation analysis was carried out by multiple sequence alignment of the retrieved sequences using the ClustalW algorithm in MEGA-X software v 10.2.320 and mutation positions were determined. This analysis was performed to check the frequency of mutations across sequences from different VOCs. After this, a particular mutation was inserted in the sequence of a VOC, followed by its modeling. As these sequences were derived from COVID-19 infected patients, they represent the actual frequency of genetic mutations acquired by SARS-CoV-2. It reveals that once a specific mutation in any variant has evolved, it remains conserved in the descendant population, which may again acquire new characteristic mutations.\n\nThe novel SARS-CoV-2 surface glycoprotein nucleotide wild type (WT) gene sequence NC_045512.2 was retrieved from the NCBI21 nucleotide database. Reported mutations were induced in the retrieved sequence. A homology model was built for the surface glycoprotein of SARS-CoV-2 VOCs using SWISS-MODEL software.22 The matched templates were Protein Data Bank (PDB) ID 7N1U, chain A for B.1.1.7; PDB ID 7N1Q, chain A for B.1.315; PDB ID 7KRS, chain A for B.1.617.2, AY.1 and P.1. The Duke University MolProbity web server23,24 and the University of California Structure Analysis and Verification Server (SAVES)25 were used to examine the modeled structure. Several other online tools such as PROCHECK,26,27 Verify3D28,29 and ERRAT25,30 were further used to check the validity of the predicted models. Minimization of the model was carried out after addition of missing hydrogens to prepare it for molecular docking.31\n\nAutoDock Vina 1.1.2 software was used for molecular docking experiments.32,33 The modeled surface glycoprotein of all SARS-CoV-2 VOCs was served as binding target and five approved drugs as ligand. All the compounds were first optimized in their active forms in physiological conditions.\n\nThe structure of investigated drugs, namely baricitinib (PubChem CID 44205240), favipiravir (PubChem CID 492405), lopinavir (PubChem CID 92727), remdesivir (PubChem CID 121304016) and dexamethasone (PubChem CID 5743) were retrieved from PubChem database.34 AutoDockTools 1.5.6, a free graphical user interface of MGL software package was used for all the required file conversions needed for the docking study.35,36 The rotatable bonds present on the ligands were treated as non-rotatable for performing the docking. All the water molecules and hetero atoms present on the receptor surface were removed, followed by the addition of Kollman charges and polar hydrogen atoms using AutoDockTools 1.5.6. The Gasteiger charge calculation method and partial charges were also applied to the ligand molecules.37\n\nMolecular Docking was performed with modeled surface glycoproteins of different VOCs as receptors and selected drugs as ligands. Grid box parameters were selected using AutoDockTools 1.5.6 (Table 2). The Lamarckian Genetic Algorithm was used for performing docking to explore the conformational space required for the ligand with a population size of 150 individuals. The total number of current grid points per map was 64,000. Other parameters were set at default.\n\nTo check the validity of molecular docking results for favipiravir against the B.1.1.7 (Alpha) variant, a molecular dynamics simulation was conducted. The simulation was conducted in GROMACS 2018,38,39 with CHARMM36 as all-atom force field.40 For the ligand-receptor complex, all receptors missing hydrogen atoms were added using Chimera.41,42 The protein-ligand complex was placed in an isotonic box with a dodecahedron cell. The box contained a neutralizing number of sodium (Na+) and chloride (Cl-) ions based on the total charge of the protein. Topology parameters for the ligand were built using the CHARMM General Force Field (CGenFF) tool43 to generate the CHARMM36 parameters. The solvation step was followed by energy minimization, equilibration number of particles volume temperature (NVT) ensemble and number of particles pressure temperature (NPT) ensemble; then, MD simulation with 2 femtoseconds (fs) integration steps for 20 ns were conducted. The output trajectory was then subjected to Periodic Boundary Conditions (PBC) correction and the system was fitted to its start position based on the backbone of the receptor. Further analysis was performed to plot the root-mean-square deviation (RMSD) of the ligand and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) energy computation.\n\n\nResults and discussion\n\nThe mutation analysis was carried out for the surface glycoprotein of SARS-CoV-2 VOC, and random clinical samples from different geographical regions were retrieved from the GISAID database18 to verify the known mutations present in variants and to reveal any other significant mutation present, if any. Multiple sequence alignment of different clinical samples with the surface glycoprotein of B.1.1.7 revealed characteristic mutations such as Del 69/70, Del 144/145, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. Similar steps were performed for another SARS-CoV-2 VOC (Supplementary Figure 1, Extended data44). However, no novel mutation was reported. The reported mutations which had a frequency of occurrence of 50% and above were considered for surface glycoprotein modeling for VOCs (Table 1). A high-quality model was constructed for the surface glycoprotein of SARS-CoV-2 based on the matching templates (7KRS, 7N1U and 7N1Q) for different SARS-CoV-2 VOCs. The 7KRS PDB accession number is a viral protein complex characterized by the mutation D614G solved by electron microscopy with a resolution of 3.20 Å.45 It has a sequence similarity of 99.14% with the B.1.617.2, AY.1 and P.1 variants. 7N1U and 7N1Q are the PDB accession numbers of viral protein structures solved by electron microscopy, with a resolution of 3.1 Å and 2.90 Å respectively.46 These were the matching templates for B.1.1.7 and B.1.351 with 99.80% and 99.92% sequence similarity respectively.\n\nThe docking results are shown in terms of binding energy (Table 3) and number of interacting amino acid residues (Supplementary Table 1, Extended data44) at the active site of SARS-CoV-2 surface glycoprotein. The binding affinity of each drug for different variants was computed by assuming 100% binding affinity with SARS-CoV-2 WT. The lowest binding energy and RMSD conformation was considered as the most suitable docking pose. The binding interactions between different drugs and surface glycoprotein of VOCs were prepared, visualized and analyzed using PyMOL v 2.5.2 and Discovery Studio 2021.47–49\n\nDexamethasone showed a sharp difference between binding energies with the SARS-CoV-2 surface glycoprotein of B.1.617.2 (the Delta VOC, binding energy of -7.7 Kcal/mol) compared to that of AY.1 (the Delta plus VOC, with a binding energy of -4.4 Kcal/mol). It formed two H-bonds with Gln851 and Val950 amino acid residues in the active site region of the surface glycoprotein of B.1.617.2. In addition, it formed two H-bonds with Asn604 and Gly650 with the surface glycoprotein of AY.1. It showed 96.25% and 55% binding affinity with B.1.617.2 and AY.1 respectively, compared to WT (reference, 100%). In contrast, favipiravir showed the highest binding affinities for all variants except AY.1 in comparison to WT. It showed a maximum binding affinity of 114.28% (binding energy: -5.6 Kcal/mol) and formed conventional H-bonds with Glu295, Tyr609 and Pro628 amino acid residues in the active pocket region of surface glycoprotein of B.1.1.7 (Alpha). Remdesivir appeared to be quite effective in binding the active pocket region of different SARS-CoV-2 variants’ surface glycoproteins. It showed a highest binding affinity of 104.28% (binding energy: -7.3 Kcal/mol) with B.1.351 (beta) as compared to WT. It formed three conventional H-bonds with amino acid residues Asn315, Arg317 and His617, one carbon-hydrogen bond with Gln319, and one alkyl bond with Ala290, Val503 and Ala621. Two amino acid residues, i.e., Gln319 and Val503 are present in the RBD of SARS-CoV-2 which facilitate its binding to the hACE2.50 Similarly, baricitinib showed a strong binding affinity of 105.97% (binding energy: -7.1 Kcal/mol) with B.1.351 and formed H-bond with Pro269 and Thr271 when compared to WT. Lopinavir also showed significant results with a binding affinity of 100% (binding energy: -10.1 Kcal/mol) with the surface glycoprotein of B.1.351 as compared to WT and other variants. It showed the formation of an H-bond with Ile622. The selected 3D structural view of SARS-CoV-2 surface glycoprotein docking with different drugs and amino acid binding residues is shown in Figure 1 and Supplementary Table 1 (Extended data44) respectively. Receptor amino acid residues of proteins are shown in blue and ligands are presented in green. Other docking images are shown in Supplementary Figure 3 (Extended data44).\n\nA) B.1.617.2 with dexamethasone; B) AY.1 with dexamethasone; C) B.1.351 with remdesivir; D) P.1 with remdesivir; E) B.1.351 with lopinavir; F) AY.1 with lopinavir; G) B.1.1.7 with favipiravir; H) AY.1 favipiravir; I) B.1.351 with baricitinib; J) B.1.617.2 with baricitinib; K) wild type; (WT) with favipiravir; L) WT with dexamethasone.\n\nThe emergence of new SARS-CoV-2 variants leads to the need for new treatment drugs development, which is a long process. However, with an increased patient mortality ratio, it is of utmost importance to repurpose existing drugs used to treat other viral diseases.51 Failure to target the gene encoding the surface glycoprotein has been observed as SARS-CoV-2 variants are detected.7 According to the latest guidelines of the Indian Council of Medical Research, approved test kits must employ multiplex RT-PCR assays, as the tests assessing only the surface glycoprotein may fail and produce false negative results.\n\nThe viral entry into the host cell is facilitated by its successful binding to the angiotensin converting enzyme (ACE2) receptor.52 Overexpression of ACE2 may lead to disease severity as observed in mice.53 Lung damage can be reversed by blocking the renin-angiotensin pathway.54 A recent study had shown that the surface glycoprotein of SARS-CoV-2 binds to ACE2 with a 10- to 20- fold higher affinity than other SARS-CoV surface glycoproteins,55 which might be the reason for the high infectivity of SARS-CoV-2. Thus, viral entry into the host cell is a vital step which must be exploited for an efficient therapeutic development. There is a rapid ongoing search for therapeutic agents against SARS-CoV-2. Various computational studies have been conducted to discover potential drugs against SARS-CoV-2.51,56,57 Recent studies have been based on the drugs targeting either surface glycoprotein or main protease of SARS-CoV-2. These approaches have led to the discovery of molecules with high binding affinities to these proteins.58\n\nThe molecular docking analysis of surface glycoproteins with selected drugs for different VOCs along (Supplementary Figure 2, Extended data44) revealed promising results for B.1.351 (Beta variant). Three drugs, namely baricitinib, lopinavir and remdesivir, showed maximum binding affinities against the Beta variant as compared to the WT and other VOCs. Other variants also expressed significant binding energies. As per a recent study,10 the combination of baricitinib and remdesivir was more effective than Remdesivir alone and thus helped to lower the recovery time and accelerate the clinical status of patients suffering from COVID-19, especially those requiring high-inflow oxygen ventilation. Remdesivir efficiently inhibits the replication of SARS-CoV-2 by causing delayed chain termination when getting incorporated into the viral RNA.59 However, it also showed considerable binding affinity when docked with the surface glycoprotein.60 Here, our molecular docking study revealed its potential as an effective drug against SARS-CoV-2 VOCs. The high energy score resulting from these docking analyses suggests that these drugs may be recommended for administration to patients with B.1.351 infection. Molecular docking revealed that two RBD residues, namely Gln319 and Val503 facilitated a strong binding. Upon comparison with the WT, favipiravir showed a significant binding affinity with B.1.1.7, B.1.617.2 and B.1.351. Favipiravir is a purine analog which inhibits the elongation phase of RNA synthesis. Favipiravir was proven to be effective in viral clearance and fast clinical improvement.61 It has shown positive results in COVID-19 patients by improving patient’s health.62 In concordance with our findings, favipiravir was successfully docked with the surface glycoprotein of B.1.1.7 (Alpha variant). Dexamethasone has been widely used as a therapeutic intervention to treat COVID-19 patients. The docking score of dexamethasone with the surface glycoprotein of B.1.617.2 (Delta variant) was the highest (binding energy: -7.7 Kcal/mol) compared to other variants of concern. In contrast, the docking score for AY.1 (Delta plus variant) showed the lowest affinity with dexamethasone (binding energy: -4.4 Kcal/mol). This observation shows that dexamethasone binding to the surface glycoprotein of the SARS-CoV-2 Delta variant (which has spread as one of the most dominant lineage worldwide) may represent an additional contribution to its efficacy in treating COVID-19.63 Lopinavir is a drug approved by the FDA and serves as a protease inhibitor commonly used in the treatment of the Human Immunodeficiency Virus (HIV); it may be considered useful in the treatment against SARS-CoV-2 infection.64 Our findings reveal that it may be a suitable choice for treatment as it shows significant binding with different VOCs, especially with B.1.351 (Beta variant; binding energy: -10.1 Kcal/mol). Remdesivir has been found to be a more potent drug than lopinavir, both in vitro and in MERS-CoV infected mice.65 In concordance with our findings, remdesivir has shown more significant binding with B.1.351 than lopinavir as compared to WT. There is evidence of lopinavir being selective against other coronaviruses.12 Despite the high binding energy with surface glycoproteins, our results encourage further in vitro and in vivo investigations. In comparison to the WT, the binding residues for different VOCs vary and some of them lie outside the receptor binding domain of the surface glycoprotein which does not have a direct role in drug affinity. However, they may impact the interaction of drug with surface glycoprotein through weak molecular interactions.\n\nThe studied favipiravir-Alpha variant (B.1.1.7) ligand heavy atoms complex showed a RMSD of 1.028 Å. The Lennard-Jones potential and the binding potential of the complex was calculated to be -129.178 kJ/mol and -137.227 kJ/mol respectively. During the initial simulation run, up to 4 ns, the ligand-receptor showed a high RMSD value, which may be due to the stearic changes the protein underwent. The ligand managed to follow the change, keeping a stable number of H-bonds. The ligand initially stabilized at the binding pocket with some additional hydrophobic interactions. At t =14 ns, the pocket was obliterated, however, the ligand was kept near to the pocket until the pocket was opened. Subsequently, the ligand was restored to its original position (Figure 2). MD simulation for favipiravir/Alpha variant was performed as it showed a significant binding affinity compared to other drugs under study. A similar procedure can be incorporated to conduct the simulation studies on other variants with drugs showing significant binding.\n\n\nConclusions\n\nDrug repurposing may help to discover and identify the potential therapeutic effect of existing drugs against the genomic targets of SARS-CoV-2 virus. This study shows that the mutations (except Gln319 and Val503) outside the RBD of the surface glycoprotein of several VOCs do not largely affect the binding affinity of these drugs. No drastic structural change has been observed in variants irrespective of binding with the residues occurring outside the RBD of the surface glycoprotein. However, favipiravir showed the highest binding affinity against the Alpha variant, whereas dexamethasone showed approximately a 50% reduction in its binding affinity with the Delta plus variant when compared to the Delta variant, revealing that dexamethasone bound to surface glycoprotein of the Delta variant more strongly than the Delta plus variant. These residual fluctuations may play a role in antibody evasion and their molecular roles should be explored further.66 However, the candidate drugs besides favipiravir and dexamethasone showed no significant alteration in the surface glycoprotein structure when compared to WT, implying that the current regimen of approved drugs can be continued in patients infected with these SARS-CoV-2 strains.\n\nFurther, molecular docking approaches offer great promise for predicting, shortlisting and quickly evaluating the anti-SARS-CoV-2 potential of candidate and existing drugs which can help timely effective interventions. The workflow depicting the study has been summarized in Figure 3.\n\n\nData availability\n\nZenodo: Interaction of Surface Glycoprotein of SARS-CoV-2 variants of concern with Potential Drug Candidates: A Molecular Docking Study, https://doi.org/10.5281/zenodo.6339952.44\n\nThis project contains the following underlying data:\n\n- Drugs.zip (tested drug pdb files)\n\n- Protein-structure-3D.zip\n\n- sequences_for_mutation_frequency_analysis.xlsx\n\n- spike_protein_sequences.docx\n\nZenodo: Interaction of Surface Glycoprotein of SARS-CoV-2 Variants of concern with Potential Drug Candidates: A Molecular Docking Study, https://doi.org/10.5281/zenodo.6339952.44\n\nThis project contains the following extended data:\n\n- Supplementary Figure 1.docx\n\n- Supplementary Figure 2.docx\n\n- Supplementary Figure 3.docx\n\n- Supplementary Table 1.csv\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CCBY 4.0).", "appendix": "Acknowledgements\n\nThe authors are thankful to Dr. PV Barde and Dr. Aparup Das for suggestions and Mr. Purushottam Patel for assistance. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "130114", "date": "25 Apr 2022", "name": "Akinwunmi Oluwaseun Adeoye", "expertise": [ "Reviewer Expertise Biomembrane", "Molecular Toxicology and 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\nThis study describes the interaction of surface glycoprotein of SARS-CoV-2 variants of concern with potential drug candidates: a molecular docking study. The authors concluded that the mutations which occurred outside the receptor-binding domains of the surface glycoprotein of several variants of concern do not affect the binding affinity of these drugs. The study is well-designed and well written. The materials and methods section is clear and detailed. Information on multiple sequence alignment and structural analysis, as well as molecular dynamics simulation, is sufficient. The results presented are good as well as the discussion section. It is an interesting study that I believe falls within the scope of this journal. The study contains stimulating information for the readers of F1000Research with an interest in repurposing existing antiviral drugs with potential activity against SARS-CoV-2 variants of concern. This paper is suitable for publication in this journal.\nI have reviewed this article with keen interest. In general, the study design seems perfect. However, I observed in the structured abstract that the objective of the study was stated in the methods. From the study, the authors reported that the binding affinity of each drug for different variants was computed by assuming 100% binding affinity with SARS-CoV-2 wild type. Also, they considered the lowest binding energy and RMSD conformation as the most suitable docking pose. I believe that the drug molecule with the highest binding affinity to different variants of SARS-CoV-2 surface glycoproteins may not necessarily have the lowest binding energy and vice versa. The authors should have reported the binding affinities of the selected drugs with the SARS-CoV-2 surface glycoproteins separately in Kcal/mol from the docking study. Probably, the affinity of the drug molecules to the surface glycoprotein of SARS-CoV-2 variants of concern could be presented or represented in fold with respect to the SARS-CoV-2 wild type. The computation of the binding affinity of each drug concerning the assumption of 100% binding affinity with SARS-CoV-2 WT led to over 100% affinity of Baricitinib with beta variant, Favipiravir with alpha, beta, and delta variants as well as Remdesivir with the beta variant. What is the justification for these increases in affinity over 100% when compared with the reference SARS-CoV-2 wild type? Do any of the drugs alter the structures of SARS-CoV-2 surface glycoproteins?\n\nIs the work 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": "148621", "date": "05 Sep 2022", "name": "Nurdjannah Jane Niode", "expertise": [ "Reviewer Expertise biology molecular (in infectious disease)", "dermatology and venereology", "medical enthomology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 explains about interactions of the surface glycoprotein of SARS-CoV-2 variants of concern (alpha, beta, delta, delta plus, gamma) with potential drugs for COVID-19 (remdesivir, baricitinib, favipiravir, lopinavir, dexamethasone) through structural modelling and molecular docking experiments.\nA. Is the work clearly and accurately presented and does it cite the current literature? Partly, because:\nIn Result and Discussion section, written that 'The reported mutations which had a frequency of occurrence of 50% and above were considered for surface glycoprotein modeling for VOCs (Table 1)'.\nPlease explain further what the author means by frequency of occurrence of 50% and above, because the frequency is not mentioned in Table 1\n\nIn MD Simulation and RSMD section written that 'MD simulation for favipiravir/Alpha variant was performed as it showed a significant binding affinity compared to other drugs under study.'\nPlease explain the reason why the authors choose Favipiravir/Alpha variant among other variants for molecular dynamic simulation in the beginning of the MD simulation and RSMD paragraph.\n\nSome references were taken from sources more than 10 years ago. If it's possible please replace the references with the latest references.\n\nB. Are the conclusions drawn adequately supported by the results?\nPartly, In conclusion section, its better to only present the results from the research (by removing the citation).\n\nIn the conclusion section in the abstract, please mention what drugs the authors mean by these drugs because there was no explanation in the beginning of the conclusion paragraph.\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": [] }, { "id": "148617", "date": "27 Sep 2022", "name": "Thomas Caulfield", "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\nNEEDS MAJOR REVISIONS.\nI like the aim and ideas. But not a very good realization of their plans.\nTime of MD 20 ns simulations is entirely too short. And for gromacs simulations, this is not sufficient sampling time for relaxation time of any protein. It should be more like 60-100 ns for relaxation and then 100-500 ns of production run (minimum). They did not provide RMSD for Spike protein in free state (we can see that). Fig2 shows relaxation period is absent…\n\nThen they try to compare free energy of docking and primitive \"The LennardJones potential and the binding potential of the complex \" -- they can continue Molecular dynamics stimulation and recalculate energy by g_mmpbsa https://rashmikumari.github.io/g_mmpbsa/ to get similar dG. But not likely because of #1.\n\nOne can wonder how such low docking energy for approved inhibitors is generated, and it is interesting to compare what the score would be from MD after g_mmpbsa; maybe something wrong was in the docking protocol.\n\nIt is interesting why the size of the box in Table 2 is so different for the same protein but just missense mutant forms?\n\nAlso, it is interesting to see conformations changes in spike structure according to interaction with inhibitors or molecular mechanism of inhibition and what is the difference with mutant forms given such short times run.\n\nIs the work 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/11-400
https://f1000research.com/articles/11-399/v1	06 Apr 22	{ "type": "Research Article", "title": "Low dose protocol for high resolution CT thorax: influence of matrix size and tube voltage on image quality and radiation dose", "authors": [ "Navish Kumar", "Abhimanyu Pradhan", "Rajagopal Kadavigere", "Suresh Sugumar", "Navish Kumar", "Abhimanyu Pradhan", "Rajagopal Kadavigere" ], "abstract": "Background: High-resolution CT (HRCT) thorax has increase demand due to its advantage in diagnosing chronic respiratory diseases. The feasibility of matrix size with different tube voltage in the HRCT protocol of thorax is unknown. Therefore, this study aimed to compare the effect of matrix sizes and tube voltage on image quality and radiation dose on adult HRCT thorax.\n\nMethods: A Phantom experiment was performed, followed by a patient scan. For phantom and patient scan, a total of six protocols with two tube voltage settings, 120 kVp and 100 kVp, with a combination of three matrix sizes, 512, 768, and 1024 were used. In this study, 180 adult patients who had HRCT thorax scan were considered. Dose data was collected, and quantitative image analysis was performed by drawing region of interests on the acquired phantom and patient images. Qualitative image analysis was performed independently by two blinded radiologists.\n\nResults: The dose report of the phantom experiment revealed that the 100kVp with selected matrix size delivered 15.64% and 15.62% less radiation dose in terms of volumetric computed tomography dose index (CTDIvol) and dose length product (DLP), respectively, compared to 120kVp settings with selected matrix sizes. Similarly, for the patient population, the CTDIvol and DLP difference noted for 120kVp and 100kVp with different matrix sizes was statistically significant (p<0.001). For quantitative image quality, the difference noted was also statistically significant among two kVp settings. The mean score for subjective image assessment was greater than 4.5 for diagnostic acceptability and streak artefacts.\n\nConclusion: The result suggests that the 100 kVp with 512 X 512 matrix size is preferable in the HRCT Lung to achieve the optimal diagnostic image quality with a reduction of almost 40% of the dose to the patients compared to 120 kVp techniques.", "keywords": [ "HRCT thorax", "low dose", "dose reduction", "matrix size", "tube voltage" ], "content": "Introduction\n\nThe prevalence of respiratory diseases in India is substantially high as per the recent global burden of disease survey data (Dandona et al., 2017). Chronic respiratory diseases, especially asthma and obstructive pulmonary disease, are of particular importance for having wide variations in morbidity and mortality in different Indian states (Salvi et al., 2018). Additionally, the novel COVID-19 outbreak declared by the World health organization (WHO) has increased the burden of respiratory disease in India as well as globally (Cucinotta & Vanelli, 2020; To et al., 2020). The outbreak of COVID-19 led to an increase in the demand for image modalities for the diagnosis of the disease. A chest radiograph plays a crucial role in diagnosing suspected respiratory diseases. High resolution computed tomography (HRCT) of the thorax is one such examination in high demand in routine use and for initial and rapid diagnosis of COVID-19 pneumonia with the low rate of missed diagnosis (Kashyape & Jain, 2021; Li & Xia, 2020). Although reverse-transcription polymerase chain reaction (RT-PCR) is the preferred tool for diagnosing novel COVID-19 infection, HRCT thorax has better sensitivity (Kalra, Homayounieh, Arru, Holmberg & Vassileva, 2020). The benefit of HRCT is not only limited to disease confirmation, but it plays a significant role in determining disease progression, treatment response, management, and decision on hospitalization and discharge of the COVID-19 patient through subsequent scans performed during these periods (Kalra et al., 2020; Rubin & Ryerson & Haramati, 2020; Azadbakht et al., 2021; Davarpanah et al., 2020).\n\nThe use of imaging modalities during the COVID-19 pandemic indeed has maximum benefit, but the fact that radiation exposure increases the risk of cancer should not be ignored. The linear-no threshold (LNT) model describes cancer risk even with a low dose of radiation (Shah, Sachs & Wilson, 2012; Mullenders, Atkinson, Paretzke, Sabatier & Bouffler, 2009). The risk of radiation-induced cancer during the pandemic is of concern, primarily due to the increase in COVID-19 infection and the need for multiple scans required during the recovery period of the disease (Wang, Liu, Yang & Chen, 2020). As recommended by International Commission on Radiological Protection (ICRP), the radiation dose delivered to the patient should be as low as reasonable achievable (ALARA) even during the pandemic, and the dose optimization for COVID-19 patients is necessary as there is no low dose HRCT protocol reported (Azadbakht et al., 2021; Yeung, 2019). There are several techniques to optimize the radiation dose for HRCT, such as reducing tube voltage, tube current, scan length, high pitch techniques, automatic exposure control (AEC), and iterative reconstruction techniques (Azadbakht et al., 2021). The feasibility of matrix size with different tube voltage in the HRCT protocol of thorax is unknown. Therefore, this study aimed to compare the effect of matrix sizes and tube voltage on image quality and radiation dose on adult HRCT thorax. The working hypothesis of this study was that the change in tube voltage could influence the image quality.\n\n\nMethods\n\nThe study approval was obtained from the institutional ethics committee (IEC: 168/2019), Kasturba hospital, Manipal. The study was carried out in department of Radio-diagnosis and Imaging, Kasturba Hospital, Manipal. An experimental study was initially tested on Phantom to understand the influence of tube voltage and matrix size on the quality of the image and dose. The outcome of the results was then tested on the patient’s population.\n\nThis experiment was conducted in a 32 cm body CT polymethyl metacrylate (PMMA) phantom (Unfors, serial no. 0143) using an iCT (Philips Medical Systems) 128-row multiple slice CT scanner. The phantom was positioned on the CT table. The height of the phantom was adjusted using the position of laser light. The experiment was performed at two different tube voltage settings (120 kVp and 100 kVp) and three matrix sizes (512, 768 & 1024) available in the CT scanner (Table 1). All the other parameters, Reconstruction mode iDose4 level 2, collimation 640.625, gantry tilt of 0 degrees, the pitch of 1, rotation time: 0.5, slice thickness: 1 mm, reconstruction increment of 0.7500, a field of view (FOV) of 350 mm, and the adaptive filter were kept constant. The automatic tube current modulation was enabled during the procedure. An axial image with 1 mm thickness was obtained. Each protocol was scanned with a 130 mm scan length. Phantom CT image obtained using different parameters was analysed for dose and quantitative image quality. Signal-to-noise (SNR) and contrast-to-noise (CNR) ratios were analysed by drawing five regions-of-interest (ROIs) of 100 mm2. Out of five ROIs, four were positioned in peripheral location (3, 6, 9, 12 o’clock) and one in the centre location of the phantom along with one ROI in the background to encompass homogeneity of measured regions (Figure 1).\n\nA total of 180 patients (Mean age 54.50 ±15.81, Male 55.06 ±17.15, Female 53.85±14.17) who were 18 years and above and prescribed for the HRCT Thorax were considered for this study (See Underlying data) (Sukumar, 2022). The written informed consent was obtained from the participant. All the scan was performed in iCT (Philips Medical Systems) 128-row multiple slice CT scanner with six different scanning protocol similar to phantom (Table 1). The automatic tube current modulation (Dose Right Index: 17) was enabled during the procedure.\n\nAfter acquiring the image using a different protocol, the subjective image assessment of the patient’s population was performed according to the European Guidelines on Quality Criteria for CT (Menzel et al., 2000). Two radiologists with a mean experience of 12.00 ± 1.41 years, reviewed images independently. The radiologists were blinded to the scanning protocols and the procedure. Images were displayed in the HRCT window (-600 WL, 1600 WL) on an Advantage Workstation (Philips Healthcare). Radiologists’ evaluation for the diagnostically acceptable image quality for HRCT thorax was considered (See Underlying data) (Sukumar, 2022). The diagnostic acceptability of the image quality was rated on five-point rating scale were, 1 = poor for diagnosis, 2 = suboptimal for diagnosis, 3 = good for diagnosis, 4 = very good for diagnosis, 5 = excellently. The streak artefacts seen anywhere on the CT image was rated on a five-point rating scale, were 1= artefact causing diagnosis impossible, 2 = artefacts affecting diagnostic information, 3 = major artefacts affecting visualization of major structures but diagnosis still possible, 4 = minor artefacts not interfering with diagnostic decision-making, and 5 = no artefacts.\n\nThe quantitative image quality assessment was assessed by drawing 100 mm2 ROIs on high density structures of thorax, such as subscapularis muscle, ascending aorta, and descending aorta (See Underlying data) (Sukumar, 2022) (Figure 2).\n\nThe SNR, CNR, and Figure of Merit (FOM) ratios for the phantom and the participant population were analysed by the following equation (Chang, Hsu, Lin & Hsu, 2017) (See Underlying data) (Sukumar, 2022).\n\nWhere CTROI and CTBackground are the average Hounsfield unit of the corresponding ROI, and SDROI and SDBackground are the standard deviations of the corresponding ROI.\n\nThe dose data (CTDIvol and DLP) for both phantom study and patient population study were collected from the dose info of the system. For the study population the obtained DLP was further used to calculate the effective dose by multiplying with a conversion factor of 0.014 (Deak, Smal & Kalender, 2010) (See Underlying data) (Sukumar, 2022).\n\nStatistical Package for the Social Sciences (SPSS) Statistics for Windows, Version 16 (Kumar, Pai & Vineetha, 2020) was used to perform the statistical analysis. For the study population the obtained data were not normally distributed. Therefore, the median and quartiles are reported, and for comparison of quantitative image analysis and dose matrix, Mann Whitney U (Kumar, Pai & Vineetha, 2020) test was performed. Mean score for each protocol was calculated for subjective image assessment. P<0.05 was considered statistically significant.\n\n\nResults\n\nThe influence of various combinations of tube voltage (120 and 100 kVp) and matrix size (512, 768 and 1024) on image quality (SNR and CNR) and dose matrix (CTDIvol and DLP) are summarized in Table 2. The difference noted (in percentage) when comparing different protocols are outlined in Table 3. In this study, the phantom was scanned with two tube voltage techniques combined with three different matrix sizes (a total of six scanning protocols). When comparing the image quality of 120 kVp with various matrix sizes, protocol A showed the highest values compared to protocols B and C. The dose changes noted were very minimal.\n\nSimilarly, when comparing the image quality of 100 kVp with various matrix sizes, protocol D showed higher values than protocol E and protocol F, and no changes were noted in terms of dose matrix (Table 2). The result of the phantom study showed that, as the matrix size increases, the SNR and CNR decreases, except for the case of protocol C, where the SNR and CNR values were slightly higher than protocol B. Therefore, when comparing the image quality of the 120 kVp technique and 100 kVp technique with various matrix sizes, the matrix size of 512 (protocol A and protocol D) produced better image quality than another matrix size. When comparing among two tube voltages with the same matrix size of 512 (protocol A and protocol D), protocol A showed better result than protocol D when considering image quality where the difference (in percentage) among these two protocols for SNR and CNR was 8% and 23.80% respectively, which is minimal as compared to the difference of other protocol (Table 3). However, the difference noted in CTDIvol and DLP among these two protocols was 15.64% and 15.62%, respectively (Table 3), which shows that 100 kVp techniques have reduced radiation dose.\n\nSimilar to the phantom study, the influence of tube voltage and matrix size on image quality and radiation dose was performed in the patient population. The collected patient data were not normally distributed. Hence the median and quartiles for image quality (SNR and CNR), radiation dose matrix (CTDIvol and DLP), and Figure of merit (FOM) are summarized in Table 4. The statistically significant value (p<0.05) for comparison of median among various protocols are outlined in Table 5.\n\n Statistical significant (p<0.05).\n\nWhen comparing the image quality of 120 kVp technique (standard dose) with three different matrix size, protocol A showed highest value when compared with protocol B and protocol C. The difference noted was statistically significant between protocol A and protocol B, and protocol A and protocol C but for protocol B and protocol C, difference in SNR was not statistically significance whereas CNR was statistically significance. There was no difference noted for radiation dose when comparing within the protocols, except for the protocol B and protocol C where DLP showed statistical significance difference and FOM showed significant difference for all the comparisons (Table 5). For 100 kVp technique (low dose), the result was in line with 120 kVp techniques, that is protocol D showed highest value in terms of image quality when compared with protocol E and protocol F, and the difference noted were statistically significant for all the comparison except for protocol E and protocol F. Similarly, there were no statistical difference noted in radiation dose matrix and FOM for all the comparisons except for comparison between protocol E and protocol F (Table 5). The overall result showed that, with the increase in matrix size, the SNR, CNR, and FOM decrease for both standard 120 kVp and a low dose of 100 kVp techniques (Figure 3).\n\nThe image quality for 512 × 512 matrix size with tube voltage settings of 120 kVp showed superior quality as compared to other combinations of tube voltage and matrix size, but when radiation dose was considered, the dose for 120 kVp with 512 matrix size (protocol A) was significantly higher than 100 kVp with 512 matrix size (protocol D) and other combinations (Table 5). Nevertheless, the mean score for the subjective image assessment did not indicate a preference for any of the protocols in terms of diagnostic acceptability or streak artefacts (Table 6).\n\n\nDiscussion\n\nThe radiation dose in the HRCT thorax is high compared to the conventional lung imaging and exceeds 100 times the radiation dose of the Chest X-ray (Ambrosino, Genieser, Roche, Kaul & Lawrence, 1994). Hence, optimising radiation dose and diagnostic accuracy of obtaining HRCT images is essential. The result of the present study state that with the use of a low dose, that is 100 kVp with the combination of 512×512 matrix size, the radiation dose to the patient can be reduced without compromising image quality.\n\nThe present study's findings on the effect of matrix size were limited to quantitative image quality, and no significant difference was noted on radiation dose. The result of the study revealed that the increase in matrix size decreased the SNR and CNR values, leading to noisier images for higher matrix size images. Similarly, Hata et al. (2018) reported the increase in noise and streak artefacts with larger matrix size, which reduces the application of larger matrix size in CT. Although the matrix size influences the spatial resolution of the image, increasing the matrix size seldom makes the spatial resolution better since the system's spatial resolution usually depends on the focal spot size of the X-ray tube and the size of the detector elements (Hata et al., 2018). Also, the data size of the acquired images is larger with high matrix size images. Therefore, using a smaller matrix size, the images of HRCT thorax can be achieved without compromising spatial resolution.\n\nIn this study, the influence of tube voltage on image quality and radiation dose was analysed and revealed that using a low tube voltage of 100 kVp significantly reduced the radiation dose compared to 120 kVp. When comparing the effect of tube voltage along with matrix size, the study revealed that low tube voltage with low matrix size could achieve images without compromising diagnostic confidence. The institute protocol for HRCT thorax uses 120 kVp with 728 × 728 matrix size as a standard protocol. Compared with a low dose protocol of 100 kVp with a lower matrix size of 512 × 512, a significant reduction in radiation dose was achieved with no significant changes noted in quantitative image quality (Protocol B and D, Table 5). However, reducing the tube voltage below 100 kVp can be beneficial if it is tailored with patient weight, as reported by Desmet J. et al. (2021), where they used 80kVp for the patient weighing below 50 kg.\n\nThe effect of tube current on image quality and radiation dose was not performed in the present study as the protocol make use of automatic tube current modulation, which modulates the tube current according to the patient size. Although the radiation dose decreases linearly with the reduction of tube current but has limited use in HRCT as reducing tube current reduces the resolution of the images (Azadbakht et al., 2021; Karabulut, Törü, Gelebek, Gülsün & Ariyürek, 2002). However, this can be overcome with the recent advancement in the reconstruction techniques, where filtered back projection (FBP) is being widely replaced by iterative reconstruction (IR) (Honda et al., 2011). Literature reports that the use of low tube current with the combination of IR, such as Model-based Iterative Reconstruction (MBIR) and Adaptive Statistical Iterative Reconstruction (ASIR), can improve the compromised image quality due to the result of reduced tube current (Yanagawa et al., 2014; Katsura et al., 2012; Tsukada et al., 2016). However, selecting the lowest possible tube current settings has benefited only over the selective examination, and it decreases the detectability of low-contrast details (Karabulut et al., 2002; Neroladaki, Botsikas, Boudabbous, Becker & Montet, 2013). Therefore, it is preferred to use automatic tube current modulation for HRCT thorax.\n\nThe low dose HRCT thorax protocol with 100 kVp and 512 × 512 matrix can be recommended for COVID-19 patients. However, according to the webinar by International Atomic Energy Agency (IAEA) on CT practices and dose optimization, it was suggested to use HRCT thorax to diagnose COVID-19 only when the availability of RT-PCR is limited (Kalra et al., 2020). Nevertheless, the use of HRCT thorax has several benefits when assessing disease progression and management of COVID-19 patients (Davarpanah et al., 2020; Rubin et al., 2020). Also, there is an increasing demand for repeat scans for the patient with comorbidities, for the recovered individuals in evaluating pulmonary complications, and for research purposes (Mahdavi et al., 2020).\n\nThe current study has a few Limitations. The present study was focused on SNR, CNR, and FOM. The correlation between the dose and quality of the image based on Body Mass Index (BMI) and chest circumference was not reported. The overall subjective diagnostic acceptability of the lung was reported. The visibility of the nodule, Broncho vascular bundle thickening, interlobular reticulation, and bronchiectasis was not reported individually.\n\n\nConclusion\n\nThis study showed that 120 kVp with 512 × 512 matrix size produces superior image quality than 100 kVp techniques. However, the tube voltage of 100 kVp with the matrix size of 512 × 512 reduced almost 40% of radiation dose compared to 120 kVp techniques. Therefore, 100 kVp with 512 × 512 matrix size can be preferred for HRCT adult lung to achieve the optimal diagnostic image quality. This low dose protocol may be well adapted for the patients requiring routine HRCT and follow-up examination.\n\n\nData availability\n\nHarvard Dataverse: Low dose protocol for high resolution CT thorax: influence of matrix size and tube voltage on image quality and radiation dose.\n\nhttps://doi.org/10.7910/DVN/P3BL0F (Sukumar, 2022)\n\nThis project contains the following underlying data:\n\nque 1(B). (Ratings for diagnostic acceptability of the image quality)\n\nque 1(E1-R2). (Combined ratings of two radiologists for diagnostic acceptability of the image quality)\n\nque 2(B). (Ratings for streak artefacts seen on image)\n\nque 2(R1-R2). (Combined ratings of two radiologists for streak artefacts seen on image)\n\nROI. (Quantitative values)\n\nSNR, CNR. (Demographic details, radiation dose value and SNR and CNR values)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAuthor contributions\n\nAll the authors have substantially contributed to the conception or design of the work. Data collection: Navish Kumar, Data analysis and Interpretation: Rajagopal Kadavigere and Abhimanyu Pradhan, drafting of article: Abhimanyu Pradhan and Suresh Sukumar, Critical Revision of the article: Suresh Sukumar and Rajagopal Kadavigere, Final Approval of the version to be published: Rajagopal Kadavigere.", "appendix": "References\n\nAmbrosino MM, Genieser NB, Roche KJ, et al.: Feasibility of high-resolution, low-dose chest CT in evaluating the pediatric chest. Pediatr. Radiol. 1994; 24(1): 6–10. PubMed Abstract \| Publisher Full Text\n\nAzadbakht J, Khoramian D, Lajevardi ZS, et al.: A review on chest CT scanning parameters implemented in COVID-19 patients: bringing low-dose CT protocols into play. Egypt. J. Radiol. Nucl. Med. 2021; 52(1): 1–10. Publisher Full Text\n\nChang KP, Hsu TK, Lin WT, et al.: Optimization of dose and image quality in adult and pediatric computed tomography scans. Radiat. Phys. Chem. 2017; 140(January): 260–265. Publisher Full Text\n\nCucinotta D, Vanelli M: WHO declares COVID-19 a pandemic. Acta. Biomedica. 2020; 91(1): 157–160. PubMed Abstract \| Publisher Full Text\n\nDandona L, Dandona R, Kumar GA, et al.: Nations within a nation: variations in epidemiological transition across the states of India, 1990–2016 in the Global Burden of Disease Study. Lancet. 2017; 390(10111): 2437–2460. PubMed Abstract \| Publisher Full Text\n\nDavarpanah AH, Mahdavi A, Sabri A, et al.: Novel Screening and Triage Strategy in Iran During Deadly Coronavirus Disease 2019 (COVID-19) Epidemic: Value of Humanitarian Teleconsultation Service. J. Am. Coll. Radiol. 2020; 17(6): 734–738. PubMed Abstract \| Publisher Full Text\n\nDeak PD, Smal Y, Kalender WA: Multisection CT Protocols: Sex- and Age-specifi c Conversion Dose from Dose-Length Product 1 Purpose: Methods: Results: Conclusion. Radiology. 2010; 257(1): 158–166. PubMed Abstract \| Publisher Full Text\n\nDesmet J, Biebaû C, De Wever W, et al.: Performance of low-dose chest CT as a triage tool for suspected COVID-19 patients. J. Belg. Soc. Radiol. 2021; 105(1): 1–8. PubMed Abstract \| Publisher Full Text\n\nHata A, Yanagawa M, Honda O, et al.: Effect of Matrix Size on the Image Quality of Ultra-high-resolution CT of the Lung: Comparison of 512 × 512, 1024 × 1024, and 2048 × 2048. Acad. Radiol. 2018; 25(7): 869–876. PubMed Abstract \| Publisher Full Text\n\nHonda O, Yanagawa M, Inoue A, et al.: Image quality of multiplanar reconstruction of pulmonary CT scans using adaptive statistical iterative reconstruction. Br. J. Radiol. 2011; 84(1000): 335–341. PubMed Abstract \| Publisher Full Text\n\nKalra MK, Homayounieh F, Arru C, et al.: Chest CT practice and protocols for COVID-19 from radiation dose management perspective. Eur. Radiol. 2020; 30(12): 6554–6560. PubMed Abstract \| Publisher Full Text\n\nKarabulut N, Törü M, Gelebek V, et al.: Comparison of low-dose and standard-dose helical CT in the evaluation of pulmonary nodules. Eur. Radiol. 2002; 12(11): 2764–2769. PubMed Abstract \| Publisher Full Text\n\nKashyape R, Jain R: The utility of HRCT in the initial diagnosis of COVID-19 pneumonia-An Indian perspective. Indian J. Radiol. Imaging. 2021; 31(5): S178–S181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatsura M, Matsuda I, Akahane M, et al.: Model-based iterative reconstruction technique for radiation dose reduction in chest CT: Comparison with the adaptive statistical iterative reconstruction technique. Eur. Radiol. 2012; 22(8): 1613–1623. PubMed Abstract \| Publisher Full Text\n\nKumar M, Pai KM, Vineetha R: Occupation-related musculoskeletal disorders among dental professionals. Med. Pharm. Rep. 2020; 93(4): 405–409. PubMed Abstract \| Publisher Full Text\n\nLi Y, Xia L: Coronavirus disease 2019 (COVID-19): Role of chest CT in diagnosis and management. Am. J. Roentgenol. 2020; 214(6): 1280–1286. Publisher Full Text\n\nMahdavi A, Haseli S, Mahdavi A, et al.: The role of repeat chest CT scan in the COVID-19 Pandemic. Acad. Radiol. 2020; 27(7): 1049–1050. PubMed Abstract \| Publisher Full Text\n\nMenzel HG, Schibilla H, Teunen D: European guidelines on quality criteria for computed tomography. Luxembourg: European Commission; 2000; 16262.\n\nMullenders L, Atkinson M, Paretzke H, et al.: Assessing cancer risks of low-dose radiation. Nat. Rev. Cancer. 2009; 9(8): 596–604. Publisher Full Text\n\nNeroladaki A, Botsikas D, Boudabbous S, et al.: Computed tomography of the chest with model-based iterative reconstruction using a radiation exposure similar to chest X-ray examination: Preliminary observations. Eur. Radiol. 2013; 23(2): 360–366. PubMed Abstract \| Publisher Full Text\n\nRubin GD, Ryerson CJ, Haramati LB, et al.: The Role of Chest Imaging in Patient Management During the COVID-19 Pandemic: A Multinational Consensus Statement From the Fleischner Society. Chest. 2020; 158(1): 106–116. PubMed Abstract \| Publisher Full Text\n\nSalvi S, Kumar GA, Dhaliwal RS, et al.: The burden of chronic respiratory diseases and their heterogeneity across the states of India: the Global Burden of Disease Study 1990–2016. Lancet Glob. Health. 2018; 6(12): e1363–e1374. PubMed Abstract \| Publisher Full Text\n\nShah DJ, Sachs RK, Wilson DJ: Radiation-induced cancer: A modern view. Br. J. Radiol. 2012; 85(1020): e1166–e1173. PubMed Abstract \| Publisher Full Text\n\nSukumar S: Low dose protocol for high resolution CT thorax: influence of matrix size and tube voltage on image quality and radiation dose.2022. Harvard Dataverse, V1. Publisher Full Text\n\nTo T, Viegi G, Cruz A, et al.: A global respiratory perspective on the COVID-19 pandemic: Commentary and action proposals. Eur. Respir. J. 2020; 56(1): 2001704. PubMed Abstract \| Publisher Full Text\n\nTsukada J, Yamada M, Yamada Y, et al.: Comparison of the diagnostic accuracy of FBP, ASiR, and MBIR reconstruction during CT angiography in the evaluation of a vessel phantom with calcified stenosis in a distal superficial femoral artery in a cadaver extremity. Medicine (United States). 2016; 95(27): e4127. PubMed Abstract \| Publisher Full Text\n\nWang YXJ, Liu W-H, Yang M, et al.: The role of CT for Covid-19 patient’s management remains poorly defined. Ann. Transl. Med. 2020; 8(4): 145–145. PubMed Abstract \| Publisher Full Text\n\nYanagawa M, Gyobu T, Leung AN, et al.: Ultra-low-dose CT of the Lung. Effect of Iterative Reconstruction Techniques on Image Quality. Acad. Radiol. 2014; 21(6): 695–703. Publisher Full Text\n\nYeung AWK: The “as Low As Reasonably Achievable” (ALARA) principle: A brief historical overview and a bibliometric analysis of the most cited publications. Radioprotection. 2019; 54(2): 103–109. Publisher Full Text" }	[ { "id": "130052", "date": "28 Apr 2022", "name": "Rahul P. Kotian", "expertise": [ "Reviewer Expertise MRI", "Fractional Anisotropy", "Diffusion tensor imaging", "Parkinson’s Disease", "Computed Tomography Radiation Dose", "Computed Tomography" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 India's National CT radiation dose and image quality related to HRCT Thorax scans. I think this is an important work that might motivate other \"smaller\" countries to do similar initiatives. Therefore I find it relevant and good to index(even if it is not the newest thing in the world scenario related to CT radiation dose). The paper is well written, the introduction section is crisp and addresses the current scenario of radiation dose and image quality in HRCT with different matrix combinations. The materials and methods section clearly defines the steps taken in data collection using a 32cm body phantom. Lastly, the results obtained are valuable and should be utilized as a routine imaging protocol to obtain optimum image quality with significant dose reduction in HRCT Thorax scans.\nIt would add value and improve the visibility of the current manuscript if the authors decide to cite the following high impact articles published in the area of CT Dose reduction and image quality:\nRawashdeh M, McEntee M, Zaitoun M, et al. Knowledge and practice of computed tomography exposure parameters amongst radiographers in Jordan.1\n\nMohamad F, Lina K, Ghida A, et al. National diagnostic reference levels have a lot of potential but a long way to go. A systematic review on the current status of adult diagnostic reference levels in head, chest and abdominopelvic Computed Tomography 2\n\nRawashdeh M, Abdelrahman M, Zaitoun M, et al. Diagnostic reference levels for paediatric CT in Jordan. 3\n\nRawashdeh M, Saade C, Zaitoun M, et al. Establishment of diagnostic reference levels in cardiac computed tomography.4\n\nRawashdeh, M, Saade C. Radiation dose reduction considerations and imaging patterns of ground glass opacities in coronavirus: risk of over exposure in computed tomography. 4\n\nEl-Merhi F, Mohamad M, Haydar A, et al. Qualitative and quantitative radiological analysis of non-contrast CT is a strong indicator in patients with acute pyelonephritis. 5\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "181038", "date": "07 Jul 2023", "name": "Pierre-Antoine Rodesch", "expertise": [ "Reviewer Expertise medical CT imaging", "image quality metrics", "thorax imaging", "photon-counting CT." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors are studying the optimization of the dose in the frame of high-resolution computed tomography (HRCT) thorax imaging. Given the advances in iterative reconstruction (IR), new possibilities to lower the dose and increase the diagnostic score can be investigated. Indeed, IR can compensate for the noise increase at lower doses. One possibility to lower the dose while maintaining the same image quality is to use a different source voltage. Here, the authors investigate two different voltages (100 and 120 kVp). In order to increase the diagnostic score, the authors study three different matrix sizes (512, 718 and 1024) to increase the spatial resolution. The combination of different voltages and matrix size are also studied to provide 6 different protocols. The authors use phantom data and a large cohort of patients to compare the 6 protocols. This represents enough data to make the work relevant and make recommendations.\nThe work is rigorously written and relevant to the field. The context and objective are nicely described and very actual with appropriate citations.\nHowever, I would have designed the methodology differently, especially on the phantom acquisition. It would be more interesting to be able to have cases with the same dose, or the same noise, or the same CNR, which is not the case for any combination of protocols. Here the authors present an increased CNR and diagnostic score for a higher dose, which is predictable and can not be used to draw conclusions.\nAs the matrix size increases, the noise increases as well. The IDose level should be increased for the 718 and 1024 cases to match the noise level.\n\nAs the voltage decreases, the contrast is expected to increase, and it would be possible to increase the IDose level as well to match the CNR. (The other possibility would be to increase the current to match the dose).\nIn table 2, instead of the SNR, the noise and contrast value would be more relevant to highlight the noise and contrast differences.\nIn table 3, it would be easier to present the results in a different way: by identifying the lowest value and calculate all percentages in comparison to the latter.\nTable 4 and Figure 3 are redundant, I think you could only keep Figure 3 and would include the scores (Table 6) in this figure. Why is there no p-values computed for the scores?\nForm Table 6, we conclude that Protocol B is the best, but you recommend protocol D, it is not clear why: why not recommending protocol E? I understand that redoing the patient acquisitions/reconstructions is not feasible. But to support your affirmation, you will need to provide more results. Could you calculate a normalized diagnostic score by dividing it by the dose, in a similar way as the FOM? Could you also provide the percentage of cases for each protocol that presented a score lower than 4 (or 4.5?) and so would not be acceptable? Please elaborate the discussion.\nMinor comments:\nI am not sure using A, B, C, D, E and F is the best way to refer to each protocol, you should use 120/512, 120/718, etc… It does not take more space and help to facilitate the comprehension.\n\nIn the abstract you don’t mention the FOV, which is important when the matrix size varies.\n\nIn this abstract sentence “For quantitative image quality, the difference noted was also statistically significant among two kVp settings.”, I don’t understand which kVp performs the best.\n\nIn material and methods, you don’t indicate the exposure (mAs) parameter in either phantom or patient acquisition.\n\nIn material and methods, you don’t explain the different patient groups: I was expecting to reconstruct each patient with 3 different matrix size but that was not the case, right? (Only one matrix size per patient?)\n\nYou could also mention photon-counting CT that could reduce further the dose of innovative denoising techniques such as deep learning methods.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "181026", "date": "13 Sep 2024", "name": "Kai Mei", "expertise": [ "Reviewer Expertise CT researcher for over 8 years. CT reconstruction", "multi-energy CT", "reconstruction algorithms", "clinical CT protocols", "CT dose reduction", "low-dose CT algorithms." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is relatively well written, and the study was adequately well performed. The main problem of this paper is the motivation of design.\nThe only parameters in a CT protocol that affect radiation dose are tube voltage, tube current, rotation speed and helical pitch. Matrix size has no effect on radiation dose and has minimal effect on diagnosis quality, compared to reconstruction filter: radiologist can always opt to sharper filters.\nThe tuning of tube voltage is mostly determined by the nature of the examination and the property of the X-ray source of that CT scanner, it is not used as a mean to reduce radiation dose. Tube voltage changes the contrast of the CT image, so specific examinations need to be done in specific tube voltage.\nIn addition, the matrix size is not really relevant in dose studies. It only changes how you store the patient data. There are so much more other ways to enhance the diagnosis quality (from reconstruction filters to post extrapolations in the DICOM viewer), when the matrix size is limited.\nThe current topic of this manuscript lacks interest in CT research fields and I would recommend the author to redesign the study to compare with pitch, scanning speed and tube current etc. instead.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-399
https://f1000research.com/articles/10-1189/v1	24 Nov 21	{ "type": "Case Report", "title": "Case Report: Therapeutic and immunomodulatory effects of plasmapheresis in long-haul COVID", "authors": [ "Dobri D. Kiprov", "Ahvie Herskowitz", "Daehwan Kim", "Michael Lieb", "Chao Liu", "Etsuko Watanabe", "Jan C. Hoffman", "Regina Rohe", "Michael J. Conboy", "Irina M. Conboy", "Ahvie Herskowitz", "Daehwan Kim", "Michael Lieb", "Chao Liu", "Etsuko Watanabe", "Jan C. Hoffman", "Regina Rohe", "Michael J. Conboy" ], "abstract": "Many patients with COVID-19 experience a range of debilitating symptoms months after being infected, a syndrome termed long-haul COVID. A 68-year-old male presented with lung opacity, fatigue, physical and cognitive weaknesses, loss of smell and lymphocytopenia. After rounds of therapeutic plasma exchange (TPE), the patient returned to normal activities and work. Mechanistically in the patient’s peripheral blood mononuclear cells (PBMCs), markers of inflammatory macrophages diminished and markers of lymphocytes, including natural killer (NK) cells and cytotoxic CD8 T-cells, increased. Circulating inflammatory proteins diminished, while positive regulators of tissue repair increased. This case study suggests that TPE has the capacity to treat long-haul COVID.", "keywords": [ "Immunomodulation", "Long Haul Covid19", "plasmapheresis", "adaptive immunity", "inflammation", "proteomics", "leukocyute subsets" ], "content": "Introduction\n\nThe symptoms of “long-haul” coronavirus disease 2019 (long COVID) are debilitating and prevent patients from working, which is projected to negatively impact the healthcare system and economic recovery.1 The most common symptoms of long COVID are dyspnea with abnormal chest radiograph (CXR) findings, extreme fatigue, cognitive impairment (described as “brain fog”), myalgias, anosmia, ageusia, headache and sleep disorder.2 Long COVID resembles myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) which is driven by autoantibodies.3,4 Therapeutic plasma exchange (TPE) was successfully tried in patients with severe COVID-195 and is typically used on patients with ME/CSF and other autoimmune disorders.6,7 Moreover, our recently published studies suggest that TPE re-sets the circulatory proteome to healthier states, attenuating the so-called cytokine storm and enhancing the systemic determinants of tissue repair.8,9 The encouraging results of these reports convinced us to try TPE on a patient with severe long COVID.\n\n\nCase\n\nA 68-year-old caucasian male attorney, who reported having been very physically active and capable of multitasking in a demanding executive position at work began to feel fatigued and short of breath on December 14, 2020. He visited an emergency room on December 18, 2020. His oxygen saturation was 90–93% on room air and he was sent home. His breathing and fatigue deteriorated, so he was admitted to the hospital on December 21, 2020. He tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by PCR and was treated with remdesivir and Decadron (dexamethasone). His status continued to worsen, and he was placed on high-flow oxygen. He was never intubated. After 11 days in the hospital, he was sent home on portable oxygen which he used intermittently.\n\nOver the following four weeks, he felt extremely fatigued. He could only walk to the bathroom and back to bed. He could not focus on anything cognitively, which he described as “brain fog”. He was unable to do any work and could not even answer his emails. He reported he had no sense of smell nor taste. Sedimentation rate, CRP, D-dimer and ferritin were abnormal in clinical lab tests (Figure 1B). At that time, chest radiographs and a chest CT scan revealed ground-glass appearance areas diffusely spread throughout his lungs (Figure 1C). A PCR test for SARS-CoV-2 was negative. A serologic test for anti-SARS-COV-2 IgG antibodies was positive.\n\nA. Schematics of the case. B. Erythrocyte sedimentation rate, CRP, D-dimer, ferritin levels were assayed before each TPE procedure and were initially elevated but normalized by the TPE. C. Chest radiographs show reduced lung opacity after TPE. D. Clouded plasma appearance was reduced by TPE.\n\nIn February of 2021, he was seen in our clinic. At that time, he was very weak and unable to walk. When he arrived at the airport, he needed a wheelchair to go from the plane to the taxi. He underwent his first TPE on February 4, 2021. One plasma volume was exchanged, using 5% albumin as an exchange fluid. The removed plasma was very dark and opaque (Figure 1D). During his first TPE treatment, he was coughing profusely. After his first treatment, he could breathe more easily, and the cough subsided. The morning after the first TPE treatment, he could walk and was not struggling for breath. Two days later, he had no difficulty breathing and was able to walk 100 feet on a level surface but still had difficulty walking uphill.\n\nHe underwent a second TPE and two days after the second treatment he was able to walk uphill with ease and even jog. His brain fog disappeared, and he was able to get back to his daily work activities. Typical biomarkers of systemic inflammation all became quickly and robustly normalized including erythrocyte sedimentation rate, CRP, ferritin, and D-dimer10 (Figure 1B). The plasma from the second TPE was clear (Figure 1D). A week later his chest radiographs showed considerable clearing of the opacities in the lungs (Figure 1C). He also reported regaining his sense of smell and taste. He was seen in the clinic for another TPE two months later and, at that time, the patient reported that he was back to work, feeling like his normal self, and able to exercise daily without shortness of breath.\n\nThe peripheral blood mononuclear cells (PBMC) and blood serum of this patient were collected and analyzed before the first round of TPE (R0) and before each subsequent round (R1–R3) (Figure 1A). Real-time qRT PCR was used to study the levels of the markers of T cells: CD3; helper T-cells: CD4; cytotoxic T-cells: CD8; NK cells: CD94; B-cells: B220 and CD19; macrophages: CD11b; inflammatory myeloid cells: CD68; and IL-2 receptor: CD25. Before TPE, there was a prevalence of CD68+ inflammatory myeloid cells’ marker, and relatively fewer lymphocyte markers, which is indicative of a lack of adaptive immunity (Figure 2). TPE increased the markers of T-cells, B-cells, NK cells, and diminished the markers of inflammatory macrophages, suggesting enhanced adaptive immunity and attenuated inflammatory response (Figure 2). CD3, CD4 and CD19 initially diminished at 5 days after the first TPE, R1, but then steadily increased during the longer intervals of R2 and R3; CD94 and CD8, the markers of cytotoxic immune cells that combat viral infections also gradually and steadily increased (Figure 2).\n\nA. The expression of T cell, B-cell NK cell markers and IL2 receptor are significantly increased in R2 and R3 compared to R0. CD11b myeloid marker increased in R1 and then returned to R0 levels. CD68 – the marker of inflammatory myeloid cells, was significantly decreased by the third round of TPE. B. The ratios of lymphoid/CD11b, lymphoid/CD68, CD25/CD11b and CD25/CD68 markers clearly demonstrate the positive effects of TPE in promoting the adaptive rather than the inflammatory immune response. Note the break in Y-axis scale. P < 0.05, P < 0.01, **P < 0.001.\n\nFive days after the first TPE and the initial increase of CD11b+, the levels of this marker stabilized, while the CD68 marker of inflammatory macrophages were never elevated by TPE and were diminished by R2 and R3 (Figure 2). Plotting the ratios of T-cell and B-cell markers to myeloid and inflammatory macrophage markers (CD11b and CD68) demonstrates a rapid and robust shift toward adaptive immunity through the rounds of TPE (Figure 2). The effects of TPE are particularly striking when looking at the relative increase in the CD8+/CD68+, e.g., anti-viral cytotoxic T cells in an inflammation reduced environment (Figure 2).\n\nBlood serum from the patient was profiled through comparative proteomics using RayBiotech antibody arrays, as we have published.8 Heatmapping clearly demonstrated the profound influence of TPE on the circulatory proteome (Figure 3A); Venn diagrams, KEGG and biological process databases uncovered 36 proteins that were commonly downregulated between the rounds of TPE, (Figure 3B–D). In agreement with the diminished inflammation and enhanced adaptive immunity that were suggested by Figures 1 and 2, this longitudinal serum proteomics revealed significant attenuation of inflammatory factors, which was stable and lasted several months (Figure 3E and F). Regulators of several growth factor pathways that are known to promote tissue repair increased in the systemic milieu: IGF, EGF, TGF-β (Figure 3E and F).\n\nA. Comparative heat mapping of 507 proteins between TPE rounds was done on RayBiotech Array. B. Venn diagram showing the overlap between the proteins, which were influenced by the TPE rounds. 36 shared proteins were diminished each TPE round (0.65> Fold change). C. The KEGG signaling pathway analysis. D. The top 20 list of biological processes. E. Heat mapping of the proteins that relate to Aging and Inflammaging and are decreased by TPE. F. Volcano plots of proteomics as per TPE rounds. More proteins are downregulated by the second round than by the first. The red dots represent differently expressed proteins (P < 0.05; \|Log2FC\|<1.5), while the grey dots represent proteins with P > 0.05.\n\nThese results establish rapid and significant changes in circulatory proteome of the patient, which are indicative of attenuated inflammation, productive immune response, and enhanced tissue maintenance.\n\nSince the COVID-19 pandemic, roughly 70–80% of patients suffer various sequelae.11 About 40% of these patients have acute respiratory distress syndrome (ARDS) and one of the main sequelae is pulmonary fibrosis.12 Although the mechanism of COVID-19-induced ARDS may be different from classical ARDS, the onset and development of pulmonary fibrosis are commonly related to elevated inflammatory cytokines, such as IL-6.13 Accordingly, anti-inflammatory drugs attenuate COVID-19-induced pneumonia.14,15\n\nIn this study, a patient who was initially healthy, quickly developed health problems after being infected with the novel coronavirus. Moreover, after two months, his health deteriorated to the point of being unable to walk, being physically short of breath, suffering mental fatigue, and diffuse ground-glass appearance was observed throughout his lungs. Clinical and biological analyses strongly suggested systemic inflammation and a lack of productive immune response. After the first TPE treatment, the dyspnea disappeared, and cough subsided. The patient was also able to walk short distances. Interestingly, these changes were observed on day 2 after the first TPE treatment. A month later, the patient was able to walk uphill easily and jog. The decrease in concentration and cognitive fog disappeared, and the patient was able to return to normal life. During the same period, inflammatory macrophage markers robustly diminished and the markers of lymphocytes increased in the blood, suggesting that TPE promoted adaptive immunity and decreased inflammatory response. Such a productive immune response shift became prominent between the second and third rounds of TPE and rounds 1 and 2 were separated by only 5 days, e.g., too quickly for changing leukocyte subset numbers. Longitudinal comparative proteomics demonstrated that many proteins that are associated with inflammaging16 were attenuated by the rounds of TPE. For instance, pro-inflammatory factors, such as IL-1β, IL-6, and IP-10, stably decreased. In contrast, lipocalin-1 that participates in the sense of taste, and regulators of EGF, IGF, and TGF-β signaling pathways increased, demonstrating a better capacity for tissue maintenance and repair. Of note, the smell/taste reception returned to normal in this patient after TPE. In summary, this case study suggests that TPE may alleviate post COVID-19 sequelae via positive shifts toward adaptive immunity and tissue repair that are concurrent with reduction of inflammation.\n\n\nMethods\n\nThe study was approved by the Diagnostics Investigational Review Board (www.dxirb.com) with study identifier - 7347. The study was performed under the written informed consent (obtained by Dr. Dobri Kiprov) from the patient for the use and publication of the patient’s data. Therapeutic plasma exchange (TPE) was performed as described.17\n\nWhole blood was collected immediately before (pre) or after (post) TPE procedure in the clinic and processed into serum, plasma and PBMCs (Histopaque density) as described.18\n\nTotal RNA was extracted from PBMC using the RNeasy mini kit (Qiagen), and the SuperScript III First-Strand Synthesis System (Invitrogen). Real-time PCR was performed on a Bio-Rad iQ5 real-time PCR machine. The primers used for PCR are listed in Table 1.\n\nSerum was analyzed on a Ray Biotech human L507 antibody capture array (AAH-BLG-1-4, Raybiotech), processed according to the manufacturer’s protocol. The array slides were imaged by a Molecular Devices 4000b scanner and data were calculated by Genepix. Normalization was done by bult-in positive and negative controls.\n\nThe gene ID of differently regulated proteins was performed using DAVID Bioinformatics Resources (version 6.8, https://david.ncifcrf.gov), as well as the GO (Gene Ontology) analysis of the biological process (BP), molecular function (MF) and cellular component (CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Heat maps were performed with ClustVis.19 Volcano plots were constructed with the Graphpad Prism software version 9 (GraphPad software Inc).\n\nAll statistical analyses were performed using GraphPad Prism software. All values are expressed as means ± SEM for independent experiments, or SD for replicates. To determine the significance of differences among groups, comparisons were made using Student’s t-test. The P < 0.05 was considered significant.\n\n\nData availability\n\nOSF: Underlying data for ‘Therapeutic and immunomodulatory effects of plasmapheresis in long-haul COVID: a case report’ https://doi.org/10.17605/OSF.IO/AXPW7.20\n\nThe project contains the following underlying data:\n\n[Dataset_1_qPCR raw data.xlsx] (Raw qPCR data).\n\n[Dataset_2_Antibody array raw data.xlsx] (Antibody array raw data).\n\n[Dataset_3_raw data of diagnosis.pdf] (Diagnosis data).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthors’ contributions\n\nDKiprov planned and performed all clinical work, established the IRB approval, provided Figure 1 and the blood samples to the Conboy laboratory and co-wrote the manuscript; DKim provided Figure 2, bio-computation and bioinformatics for Figure 3, and participated in the manuscript writing; ML contributed to Figure 2; CL and EW performed the comparative proteomics that is shown in Figure 3 and CL provided the schematic of Figure 1A; JH, RR and AH provided clinical support; MJK contributed to the planning of this work and co-wrote the manuscript; IC planned, directed, and integrated the study, interpreted the data, and co-wrote the manuscript. All authors agreed with publication of this work.", "appendix": "Acknowledgements\n\nWe would like to thank William Hou, Zhixin Zhang and Xiaoyue Mei for formatting the references, and Wu Love for logistics help.\n\n\nReferences\n\nPhillips S, Williams MA: Confronting Our Next National Health Disaster — Long-Haul Covid. N Engl J Med. 2021; 385(7): 577–579. Publisher Full Text\n\nDavis HE, Assaf GS, McCorkell L, et al.: Characterizing long COVID in an international cohort: 7 months of symptoms and their impact. EClinicalMedicine. 2021; 38: 101019. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nYong SJ: Long COVID or post-COVID-19 syndrome: putative pathophysiology, risk factors, and treatments. Scand J Infect Dis. 2021; 53(10): 737–754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWirth K, Scheibenbogen C: A Unifying Hypothesis of the Pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): Recognitions from the finding of autoantibodies against ß2-adrenergic receptors. Autoimmun Rev. 2020; 19(6): 102527. PubMed Abstract \| Publisher Full Text Reference Source\n\nKhamis F, Al-Zakwani I, Al Hashmi S, et al.: Therapeutic plasma exchange in adults with severe COVID-19 infection. Int J Infect Dis. 2020; 99: 214–218. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nTölle M, Freitag H, Antelmann M, et al.: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Efficacy of Repeat Immunoadsorption. J Clin Med. 2020; 9(8): 2443. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nWunsch E, Kruk B, Snarski E, et al.: Plasmapheresis improves chronic fatigue in patients with primary biliary cholangitis. Polish Arch Intern Med. 2020. Publisher Full Text Reference Source\n\nMehdipour M, Skinner C, Wong N, et al.: Rejuvenation of three germ layers tissues by exchanging old blood plasma with saline-albumin. Aging (Albany NY). 2020; 12(10): 8790–8819. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMehdipour M, Mehdipour T, Skinner CM, et al.: Plasma dilution improves cognition and attenuates neuroinflammation in old mice. GeroScience. 2021; 43(1): 1–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoudel A, Poudel Y, Adhikari A, et al.: D-dimer as a biomarker for assessment of COVID-19 prognosis: D-dimer levels on admission and its role in predicting disease outcome in hospitalized patients with COVID-19. PLoS One. 2021; 16(8): e0256744. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarfì A, Bernabei R, Landi F: Persistent Symptoms in Patients After Acute COVID-19. JAMA. 2020; 324(6): 603–605. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nLiu J, Zheng X, Tong Q, et al.: Overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses SARS-CoV, MERS-CoV, and 2019-nCoV. J Med Virol. 2020; 92(5): 491–494. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRai DK, Sharma P, Kumar R: Post covid 19 pulmonary fibrosis. Is it real threat?. Indian J Tuberc. 2021; 68(3): 330–333. Publisher Full Text Reference Source\n\nCollins BF, Raghu G: Antifibrotic therapy for fibrotic lung disease beyond idiopathic pulmonary fibrosis. Eur Respir Rev. 2019; 28(153): 190022. PubMed Abstract \| Publisher Full Text\n\nLi Y, Li H, Liu S, et al.: Pirfenidone ameliorates lipopolysaccharide-induced pulmonary inflammation and fibrosis by blocking NLRP3 inflammasome activation. Mol Immunol. 2018; 99: 134–144. PubMed Abstract \| Publisher Full Text Reference Source\n\nGuan W, Ni Z, Hu Y, et al.: Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med. 2020; 382(18): 1708–1720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoada M, López OL, Olazarán J, et al.: A randomized, controlled clinical trial of plasma exchange with albumin replacement for Alzheimer’s disease: Primary results of the AMBAR Study. Alzheimer’s Dement. 2020; 16(10): 1412–1425. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMallone R, Mannering SI, Brooks-Worrell BM, et al.: Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society. Clin Exp Immunol. 2011; 163(1): 33–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMetsalu T, Vilo J: ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Res. 2015; 43(W1): W566–W570. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKiprov DD, Herskowitz A, Kim D, et al.: Underlying data for ‘Therapeutic and immunomodulatory effects of plasmapheresis in long-haul COVID: a case report’.2021. Publisher Full Text" }	[ { "id": "119963", "date": "28 Jan 2022", "name": "Bernd Stegmayr", "expertise": [ "Reviewer Expertise Long term experience with apheresis therapies and research. Specialist Internal Medicine and Nephrology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 using Plasmapheresis (apheresis) for long-haul Covid increases awareness of the complexity of the disease and the possibilities to immunomodulate by apheresis.\n\nThe topic is very important and the concept well described. The case is also well described.\n\nThere is a lack of information that should be added:\nWhat type of apheresis procedure (centrifuge or filtration, leukapheresis?) was used?\n\nWhat type and doses of anticoagulation during apheresis was used? (citrate, LMWH, heparin?)\n\nWas a maintenance pharmacological therapy used also after initiation of apheresis to prohibit antibodies to increase?\n\nIf so, what were the pharmacological doses/kg body weight and/day of such medication (or interval)?\n\nWhat were the time intervals between the apheresis procedures?\n\nDid you try to investigate for specific antibodies?\n\nYou could add the concept of apheresis for antibody removal such as described for VITT by Rock et al. 20211\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": [] }, { "id": "123772", "date": "25 Feb 2022", "name": "Prashant Nasa", "expertise": [ "Reviewer Expertise Management of COVID-19 related respiratory failure." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCongratulations authors for the successful outcome of this exploratory study. I have the following points for consideration to make the discussion comprehensive.\nThere is a recent review published in Nature on the pathology of long-haul COVID-19. It will be worth using that in the discussion. (https://doi.org/10.1038/s41590-021-01104-y). The pathophysiology of long-haul COVID-19 involving imbalance between persistent inflammation on homeostasis should be discussed and provide prospects for future controlled studies.\n\nWas plasma removed through therapeutic plasma exchange profiled through comparative proteomics? This would have helped in understanding the degree of reduction of the biomarkers with each session of TPE.\n\nRole of TPE and Plasma exchange has shown variable effects and investigators have used them in different combinations. It is worth including them in discussing (https://www.ijidonline.com/article/S1201-9712(20)32289-X/fulltext)\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/10-1189
https://f1000research.com/articles/11-279/v1	04 Mar 22	{ "type": "Research Article", "title": "Recurrence in Plasmodium vivax malaria: a prospective cohort study with long follow-up from a coastal region in South-West India", "authors": [ "Divya Gandrala", "Nitin Gupta", "Alekhya Lavu", "Vishnu Teja Nallapati", "Vasudeva Guddattu", "Kavitha Saravu", "Divya Gandrala", "Nitin Gupta", "Alekhya Lavu", "Vishnu Teja Nallapati", "Vasudeva Guddattu" ], "abstract": "Background: India is endemic for Plasmodium vivax (Pv) malaria. Despite a decrease in incidence, its elimination is hampered by recurrences. This study aimed to characterize recurrences in Pv malaria and study its association with primaquine (PQ) usage. Methods: Symptomatic adult Pv patients were followed-up for up to 23 months for recurrences. The time to recurrence was compared by the PQ dosage they received using a log-rank test. Results: Of the 294 malaria patients, 206 (70%) patients had Pv infection during the study period. A total of 20 (9.7%) recurrences were seen in 17 (8.2%) patients of Pv. The percentage of first-time recurrences were highest in the no PQ group (25%), followed by the weekly PQ group (20%), low dose daily PQ (8.2%) group, and high dose daily PQ group (3.1%). Conclusions: Recurrence in Pv malaria is common, especially in those who receive an inappropriate prescription of primaquine.", "keywords": [ "Primaquine", "relapse", "severe malaria" ], "content": "Introduction\n\nMalaria is a major global health problem, with around 228 million reported cases alone in 2018, most due to Plasmodium falciparum (Pf).1 Consequently, most reports on malaria concentrate on Pf. Traditionally, Pf has been described as the causative agent for severe malaria. However, recent reports have shown that malaria caused by Plasmodium vivax (Pv) can also be severe. Although India represents a small percentage of the overall global malaria cases, it is responsible for nearly half of the total cases of Pv.2,3 Despite a decline in the number of Malaria cases in India, the major roadblock to elimination is the tendency of Pv to relapse frequently, mainly when primaquine (PQ) is not prescribed or prescribed in sub-therapeutic dosage.4,5 Therefore, the objective of the study was to calculate the incidence of recurrence in patients with Pv malaria and find the impact of PQ prescription practices on recurrence.\n\n\nMethods\n\nA prospective observational cohort study was conducted at Kasturba Hospital, Manipal in Udupi district of Karnataka State, India, for two years, from October 2016 to August 2018. The study was commenced after taking approval from the Institute's Ethical Committee (IEC 636/2016). All patients of either sex above 18 years of age who presented during the study period with fever and had malarial parasites on the quantitative buffy coat (QBC) examination were included in the study after taking written informed consent. The article was reported according to the STROBE guidelines and all the criteria in the STROBE checklist were met. The sample size was calculated as 206 cases of Pv, considering recurrence prevalence as 31.5%, 95% level of confidence and 6.5% precision.6\n\nThey were categorized into Pv, Pf or mixed based on the results of peripheral smear. The patients were classified as having severe disease if they met the criteria for severity laid down by World Health Organisation.7 A detailed history (including comorbidities), physical examination, and laboratory parameters were noted in a predefined case study form. In addition, the worst value of the variables during hospitalization was recorded. Since the study aimed to record the prescription practices of treating physicians, the study objectives were not disclosed to them to avoid bias. The diagnosed cases were treated by the treating team. Glucose-6 Phosphate dehydrogenase (G6PD) levels were requested by the treating physician’s discretion. The enzyme activity was quantified by the manual spectrophotometric kinetic 'gold standard' method in the institutional biochemistry laboratory. G6PD deficiency was defined as less than 30% of mean G6PD activity.\n\nThe treating physicians decided the dosage of antimalarials, including PQ. The details of treatment, supportive care hospitalization days and mortality during hospital stay were noted. The primary outcome was microbiologically-confirmed recurrence at the end of the study period. Individuals were followed up telephonically every two months until the end of the study period for the development of fever recurrence. Additionally, individuals were asked to report if the fever recurred and were classified as recurrence if they were microscopically proven to have malaria again.\n\nStatistical analysis was performed using Statistical Package for the Social Sciences version 23.0 (SPSS, RRID:SCR_002865, http://www-01.ibm.com/software/uk/analytics/spss/). Continuous variables were summarized as mean with standard deviation (SD) or median with interquartile range (IQR) (in skewed data). Categorical variables were summarized as the frequency with proportion. Overall, patients with Pv were divided into four groups according to PQ dosage- no PQ, weekly PQ, low dose daily PQ (0.25 mg/kg/day), and high dose daily PQ (0.5 mg/kg/day). The number of recurrences in each group were calculated. A Kaplan-Meier survival plot was generated to determine the survival function of recurrences according to PQ categories until 23 months' follow-up duration. Log-rank test was used to compare the survival function. A Chi-square test was used to compare qualitative variables, whereas an independent t-test was used to compare quantitative variables. A p-value of less than 0.01 was considered significant.\n\n\nResults\n\nA total of 294 malaria cases were enrolled during the study period, of which 206 (70%) were Pv, 79 (27%) were Pf, and 9 (3%) were mixed (pv+pf). A total of 29.6 % (87/294) cases had severe malaria. The proportion of severity, the requirement of supportive care, and mortality were comparable in both groups and summarized in Figure 1. The baseline clinical and laboratory features of patients with Pv and Pf malaria have been summarized in Table 1.\n\nPv: Plasmodium vivax; Pf: Plasmodium falciparum; ICU: Intensive care unit.\n\n* Categorical variables are summarized as the frequency with proportion whereas continuous variables are summarized as either mean (±SD) or median (IQR). Chi-square or Fischer's exact test and Independent sample t-test or Mann Whitney U test were performed, p-value less than 0.05 shows the statistically significant difference and shown in bold font. ARDS: Acute Respiratory Distress Syndrome.\n\nOf 294 cases included in the study, there were 21 recurrences in 18 (6.12%) patients. All patients with recurrent disease had non-severe malaria with good clinical recovery. Twenty recurrences (20/206, 9.7%) belong to the Pv group and 1 (1/79, 1.3%) patient from the Pf group. Of the 20 recurrences in the 17 patients were in the Pv group, three patients had a recurrence for the second time. The median time to follow-up was 388 (293–567) days. The median time to the first recurrence in the Pv group was 83 (66.5–242.5) days.\n\nOf the 206 patients with Pv, G6PD levels could be done in 196 patients only, out of which nine patients were found to have low G6PD levels (Table 2). No case of PQ-induced hemolysis was noted in our cohort. The dose of PQ was significantly associated with recurrences on the Chi-square test (p<0.001). The percentage of first-time recurrences were highest in the no PQ group (25%), followed by the weekly PQ group (20%), low dose daily PQ (8.2%) group, and high dose daily PQ group (3.1%) (Table 2). A Kaplan-Meier curve was plotted to compare the median time to recurrence in each of the PQ-based groups, and the difference was found to be significant on the log-rank test (p=0.009) (Figure 2).\n\n\nDiscussion\n\nUdupi district has a population of 1,177,908 with an area of 3,582 sq. km and is located 13°32′ 24.43′′ N latitude and 74°52′26.78′′ E longitude, with typical tropical climatic conditions. The monsoon in this region starts in June and extends till October, with an average rainfall of more than 4000mm every year. The catchment area of our hospital encompasses both the rural and urban populations of coastal and interior Karnataka, Goa and Kerala. Pv is the largest infecting species in this region, followed by Pf.8,9 The same trend is noted in other parts of India.10,11\n\nIn this study, around 30% of the cases had severe malaria with a similar incidences of severity in Pv and Pf. In the last decade, the severity of malaria has ranged from 6.5% to 48% across the world.12,13 Classically, Pf is supposed to be one with a higher frequency of severe manifestation, whereas Pv is apparently the benign form.14 This dogma has been challenged more and more as reports emerge from Pv endemic areas. Like other reports, hepatic and renal dysfunction were the commonest manifestation of severity in our study.14 Central nervous system (CNS) manifestations, which were initially thought to be exclusive to Pf, were seen in Pv and Pf in our study. This study reiterates that severe Pv malaria cases presented with similar phenotypic features as Pf malaria. Although previous studies have reported variable mortality with malaria cases, mortality in our study was low, with one mortality each in Pv and Pf patients.10\n\nAs expected, all but one recurrence were seen in patients with Pv. The percentage recurrence in Pv cases was close to 10%, which was considerably lower than recurrences reported in the previous series (24–38%).15,16 Like a previous study, all recurrent cases had mild symptoms, presumably due to the development of acquired immunity from the previous episode.17 The median time to recurrence was 83 days in our study, similar to previously published studies.15 Those patients for whom PQ was not used had higher rates of recurrence. Interestingly, the 16 patients for whom no PQ was used, only one patient had proven low levels of G6PD. In all the other cases, the levels were either not done or were normal. This reflects the need to reinforce the importance of PQ prescription in patients with Pv. The rates were lower in the daily PQ group even when they were used at a lower dose. Similar results were observed in other studies as well.18 Since the study was done in a tertiary care hospital where G6PD levels and specialist referrals are available, the study cannot be generalized to primary care settings. Similar widespread prescription audits are required all over the country to understand the practices and pattern of recurrences in patients with Pv.\n\nSelf-limiting intermittent recurrences that are asymptomatic could not be ruled out as symptom-based screening for recurrence was done. The genotyping of recurrences could not be done to discern relapse and reinfection. The possibility of non-compliance cannot be ruled out as PQ therapy was unsupervised.\n\n\nConclusions\n\nThe study reiterates that Pv is the dominant species in this part of India with similar frequencies of severity. Moreover, it is associated with recurrences, especially when PQ prescription is inappropriate. Therefore, there is a need for improving prescription practices amongst primary care physicians through regular educational interventions.\n\n\nData availability\n\nData cannot be shared due to ethical and security concerns, however a de-identified dataset with all the details can be shared with reviewer or readers at reasonable request to corresponding author.\n\n\nAuthor's contributions\n\nAll authors have read and approved the final manuscript. The requirements for authorship have been met, and each author believes that the manuscript represents honest work.\n\nDivya Gandrala: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing-original draft preparation, Writing-Review & Editing\n\nNitin Gupta: Formal analysis, Validation, Writing-original draft preparation, Writing-Review & Editing\n\nAlekhya Lavu: Data curation, Formal analysis, Investigation, Methodology\n\nVishnu Teja Nallapati: Writing-original draft preparation, Writing-Review & Editing\n\nVasudeva Guddattu: Formal analysis, Software, Writing-original draft preparation, Writing-Review & Editing\n\nKavitha Saravu: Conceptualization, Data curation, Formal analysis, Project administration, Supervision, Validation, Writing-original draft preparation, Writing-Review & Editing", "appendix": "Acknowledgements\n\nAuthors gratefully acknowledge the seed grant funding and publication support from Manipal Center for Infectious Diseases, Prasanna School of Public Health, Manipal Academy of Higher Education, Manipal, India.\n\n\nReferences\n\nWorld Health Organization: World Malaria Report, World Health, vol. WHO/HTM/GM, no. December.2019; p. 238. ISBN 978 92 4 1564403.\n\nSaravu K, Kumar R, Ashok H, et al.: Therapeutic Assessment of Chloroquine-Primaquine Combined Regimen in Adult Cohort of Plasmodium vivax Malaria from Primary Care Centres in Southwestern India. PLoS One. 2016 Jun 17; 11(6): e0157666. PubMed Abstract \| Publisher Full Text\n\nRahi M, Sharma S, Das P, et al.: Connecting the dots to strengthen malaria elimination strategies in India: A Malaria Elimination Research Alliance - India initiative. Indian J. Med. Res. 2021 Jul; 154(1): 19–23. PubMed Abstract \| Publisher Full Text\n\nKumar R, Guddattu V, Saravu K: Therapeutic assessment of primaquine for radical cure of plasmodium vivax malaria at primary and tertiary care centres in Southwestern India. Korean J. Parasitol. 2016; 54(6): 733–742. PubMed Abstract \| Publisher Full Text\n\nRishikesh K, Kamath A, Hande MH, et al.: Therapeutic assessment of chloroquine–primaquine combined regimen in adult cohort of Plasmodium vivax malaria from a tertiary care hospital in southwestern India. Malar. J. 2015; 14(1): 1–6. Publisher Full Text\n\nDouglas NM, Nosten F, Ashley EA, et al.: Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and effect of antimalarial kinetics. Clin. Infect. Dis. 2011 Mar 1; 52(5): 612–620. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: WHO-Severe malaria. World Health Organization; 2014. Reference Source\n\nSaravu K, Docherla M, Vasudev A, et al.: Thrombocytopenia in vivax and falciparum malaria: an observational study of 131 patients in Karnataka, India. Ann. Trop. Med. Parasitol. 2011; 105(8): 593–598. PubMed Abstract \| Publisher Full Text\n\nSaravu K, Rishikesh K, Parikh CR: Risk Factors and Outcomes Stratified by Severity of Acute Kidney Injury in Malaria. PLoS One. 2014; 9(3): e90419. PubMed Abstract \| Publisher Full Text\n\nNadkar MY, Huchche AM, Singh R, et al.: Clinical profile of severe Plasmodium vivax malaria in a tertiary care centre in Mumbai from June 2010-January 2011. J. Assoc. Physicians India. 2012 Oct; 60: 11–13. PubMed Abstract\n\nZubairi AB, Nizami S, Raza A, et al.: Severe Plasmodium vivax malaria in Pakistan. Emerg. Infect. Dis. 2013 Nov; 19(11): 1851–1854. PubMed Abstract \| Publisher Full Text\n\nKochar DK, Das A, Kochar A, et al.: A prospective study on adult patients of severe malaria caused by Plasmodium falciparum, Plasmodium vivax and mixed infection from Bikaner, northwest India. J. Vector Borne Dis. 2014 Sep; 51(3): 200–210. PubMed Abstract\n\nAbdallah TM, Abdeen MT, Ahmed IS, et al.: Severe plasmodium falciparum and plasmodium vivax malaria among adults at Kassala Hospital, eastern Sudan. Malar. J. 2013; 12(1): 1–7. PubMed Abstract \| Publisher Full Text\n\nSaravu K, Rishikesh K, Kamath A, et al.: Severity in Plasmodium vivax malaria claiming global vigilance and exploration - A tertiary care centre-based cohort study. Malar. J. 2014; 13(1). Publisher Full Text\n\nZuluaga-Idárraga L, Blair S, Akinyi Okoth S, et al.: Prospective Study of Plasmodium vivax Malaria Recurrence after Radical Treatment with a Chloroquine-Primaquine Standard Regimen in Turbo, Colombia. Antimicrob. Agents Chemother. 2016 Jul 22; 60(8): 4610–4619. PubMed Abstract \| Publisher Full Text\n\nKim JR, Nandy A, Maji AK, et al.: Genotyping of Plasmodium vivax reveals both short and long latency relapse patterns in Kolkata. PLoS One. 2012; 7(7): e39645. PubMed Abstract \| Publisher Full Text\n\nKochar DK, Saxena V, Singh N, et al.: Plasmodium vivax. Emerg. Infect. Dis. 2005; 8(11): 11–12. Publisher Full Text\n\nGanguly S, Saha P, Guha SK, et al.: Recurrence Pattern of P. Vivax Malaria Following Treatment with Chloroquine Either Alone or in Combination with Primaquinein Urban Kolkata, India. Int. J. Recent Sci. Res. 2014; 5(6): 1046–1049." }	[ { "id": "126318", "date": "16 Mar 2022", "name": "Vettakkara Kandy Muhammed Niyas", "expertise": [ "Reviewer Expertise Infectious Diseases", "Tropical Infections" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGandrala et al. has performed a prospective cohort study in which malaria patients were followed up for a median duration of 388 days. There were 20 cases (9.7%) of recurrences in 206 cases of P. vivax malaria, occurring at a median time of 83 days. The authors observed that recurrences were highest in the no primaquine (PQ) group and were least in the high dose daily PQ group. The authors also use a Kaplan-Mier analysis to show that there was a significant difference in the median time to recurrence among various PQ-based groups. However, the median time to recurrence in each of these groups is not mentioned.\nThe authors mention that no PQ-related haemolysis occurred in the cohort. However, it may be noted that no patient with low G6PD levels received a high dose of daily primaquine (0.5mg/kg). It is interesting to note that despite normal G6PD levels majority of patients did not receive a high dose PQ regimen. This data calls for awareness programmes among physicians regarding the optimal dosing of primaquine to prevent relapse. The authors rightly conclude that the results of the study cannot be generalised to primary care settings due to limited access to G6PD levels.\n\nIs the work 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": "126315", "date": "18 Mar 2022", "name": "Cindy Chu", "expertise": [ "Reviewer Expertise Plasmodium vivax", "radical cure with 8-aminoquinolines", "G6PD deficiency" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the prevalence and some epidemiologic aspects of Plasmodium vivax (Pv), Plasmodium falciparum (Pf), and mixed-species infection in Southwest India. The study is a prospective observational study and appears to be part of a prescription audit. There is a sub-analysis that compares the recurrence rates of Pv malaria among four groups: no primaquine, weekly primaquine, low dose primaquine, and high dose primaquine. Primaquine (PQ) was not supervised. The data are useful, however, the presentation of the study design, sample size, and methods need more explanation and some of the results need more precision so that the correct conclusions can be made.\nABSTRACT:\nThe conclusion states that recurrence in Pv is common especially when PQ prescription is inappropriate. I suggest using a more precise word instead of ‘inappropriate’.\n\nINTRODUCTION:\nThe last sentence of the introduction states that the objective of the study was to calculate the incidence of recurrence in patients with Pv malaria and find the impact of PQ prescription practices on recurrence. The authors then conduct a prospective observational study. However, this analysis appears to be a part of a prescription audit (as stated in the Discussion) rather than a study specifically designed to compare drug groups. This becomes more apparent in the study methods where it is stated: “the study aimed to record the prescription practices of treating physicians”. Moreover, the inclusion of Pf and mixed cases does not match the study objective or the sample size calculation. Please specify the study design and study outcome(s).\n\nMETHODS:\nIn the methods section, it is stated that the treating physicians decided on the dosage of antimalarials. Since PQ is being used, can the authors describe how G6PD testing is used before prescribing PQ? Which schizonticidal treatments are prescribed?\n\nRESULTS:\nIn the results section, 294 malaria cases were enrolled. The sample size is specified to be 206. Do the authors mean that 294 malaria cases were screened?\n\nFigure 1 describes the data by malaria species but the analysis is by drug group. Please modify so that the trial diagram is consistent with the data analysis.\n\nThe chi-squared test would normally be used to analyse 2x2 groups. The chi-squared test as used in the results section is for 2x4 groups (Pv yes/no and 4 different drug regimens). The Kaplan Meier figure is the correct analytic tool so the chi-squared result could be removed.\n\nDISCUSSION:\nThere is one paragraph dedicated to severe malaria, however, to this point in the manuscript, the outcome of the study was Pv recurrence. This paragraph (the Pf part) does not seem related the stated study outcome. Please clarify.\n\nWhat is the relevance of G6PD activity levels in this study for Pv recurrence? I understand the G6PD test would be used to determine which PQ regimen should be prescribed, but that has not yet been described in the manuscript and it should be added. There is a statement that only one patient in the no PQ group had low G6PD levels. Presumably any participant with low G6PD activity should have been in the no PQ or weekly PQ groups. Please explain.\n\nThe following sentence “This reflects the need to reinforce the importance of primaquine prescription in patients with Pv”, needs more specific language. Do the authors mean low G6PD levels reflect the need to reinforce the importance of PQ prescription? Or the need for G6PD testing before prescribing PQ?\n\nThe following sentence “The rates were lower in the daily PQ group even when they were used at a lower dose”, needs more specific language. What rates were lower? What is 'they' - the patients or PQ?\n\nSTUDY LIMITATIONS:\nNew Pv infections cannot be differentiated from relapses in this study. The study follow-up has a very long duration so recurrences at 1-2 years may or may not be related to the PQ dose. This should be included in study limitations and appropriate citations included.\n\nCONCLUSION:\nWhat do the authors mean by inappropriate PQ prescription? How do they determine whether recurrences are associated with inappropriate prescription when new infections cannot be differentiated from relapses or if results are affected by non-compliance? What prescription practices need to be improved? It seems that if the study design and methods are clearer, then the results and conclusion will be better supported.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "8039", "date": "07 Apr 2022", "name": "Kavitha Saravu", "role": "Author Response", "response": "This manuscript describes the prevalence and some epidemiologic aspects of Plasmodium vivax (Pv), Plasmodium falciparum (Pf), and mixed-species infection in Southwest India. The study is a prospective observational study and appears to be part of a prescription audit. There is a sub-analysis that compares the recurrence rates of Pv malaria among four groups: no primaquine, weekly primaquine, low dose primaquine, and high dose primaquine. Primaquine (PQ) was not supervised. The data are useful, however, the presentation of the study design, sample size, and methods need more explanation and some of the results need more precision so that the correct conclusions can be made. Reply: We appreciate the constructive suggestions by the reviewer. We have modified our manuscript as per the suggestions. A point-wise reply, explanation, and modification are as follows. ABSTRACT: The conclusion states that recurrence in Pv is common especially when PQ prescription is inappropriate. I suggest using a more precise word instead of ‘inappropriate’. Reply: The inappropriate word was changed to ‘Incorrect’. INTRODUCTION: The last sentence of the introduction states that the objective of the study was to calculate the incidence of recurrence in patients with Pv malaria and find the impact of PQ prescription practices on recurrence. The authors then conduct a prospective observational study. However, this analysis appears to be a part of a prescription audit (as stated in the Discussion) rather than a study specifically designed to compare drug groups. This becomes more apparent in the study methods where it is stated: “the study aimed to record the prescription practices of treating physicians”. Moreover, the inclusion of Pf and mixed cases does not match the study objective or the sample size calculation. Please specify the study design and study outcome(s). Reply: The study was designed as a prospective observational study where patients with malaria were enrolled at baseline. A follow-up component was added to estimate the percentage of patients with recurrence. Prescription audit was a main component of the study which is a type of observational study. Since there is a follow-up component built in the study to estimate the percentage of recurrences, we described our study as a prospective observational study. We included all malaria cases but the analysis was restricted to Pv as recurrences in Pf are not common. The primary objective was to study the recurrences in Pv. The sample size was also calculated based on that primary objective. Baseline parameters were collected at presentation and prescription patterns were recorded. The patients were followed up for recurrences. The inclusion of other cases was shown for the sake of comprehensiveness but as the reviewer suggests, they appear to be distractors. We have therefore removed all data on Pf and mixed malaria from this study. METHODS: In the methods section, it is stated that the treating physicians decided on the dosage of antimalarials. Since PQ is being used, can the authors describe how G6PD testing is used before prescribing PQ? Which schizonticidal treatments are prescribed? Reply: We appreciate the suggestion and have incorporated the following statement, “Chloroquine was used in all patients for the treatment of Pv malaria. In an ideal situation, G6PD levels should be done prior to initiation of primaquine. If the levels are within normal range, WHO recommends 0.5 mg/kg primaquine to prevent relapse in tropical areas. The national guidelines in India, however, recommend 0.25 mg/kg according to their last available guidance. If the levels are low, weekly primaquine is recommended for 8 weeks.” RESULTS: In the results section, 294 malaria cases were enrolled. The sample size is specified to be 206. Do the authors mean that 294 malaria cases were screened? Reply: We enrolled 294 cases with 206 being Pv. For the other patients who had Pf or mixed, the baseline parameters were recorded. However, as mentioned earlier, we have removed the data on Pf and mixed malaria for clarity as per the reviewer’s suggestion. Figure 1 describes the data by malaria species, but the analysis is by drug group. Please modify so that the trial diagram is consistent with the data analysis. Reply: The table shows the trial diagram in a more succinct manner so Figure 1 is removed to avoid confusion. Since we have removed Pf and mixed malaria, Figure 1 seemed redundant anyways. The chi-squared test would normally be used to analyse 2x2 groups. The chi-squared test as used in the results section is for 2x4 groups (Pv yes/no and 4 different drug regimens). The Kaplan Meier figure is the correct analytic tool so the chi-squared result could be removed. Reply: Chi-square test was meant for comparing severe and non-severe malaria at baseline. However, we have removed that from the main table as suggested. DISCUSSION: There is one paragraph dedicated to severe malaria, however, to this point in the manuscript, the outcome of the study was Pv recurrence. This paragraph (the Pf part) does not seem related the stated study outcome. Please clarify. Reply: Since we classified the baseline features into severe and non-severe, we had discussed this in the discussion section. The paragraph has now been removed as suggested. What is the relevance of G6PD activity levels in this study for Pv recurrence? I understand the G6PD test would be used to determine which PQ regimen should be prescribed, but that has not yet been described in the manuscript and it should be added. There is a statement that only one patient in the no PQ group had low G6PD levels. Presumably, any participant with low G6PD activity should have been in the no PQ or weekly PQ groups. Please explain. Reply: We agree with the suggestion and have added the following statement. “If the levels are within normal range, WHO recommends 0.5 mg/kg primaquine to prevent relapse in tropical areas. The national guidelines in India, however, recommend 0.25 mg/kg according to their last available guidance. If the levels are low, weekly primaquine is recommended for 8 weeks.” We classified the patients according to the G6PD levels because, in those patients where G6PD levels were not done, we couldn’t judge the correctness of the prescription choices. The idea was to show that G6PD levels were not even offered to some patients. On top of that, many patients were given incorrect prescriptions despite G6PD levels indicating otherwise. Since the treatment plans were not decided by the study team and the objective of the study was to study the correctness or incorrectness of the prescription, we couldn’t presume that the participant with low G6PD activity would be in the no PQ group or weekly PQ group. Therefore, we mentioned in the statement that only one patient in the no PQ group had low G6PD levels. PQ prescription pattern in patients with low G6PD has been depicted in Table 2. The following sentence “This reflects the need to reinforce the importance of primaquine prescription in patients with Pv”, needs more specific language. Do the authors mean low G6PD levels reflect the need to reinforce the importance of PQ prescription? Or the need for G6PD testing before prescribing PQ? Reply: We meant that G6PD levels should be done in all patients and the prescriptions should be guided by the G6PD levels. Those with low levels should be given weekly prescriptions and those with normal levels should receive 0.5 mg/kg prophylaxis. The following sentence “The rates were lower in the daily PQ group even when they were used at a lower dose”, needs more specific language. What rates were lower? What is 'they' - the patients or PQ? Reply: The recurrence rates were lower in the daily PQ group even when the PQ was used at a lower dose i.e 0.25 mg/kg. STUDY LIMITATIONS: New Pv infections cannot be differentiated from relapses in this study. The study follow-up has a very long duration so recurrences at 1-2 years may or may not be related to the PQ dose. This should be included in study limitations and appropriate citations included. Reply: We agree with the suggestion. The same has been added. CONCLUSION: What do the authors mean by inappropriate PQ prescription? How do they determine whether recurrences are associated with inappropriate prescription when new infections cannot be differentiated from relapses or if results are affected by non-compliance? What prescription practices need to be improved? It seems that if the study design and methods are clearer, then the results and conclusion will be better supported. Reply: We have revised the paper so that the conclusion makes more sense. The meaning of inappropriate prescription practices has been described in the discussion section. Examples included, not giving PQ or giving them at a lower dosage. The significant log-rank test between the groups suggests that poor prescription practices may have some role. Prescription practices are required to be aligned with the current evidence-based recommendations." } ] } ]	1	https://f1000research.com/articles/11-279
https://f1000research.com/articles/10-1223/v1	01 Dec 21	{ "type": "Research Article", "title": "Nursing home-sensitive conditions: analysis of routine health insurance data and modified Delphi analysis of potentially avoidable hospitalizations", "authors": [ "Sabine Bohnet-Joschko", "Maria Paula Valk-Draad", "Timo Schulte", "Oliver Groene", "Maria Paula Valk-Draad", "Timo Schulte", "Oliver Groene" ], "abstract": "Background: Hospitalizations of nursing home residents are associated with various health risks. Previous research indicates that, to some extent, hospitalizations of this vulnerable population may be inappropriate and even avoidable. This study aimed to develop a consensus list of hospital discharge diagnoses considered to be nursing home-sensitive, i.e., avoidable. Methods: The study combined analyses of routine data from six statutory health insurance companies in Germany and a two-stage Delphi panel, enhanced by expert workshop discussions, to identify and corroborate relevant diagnoses. Experts from four different disciplines estimated the proportion of hospitalizations that could potentially have been prevented under optimal conditions.\n\nResults: We analyzed frequencies and costs of data for hospital admissions from 242,236 nursing home residents provided by statutory health insurance companies. We identified 117 hospital discharge diagnoses, which had a frequency of at least 0.1%. We recruited experts (primary care physicians, hospital specialists, nursing home professionals and researchers) to estimate the proportion of potentially avoidable hospitalizations for the 117 diagnoses deemed avoidable in two Delphi rounds (n=107 in Delphi Round 1 and n=96 in Delphi Round 2, effective response rate=91%). A total of 35 diagnoses with high and consistent estimates of the proportion of potentially avoidable hospitalizations were identified as nursing home-sensitive. In an expert workshop (n=16), a further 25 diagnoses were discussed that had not reached the criteria, of which another 23 were consented to be nursing home-sensitive conditions. Extrapolating the frequency and mean costs of these 58 diagnoses to the national German context yielded total potentially avoidable care costs of €768,304,547, associated with 219,955 nursing home-sensitive hospital admissions. Conclusion: A total of 58 nursing home-relevant diagnoses (ICD-10-GM three-digit level) were classified as nursing home-sensitive using an adapted Delphi procedure. Interventions should be developed to avoid hospital admission from nursing homes for these diagnoses.", "keywords": [ "geriatrics", "health services research", "potentially avoidable hospitalization", "hospitalization", "long-term care", "nursing homes", "nursing home-sensitive conditions" ], "content": "Introduction\n\nHospitalizations pose various risks to nursing home residents. Residents may experience reduced functioning upon their return to the nursing home (post-hospital syndrome).1 Hospital-acquired conditions may occur, for example, when specific hospital pathogens lead to infections.2 Adverse drug effects can also develop, such as medication overdose or administration of the wrong medication.3 Cognitively impaired patients often experience loss of orientation as well as confusion in an unfamiliar hospital setting.4 Therefore, there should be intense efforts to avoid unnecessary hospitalizations amongst nursing home residents in health systems across the globe.\n\nThe issue of preventing hospital admissions has been intensely discussed regarding ambulatory care-sensitive conditions, for which specific indicator sets have been developed to measure the extent and preventability.5–13 Ambulatory care (or primary care)-sensitive conditions are those which by expert consensus should not require a hospital admission in the presence of effective primary care. Conditions frequently referred to in existing indicator sets include asthma, chronic obstructive pulmonary disease, congestive heart failure, diabetes mellitus and hypertension, among others.14 Indicator sets to measure ambulatory care-sensitive conditions received substantial attention in policy, research, and clinical practice.15 Such indicator sets may identify variations in regional hospital admission rates, which can lead to investigations about appropriate care structures, as well as interventions to reduce unnecessary hospital admissions.\n\nWhether ambulatory care-sensitive indicator sets can be applied to hospital admissions from nursing homes has been subject to debate. One argument against the use of ambulatory care-sensitive indicator sets is that nursing home populations differ significantly from the general population. The age structure of nursing home residents, the number of comorbidities, the geriatric disease spectrum, as well as the healing process of the elderly population and typical medical interventions, differ from those of community-dwelling residents.10,12 Moreover, the care setting in long-term care facilities, where care is provided by trained nurses and allied professions 24 hours a day, contrasts with community or outpatient care. The characteristics of the nursing home resident population as well as the long-term care setting influence the type of diagnosis that may require hospital admission, its frequency, and its preventability. Therefore, as existing indicators regarding ambulatory care-sensitive conditions are unlikely to be applicable to long-term care settings or patient populations who are nursing home residents, others have urged the need to develop additional nursing home-sensitive indicator sets.16\n\nVarious studies have investigated potentially preventable hospital admissions from the long-term care situations/nursing home setting.8,16–19 Earlier studies conducted medical chart reviews and convened panels to gauge the preventability of a hospital admission. Using such an approach, Ouslander et al.8 estimated that 67% of hospital admissions in the USA were preventable. Others, such as Walker et al.17 in Canada, adopted existing indicator sets for ambulatory care-sensitive hospital admissions, and used administrative databases to calculate that 55% of hospital admissions were preventable. In Germany, Leutgeb et al.10 compared ambulatory care-sensitive hospital admission rates amongst nursing home and community-dwelling residents, and found that admission rates were significantly higher amongst nursing home residents. Allers et al. cautioned in their systematic review that interventions to reduce hospitalization of nursing home residents should be tailored to health care systems: for policy and clinical practice, it is critical that indicator sets are based on consensus of experts working in the field, that they reflect the characteristic of the national/regional nursing home population and settings, and that they take into account the available health system resources, such as the nursing skills available in the facility, or access to family doctor and specialist visits to the nursing home.20\n\nIn order to inform policy debate and practical improvement actions to reduce hospital admissions from nursing homes in Germany, this research project aimed to address the following questions: 1. How often are nursing home residents treated in hospital and what are the main diagnoses and associated costs of these hospital admissions? 2. Which hospital cases are nursing home-sensitive, i.e., at least partially preventable under optimal conditions? 3. What is the impact of the estimated preventability, in case these optimal care conditions existed, at the national level?\n\n\nMethods\n\nFor this study, a quantitative mixed-method approach was used, in three consecutive phases. First, an analysis was conducted based on health insurance claims data to identify frequent diagnoses amongst nursing home residents admitted to a hospital. The recommendations of the Working Group for the Survey and Utilization of Secondary Data for the analysis of German health insurance claims data were considered.21 These include data quality issues and recommendations on contractual details between researchers and data owners, among others. Second, a RAND/UCLA Appropriateness Method22 was executed, in which a Delphi expert panel and expert workshop were combined, to reach expert consensus regarding the extent to which hospital admissions might be prevented. A randomized clinical trial was not appropriate for our nursing home resident population: the decision to hospitalize or not, often influences the patient’s survival itself in this vulnerable population. Our method yielded the best available scientific evidence with the collective judgment of experts regarding the appropriateness of hospitalization in nursing home residents. And third, an analysis of routine health insurance data was extrapolated to the total German nursing home resident population, based on which the expenses associated with potentially preventable hospitalizations were estimated. The quantitative data analyses were performed with Microsoft Excel 2016 and IBM SPSS Statistics 26.\n\nAn ethical approval was not required and therefore waived. We relied on secondary use of anonymous, aggregated routine data and expert health professionals’ assessments. We did not conduct human research, interventional and non-interventional clinical studies nor clinical trials. We did not collect nor use data in the form of direct health care data, nor did we use (residual) human material for scientific purposes.\n\nAll experts provided their written informed consent to participate voluntarily. The Delphi procedure was conducted pseudonymously: towards the end of the data collection, experts were requested to provide their name and email address in a separate database, so that they could be reached for subsequent Delphi rounds and payment of the incentive. Neither the name nor e-mail address given could be linked to the data collection, although the names of the participants were known to the research team. Nonetheless, the assessments towards the potential of preventability were completely anonymized and uninfluenced by the research team. The voluntary nature and pseudonymity of participation was pointed out, together with complete information on the EU General Data Protection Regulation.\n\nWe obtained claims data from six statutory health insurance companies, which together provide a representative data set of about 29.6% of all nursing home residents in the German population. A data request was agreed between the researchers and health insurance companies, whereupon aggregated data on hospital discharge diagnoses were provided. Included were insured persons living in a nursing home in the calendar year 2017, with a hospital discharge diagnosis (coded as ICD-10-GM, three digits) that occurred in more than 0.1% of this population. Nursing home residents whose insurance period ended in 2017 due to a change in health insurance fund, or who did not have a valid insurance period were excluded from the analysis, to be able to consider insurance utilization without gaps. Insured persons who provided implausible information were also excluded from further analysis. Deceased insured persons, however, were not excluded, because otherwise serious illnesses associated with death in hospital might have been underrepresented, and because nursing home patients have a higher risk of death in hospital than comparable populations.23 For the purposes of this study, nursing home residents are those insured persons for whom a start date prior to Jan 1, 2017, was documented for both a need for long-term care and for full inpatient care in an approved nursing home, pursuant to Section 43 of the German Social Code, Book XI, and who – except for deceased insured persons - consistently had these care services in 2017.\n\nThe hospital discharge diagnoses had to have a discharge date within the calendar year 2017. This excluded cases who were admitted to hospital in 2017 or before, but discharged in a later calendar year, which was considered unproblematic as, in contrast, cases were included that were admitted before 2017 but discharged in 2017. In addition, the average costs per hospital case were evaluated. Here, the average total amount paid by social health insurance for the case was used, not just the cost share that would result from the diagnosis-related group (DRG) of the principal diagnosis. The total amount was evaluated entirely on the discharge date. The list of principal hospital diagnoses was aggregated from all insurance companies and sorted in descending order for the analysis, to determine ICD-10 diagnoses with highest frequency and cost. The exact data request is included in our Extended data for this publication (see Data availability section).\n\nWe conducted the modified Delphi study between December 2019 and July 2020 as a two-round online tool followed by an expert workshop in September 2020, combining the strengths of the anonymous questioning of experts with the deeper insight emerging from the discussions at the moderated workshop. The online questionnaire and the expert workshop background information and workshop task are included in the Extended data.\n\nIn the online tool, experts of four different disciplines (physicians working in outpatient and inpatient care, nursing professionals, and researchers) were asked to estimate the proportion of potentially avoidable hospitalizations identified in the health insurance claims data. The assessment of preventability was under the assumptions of optimal, but still realistic conditions: access to trained personnel, resources and infrastructure for monitoring and nursing, and cooperation agreements with ambulatory general and specialist care providers where needed. For each discharge diagnosis, a short description and a link to the official definition on the German ICD-10 classification were added, to facilitate the assessment for all experts. ICD-10 codes were restricted to the three-digit level to ensure comprehension and usability of the list of diagnoses by all professions. The differentiation for subgroups of the ICD-10 three-digit code was indicated by experts via the numerical estimation of the potential preventability. The experts made their assessments on a scale from 0% to 100% in 5% increments (0% meaning hospitalization was unavoidable, 100% meaning all patients could have been treated in the nursing home). In addition, Delphi participants were provided the opportunity to offer voluntary comments for each ICD-10 code, which became mandatory in the event that experts were unable to give a quantitative estimate of the preventability. LimeSurvey (Version 3.27.20+211012) was selected as the tool for online data collection and was customized specifically for this study. Feedback was obtained and processed from four experts regarding the questionnaire tool, to improve overall presentation and instructions for the experts as well as the presentation of the list of ICD-10 codes. Comprehension and practicability of the online evaluation were tested in a pilot group, technical errors were corrected and ambiguities in the content were clarified.\n\nTo recruit the experts, we used the Delphi funnel, a panel management model, according to Donohoe et al.24 Funneling constituted in first identifying and selecting potential experts or expert groups, after which we approached them. Additionally, gatekeepers were identified to help pinpoint those individuals who would have knowledge of the topic under study.25 The professional networks of the research team were consulted, and potentially eligible experts were contacted through posts on websites and social media such as LinkedIn, via newsletter announcements, through personal email as well as email distribution lists, on personal recommendation, by phone, and through personal visits.\n\nA Delphi group size depends on group dynamics for arriving a consensus among experts.26 It is also subject to the expected loss-to follow up because of attrition.24 The literature recommends 10 to 1826 or, in case of high attrition, up to 90 experts24 on a Delphi panel. The Delphi panel should be large enough to reach a sufficient number of perspectives from the “inside”.26 A detailed expert selection criteria list was developed, as purposeful panelist selection can reduce attrition due to loss of interest or frustration.24 For our study, experts should either have practical experience from the sectors involved in the treatment of nursing home residents, play an important role in the decision about hospital treatment, or have published scientific research related to the care of elderly patients. According to these recommendations and in order to ensure a large, methodologically robust and balanced sample of experts for the consensus ratings, we chose to reach out to heterogeneous experts from four different disciplines and planned to recruit 100 experts: 30 outpatient/clinical physicians each, 30 nursing professionals and 10 scientists.\n\nSecond, we supported the participants’ engagement by distributing an introductory package along with the invitation to participate in the assessment.24 The preparation of the experts was important in order not to compromise the response rate in future rounds.25 Therefore, before the first Delphi round, experts were informed about the content, the objectives of the research project, what they would be asked to do, how much time they would be expected to contribute, what use would be made of the information they provided, the voluntary nature of participation, and data confidentiality.25 Additional information was also given to pseudonymity of the study (see the Ethical considerations section). After giving their informed consent, experts were invited to participate in this study.\n\nThird, results of the first round should be distributed to the panel.24 Therefore, the data collection tool for the second round integrated the results of the expert ratings from the first round. The RAND/UCLA-Appropriateness Method22 is visualized in Figure 1. Expert panel management for the Delphi process was integrated as shown in the Delphi funnel.24\n\nSource: Delphi funnel24; minimum group size.32–35\n\nTo increase the willingness to participate, a compensation of 100€ was offered for successful participation in both rounds of the survey. For each of the Delphi rounds, the acquisition of experts in that group was discontinued as soon as the required number of participants in each group was reached.\n\nThe data from the first Delphi round were analyzed as follows: the median and its interquartile range (containing 50% of all assessments around the median) was reported for each ICD-10-GM three-digit code. Based on this information, the participants were asked to quantify the proportion of potentially avoidable hospitalization again in the second round. If the estimation of the proportion either fell outside the given interquartile range or could not be estimated, the comment field was mandatory again; otherwise, the participants could include extra comments voluntarily. Comments would reveal information about the reasons behind a deviating answer, possible disruptors or problematic conditions complicating the avoidance of hospitalization. Only the comments from the first and second rounds of those ICD-10 codes reaching relevant but dispersed preventability estimates after both online Delphi rounds (conditions described in workshop section) were looked at. This was solely done to be able to identify possible difficulties in the estimation of preventability to be discussed at the expert workshop. Content analysis on this subpart of comments identified themes, which were grouped and described close to the original text, to stimulate the subsequent discussion in the expert workshop.27 We followed SRQR guidelines for this small qualitative component of our research: the analysis of a subpart of free text comments collected as part of our Delphi study. However, this only corresponds to a fraction of our research, the much larger part of our study being based on quantitative consensus techniques and the analysis of administrative health insurance claims data. These were analyzed and reported according to STROBE.\n\nA multidisciplinary expert workshop was convened to discuss those diagnoses, for which the Delphi panel generated highly dispersed data. For face-to-face discussions, it is recommended to have a panel size that permits sufficient diversity, while ensuring that all have a chance to participate.22 This panel consisted of sixteen experts. Again, all four disciplines were represented in equal proportions. A total of 10 out of 16 experts had taken part in the online Delphi rounds. The remaining six experts were provided with the same information on the project from the online rounds. All experts received the same information about the goal and content of the workshop, and all gave their informed consent to take part in course of registration for the online workshop. ICD-10 codes with an assessed preventability potential (median ≥ 75%) and a narrow range of dispersion (dispersion around median ≤ 15%), were considered directly eligible for the list of nursing home-sensitive conditions. In case the preventability potential of at least 75% was not comprised in the range of 75% of all expert assessments, the ICD-10 codes were excluded. For all other ICD-10 codes, statistical data (median, modal values, dispersion parameters, bar charts) and categorized comments from the Delphi questionnaire were provided for the expert workshop. The ICD-10 codes were thematically clustered where possible and distributed across three working groups, while the experts were assigned to the working groups according to their expertise.\n\nAfter preventability estimates were corroborated, the composition and the preventability estimates of these conditions were compared to the results of Sundmacher et al.7 They developed a list of ambulatory care-sensitive conditions for the outpatient-German setting. They sorted 258 ICD-10 conditions, expected to be ambulatory care-sensitive by experts, into 40 groups according to disease categories. Each group comprised three- and four-digit ICD-10, because, in some ICD-10, potential preventability was attributed to subcategories only. After that, the potential preventability for each of these groups was estimated. The estimates of preventability for these groups of ICD-10 ranged between 55-96%.7 Of these groups, 22 had an estimated preventability of more than 85% and were posed as core ambulatory care-sensitive conditions.7 The more common ICD-10 discharge diagnoses among nursing home residents, i.e., with a frequency of at least 0.1%, as well as the nursing home-sensitive conditions we found in our study, were then compared to both groups: the ambulatory care-sensitive conditions and the core ambulatory care-sensitive conditions.\n\nFollowing the expert consensus process, based on which consensus on a list of nursing home-sensitive hospital admissions was obtained, we used health insurance claims data to calculate the total amount of costs associated with preventable hospital admissions from nursing homes in Germany. We used data from six health insurance companies to assess the total costs associated with the hospitalization case for each diagnosis, and data from the Federal Statistical Office to identify the total number of people living in long-term care facilities in Germany.28 We calculated the number of hospital cases and multiplied those with the average case costs for each diagnosis, which resulted in the total costs of cases with the respective discharge diagnoses. This, further multiplied by the proportion of potential preventability then resulted in the total amount of health care system costs that could potentially be avoided (given optimal care conditions).\n\n\nResults\n\nWe received data from six German statutory health insurance companies according to our data request, covering information on 242,236 nursing home residents. Where data was provided in deviating format, we cleaned and prepared the datasets so they could be aggregated into a global data set. Data included the ICD-10 code, the proportion of hospital admission from nursing-home/long-term care, the gender and age distribution (categorized in 5-year strata) and the mean total cost per case. The age and gender distributions are presented in Table 1 and the gender-differentiated number of hospitalizations, hospitalization proportions and ratios are presented in Table 2. The data received from six health insurance companies were merged and the results are presented in Table 3. This table shows all the hospital discharge diagnoses with a frequency of at least 0.1% in our sample, sorted according to their frequency in descending order, together with a short description of the ICD-10 code, case counts, percentage as well as cumulative percentage of hospital discharge diagnoses from long-term care, and average cost per case for each ICD-10 code.\n\nOver 85% of nursing home residents in our sample were at least 65 years old (206,503 persons of the total sample; Table 1). In the aggregated data set, 44% of fully insured nursing home residents were hospitalized. Thus in 2017, there were 79 hospital admissions for every 100 nursing home residents (Table 2). The percentage of persons with one or more hospitalizations was slightly higher among men (48%) than among women (44%; Table 2).\n\n NHR: nursing home residents.\n\nIn total, the top 25 most common discharge diagnoses accounted for 97,378 cases (Table 3). They covered about half of all hospital cases. About one-third of all hospital cases accounted for one of the following diagnoses: heart failure, pneumonia, fracture of the femur, dehydration, diseases of the urinary tract, intracranial injuries, sepsis, cerebral infarction, and epilepsy. Diseases of the central and peripheral nervous system most frequently led to inpatient treatment of nursing home residents (18%), closely followed by diseases of the respiratory tract (17%) and the digestive tract (15%). The distribution of treatment costs showed a partly different ranking. Neurological diseases such as stroke and epilepsy accounted for the largest share of costs (21%), followed by diseases of the musculoskeletal system (17%), the respiratory system (16%), and the cardiovascular system (13%). From a macroeconomic perspective, it is particularly interesting to note that in the individual organ system groups, a particularly high proportion of costs could be allocated to a few discharge diagnoses. These included fractures in musculoskeletal diseases (over 80%) and pneumonia in respiratory diseases (almost 60%).\n\n* Table 3 is based on the data request (see Extended data in the data availability section).\n\nThe average cost of a nursing home resident hospital case in 2017 in the sample was €4,030 (191,174 hospitalizations with hospital costs of €770,368,090; Tables 3 and 7). Extending Table 3 to the defined cut-off point of 0.1% share of all hospital cases, 117 different ICD-10 diagnoses were considered for the Delphi study (totaling 157,322 cases).\n\nWe were able to exceed our recruitment goal of n = 100 experts for the Delphi study, as defined in the protocol, to a number of 107. Of the 107 experts participating in the first round of the Delphi study, 104 (97.2%) had indicated their name and e-mail address and were invited to the second round. Of these, 96 (92.3%) followed the invitation (Table 4) and 95 completed the second round successfully so that responses were usable (effective response rate 91%). There were no significant differences in age, gender, and years of experience between responders and non-responders.\n\nDetails of the experts’ estimate of the preventability of hospitalizations from the nursing home following the Delphi rounds and the expert workshop are reported in Table 5. In the first Delphi round, experts estimated the proportion of potentially avoidable hospitalizations for 117 ICD-10 codes which were identified in the previous step, based on the analysis of routine health insurance data. Experts were asked to provide their estimations assuming optimal structural and care conditions. Where ICD-10 codes were not assessed by six or more experts, we reviewed the comments to identify why experts had difficulties in assessing the potential preventability of individual ICD-10 codes. This concerned 20 of the 117 ICD-10 codes (17%). To avoid further assessment difficulties, notes were included in the explanations for these ICD-10 codes, or their lay-out was changed for the second Delphi round. Furthermore, ICD-10 codes were planned to be excluded when at least 75% of the experts estimated the preventability as zero in the first round. This condition was never met. Secondly, ICD-10 codes were planned to be combined if the diseases were very similar (in terms of symptoms, diagnosis, prognosis, treatment) and the proportions of potential preventability were nearly identical. Two conditions for “nearly identical potential preventability” had to be fulfilled: the medians of the estimated preventability should not differ by more than 5% and the limits of the interquartile range had to be less than 10% apart). Neither conditions were met sufficiently. Therefore, for the second round, all 117 hospital discharge diagnoses were assessed again.\n\n1 p-value of the Kruskal-Wallis test; H0: There was no difference in the department-specific assessment of avoidance potential.\n\n* 21 ICD-10-GM three-digit tending to have statistically significantly different estimates of potentially avoidable hospitalization by specialty: 0.05≤Kruskall-Wallis p-value<0.10.\n\n** 34 ICD-10-GM triplicates with statistically significantly different assessment of potential avoidable hospitalization by specialties: Kruskall-Wallis p-value<0.05.\n\n^ ICD: International Classification of Diseases, 10th revision, German Version (ICD-10-GM), a direct translation in German language of the ICD-10 of WHO.\n\n° R2: second Delphi round.\n\n°° R1: first Delphi round.\n\n+ N: Number of assessments in the second Delphi round.\n\n++ 258 ambulatory care-sensitive ICD-10 conditions, comprising three- and four-digit codes, grouped according to disease categories in 40 groups; estimated preventability between 55-96%.7\n\n+++ 22 core ambulatory care-sensitive condition groups; estimated preventability at least 85%.7\n\nComparing the responses to the Delphi rounds, we found no differences in the median estimates of the preventability of the 117 ICD-10 codes; however, the scatter range decreased significantly. The width of the interquartile range decreased, for all ICD-10 codes together, from 42.3% to 5.5% on average. Comparing the respective participants’ assessments in both online questionnaire Delphi rounds, both assessments were very close together: the median difference between round 1 and 2 was maximum 5% for 114 of the 117 ICD-10 codes, and maximum 10% for the three remaining ICD-10 codes.\n\nFor 34 of the 117 ICD-10 codes, the four groups of experts gave statistically significant different preventability estimations. On average, estimations differed by only 5%, and by a maximum of 15% for individual ICD-10 codes. In most of these 34 cases, the clinicians indicated slightly lower estimates. A statistically significant difference of 5% in the median between men (median estimate at 25%) and women (median estimate at 30%) was found for only one of the 117 ICD-10 codes. All age groups of experts (under 40 years old (n = 29), 40-49 years old (n = 20), 50-59 years old (n = 25), and 60 years or older (n = 20)) were consistent in their estimates of potential avoidability. For 114 of 117 ICD-10 codes the difference in the estimated proportion of potentially avoidable hospitalizations between individual age groups (median) amounted to maximum 5%. The maximum difference found was 10% (only for one ICD-10 code). Age groups differed in their estimates for six conditions, although for these the average assessments were only 2.5 % and never more than 7.5% apart.\n\nFor 38 ICD-10 codes, the potentially avoidable hospitalization rate was estimated to be at least 75%; for 12 ICD-10 codes it was estimated to be at least 90%. For 35 of these 38 ICD-10 codes, the unambiguous assessment of a condition as potentially nursing home-sensitive was already clear after the second round of questioning: the range of dispersion around the median of three quarters of all expert’s assessments for the respective ICD-10 code was 15% or less. The three for which the latter did not apply were discussed at the expert workshop. Further, a total of 22 ICD-10 codes had a median preventability proportion below 75%, but their scatter spectrum for three quarters of all assessments contained the relevant preventability proportion of 75%, signaling a relevant but still ambiguous preventability proportion. Thus, a total of 25 ICD-10 codes were prepared for the expert workshop consensus process (Table 5; ICD-10 codes discussed in workshop are shown in italic font).\n\nOverall, after the workshop with 16 experts, all proportions for potentially avoidable hospitalizations could be corroborated, and 58 ICD-10 codes with an estimated potential avoidability of at least 70%, were selected for the list of nursing home-sensitive conditions. In Table 5 the nursing home-sensitive conditions are shown in the greyed table part. Table 6 shows these conditions sorted by disease category.\n\n^ ICD: International Classification of Diseases, 10th revision, German Version (ICD-10-GM), a direct translation in German language of the ICD-10 of WHO.\n\nFor the comparison of nursing home-sensitive diagnoses with ambulatory care-sensitive diagnoses, the occurrence of the 117 ICD-10 codes in the ambulatory care-sensitive groups was reviewed. In Table 5, the last three columns were added to show the results of this review. Comparing the 58 nursing home-sensitive conditions with the ambulatory care-sensitive conditions, it appeared that only 28 three-digit and seven four-digit nursing home-sensitive ICD-10 conditions were also included in the ambulatory care-sensitive ones. Therefore, 60% (35/58) of the nursing home-sensitive conditions were also partly or completely ambulatory care-sensitive, and 40% were not.\n\nExtending this comparison to all common conditions in nursing home residents, only 29 three-digit and another 10 four-digit of the 117 ICD-10 codes were ambulatory care-sensitive. Thus, only 33% (39/117) of the ICD-10 codes relevant to the nursing home population were partially or wholly ambulatory care-sensitive, and 67% were not.\n\nSundmacher published preventability estimates for the core ambulatory care-sensitive groups of ICD-10 codes. A total of 27 (47%) nursing home-sensitive conditions belong to these core ambulatory care-sensitive conditions (23 completely [three-digit ICD-10] and four only partially [four-digit ICD-10]), and 53% do not. Of all 117 ICD-10 codes, 31 (26%) conditions appeared partly (seven four-digit ICD-10 codes) or wholly (24 three-digit ICD-10 codes) in this core list, and 74% do not.\n\nOn the other hand, 178 of the 258 ambulatory care-sensitive hospitalizations (three- and four-digit level) were not listed in the nursing home-sensitive list (69%).\n\nThe preventability estimates were only known to the authors for the core ambulatory care-sensitive condition groups. Therefore, for only 31 out of 117 ICD-10 common nursing home hospitalizations, the preventability potential for the nursing home common as well as nursing home-sensitive ICD-10 codes were compared to their counterpart in the core ambulatory care-sensitive groups. For the 27 nursing home-sensitive conditions, the minimum and maximum differences between both settings was −9% and +23%, respectively. For 12 ICD-10 codes, the preventability potentials of both lists were only 5% apart. For the remaining four nursing home common conditions, the difference was −18% to −41% apart.\n\nThus, our results show that nursing home-sensitive conditions are to be distinguished from ambulatory care-sensitive conditions: both diagnoses and preventability estimates differed between the settings.\n\nIn 2017, there were 3.4 million persons in need of long-term care in Germany, of which 818,289 were nursing home residents.28 With 242,236 insured persons in inpatient care, our study population represented about 29.6% of all nursing home residents in Germany. Our sample yielded 191,174 hospital cases in one year. The annual incidence was therefore around 0.79 hospital cases per insured person in stationary care (191,174/242,236). Table 7 shows the extrapolation of the results from the analysis of routine health insurance data for Germany. For this purpose, the total costs per ICD-10 code were first calculated, weighted according to their proportion of cases (number of casescost per hospital case). The total cost for each ICD-10 code was than multiplied by the extrapolation factor and summed to obtain the total costs incurred in Germany. Calculating the costs for nursing home-sensitive conditions was done accordingly. Total costs per nursing home-sensitive condition were multiplied by the proportion (in %) of potentially avoidable hospitalizations agreed to during the Delphi process, and then summed to allow a weighted calculation by case proportion and prevention potential. This was done to estimate the number of cases and costs that would potentially be avoidable, if certain cross-sectoral and structural conditions were in place for the provision of needs-based care for nursing home residents. Table 7 shows the sample results, the extrapolation for Germany and finally the calculations respective to nursing home-sensitive conditions.\n\n Multiplication factor for the extrapolation of results of the health insurance data analysis to the situation in Germany: number of nursing home residents in Germany/sample size (818,289/242,236).\n\n^ ICD: International Classification of Diseases, 10th revision, German Version (ICD-10-GM), a direct translation in German language of the ICD-10 of WHO.\n\nThe extrapolation forecasts a total of about 646,000 hospital cases per year for all nursing home residents in Germany (818,289/242,236*191,174) with a total cost expense for hospital admissions in the nursing home population of over 2,600,000,000€ (2,6 billion €). Approximately 220,000 hospitalizations might have been prevented if interventions were implemented in favor of greater needs-based care for nursing home residents. If measures were effective, the expenses required to establish them could be met from the funds saved (about three quarters of a billion Euros). The relevance of common nursing home hospitalizations as well as nursing home-sensitive conditions is shown in Figure 2.\n\n\nDiscussion\n\nWe used routine health insurance data from 242,236 nursing home residents to assess frequencies and costs of hospital admissions amongst nursing home residents; we identified 117 hospital discharge diagnoses which had a frequency rate of at least 0.1%. In a two-stage Delphi study, 107 and 96 experts in round 1 and 2, respectively, estimated the potential to avoid a hospitalization for these diagnoses. After two Delphi rounds and an expert workshop, we were able to identify 58 diagnoses considered to be nursing home-sensitive conditions in the context of the German health care system. The frequency of hospital admissions for these diagnoses and associated costs to the care system were substantial, and warrant further discussion on strategies to decrease hospitalizations for these diagnoses.\n\nThere are few comparable studies on nursing home-sensitive hospitalizations. Most of the research in this field focused on ambulatory care-sensitive hospital admissions, which cannot be directly applied to the nursing home context as the nursing home population differs regarding frequency of diseases, healing process, nursing care and systematic care conditions. However, the seminal studies by Purdy et al.,5 Ouslander et al.8 and Walker et al.17 are noteworthy, for developing the concept of avoidable hospital admissions and using various methods established in health services research, from medical chart reviews to analysis of administrative datasets, to quantify the impact of avoidable hospital admissions, and indicate that a large proportion of hospital activity might in fact be avoidable. For the German health system context, Leutgeb et al.10 demonstrated that hospital admission rates are much higher amongst nursing home residents compared to community-dwelling residents.\n\nThe number of people in need of nursing home care has steadily increased over 17% in the last ten years in Germany and, considering the country’s population is aging, it is expected to increase further.29 Similar trends hold true for other high-income countries.30\n\nThis is the first study to compare nursing home-sensitive with ambulatory care-sensitive conditions for the German setting. It showed that nursing home-sensitive conditions are to be distinguished from ambulatory care-sensitive conditions: 74% and 53% of common nursing home and nursing home-sensitive conditions, respectively, do not appear on the ambulatory care-sensitive list; for the core ambulatory care-sensitive list these numbers were 67% and 40%, respectively. In contrast, 69% of all ambulatory care-sensitive conditions did not appear on the list with 117 common nursing home conditions. Second, the number of hospitalizations that could have been prevented differed in case of optimal care conditions for the nursing home and outpatient setting, respectively. This may be due, in part, to the fact that Sundmacher et al.7 estimated preventability for groups of ICD-10 conditions, clustered by disease category, while we estimated the preventability for every ICD-10 code individually. Although ICD-10 conditions and their estimated preventability differed in both health care settings, the main goal of these lists lies in raising awareness for which hospitalizations may be preventable.\n\nThe focus on nursing home-sensitive hospital admissions is therefore a critical issue for health care organization and reform. In addition to the financial implications associated with the potential of the diagnoses on our consensus list to avert hospitalization, reducing hospital admissions amongst nursing home residents would have a substantial impact on the person-centeredness of health care and quality of life of residents.\n\nA strength of our study is that we were able to combine the analysis of routine health insurance data with a two-stage Delphi study and expert workshop. The health insurance data covered nearly 30% of the statutory health insured persons in Germany. For the Delphi study, we were able to recruit a high-calibre expert group and succeeded in ensuring a very high response rate of over 90%. Our subgroup analysis demonstrated that the assessment of experts was robust and not biased towards specialization, age or gender. This supports widespread recognition and applicability of our indicator list. By using the RAND/UCLA Appropriateness Method,22 enhancing the Delphi method with an expert workshop, we were able to introduce direct interaction between experts as in other consensus development methods,31 thereby combining the strengths of the Delphi methods with others.\n\nThe external validity of the results of the modified Delphi method, in general, is dependent on the representativeness of the panel of experts. The validity of the Delphi method depends, among other things, on the response rate,25 with response rates between 51%-80% being recommended in the literature.32–35 The commitment of participants to complete the Delphi process is often related to their interest and involvement with the question being examined.25 In our study, we observed a high intrinsic motivation of the experts to participate, as many waived the incentive offered, and as evidenced by the extremely high response rate of 91%, with unlikely bias resulting from the minor loss-to follow-up. The validity of the Delphi method also depends on the included experts.26 The more diverse and heterogeneous the expert panel is, the higher the quality of the decisions.36 Four disciplines were represented in our panel, but no lay persons, such as nursing home residents or relatives. Another factor to consider when assessing the validity of the findings from the Delphi study is selection bias. If participants dropped out because of pseudonymity, there would only be a selection bias, if these individuals assessed the avoidance potential differently than the participants in this study. This is highly unlikely, as our results across Delphi rounds as well as various disciplines were very stable. The stability of the assessments in the individual Delphi rounds is considered more important than the response rate with regard to the occurrence of consensus.37 We observed specialty-specific statistically significant, though minimal and therefore irrelevant, differences in estimated hospitalization preventability after the second round of surveys, for a subset (n= 34) of the 117 ICD-10 codes. Clinicians had slightly lower avoidability estimates, which could be due to the fact that they see the more serious cases in the clinic and are therefore more cautious with their assessment. Gender and age effects in estimating the proportion of potentially avoidable hospitalizations were low. Because of multidisciplinarity, the stable assessments of and the low dispersion in the estimates of the potential avoidability of hospitalization of nursing home residents and the relatively high number of included experts compared to other Delphi methods, it is rather unlikely that another panel would have come to different results. For these reasons, together with the high response rate, we expect the list of 58 nursing home-sensitive conditions to be generalizable.\n\nThe key limitation of our study, like comparable studies, is that preventability assessments were made assuming optimal structural and nursing home care conditions. We are aware that these conditions are currently not always met, and that interventions and improvement efforts are required to reduce or avoid hospital admission in the current health care setting.\n\nAll researchers were qualified health services researchers, familiar with the different methodological components of our study. However, the relationships between researchers and study participants were to a large extent limited to time-restricted conversations in a series of workshops. Given that participants were specialists invited for their specific expertise, researchers had no specific assumptions or presuppositions about the experts’ input. In the manuscript, no further interpretations of the experts’ inputs have been added.\n\nThe expert workshops and Delphi questionnaire – the only component of the research where qualitative comments were made – were all conducted online. No salient contextual factors impacting the research were identified.\n\nOur study has various implications for policy, practice and research. Similar to the research on ambulatory care-sensitive hospital admissions, we assume that our study will lead to substantial debate and controversy about variations on nursing home-sensitive hospital admission rates and on the policy response to this variation. Such debate is likely to lead to proposals for improvements of the organization of the health care delivery system, and to constitute new indicators to monitor health system performance. For all stakeholders (e.g., medical and nursing providers, policy makers, health economists), the list of nursing home-sensitive conditions can inform the development of interventions and facilitate local quality improvement efforts. Practitioners and researchers should collaborate to identify the type of interventions (including personnel, staffing, skill mix, continuing professional education, infrastructure/resources, technology) required to reduce hospital admissions. Research should further address a costing of these interventions and calculations on the headroom (the maximum cost of the intervention to be cost-effective) to inform managers of nursing homes, hospitals and delivery systems. Finally, internationally comparative research should aim to identify a robust core basket of indicators for nursing home-sensitive hospital conditions for different health care system contexts.20\n\n\nData availability\n\nAll data that can be directly shared has already been included in the manuscript. Further, we have (i) used relevant keywords and descriptions for other researchers to identify our research (Findable), (ii) described our routes to data access for other researchers to grant access to similar data (Accessible), (iii) have used international nomenclature (ICD-10 codes) to facilitate merging of our datasets with those of other researchers (Interoperable) and (iv) we encourage other researchers to build on and reuse our methodological approach and data for further exploitation of the research findings (Reusable).\n\nDue to the provision of the data by the statutory health insurance (SHI) companies within the framework of a data evaluation contract, a direct publication is not possible. However, readers and reviewers may apply to access the data by contacting the following SHI companies. Several factors will be considered before access to data is granted, including the adherence to the EU General Data Protection Regulation.\n\n- General Local Health Insurance Fund (AOK) in Rhineland/Hamburg (aok@rh.aok.de)\n\n- General Local Health Insurance Fund (AOK) Baden-Württemberg (info@bw.aok.de)\n\n- General Local Health Insurance Fund (AOK) Rhineland-Palatinate/Saarland (service@rps.aok.de)\n\n- BARMER Health Insurance Fund (service@barmer.de)\n\n- German Employees' Health Insurance Fund (DAK; service@dak.de)\n\n- Health Insurance Fund (BKK) Werra-Meissner (info@bkk-wm.de)\n\nOpen Science Framework: “Nursing home-sensitive conditions”, https://doi.org/10.17605/OSF.IO/EAJ5838\n\nThis project contains the following extended data:\n\n- f1000Extended data_2021-11-03.pdf (questionnaire, workshop and data request documentation)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank researchers and postgraduate students at the Chair of Management and Innovation in Health Care at the Witten/Herdecke University, Germany; Prof. Dr. Hagen Bachmann, Chair of Pharmacology and Toxicology at the Witten/Herdecke University, Germany; Prof. Dr. Petra Thürmann, Chair of Clinical Pharmacology at the Witten/Herdecke University, Germany; Prof. Dr. Hans-Jürgen Heppner, Chair of Geriatrics at the Witten/Herdecke University, Germany; Prof. Dr. Thomas Klie, FIVE- Registered Association for Research and Innovation of the Protestant University of Applied Sciences Freiburg, Research focus group Social Research in Gerontology and Nursing, Freiburg, Germany; Dr. honoris causa Helmut Hildebrandt, OptiMedis Inc., Hamburg, Germany; Prof. Dr. Christel Bienstein, Registered Association for Nursing, Berlin, Germany; Prof. Dr. Andreas Sönnichsen, Department of General Practice and Family Medicine, Centre for Public Health at the Medical University of Vienna, for their academic support. We also thank Philip Lewin, OptiMedis Inc., Hamburg, Germany, for his support in programming the online tools.\n\nWe acknowledge the following German statutory health insurance companies, for providing the anonymized hospital release data on nursing home residents: General Local Health Insurance Fund (AOK) in Rhineland/Hamburg, Baden-Württemberg, Rhineland-Palatinate/Saarland, BARMER Health Insurance Fund, German Employees' Health Insurance Fund (DAK), and the Health Insurance Fund (BKK) Werra-Meissner.\n\nLast but not least, we would like to thank all ambulatory and clinical physicians, nurses, scientists, and other experts who, despite the COVID-19 pandemic, took the time to participate in both lengthy surveys and/or workshop for the adjusted Delphi process.\n\n\nReferences\n\nPalleschi L, De Alfieri W, Salani B, et al.: Functional recovery of elderly patients hospitalized in geriatric and general medicine units. The PROgetto DImissioni in GEriatria Study. J. Am. Geriatr. 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Ther. 1998; 61(4): 153–156. Publisher Full Text\n\nPowell C: The Delphi technique: myths and realities. J. Adv. Nurs. 2003; 41(4): 376–382. PubMed Abstract \| Publisher Full Text\n\nCrisp J, Pelletier D, Duffield C, et al.: The Delphi Method?. Nurs. Res. 1997; 46(2): 116–118. PubMed Abstract \| Publisher Full Text\n\nBohnet-Joschko S, Valk-Draad MP, Schulte T, et al.: Nursing home-sensitive conditions.2021. Publisher Full Text" }	[ { "id": "119120", "date": "24 Feb 2022", "name": "Daniela Holle", "expertise": [ "Reviewer Expertise My focus is on nursing research", "particularly in the area of nursing home care. I have no expertise in conducting economic 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\nThank you for the option to review the manuscript entitled “Nursing home-sensitive conditions: analysis of routine health insurance data and modified Delphi analysis of potentially avoidable hospitalizations“.\nIn a first research question, the study examines how often nursing home residents are treated in hospitals and what are the main diagnosis and associated costs of these hospital admissions. The second research question addresses potentially avoidable hospitals admission of nursing home residents. The third research question asks for the impact of the estimated preventability of hospital admissions. To answer the questions a secondary analysis of routine data based on health insurance claims data were used. In addition, a Delphi survey in combination with an expert workshop was conducted.\nIn view of rising healthcare costs and increasingly scarce healthcare resources, answering the research questions is essential to ensure the best possible patient care while maintaining patient safety. A striking feature of the study is the very large data set of approximately €240,000 nursing home residents, which includes one-third of all statutorily insured nursing home residents. This is particularly noteworthy with regard to the representativeness of the study finding. The large number of experts who participated in the Delphi survey should also be emphasized.\nOn closer inspection, however, I noticed a few aspects that I would like to briefly outline here: The introduction notes that interventions to reduce hospitalizations of nursing home residents should be tailored to health care systems. As an example of possible interventions, studies on the introduction and testing of nurse-led care models in nursing homes could be listed here. For example, the recent study by Zuniga et al (see Zúñiga et al. Positive effect of the INTERCARE nurse-led model on reducing nursing home transfers: A nonrandomized stepped-wedge design.1) demonstrates that with an introduction of a nurse-led care model to hospital admissions of nursing home residents can be significantly reduced. Similar positive approaches can be found, for example, on the recent scoping review by Schmüdderich et al. (see Schmüdderich et al. Core elements and potential of nurse-led care models in residential long-term care: A scoping review. J Clin Nurs. 2022 Feb 4.2). These efforts in the reduction of hospital admissions from nursing homes are worth noticing in the introduction.\nThe methodology of the study is presented in a structured and comprehensible manner. However, from a content perspective, the study provides some limitations that I would like to outline briefly here and that should be critically presented and discussed in the course of the study. The routine data on discharge diagnoses were used to describe the admission diagnosis of nursing home residents. It should be critically reflected that there is a process between admission and discharge of a hospital patient, which can lead to confirmation, extension, or refutation of the admission diagnosis. Thus, the admission diagnosis is a limited indicator to describe the frequency and nature of hospital admissions of nursing home residents.\nWith regard to the avoidability of hospital admissions, it must also be taken into account that the admission diagnosis alone is not necessarily the decisive factor for a corresponding admission, but also resident-specific criteria, which, in combination with a diagnosis, can lead to hospitalization. Thus, hospitalization may not be avoidable even with optimal care due to specific characteristics of nursing home residents.\nIn this study, the significance of the results on the estimated avoidability of hospital admission mainly depends on the definition of optimal care for nursing home residents. In this manuscript, optimal care is defined by access to trained personnel, resources, and infrastructure for monitoring and nursing, and cooperation agreements with ambulatory general and specialists care providers (page 4/29, manuscript). Further explanations for the definition of optimal care are given within the attachment of the extended data (page 21/359):\nwell-trained nursing staff the possibility or the equipment for a preliminary examination, for monitoring a possible crisis in the condition of the nursing home resident and for initiating treatment measures good cooperation with outpatient medical care the possibility of consultation with a specialist\nIn the definition of optimal care, it must be critically appreciated that both the qualification of the nursing staff and the cooperation of different professions were included, although well–trained outpatient medical care staff is also needed for optimal care.\nNevertheless, the criteria for optimal care are very general, which is why a later discussion on the impact of avoidable hospital admissions also proves to be very difficult. With regard to the nursing staffing, the ratio between residents and nursing staff is relevant on the one hand, but also the skill and grade mix of the nursing staff against the background of the current resident structure in nursing homes. A good cooperation with the outpatient medical care could also have been specified in more detail, e.g. how often the individual resident is visited by an outpatient medical care service, how often the resident is also cared for by a specialist.\nThe more specifically optimal care would have been described here, the more specifically the experts' assessments could have been estimated and the costs for the expenditure of optimal care could have been calculated and compared with the costs for preventable hospital admissions. In the course of the conclusion, it is stated that interventions should be developed that contribute to the reduction of hospitalizations of nursing home residents. In this context, it would be useful to refer to existing concepts (see comment introduction).\nI hope my comments help to strengthen 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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "8043", "date": "06 Apr 2022", "name": "Sabine Bohnet-Joschko", "role": "Author Response", "response": "Dear Editors, Dear Daniela Holle, We kindly thank you for your reply and for offering us the revision of our manuscript for consideration in the F1000Research Platform. We carefully considered the comments offered by you to improve the quality of the initial draft of the manuscript. Please find below a point-by-point response, in which we describe how we have addressed the suggestions. All changes or adjustments are shown in italic font in the table below and in track changing mode in the revised paper accordingly. Again thank you for taking the time to critically review the paper. We truly appreciate the constructive input and hope that our revisions meet your approval. Yours sincerely, from the research team, Prof. Dr. Sabine Bohnet-Joschko Comment 1: “As an example of possible interventions, studies on the introduction and testing of nurse-led care models in nursing homes could be listed here. For example, the recent study by Zuniga et al (see Zúñiga et al. Positive effect of the INTERCARE nurse-led model on reducing nursing home transfers: A nonrandomized stepped-wedge design.) demonstrates that with an introduction of a nurse-led care model to hospital admissions of nursing home residents can be significantly reduced. Similar positive approaches can be found, for example, on the recent scoping review by Schmüdderich et al. (see Schmüdderich et al. Core elements and potential of nurse-led care models in residential long-term care: A scoping review. J Clin Nurs. 2022 Feb 4.). These efforts in the reduction of hospital admissions from nursing homes are worth noticing in the introduction.” Authors’ response to comment 1: Thank you for this valuable comment. We have now added the following sentence in the “Introduction” section, at the end of the second to last paragraph, and incorporated both references: “For instance, nurse-led care models with higher qualified nurses in expanded roles have been introduced and showed a positive impact on reducing hospitalization of nursing home residents and on advancing nursing practice in nursing homes, (Zúñiga et al. (2022), Schmüdderich et al. (2022)).” Comment 2: \"The routine data on discharge diagnoses were used to describe the admission diagnosis of nursing home residents. It should be critically reflected that there is a process between admission and discharge of a hospital patient, which can lead to confirmation, extension, or refutation of the admission diagnosis. Thus, the admission diagnosis is a limited indicator to describe the frequency and nature of hospital admissions of nursing home residents.\" Authors’ response to comment 2: Thank you for this comment: You are absolutely right, and therefore our study was based on discharge diagnoses of the nursing home residents instead of on admission diagnoses. To clarify this, we have added the following sentence to the “Methods” section, subsection “Analysis of routine health insurance data”, at the end of the second paragraph: “In line with previous studies, our analysis of routine health insurance data focused on the hospital discharge diagnosis, not on the admission diagnosis, as the latter is often subject to confirmation, extension or refutation during the hospital stay.” Comment 3: \"With regard to the avoidability of hospital admissions, it must also be taken into account that the admission diagnosis alone is not necessarily the decisive factor for a corresponding admission, but also resident-specific criteria, which, in combination with a diagnosis, can lead to hospitalization. Thus, hospitalization may not be avoidable even with optimal care due to specific characteristics of nursing home residents.\" Authors’ response to comment 3: This is absolutely correct and is well reflected by nursing home-sensitive conditions only expected to be avoidable in at least 70% of all cases with this condition. Conversely, this automatically means that up to 30% of the remaining cases with a disease represented in the nursing home-sensitive conditions catalogue are unavoidable hospitalizations. We included the following sentence under “Key findings” to clarify this: “After two Delphi rounds and an expert workshop, we were able to identify 58 diagnoses considered to be nursing home-sensitive conditions in the context of the German health care system, i.e., at least seventy percent of all hospitalizations with such a condition is expected to be preventable, depending on the individual health status and advance directive of the nursing home resident.” Comment 4: In the definition of optimal care, it must be critically appreciated that both the qualification of the nursing staff and the cooperation of different professions were included, although well–trained outpatient medical care staff is also needed for optimal care. Nevertheless, the criteria for optimal care are very general, which is why a later discussion on the impact of avoidable hospital admissions also proves to be very difficult. With regard to the nursing staffing, the ratio between residents and nursing staff is relevant on the one hand, but also the skill and grade mix of the nursing staff against the background of the current resident structure in nursing homes. A good cooperation with the outpatient medical care could also have been specified in more detail, e.g. how often the individual resident is visited by an outpatient medical care service, how often the resident is also cared for by a specialist. The more specifically optimal care would have been described here, the more specifically the experts' assessments could have been estimated and the costs for the expenditure of optimal care could have been calculated and compared with the costs for preventable hospital admissions. Authors’ response to comment 4: Thank you for this detailed evaluation of this aspect of our study. All mentioned aspects do matter indeed. We had some practical restraints, that made us decide to define optimal care conditions the way we did, which we have explained now in more detail in the “Discussion” section under “Strengths and limitations”: “The description of optimal care conditions was rather short, and perhaps therefore rather general. This could have led to less accurate preventability estimates. By addressing optimal care conditions in more detail (e.g., the importance of well-trained nursing staff both in the out- as well as the inpatient setting, special geriatric care knowledge and skills, resident structure in the nursing home, resident/ nursing staff ratio, skill and grade mix of staff, good cooperation with outpatient medical care, frequency of medical care visits by an outpatient medical care service/ specialist), experts might have given other (higher) estimates of preventability. This might have influenced the extrapolation of costs as well. As we already had a very long list of instructions, explanations and conditions to be assessed, we decided not to overstrain clarity and longevity for the Delphi experts hereupon, and kept the information provided on optimal care conditions to the point.” Comment 5: In the course of the conclusion, it is stated that interventions should be developed that contribute to the reduction of hospitalizations of nursing home residents. In this context, it would be useful to refer to existing concepts (see comment introduction). Authors’ response to comment 5: We addressed this significant input in the “Discussion” section under “Implications for policy, research and clinical practice” as follows: “For all stakeholders (e.g., medical and nursing providers, policy makers, health economists), the list of nursing home-sensitive conditions can inform the development of interventions and facilitate local quality improvement efforts, also taking into account existing evidence-based concepts.”" } ] }, { "id": "126098", "date": "21 Mar 2022", "name": "Tim Badgery-Parker", "expertise": [ "Reviewer Expertise I am a biostatistician with considerable experience in using administrative data to examine low-value hospital care and patient safety." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the development of a list of \"nursing home sensitive\" conditions, and the use of the list to estimate costs of potentially preventable admissions from nursing homes to hospitals in Germany. The authors demonstrate differences between their list and an existing list of ambulatory sensitive conditions, confirming that the nursing home setting is sufficiently different, that the use of ambulatory care sensitive conditions is unlikely to be appropriate.\nThe candidate diagnoses are derived from a large administrative dataset (30% of nursing home residents in Germany) and the process of developing the list is appropriate and comprehensively described, as is the cost estimation. The language is generally quite good, with some stray punctuation and a few unclear sentences.\nThe results include a comparison of the preventability estimates between subgroups of the Delphi panel, this does not appear to be described in the methods.\nThe results also describe criteria for excluding or combining diagnoses after the first round for presentation in the second round. (The criteria were not met for any diagnosis.) These criteria should really be in the methods.\nOverall this is a strong study that makes a valuable contribution to the field.\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": "8044", "date": "06 Apr 2022", "name": "Sabine Bohnet-Joschko", "role": "Author Response", "response": "Dear editors, Dear Tim Badgery-Parker, We kindly thank you for your reply and for offering us the revision of our manuscript for consideration in the F1000Research Platform. We carefully considered the comments offered by you to improve the quality of the initial draft of the manuscript. Please find below a point-by-point response, in which we describe how we have addressed the suggestions. All changes or adjustments are shown in italic font and in revision mode in the revised paper accordingly. Again thank you for taking the time to critically review the paper. We truly appreciate the constructive input and hope that our revisions meet your approval. Yours sincerely, from the research team, Prof. Dr. Sabine Bohnet-Joschko Comment 1: \"The results include a comparison of the preventability estimates between subgroups of the Delphi panel, this does not appear to be described in the methods.\" Authors’ response to comment 1: Thank you very much indeed for your review and the comments. We have now added two sentences to address this aspect: In the “Method” section, subsection “Delphi study and expert workshop”, fourth paragraph, last sentence: “…and planned to recruit 100 experts: 30 outpatient/clinical physicians each, 30 nursing professionals and 10 scientists. This also allowed for comparison of preventability assessments by expert group.” And in the ninth paragraph: “In case the preventability potential of at least 75% was not comprised in the range of 75% of all expert assessments, the ICD-10 codes were excluded. For all other ICD-10 codes, statistical data (median, modal values, dispersion parameters, bar charts), results of expert group comparison on preventability assessments (Kruskal-Wallis (K-W)-Test), and categorized comments from the Delphi questionnaire were provided for the expert workshop.” Comment 2: \"The results also describe criteria for excluding or combining diagnoses after the first round for presentation in the second round. (The criteria were not met for any diagnosis.) These criteria should really be in the methods.\" Authors’ response to comment 2: Thank you for bringing this to our attention, which we, unfortunately, overlooked before. We have now moved the criteria to the methods section and only referred to them in the results section, as follows: In the “Method” section, subsection “Delphi study and expert workshop”, eighth paragraph, second sentence: “ICD-10 codes were planned to be excluded for assessment in the second round, when at least 75% of the experts estimated the preventability as zero in the first round. Secondly, ICD-10 codes were planned to be combined if the diseases were very similar (in terms of symptoms, diagnosis, prognosis, treatment) and the proportions of potential preventability were nearly identical. Two conditions for “nearly identical potential preventability” had to be fulfilled: the medians of the estimated preventability should not differ by more than 5% and the limits of the interquartile range had to be less than 10% apart. Based on this information, the ICD-10 codes for the second round were identified and the participants were asked to quantify the proportion of potentially avoidable hospitalization again in the second round.” And in the “Results” section, subsection “Delphi study and expert workshop”, second paragraph, rather at the end, we changed the text \"Furthermore, ICD-10 Codes... . Neither conditions were met sufficiently.\" was changed as follows: \"The condition that at least 75% of the experts estimated the preventability as zero in the first round was met for none of the ICD-10 codes. Furthermore, none of the ICD-10 codes could be combined as the conditions for “nearly identical potential preventability” were not fulfilled.\"" } ] } ]	1	https://f1000research.com/articles/10-1223
https://f1000research.com/articles/11-396/v1	06 Apr 22	{ "type": "Research Article", "title": "Drug utilization pattern and adverse drug reactions of chemotherapy in pediatric patients at Muhimbili National Hospital, Tanzania", "authors": [ "Josephine Efraim", "Castory Munisi", "Auson Magige", "Kelvin Msuya", "Alphonce Ignace Marealle", "Manase Kilonzi", "Hamu Mlyuka", "Wigilya Mikomangwa", "Bertha Mallya", "Wema Aswile", "Kauke Bakari Zimbwe", "Ritah Francis Mutagonda", "Josephine Efraim", "Castory Munisi", "Auson Magige", "Kelvin Msuya", "Alphonce Ignace Marealle", "Manase Kilonzi", "Hamu Mlyuka", "Wigilya Mikomangwa", "Bertha Mallya", "Wema Aswile", "Kauke Bakari Zimbwe" ], "abstract": "Background: Cancer is a highly debilitating non-communicable disease and an essential contributor to the global burden of disease. Pediatric patients are highly exposed to multiple drugs for the management of cancer. Monitoring drug utilization patterns helps to provide feedback to healthcare providers to ensure the rational use of medicines; as a result, it increases the therapeutic efficacy and decreases the frequency and severity of adverse drug reactions (ADRs). Therefore, this study assessed the utilization pattern and ADRs of chemotherapy in pediatric patients at Muhimbili National Hospital (MNH). Methods: A descriptive cross-sectional study was conducted for three months from February to April 2021 in pediatric cancer patients undergoing chemotherapy at MNH. A total of 123 children diagnosed with cancer and on chemotherapy were enrolled in this study. Patients’ socio-demographics, clinical information, chemotherapy status, prescribed medications, and prevalence of ADRs were collected. Descriptive statistics was used in data analysis, whereby frequency and proportions were used to summarize data. Results: Out of 123 patients, 62.6% were male. Most patients received an average of four anticancer drugs. Vincristine (55.3%) was the most used anticancer drug, followed by cytarabine (44.7%) and methotrexate (42.3%). The most used adjuvant drugs were ondansetron (30.9%), hydrocortisone (27.6%), and piperacillin/tazobactam (23.6%). The percentage of drugs prescribed from the Tanzania Essential Medicine List (TEML) and World Health Organization (WHO) list was 66.4% and 93%. Most (87%) of the patients reported having experienced ADRs whereby nausea and vomiting (45.8%), hair loss (33.6%), and neutropenia (32.7%) were more prevalent ADRs reported. Conclusions: This study found the drug prescribing pattern to be in line with the essential medicine list, but the average number of drugs prescribed was higher than recommended. ADRs were prevalent among pediatric cancer patients.", "keywords": [ "Pediatric chemotherapy", "Drug utilization pattern", "Adverse Drug Reactions" ], "content": "Introduction\n\nCancer is a non-communicable disease (NCD) that reduces the quality of life.1 It is a group of diseases that involves abnormal cell growth that invades or spreads to other parts of the body.2 Cancer is a disease of public health importance, and it is reported to be among the leading causes of death globally in both developing and developed countries.3 As per the World Health Organization (WHO) survey report, the global cancer incidence in 2012 increased to 14 million new cases. It is estimated that the incidence may rise to 19.3 million by 2025.4 Worldwide, an estimated number of 250,000 children are diagnosed with cancer yearly, whereby most diagnoses occur in low and middle-income countries.5\n\nIn Tanzania, the incidence of pediatric cancer is unknown due to the lack of a national cancer registry, but it has been estimated to be at 134 occurrences per million.6 The likelihood of surviving a diagnosis of childhood cancer depends on the country; more than 80% of children with cancer are cured in high-income countries while in low-middle income countries only 30% are cured.7,8 Cancer has contributed to 5.1% of all in-hospital deaths in Tanzania in 2006-2015. The mortality rate was 47.8 per 100000 population and the number of deaths was high among individuals 15-59 years of age.9\n\nMost used chemotherapy agents in cancer are cytotoxic, meaning that they function by killing fast-dividing cells. The most immediate adverse drug reactions (ADRs) of chemotherapy are due to the cytotoxic effect on the normal cells. Cancer chemotherapy's common ADRs include hair loss, nausea and vomiting, anemia, febrile neutropenia, thrombocytopenia, tiredness, confusion, mood changes, tingling, burning, weakness, numbness, and pain in the hands and feet and mucositis.10\n\nThe utilization pattern of anticancer drugs has changed significantly in recent years because of better enhancement in carcinomas' pathophysiology and the introduction of newer drugs. Significant inter-individual variability in the response rate of anticancer drugs, availability of different regimens, and combination regimens intolerability necessitate monitoring and evaluation of cancer chemotherapy.\n\nLike many other low- and middle-income countries, the pediatric cancer outcomes in Tanzania are poor, and there are limited diagnostic and treatment capacities.5 Moreover, it is unknown whether pediatric cancer patients are being managed rationally in Tanzania. Poor drug utilization among pediatric cancer patients will increase the occurrence of drug toxicity and ADRs hence decreasing the survival rates even further.11 Therefore, this study assessed the drug utilization pattern and reported ADRs among pediatric cancer patients at Muhimbili National Hospital (MNH).\n\n\nMethods\n\nThis hospital-based descriptive cross-sectional study was conducted from February to April 2021.\n\nThe study was conducted at MNH which is the National Referral Hospital, a research center and university teaching hospital with 1,500 bed facility, attending 1,000 to 1,200 outpatients per day, admitting 1,000 to 1,200 inpatients per week. There are five government referral hospitals for dealing with cancer which are Muhimbili National Hospital, Ocean Road Cancer Institute, The Benjamin Mkapa Hospital, Mbeya Zonal Referral Hospital, Kilimanjaro Christian Medical Centre and Bugando Medical Centre which are mainly specialized in adult and pediatric malignancies. MNH has a special ward known as the Pediatric oncology ward that attends 60 to 70 pediatric cancer patients per month.\n\nThe study was carried out on pediatric cancer patients admitted to the pediatric oncology ward and diagnosed with malignancy during the study period. The list of eligible participants was obtained from Tumaini and Upendo wards registers at MNH. These patients were then followed in their admission cubes whereby the parents or guardians who attend to them were told the details of the study. The consent was requested from parents followed by assent from children.\n\nAll pediatric cancer patients receiving chemotherapy, aged less than 18 years old were included in the study. Exclusion criteria were patients whose diagnosis has not been well established and those with incomplete records in their files.\n\nA total of 126 patients were enrolled in the study. The estimated sample size N was computed using Kish and Leslie formula given below:\n\nN = estimated sample size\n\nZ is percentage point of the normal distribution corresponding to the level of significance <5%,\n\nTherefore, Z = 1.96.\n\nP = Proportion of pediatric cancer patients on chemotherapy, from a study done in northern Tanzania on pediatric cancer patients, whereby a proportion of 93% was reported.5\n\nε = margin of error, which is approximately 5%.\n\nSystematic random sampling was used to select 126 patients out of the 240 patients. The IDs of 240 patients fulfilling the inclusion criteria were entered in Microsoft Excel followed by systematic random sampling whereby the sampling interval was 2.\n\nA structured questionnaire was used to collect data. This tool was adapted from previous studies by Bepari et al and Kamlekar et al12,13 with addition of demographic information to reflect the Tanzania context. The questionnaire consisted of socio-demographic information, clinical characteristics of the patients, drugs used, and reported ADRs. Patient socio-demographics included age, gender and residence. Clinical characteristics included the admission date, referral status, diagnosis, comorbidities, body mass index (BMI), hemoglobin levels. In addition, both anticancer, adjuvants drugs used and side effects at the time of data collection were recorded. The data collection tool can be found as Extended data.28\n\nData collected was entered, cleaned, and analyzed using Statistical Package for Social Sciences (SPSS, RRID:SCR_016479) version 24, and R statistical software version 4.0.3 (RRID:SCR_001905) was used for plotting. The data was summarized using frequency distribution and proportion. The continuous variables were summarized using median and interquartile range (IQR). The R scripts used in the analysis can be found as Extended data.\n\nEthical clearance with reference number DA.25/11/01/dated 28th January 2021 was obtained from the Muhimbili University of Health and Allied Sciences (MUHAS) Institutional Review Board (IRB). Permission to collect data from the hospital was obtained from the MNH administration. A signed informed consent was obtained from all parents/guardians before interview followed by assent obtained verbally from older children who were asked whether they would like to participate in the study and they said either a yes or no as an assent. For younger children 1-6 years which were majority of the study population, consent from the patient was enough for participation. Privacy and confidentiality were highly observed in data collection and person identifying information was not collected from the patient’s files.\n\n\nResults\n\nOverall, 240 patients were potentially eligible for the study. Systematic random sampling using Microsoft Excel was used to select 126 patients. Of the 126 patients, 123 were eligible and included in the final analysis and 3 patients were excluded.28 Of the 3 excluded one refused to participate in the study and 2 had incomplete information in their files (Figure 1).\n\nOut of the 123 patients, the majority (62.6%, n = 77) were male. The median age was 5.2 (IQR = 6.4) years with half of patients (50.4%, n = 62) in the range of 0 – 5 years. The median weight was 16.7 (IQR = 10.4) kg, and almost half of the patients (48.8%, n = 60) were diagnosed in 2020. Based on hematological parameters, the median hemoglobin (Hb) level was 9.6 (IQR = 2.9) g/dl, median absolute neutrophil count (ANC) was 2.3 (IQR = 3.5) and the median platelet count (PLT) was 288 (IQR = 251). Most patients (79.3%, n = 98) were from regions outside of Dar es salaam (Table 1).\n\nMost patients (64.1%) had bone marrow and kidney malignancies with the prevalence of 30.1% (n = 37) and 27.6% (n = 34), respectively. The most dominant tumors were Wilms Tumor with 23.6% (n = 29) followed by B Cell Acute Lymphoblastic Leukemia (B Cell ALL) (17.1%) and Burkitt Lymphoma (17.1%) (Table 2).\n\nThe average number of drugs prescribed per prescription was 7 and the average number of cytotoxic drugs prescribed per prescription was 4. The percentage of drugs prescribed from the National Essential Medicines List (NEMLIT) and WHO Model Lists of Essential Medicines was 66.4% and 93%, respectively. More than a quarter (30.4%) of the prescribed drugs were injectables, 93.9% were prescribed using generic names and 19.0% of the medications were antibiotics. The most used class of anticancer agents were the antimetabolites (31.9%, n = 138) followed by vinca alkaloids (17.6%, n = 76) and antitumor antibiotics (17.4%, n = 75) (Figure 2). Enzyme cytotoxic drugs were the least used 5.1% (n = 22). The commonly used anticancer drugs were Vincristine (55.3%, n = 68), followed by Cytarabine (44.7%, n = 55) and Methotrexate Injection (42.3%, n = 54) (Table 3).\n\nVarious adjuvants were given whereby the most commonly used adjuvants were antiemetic ondansetron injection (30.9%, n = 38), followed by steroids hydrocortisone injection (27.6%, n = 34) and cytoprotective agents such as dexrazoxane 24 (19.5%, n = 24) and mesna (21.9%, n = 27) (Table 4).\n\n* Compounded in ratio of 4:4:1, Benylin cough syrup 100 mls, Maalox antacid syrup 100 mls and Xylocaine local anaesthetic 25 mls.\n\nOver three-quarters of the patients (87%) reported having experienced ADRs upon using chemotherapy medications. The most prevalent ADRs were nausea and vomiting reported by almost half of the study patients (45.8%) followed by hair loss and neutropenia with the prevalence of 33.6% and 32.7%, respectively (Figure 3).\n\n\nDiscussion\n\nAssessment of drug utilization pattern is important as it provides information that will help in promoting the rational use of medication. Unlike the adult population, there is limited information on drug utilization pattern and ADRs experienced by pediatrics undergoing cancer chemotherapy in Tanzania. Therefore, this study assessed drug utilization pattern and reported ADRs of chemotherapy among pediatric cancer patients undergoing chemotherapy at MNH.\n\nIn this study the most predominant malignancies were Wilms Tumor, B Cell Acute Lymphoblastic Leukemia, Burkitt Lymphoma and Retinoblastoma. This is comparable to a study by Schroeder et al. which reported on the most prevalent pediatric cancers in northern Tanzania were Burkitt Lymphoma 18% and Wilms tumor 14%.5 Similar malignancy types among pediatric patients have also been reported in other African countries.14,15\n\nThe average number of drugs prescribed per prescription in this study was 7 which is higher than the WHO recommended range of 1.6 – 1.8.16 This is comparable with the range of 6.0-6.9 which was reported in previous studies.12,13 The average number of cytotoxic drugs prescribed per prescription was 3.5 which is higher than a study by Sandeep et al. and Bepari et al. in which it was 1.94 and 1.27, respectively.12,13 This could be explained by differences in prescribing pattern from country to country influenced by existing cancer management guidelines, medicines availability, clinicians’ preferences, cost of medicines, diseased population, and disease status in the area.17\n\nThe percentage of drugs prescribed from NEMLIT and WHO Model Essential Medicines List were 66.4% and 93.0%, respectively. The discrepancy observed in compliance to these two lists is attributed by the fact that the NEMLIT used during the study was not updated since 2017 compared to the WHO Essential Medicines List which has been updated in 2019.18,19 After the study completion there was a release of updated NEMLIT in 2021 whereby most drugs have now been included in the management of cancer in pediatric patients.\n\nIn this study vincristine was the most used anticancer drug, followed by cytarabine and methotrexate which are both antimetabolites. Vincristine is the drug for most pediatric malignancies which was the target population in this study.20 Similar findings were reported in Ethiopia whereby 85.4% of the pediatric patients were using vincristine.14 The results differ from those obtained from the adult population in India in which carboplatin was the most prescribed drug, followed by paclitaxel and gemcitabine.12 Also, they differ to those in another study conducted in India by Vijayalakshmi et al. on drug utilization pattern reported that cisplatin 58% and 5-fluorouracil 41% were most prescribed among all anticancer drugs, followed by doxorubicin.21\n\nIn our study the most used adjuvant drugs were ondansetron, hydrocortisone and piperacillin/tazobactam. This is comparable to other studies in which similar adjuvants were also found to be commonly used for reduction of the ADRs of chemotherapy medications.12,13,22\n\nIn the present study, over three-quarters (87%) of the study patients, reported to have experienced at least one side effect upon using chemotherapy medications. This is comparable to a study by Pearce et al. which looked at the incidence and severity of self-reported chemotherapy side effects in routine care in which 86% of the study patients reported at least one side effect during the study period.23 The findings are also comparable to a cross-sectional national survey done in the U. S by Henry et al. in which 88% of the study patients reported at least one side effect.24 Furthermore, a study done in Kenya by Opanga et al. looking at side effects of chemotherapy 93 % of the study reported to have experienced at least one side effect during chemotherapy treatment.25\n\nIn our study the most prevalent side effects were nausea and vomiting, which were reported by about half of the study patients, followed by hair loss and neutropenia. This is comparable to the studies conducted in Malaysia and India, where the most common side effects of anticancer drugs included nausea and vomiting, hair loss, loss of appetite, and tiredness or weakness.26,27 This could be explained by the fact that most anticancer drugs are associated with nausea and vomiting.\n\nThis study is limited in its scope to a single institution. However, MNH being the only national hospital has capacity in terms of human resources and technology required in management of cancer patients in the country. Therefore, information obtained from this center provide the best indicator of the drug utilization pattern and ADRs experienced by pediatric cancer patients in the country. Some important information was missing in the patients’ files and prescriptions like duration of treatment, height and weight of patients, hence limiting patients’ enrollment in the study. Moreover, factors influencing prescription patterns by the clinicians were not examined.\n\n\nConclusions and recommendations\n\nThe prescribing pattern among the pediatric cancer patients at MNH was highly adherent to the WHO Model Essential Medicines List. However, the average number of drugs per prescription was very high. Vincristine was the most used anticancer drug and ondansetron was the most used adjuvant drug. The prevalence of side effects was very high indicating a need for improvement in prescribing for pediatric cancer patients to avoid ending up in irrational medicine use which could hinder the achievement of the treatment goals.\n\nSince the average number per prescription was very high, we recommend multidisciplinary teamwork between prescribers and dispensers to reduce polypharmacy which could in turn can improve the management of pediatric cancer patients. Moreover, high prevalent of ADRs among these patients requires a vigilant ADR monitoring system to ensure early detection, management and reporting of ADRs experienced by pediatric cancer patients.\n\n\nData availability\n\nMendeley Data: Dataset for a cross-sectional study on “Drug Utilization Pattern and Adverse Drug Reactions of Chemotherapy in Pediatric Patients at Muhimbili National Hospital”. https://doi.org/10.17632/gjcyvs5nfx.3.28\n\nThis project contains the following underlying data:\n\n- paediatric.xlsx\n\n- combined.xlsx\n\nMendeley Data: Dataset for a cross-sectional study on “Drug Utilization Pattern and Adverse Drug Reactions of Chemotherapy in Pediatric Patients at Muhimbili National Hospital”. https://doi.org/10.17632/gjcyvs5nfx.3.28\n\nThis project contains the following extended data:\n\n- analysis. R (analysis script)\n\n- data_analysis.Rproj (analysis script)\n\n- Data Collection Tool.docx\n\n- Consent Forms English and Kiswahili Versions.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank enrolled children and their parents/guardians for providing their cooperation throughout the study period. We acknowledge the support we received from the management and healthcare providers at Muhimbili National Hospital.\n\n\nReferences\n\nNayak MG, et al.: Quality of life among cancer patients. Indian J. Palliat. Care. 2017; 23: 445–450. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization (WHO): Cancer.Reference Source\n\nWHO: The Global Burden of Disease: 2004 Update. 2008.\n\nVijayalakshmi SRB, Usharani M, Latha KS: (PDF) Assessment of drug utilization pattern in patients undergoing chemotherapy for various types of metastatic cancers in a tertiary care government hospital.2018.\n\nSchroeder K, et al.: Pediatric cancer in northern Tanzania: Evaluation of diagnosis, treatment, and outcomes. J. Glob. Oncol. 2018; 2018: 1–10. Publisher Full Text\n\nInstitute, N. cancer (NIH): Cancer Statistics Review, Previous Version - SEER Cancer Statistics Review.2016.\n\nWorld Health Organization: Assessing national capacity for the prevention and control of noncommunicable diseases: report of the 2019 global survey. World Health Organization; 2020. Reference Source\n\nLam CG, Howard SC, Bouffet E, et al.: Science and health for all children with cancer. Science. 2019 Mar 15; 363(6432): 1182–1186. PubMed Abstract \| Publisher Full Text\n\nLyimo EP, et al.: Cancer mortality patterns in Tanzania: A retrospective hospital-based study, 2006-2015. J. Glob. Oncol. 2020; 6: 224–232. Publisher Full Text\n\nDrugs: Side Effects of Chemotherapy - ACCO.\n\nMelku L, Wubetu M, Dessie B: Irrational drug use and its associated factors at Debre Markos Referral Hospital’s outpatient pharmacy in East Gojjam, Northwest Ethiopia. SAGE Open Med. 2021; 9: 205031212110251. PubMed Abstract \| Publisher Full Text\n\nKumar Kamlekar S, Agarwal A, Latha PA, et al.: Evaluation of drug utilization pattern of anticancer drugs in oncology department of a tertiary care teaching hospital of southern Rajasthan. Natl. J. Physiol. Pharm. Pharmacol. 2020; 10: 1. Publisher Full Text\n\nBepari A, Sakre N, Rahman I, et al.: The assessment of drug utilization study of anticancer drugs using who prescribing indicators in a government tertiary care hospital of the Hyderabad-Karnataka Region of India. Open Access Maced. J. Med. Sci. 2019; 7: 1203–1208. PubMed Abstract \| Publisher Full Text\n\nWorkalemahu G, Abdela OA, Yenit MK: Chemotherapy-Related Adverse Drug Reaction and Associated Factors Among Hospitalized Paediatric Cancer Patients at Hospitals in North-West Ethiopia. Drug Healthc. Patient Saf. 2020; 12: 195–205. PubMed Abstract \| Publisher Full Text\n\nStefan DC: Patterns of distribution of childhood cancer in Africa. J. Trop. Pediatr. 61: 165–173. 201. PubMed Abstract \| Publisher Full Text\n\nBiradar SM, Khaja Hussain BS, Urmila G, et al.: Assessment of drug utilization patterns in paediatric patients in comparison with WHO core indicators. GSC Biological and Pharmaceutical Sciences. 2019; 9(2): 084–092. Publisher Full Text\n\nAbolfazl Abolfazli S, Mohammadzadeh M, Peiravian F, et al.: Chemotherapy Drugs in Cancer Treatment in Iran: an Interview Questionnaire Study. Iran. J. Pharm. Sci. 2017.\n\nHealth, M. of. Standard Treatment Guidelines & National Essential Medicines List Tanzania Mainland.2017.\n\nOMS: World Health Organization Model List of Essential Medicines. Mental and Holistic Health: Some International Perspectives. 2019; 21: 119–134.\n\nDyer O: US paediatric oncologists are forced to prioritise patients for vincristine treatment as supplies run short. BMJ (Clinical research ed.). 2019; 367: l6086. Publisher Full Text\n\nVijayalakshmi, Bendi SR, Usharani M, Latha KS: (PDF) Assessment of drug utilization pattern in patients undergoing chemotherapy for various types of metastatic cancers in a tertiary care government hospital.2018.\n\nKumar BS, Maria S, Shejila CH, et al.: Drug utilization review and cost analysis of anticancer drugs used in a tertiary care teaching hospital. Indian J. Pharm. Sci. 2018; 80: 686–693. Publisher Full Text\n\nPearce A, et al.: Incidence and severity of self-reported chemotherapy side effects in routine care: A prospective cohort study. PLoS ONE. 2017; 12: e0184360. PubMed Abstract \| Publisher Full Text\n\nHenry DH, et al.: Symptoms and treatment burden associated with cancer treatment: Results from a cross-sectional national survey in the U.S. Support. Care Cancer. 2008; 16: 791–801. Publisher Full Text\n\nOpanga L, Mulaku MN, Opanga SA, et al.: Adverse effects of chemotherapy and their management in Pediatric patients with Non-Hodgkin’s Lymphoma in Kenya: A descriptive, situation analysis study. Expert. Rev. Anticancer. Ther. 2019; 19: 423–430. PubMed Abstract \| Publisher Full Text\n\nSunny S, et al.: Assessment of Adverse Effects of Most Commonly Prescribed Anticancer Drugs in a Tertiary Care Teaching Hospital. Indian Journal of Pharmacy Practice. 2018; 10: 270–275. Publisher Full Text\n\nChan HK, Ismail S: Side effects of chemotherapy among cancer patients in a Malaysian general hospital: Experiences, perceptions and informational needs from clinical pharmacists. Asian Pac. J. Cancer Prev. 2014; 15: 5305–5309. PubMed Abstract \| Publisher Full Text\n\nMassawe J, Munishi C, Mutagonda R: Dataset for a Study titled “Drug Utilization Pattern and Adverse Drug Reactions of Chemotherapy in Pediatric Patients” at Muhimbili National Hospital Data. Mendeley Data, V3, [Dataset]. 2022. Publisher Full Text" }	[ { "id": "136029", "date": "05 May 2022", "name": "Divya Subramonian", "expertise": [ "Reviewer Expertise Pediatric Cancer - 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\nIn this article, the authors have addressed a critical and important issue by looking at drug utilization and adverse drug reactions in pediatric cancer patients. The authors have provided a clear and sound introduction covering relevant literature. The paper is well presented and could be used as a good tool for clinicians and researchers while prescribing drugs to pediatric patients.\nMethods:\nAlthough the study was done only in one hospital, the sample size, distribution between male and female, and distribution of age group seems reasonable. The process of sample collection is clear with inclusion and exclusion criteria. However, it would be great if the authors can address this:\nIs there a reason for collecting data during the mentioned 3-month period (Feb-April 2021)? Is it possible to include/get data for a longer period of time?\nResults:\nThe tables are well presented, taking into consideration the demographic and clinical characteristics of the patients. The chemotherapy and adjuvant medications are well tabulated indicating the frequency of intake. The figures give a clear representation of the cytotoxic drugs used in the study pointing out the most commonly used ones. The most common side effects are also shown in Figure 3. All of this information can be used as a reference for physicians and researchers alike.\nNevertheless, it would be nice to have a couple of things addressed/discussed.\nIn Table 1, the number of males and females does not add up to 123. I believe the numbers 77 males and 23 females should be revisited.\n\nThis is probably beyond the scope of the paper, but it would be nice to see the number or percentage of pediatric patients recovering from the disease given the adverse effects.\nDiscussion:\nAs the clinical treatment of pediatric patients has changed significantly over the last few decades, this study points out the adverse effects the drugs have on patients.\nIt would be useful if the authors can address how long the side effects last and discuss a little bit about the risk-benefit analysis for the most commonly used chemotherapy drugs.\nIt is alarming to note that the average number of drugs prescribed per prescription is 7, which is several times higher than recommended by WHO.\nFinally, do the authors have a hypothesis for this discrepancy and if that is true in other parts of Tanzania and the world? A little more insight on this would be a great eye-opener.\n\nIs the work 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": "261864", "date": "13 Apr 2024", "name": "Sidharth Totadri", "expertise": [ "Reviewer Expertise Pediatric Hematology Oncology practice in a low- and middle-income country", "supportive care", "thalassemia", "common childhood cancers" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDrug utilization pattern and adverse drug reactions of chemotherapy in pediatric patients at Muhimbili National Hospital, Tanzania General comments\n\nThe authors describe a single-center cross-sectional study of drug utilization and adverse reactions in children with cancer. Studies from regions such as the sub-Saharan are the stepping stones for capacity building in low- and middle-income countries. The authors can address the following comments.\nIntroduction: The first paragraph on cancer can focus more on childhood cancer rather than describing what cancer is. It would be good to mention the global initiative for childhood cancer from the World Health Organization to set the stage for a study on children being treated for cancer. Results:\n\nIt would be good to know what proportion of patients were underweight or malnourished rather than the median weight. The term bone marrow malignancy is vague as solid tumors like neuroblastoma and lymphomas can disseminate to marrow too. The term ‘leukemias’ may be better and also represent the disease site well. It is interesting to note that Wilms tumor surpassed B cell ALL in being the commonest malignancy. Do the authors have an explanation for this? Was any grading system like CTCAE used for adverse events? It would be good to know the proportion of severe or grade 3 or 4 toxicity. All scientific terms in results must be predefined in methods. For example, anemia and neutropenia must be defined with the cut-off used. Again, this reiterates the need to use a classification system like CTCAE or an indigenous system if used by the center, for all toxicity events. Since the study is aimed at evaluating drug utilization and capacity building there should be more details on the route of administration and doses. This is particularly relevant for drugs such as methotrexate which can be administered orally, intravenously, and intrathecally. Drugs such as methotrexate and cytarabine can be administered at high doses and low doses, and the corresponding vial strengths needed would be different. With this large sample size, one would like to know of treatment-related mortality during the study period. How many patients succumbed to adverse events of chemotherapy and what were the specific etiologies? The term ‘adjuvant therapy’ in oncology typically refers to the cytotoxic therapy that follows local therapy in a solid tumor. ‘Supportive care’ medication is an alternative term. Again, the authors must define what constitutes adjuvant therapy in the methods section, with a brief description of the center’s supportive care protocols. There is no mention of antimicrobial prophylaxis such as co-trimoxazole which would be an indispensable part of supportive care. The indication of steroids such as hydrocortisone must be mentioned, as they can be part of the chemotherapy regimen too. Figure 3: the side effects that comprise ‘others’ must be listed in the legend or in the main text. A singular unexpected or severe adverse event would be as important as a common adverse event.\n\n3.It would be interesting to see if there was an unavailability of any specific drug during the study period that led to a delay in therapy, or a need to modify the regimen to\n\nexclude or replace the unavailable drug.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "217377", "date": "26 Apr 2024", "name": "Paola Muggeo", "expertise": [ "Reviewer Expertise pediatric hematolgy 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\nThe Authors report data about childhood cancer in Tanzania and the use of antineoplastic drugs, together with the ADRs. The importance of this paper is to report incidence and outcome of childhood cancer in a developing Country, which is the first step to work for a better medical support and outcome. However the paper needs major adjustment to be improved. First of all, about methods. Diagnosis: the authors report that the most frequent diagnosis is Wilms' tumor, which is a noteworthy result since it may depend on differences in genetic background, being this tumor a genetically influenced cancer. However diagnosis should be categorized according to international WHO classification. Moreover how is the diagnosis and staging of disease performed? This should be reported in the methods. The modality of drug association and delivery should be detailed. Are there any prescribed protocols to administer polychemotherapy? which is the basis to deliver polychemotherapy? usually standardized protocols should be used. If this is the case, they should be cited in the methods. Results: The authors conclude about irrational medicine use, however in the field of polychemotherapy detailed protocols guide the correct use of antitumor drugs based on mechanism of action, pharmacokinetics, and pharmacodynamics. Is the 30% survival rate caused by poor response to chemotherapy or to severe side effects (such as infectious complication or other severe side effects). In other words, is there any possibility to check the tumor response after chemotherapy? The paper needs major reorganization to offer a key of interpretation of an important and urgent problem in developing Countries.\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? No", "responses": [] } ]	1	https://f1000research.com/articles/11-396
https://f1000research.com/articles/11-394/v1	05 Apr 22	{ "type": "Systematic Review", "title": "A systematic review: Male engagement in adolescent and young adults’ sexual and reproductive health in the Americas", "authors": [ "Ivonne Salinas", "Erick Freire", "Jane Guevara", "Keren Herrán", "Andrea Ortiz", "Iván Palacios", "Erick Freire", "Jane Guevara", "Keren Herrán", "Andrea Ortiz", "Iván Palacios" ], "abstract": "Progress towards sexual and reproductive health (SRH) goals for adolescents across the Americas has stagnated. Of all the regions worldwide, Latin America has experienced the slowest decline in adolescent fertility rates. Reports published by the United Nations and multiple nongovernmental organizations demonstrate a growing consensus for a masculinities framework that engages men and boys in public health and social change. Male engagement acts as a complement - and not a replacement - of current SRH. Emerging evidence indicates that Coronavirus disease in 2019 has worsened SRH outcomes, especially related to gender-based violence; new evidence-based interventions are ever more urgent. This systematic review includes a focus on education-based male engagement, a special consideration of gender equity, and systematic searches by fluent speakers in three most populous languages in the Americas (English, Spanish, and Portuguese). PubMed, EBSCO, SCOPUS, and Google Scholar databases were digitally searched. Publications were excluded if their focus did not align directly with sexual reproductive health, their location was outside the scope of study, its content derived from information collected before 2010, or its study’s population’s age of focus was not between 15-24 years of age. After abstract screening and full-text review, the original 10,721 articles identified were narrowed down to 13 articles whose references were further examined through hand searching, leading us to a total of 32 final articles chosen for analysis. The results were classified by geographic regions of the American continent. The literature emphasized that society often defines masculinity as a hegemonic role grounded in aggressive high-risk sexual behavior. Adolescent males internalize this and hold their peers to these expectations. These beliefs have detrimental SRH consequences that have yet to be fully understood among adolescent boys and males. The efficacy of future interventions will depend on further exploration of these topics, especially among minority populations.", "keywords": [ "Sexual and Reproductive Health", "Male Engagement", "Masculinities", "Gender Equity", "Health Education", "Rights-Based Approach", "Community Health" ], "content": "Introduction\n\nSexual and reproductive health (SRH) is intertwined with the physical, mental, social, and economic well-being of individuals and society. SRH issues include, but are not limited to, “unintended pregnancy, complications of pregnancy and childbirth, unsafe abortion, gender-based violence, sexually transmitted infections (STIs).and reproductive cancers.”1 Since the 1994 International Conference on Population and Development in Cairo, rights-based research and interventions have encouraged better sexual and reproductive health outcomes.2 A gender transformative approach, which promotes attitudes of gender equity with men/boys and women/girls, involves reducing harmful masculinities and femininities that limit a person’s full potential; a gender equity approach is essential to further SRH rights.2,3 However, engagement of men and boys with SRH topics has been limited to less frequent, smaller, and more short-term projects compared to those involving women.2,3\n\nSRH in adolescents is of particular concern. Teenage pregnancy (mothers aged 10–19) poses an increased risk for complications, including eclampsia and systemic infections as well as preterm delivery and low birth weight.4 The World Health Organization cited that for females aged 15–19 years, complications with pregnancy and childbirth remain the leading cause of death.5 Pregnancy in adolescence, especially early adolescence, impacts school attendance as well as the economic trajectory of women, their communities, and even their countries.6 Beyond pregnancy, concerns of STIs, gender-based violence, and unsafe abortions particularly threaten the well-being of adolescents, especially teen girls.5,7\n\nWhile the adolescent brain increases the propensity for risk-taking,8 adolescence is also an important stage in developing lifelong knowledge, attitudes, and practices.9 Health-related attitudes in teenage years have both immediate as well as life-long effects – a point emphasized in the life course perspective.9 Adolescence is an important moment for normative and educational interventions related to gender equity and SRH.9,10 Despite this understanding, progress towards SRH goals for adolescents across the Americas has stagnated. For example, countries in Latin America and the Caribbean [LAC] have, “experienced the slowest decline in adolescent fertility for the 15–19-year age group of all regions in the world”. Additionally, LAC is the only region with, “a rising trend in pregnancies in adolescents younger than 15 years”.6\n\nReports from the US Aid and International Development (USAID) agency and the Pan-American Health Organization, among others, demonstrate a growing consensus for a masculinities framework that engages men and boys in public health and social change.3,11 Male engagement acts as a compliment – not a replacement – of current SRH and empowerment work with women.12 Such work welcomes men to become better agents of their own health and the health of their communities. While a large metadata review of literature in 2018 addresses a similar topic globally, the study faces limitations.2 Emerging evidence indicates that Coronavirus Disease-19 (COVID-19) has frustrated and even worsened SRH outcomes, especially related to gender-based violence;13 new interventions are ever more urgent. The first of its kind focused on the Americas region, this current and inclusive review of male engagement in SRH can drive community-based and larger-scale interventions. Thorough in nature, this systematic review includes a focus on education-based male engagement, consideration of gender equity, and systematic searches by fluent speakers in the three most populous languages of the Americas (English, Spanish, and Portuguese).\n\n\nMethods\n\nBefore database exploration, all authors thoroughly reviewed the PROSPERO registry at the end of December 2020, to verify that a systematic review focused on the nexus of masculinity norms and SRH within the Americas did not already exist. Once this was confirmed the database research commenced for this systematic review. In January 2021, the investigators scrutinized four large multidisciplinary databases: PubMed, EBSCOhost, SCOPUS, and Google Scholar. The specific databases within the mega databases of EBSCOhost that were chosen for search are detailed in Table 1. Furthermore, within PubMed and SCOPUS, advanced search filters were applied to tailor the search such that articles only focused on countries within the Americas region and published from 2010 and onward were outputted in the search results. Database access was enabled by the Universidad San Francisco de Quito’s online library services for students.\n\nAll authors collaborated in developing and finalizing the keyword formula utilized for database search. The following is the English representation of the algorithm:\n\n(“America* region” OR “America*” OR “Caribbean”) AND (“masculinity” OR “manhood” OR “the role of men”) AND (“teenager” OR “young adult” OR “adolescent” OR “young men” OR “early adolescence”) AND (“sexual health” OR “sexual health education” OR “reproductive health”).\n\nThis formula was translated into Spanish and Portuguese in order to broaden the number of publications found in each database. The research team consisted of native Spanish speakers as well as members fluent in Portuguese, thus a translation of the keyword formulas did not require external support. Across all databases and languages, the use of Boolean terms, truncation, and parenthesis was consistent.\n\nIn order to best understand how male-engagement interventions and internalizations of masculinities impact SRH throughout the Americas region, the keyword search was limited to literature from the timeframe range of 2010 until January 2021. Publications were excluded if they met one of the following criteria: 1) the focus of the article did not align with sexual and reproductive health topics, 2) the study’s location was outside the scope of the Americas region, 3) the publication’s content only described information collected before 2010, 4) the article’s population of focus did not fall between the ages of 15 and 24, 5) the publication was not an original science research article but rather an editorial/commentary piece or report from a governmental, non-governmental, or intergovernmental entity.\n\nPlease refer to Figure 1 for visual aid in understanding the screening process. All the investigators searched the previously explained keyword formula in three languages (English, Spanish, and Portuguese) across all four databases. The total sum of initial results was 10,721 sources. Article titles were compared to remove duplicates, resulting in 7,803 publications. However, since Google Scholar produces results that are organized by relevance according to the keyword inputs, publications listed after page number 5 of the search results were clearly beyond the scope of interest. It was deemed to eliminate these before the screening process, resulting in 7,548 articles removed from the total records. Duplicates across database results were then removed, narrowing the number of records to be formally screened to 255 articles.\n\nThis figure shows the total number of articles obtained for this systematic review. After filtration by inclusion and exclusion criteria along with the application of snowballing, 32 articles were selected for analysis.\n\nArticles were scrutinized for relevance according to the pre-defined exclusion and inclusion criteria. Each of the 255 article’s basic identification information was organized into a comprehensive spreadsheet, documenting the following parameters: article link, title, abstract, authors, journal, total pages, as well as an outcome decision for the article’s inclusion in the review. After eliminating articles by reviewing the 255 articles via abstract review and application of the exclusion criteria, a total of 22 articles remained. A full-text article review of these remaining publications was performed. During the full-text review, articles were removed if they met previous exclusion criteria and were not peer-reviewed. To ensure valid decision-making, the researchers consulted all team members for clarity when a disagreement arose regarding exclusion criteria fulfillment. This full-text discussion resulted in nine other articles being excluded, leaving 13 articles for final synthesis.\n\nIn February 2021, the research team decided to implement the snowball technique in order to include additional relevant articles and expand investigation content. The applied snowball method involved hand searching all the 533 bibliographical references from the eligible 13 papers identified via systematic review. For each reference, the previously established systematic review parameters of inclusion and exclusion criteria were used. Snowball searching completed after conducting the systematic keywords formula search led to the identification of 19 additional eligible publications for synthesis. Hence, a total of 32 articles were analyzed thematically to generate findings.\n\n\nResults\n\nStudies included in this systematic review, and their baseline information, are outlined in Table 2. It was decided to divide the Americas into four main regions for the interpretation of results: North America, South America, Central America, and the Caribbean. The design of the scientific publications found included qualitative-based studies, cross-sectional, literature reviews, instrument validation assessment, intervention evaluations, article reviews, and community-based participatory research. The distinct forms of gathering primary data within these included focus groups, in-depth interviews, semi-structured interviews, and electronic surveys. Figure 2 details the distribution of the regions and study designs of the publications synthesized.\n\nThe figure shows the geographical distribution of the selected articles alongside their study design.\n\nThe perception, influence and relationship of masculinity within sexual and reproductive health addressed in the North American articles included in this systematic review highlight important considerations as shown in Table 3.\n\nConventional “guy talk” boosts idealized masculine expectations and the generation of masculine norms based on men’s abilities to communicate sexual behavior with peers. Masculinity standards assign sexual health care to women and the discussion of STIs to subordinated men thus leading to the discussion of these issues in private and with people whom men consider trustworthy.14\n\nIn terms of sexual and reproductive health, studies established the need to implement gender transformative programs that aim to reshape gender norms that encourage risky sexual behaviors, violence, and sexually transmitted infections (STIs).15 Awareness and attitudes of gender equality in adolescent populations is key for sustainable public health interventions.16\n\nWithin the African American populations studied, masculinity is highly related to cultural and community standards; this relationship can be measured by a Masculine Attributes Questionnaire (MAP).17 Young African American men have a higher incidence of sexually transmitted infections than any other population in the United States which may be due to the influence of “perceived peer norms” related to risky sexual behaviors such as the wrong and “shameful” use of condoms.18 Social recognition for having multiple partners, intertwined social networks, limited community of sexual partners along with the lack of relationship commitment are important determinants of masculinity expression in these populations.19–21\n\nIn terms of contraception, one study centering on African American men recognized their lack of knowledge regarding contraceptive methods besides condoms.22 Also, it was found that condom use errors were frequent and included failure to review some type of condom damage, conscious use of damaged or expired condoms, lack of discussion about the use of condoms, problems with condom sensation, adjustment, ruptures and premature removal.23 These mistakes were associated with African American men with multiple sexual partners. This population along with a sample of young aboriginal Canadians asserted women as the primary active negotiators of condom use.24\n\nMen’s romantic relationships in early adolescence increase the influence of sexual health and develop a process characterized by curiosity and anticipation of sexual behaviors.25 Preventive clinical interventions must extend beyond mere recommendations of condom use or abstinence, including the promotion of strategies that help young men self-assess “personal disposal” for sex.26 The development of values as well as sexual and romantic relationship dynamics in adolescence occurs alongside family and peer experiences.27 These fundamental components of a sexual history implicate the risk of STIs as well as early fatherhood.26,27\n\nOne study associated the influence of the perception of young African American on their partner’s desire to conceive their child and the practice of risky sexual behaviors as a tool for compensating masculinity in the context of lack of schooling or job opportunities that prevents them from expressing other male responsibilities such as financial or sentimental support.28 Then, the development of couple communication skills regarding pregnancy intentions plays a crucial role in pregnancy prevention interventions and father’s involvement during pregnancy, and thus future child health and social outcomes.29\n\nAdolescent parenthood is filled with life development limitations such as decreased years of schooling, decreased human capital and balk psychological development.20,30 These factors may contribute to dysfunctional atmospheres and may start an intergenerational cycle of early fatherhood in which children are raised in low-income environments and are exposed to abuse and neglect.31,32\n\nAdolescent men have less involvement with primary health systems and unmet health care needs.33 Non-clinical youth-serving professionals represent a bridge between adolescent primary close up to sexual health and their access to clinical interventions; however, there are multiple gaps that limit this linkage such as the poor training of these professionals in terms of sexual health.34\n\nAdolescence is a period of transition in which the personality is influenced by hegemonic masculinities and intersectionality.31 Understanding how risk factors such as poverty, school failure, drug use and abuse, and sexual abuse contribute to early parenthood is the first step to prevention.31,35 The construction of identities at this stage should encourage adolescent men to understand the sense of responsibility of parenthood and promote their talents.33\n\nFurthermore, one particular study showed that there is a greater range of participation in education campaigns and prevention of sexually transmitted diseases when applying social activities.36 The role of media campaigns and the use of social media and networks that support health interventions can contribute to community acceptance and the reduction of stigma of certain aspects of sexual health.36\n\nThe masculinity in adolescent relationships of the African Americans studied set precedents for their adult relationships.27 Difficulties in regulating emotions were associated with the practice of risky sexual behaviors.21 Redefining masculinity towards egalitarian terms that promote healthy sexual practices, prevent violence, reduces the transmission of HIV and STIs and enhances the physical and mental health of both men and women.37–39\n\nOne article based within Panama represents the results of the Central American region and shows important considerations reflected in Table 3.\n\nA “real man” within the Panamanian sample studied represents a sexually active, independent, dominant male. Adolescents of Bañado Sur tend to replicate this socially acceptable masculine role exclusively with casual relationships or peers even if these behaviors do not match their personal beliefs. Men that do not follow acceptable masculinity trademarks are related to a certain degree of exclusion or homosexual tags. Family and support networks influence adolescent sexual behaviors directly. In the context of the Panama sample studied, adolescents attribute their risky sexual practices and promiscuity to poor parent and school sexual health education which has motivated them to experiment these topics by themselves. Machismo and taboos are important determinants of risky behaviors and can reflect relationship dynamics such as sex restriction until marriage and trivialization of sex.40\n\nTwo articles within the context of Cuba and one in Jamaica represent the results of the Caribbean region and show important considerations reflected in Table 3.\n\nIn Cuban samples where sex-related roles were assigned, women were labeled as dependent, “soft,” and sentimental whereas men represented power and resistance. Within these populations, men have a low incidence of condom use. Knowledge about sexual education is minimal and mostly acquired in out-of-school settings. In terms of contraception, it was found that, for some men, willingness to engage in safe sex practices such as the use of condoms was only related to STI prevention and not pregnancy prevention. Men’s tendencies to practice condomless sex, deny readiness for parenting responsibilities, and consider pregnancy as primarily a woman’s responsibility.41 Men attributed their lack of condom use specifically at loss of pleasure and were more likely to engage sexual practices with older women who prefer condomless sex. The relationship between dominance/virility and procreative desire can be measured by a “Macho Scale” described within the Jamaican study included in this systematic review.42\n\nOne investigation that occurred in Chile and another which took place in Paraguay represent the results of the South American region and show important considerations reflected in Table 3.\n\nBased on results from the two South American publications selected, evidence demonstrates that several programs with goals of advancing SRH have been carried out focusing solely on female populations. The article from Chile investigates masculinity perceptions as related to participation in SRH services. It includes the perceptions of healthcare personnel and that of male adolescents about masculinity and its link with SRH services. Likewise, it is appreciated that the variable masculinity or ideology of masculinity intervenes in several aspects such as the use of condoms, contraceptives, in general to the SSR. The context of creation of friendly spaces is relevant to understand the sense of masculinity in young people nevertheless, despite the existence of these spaces, young men go to hospitals and clinical spaces in cases of extreme urgency only.43 According to the findings of a study conducted in Paraguay, young people believe stereotypes and openly express them. The study points out that gender, relationships, and masculine norms interact in a similar way in disadvantaged and marginalized communities all around the world.44\n\n\nDiscussions\n\nThis systematic literature review is the first study that has explored the role and influence of masculinity in adolescent sexual and reproductive health in the Americas in the three most popular languages of this continent: English, Spanish, and Portuguese. The traditional belief in the role of women as solely responsible for sexual health has stagnated male involvement in family planning, contraception, communication skills between partners, prevention of sexually transmitted diseases and pregnancy.45 For these reasons, the need to implement gender-transformative interventions is an opening to the practice of safe sexual behaviors that will decrease the prevalence of STIs and gender-based violence.\n\nIn this study, we took into consideration the geographical and cultural context of the results since harmful masculinity foundation goes beyond sexual health education. In fact, it includes the beliefs, perceptions, and social practices that men have regarding their sexuality as reflected in past research.46 Under the social pressure to become a “real man”; understood as a sexually active, dominant masculine type; men’s support networks are important as they exert a significant influence on sexual behaviors. The influence can represent a place free from social constructions that stigmatize and affect men’s health or maximize harmful gender norms.14,37\n\nConsidering the cultural background of the populations studied in the articles helped us identify the risk of STIs in minorities. The results positioned African American and Canadian aboriginal young males as more pressured to have a hypersexual state and not use condoms, respectively.21,18 Hence, we recommend that further strategies for targeting minorities should include a global understanding of the socio-cultural and economic context of specific populations.\n\nOur findings enhance the importance of innovative educational interventions. School sexual education remains as the most used tool to identify focal groups of young males and create support systems that strengthen non-toxic masculinity perceptions. However, in today’s global context, it is crucial to use educational platforms that can be spread throughout social media.19 This strategy will allow rapid dissemination of information on STIs, pregnancy prevention programs and beneficial gender perceptions.\n\nUnlike past reviews related to this topic, the search terms were translated to the three most populous languages in the Americas (English, Spanish and Portuguese). While searches in Spanish and Portuguese did not produce many additional results, it clarifies that the literature is largely published in English, followed by Spanish. Additionally, the consulted search engines were four reliable public health and social science research databases. The precision of specific inclusion and exclusion criteria allowed the search to reveal the most pertinent information for future community health practice in the topic area of adolescent SRH. We included the latest data using studies performed since 2010 in order to provide an update on our topic. A “snowball approach”, utilizing the referenced citations of searched publications allowed for a more complete review of the relevant literature.\n\nSince many studies initially produced by database search were eliminated due to not meeting the inclusion criteria, the research team questioned whether the focus of the systematic review was too narrow. The rigid search criteria, while positing only the most rigorous results, may exclude important strides in the work of interest completed through thesis or dissertation studies as well as through government and/or non-governmental research reports. Certain search terms such as “gender equity,” “gender transformative,” “sexual and reproductive rights,” among others could have been added to broaden relevant language to the study’s interest. An expanded search of current literature might be specifically helpful in regions of the Americas where such little original research has been published on the topic, specifically Central and South America.\n\n\nConclusions\n\nSexual and reproductive health interventions with adolescents must include male engagement as part of a more integrated approach towards gender equity. Project implementers should take into consideration the local context of studied populations, utilizing methods such as focus groups or in-depth interviews to understand social norms. Short-term approaches may provisionally motivate change related to perceptions of masculinity and SRH practices. Current interventions have been completed on small populations and scales, lacking the wherewithal for long-term impact related to positive youth development and social change. Without a sustainable infrastructure to maintain interventions, all the improvements may be lost as the global context of the participants remains quite similar after the study. The current literature contains some focus on vulnerable and/or historically marginalized populations, especially in North America. Future investigations of the link between masculinity and adolescent SRH should be extended to populations experiencing both physical and mental disabilities, a vulnerable population across the Americas that has not been formally incorporated into these research and interventions. The precedent has been set for the important work to improve sexual and reproductive health outcomes for all in the coming decades. Male engagement as a solid base for SRH education is the first step towards new interventions that would help eradicate gender inequity in the Americas. This study serves as a foundation for a new field in SRH that needs to be explored. Only through engaging all people, men and women alike, can our communities become healthier, safer, and more agentive.\n\n\nFunding source\n\nPublication of this article was funded by the Universidad San Francisco de Quito’s $675 grant.\n\n\nConflict of interest statement\n\nThe authors have no competing interests to declare.\n\nIvonne Salinas, Corresponding Author Contributions =\n\n- Contributed significantly to Abstract, Methods, Discussion and Conclusion section\n\n- Collaborate with co-authors in overall conceptualization and writing of manuscript\n\n- Grant final approval of manuscript version to be published\n\n- Agree to be held accountable for accuracy and integrity of work\n\n- Holds primary responsibility for proper submission of manuscript and correspondence with journal\n\nErick Freire, Co-Author Contributions =\n\n- Contributed significantly to Abstract, Introduction, Methods and Discussion section\n\n- Collaborate with co-authors in overall conceptualization and writing of manuscript\n\n- Grant final approval of manuscript version to be published\n\n- Agree to be held accountable for accuracy and integrity of work\n\nJane Guevara, Co-Author Contributions =\n\n- Contributed significantly to Results and Discussion section\n\n- Collaborate with co-authors in overall conceptualization and writing of manuscript\n\n- Grant final approval of manuscript version to be published\n\n- Agree to be held accountable for accuracy and integrity of work\n\nKeren Herrán, Co-Author Contributions =\n\n- Spearhead study design, data collection, analysis of results, and writing of manuscript\n\n- Contributed significantly to abstract and conclusion\n\n- Grant final approval of manuscript version to be published\n\nGabriela Ortiz, Co-Author Contributions =\n\n- Contributed significantly to Results and Discussion section\n\n- Collaborate with co-authors in overall conceptualization and writing of manuscript\n\n- Grant final approval of manuscript version to be published\n\n- Agree to be held accountable for accuracy and integrity of work\n\nIván Palacios, Co-Author Contributions =\n\n- Provide main expert insight and oversight on publication content\n\n- Initiate project conception and lead formation of team\n\n- Collaborate with co-authors in overall conceptualization and writing of manuscript\n\n- Grant final approval of manuscript version to be published\n\n- Agree to be held accountable for accuracy and integrity of work\n\n- Secure publication funding", "appendix": "Acknowledgements\n\nThe authors appreciate and recognize the valuable edits of Fehintola Bright, Het Desai, John DiBello, Jonathan Guillemot, and Ashley Romo in the formation of this piece.\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nStarrs AM, Ezeh AC, Barker G, et al.: Accelerate progress-sexual and reproductive health and rights for all: report of the Guttmacher-Lancet Commission. 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Publisher Full Text\n\nKogan SM, Cho J, Barton AW, et al.: The influence of community disadvantage and masculinity ideology on the number of sexual partners: A prospective analysis of young adult, rural black men. J Sex Res. 2017; 54(6): 795–801. PubMed Abstract \| Publisher Full Text\n\nHicks MR, Kogan SM, Cho J, et al.: Condom use in the context of main and casual partner concurrency: individual and relationship predictors in a sample of heterosexual african american men. Am J Mens Health. 2017; 11(3): 585–591. PubMed Abstract \| Publisher Full Text\n\nWoodhams E, Sipsma H, Hill BJ, et al.: Perceived responsibility for pregnancy and sexually transmitted infection prevention among young African American men: An exploratory focus group study. Sex Reprod Healthc. 2018; 16: 86–91. PubMed Abstract \| Publisher Full Text\n\nCrosby RA, Milhausen RR, Sanders SA, et al.: Condom use errors and problems: a study of high-risk young Black men residing in three Southern US cities. Int J STD AIDS. 2014; 25(13): 943–948. PubMed Abstract \| Publisher Full Text\n\nDevries KM, Free C: “I told him not to use condoms”: masculinities, femininities and sexual health of Aboriginal Canadian young people. Sociol Health Illn. 2010; 32(6): 827–842. Publisher Full Text\n\nCummings T, Auerswald CL, Ott MA: Factors influencing abstinence, anticipation, and delay of sex among adolescent boys in high-sexually transmitted infection prevalence communities. J Adolesc Health. 2014; 54(5): 593–598. PubMed Abstract \| Publisher Full Text\n\nOtt MA: Examining the development and sexual behavior of adolescent males. J Adolesc Health. 2010; 46(4 Suppl): S3–S11. PubMed Abstract \| Publisher Full Text\n\nBell DL, Rosenberger JG, Ott MA: Masculinity in adolescent males’ early romantic and sexual heterosexual relationships. Am J Mens Health. 2015; 9(3): 201–208. PubMed Abstract \| Publisher Full Text\n\nCrosby RA, Graham CA, Milhausen RR, et al.: Desire to father a child and condom use: a study of young black men at risk of sexually transmitted infections. Int J STD AIDS. 2015; 26(13): 941–944. PubMed Abstract \| Publisher Full Text\n\nLewin A, Mitchell SJ, Hodgkinson S, et al.: Pregnancy intentions among expectant adolescent couples. J Pediatr Adolesc Gynecol. 2014; 27(3): 172–176. PubMed Abstract \| Publisher Full Text\n\nFletcher JM, Wolfe BL: The effects of teenage fatherhood on young adult outcomes. Econ Inq. 2012; 50(1): 182–201. Publisher Full Text\n\nShade K, Kools S, Weiss SJ, et al.: A conceptual model of incarcerated adolescent fatherhood: adolescent identity development and the concept of intersectionality. J Child Adolesc Psychiatr Nurs. 2011; 24(2): 98–104. PubMed Abstract \| Publisher Full Text\n\nSipsma H, Biello KB, Cole-Lewis H, et al.: Like father, like son: the intergenerational cycle of adolescent fatherhood. Am J Public Health. 2010; 100(3): 517–524. PubMed Abstract \| Publisher Full Text\n\nBell DL, Breland DJ, Ott MA: Adolescent and young adult male health: a review. Pediatrics. 2013; 132(3): 535–546. PubMed Abstract \| Publisher Full Text\n\nMarcell AV, Gibbs SE, Howard SR, et al.: Do Nonclinical Community-Based Youth-Serving Professionals Talk With Young Men About Sexual and Reproductive Health and Intend to Refer Them for Care?. Am J Mens Health. 2017; 11(4): 1046–1054. PubMed Abstract \| Publisher Full Text\n\nOtt MA, Wells M, Imburgia TM, et al.: The sexual health needs of adolescent boys involved in a pregnancy. J Adolesc Health. 2019; 64(4): 537–540. PubMed Abstract \| Publisher Full Text\n\nOtt MA, Campbell J, Imburgia TM, et al.: Community Engagement and Venue-Based Sampling in Adolescent Male Sexually Transmitted Infection Prevention Research. J Adolesc Health. 2018; 62(3S): S58–S64. PubMed Abstract \| Publisher Full Text\n\nFleming PJ, Lee JGL, Dworkin SL: “Real men don’t”: constructions of masculinity and inadvertent harm in public health interventions. Am J Public Health. 2014; 104(6): 1029–1035. PubMed Abstract \| Publisher Full Text\n\nVagi KJ, O’Malley Olsen E, Basile KC, et al.: Teen dating violence (physical and sexual) among US high school students: findings from the 2013 national youth risk behavior survey. JAMA Pediatr. 2015; 169(5): 474–482. PubMed Abstract \| Publisher Full Text\n\nRink E, FourStar K, Medicine Elk J, et al.: Pregnancy prevention among American Indian men ages 18 to 24: the role of mental health and intention to use birth control. Am Indian Alaska Native Ment Health Res. 2012; 19(1): 57–75. PubMed Abstract \| Publisher Full Text\n\nCantos NC: Modelo cultural implicado en las relaciones de género y su influencia en la desigualdad de poder en la salud sexual y reproductiva de los adolescentes de la ciudad de Panamá. Societas. 2012.\n\nAlvaré LEA, Pastrana DL, Victores MM, et al.: Percepción del adolescente varón frente a las conductas sexuales de riesgo. Revista de Especialidades Médico-Quirúrgicas. 2011.\n\nThe impact of masculinity ideologies and conjugal involvement on sexual risk-taking among young Jamaican males. Accessed October 21, 2021. Reference Source\n\nObach A, Sadler M, Aguayo F, et al.: Sexual and reproductive health in young men in Chile: results of a qualitative studySaúde sexual e reprodutiva de jovens do sexo masculino no Chile: resultados de um estudo qualitativo. Rev Panam Salud Publica. 2018; 42: e124. PubMed Abstract \| Publisher Full Text\n\nFleming PJ, Andes KL, DiClemente RJ: “But I’m not like that”: young men’s navigation of normative masculinities in a marginalised urban community in Paraguay. Cult Health Sex. 2013; 15(6): 652–666. Publisher Full Text\n\nRodríguez Morales V, Castañeda Abascal IE, Rodríguez Cabrera A, et al.: Necesidad del abordaje de los estudios de la salud sexual y reproductiva en el hombre. Scielo. Rev Cubana Salud Pública. 2013; 39.\n\nOchoa-Marin SC, Vásquez-Salazar EA: Salud sexual y reproductiva en hombres. Scielo. Rev Salud Pública. 2012; 14(1): 15–27. PubMed Abstract \| Publisher Full Text" }	[ { "id": "222750", "date": "30 Nov 2023", "name": "Alison Kutywayo", "expertise": [ "Reviewer Expertise Adolescent sexual and reproductive health and gender based violence" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 systematic review of male engagement in SRH in the Americas. It is an interesting piece of work.\n\nHaving reviewed this manuscript, my main comments are related to the structure of the Methods, Results and Discussion. The Results need to be thematically analyzed by theme, rather than by geographical area and there are many things in the Methods that need to be in the Results.\n\nI suggest that the authors please carefully review the following manuscript ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005924/ ) for guidance on what to include in the respective sections. Table 1 in this PRISMA manuscript provides a clear guide that will help you strengthen your manuscript.\nIn addition to these main comments, I have a 61 editorial comments throughout the manuscript for your consideration. (See attached PDF)\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/11-394
https://f1000research.com/articles/10-1142/v1	11 Nov 21	{ "type": "Case Report", "title": "Case Report: Isolated, unilateral oculomotor palsy with anti-GQ1b antibody following COVID-19 vaccination", "authors": [ "Takafumi Kubota", "Takafumi Hasegawa", "Kensuke Ikeda", "Masashi Aoki", "Takafumi Kubota", "Kensuke Ikeda", "Masashi Aoki" ], "abstract": "Neurological complications following vaccinations are extremely rare, but cannot be eliminated. Here, we report the first case of unilateral oculomotor nerve palsy (ONP) with anti-GQ1b antibody after receiving the Pfizer-BioNTech COVID-19 (BNT162b2) mRNA vaccine. A 65-year-old man developed diplopia and ptosis in the right eye 17 days after vaccination, without preceding infection. Neurological examination revealed mild blepharoptosis, limitation of adduction, and vertical gaze on the right side. Increased levels of anti-GQ1b ganglioside antibody in the serum and albuminocytologic dissociation in the cerebrospinal fluid were detected. Cranial magnetic resonance imaging showed swelling and enhancement of the right oculomotor nerve. The patient was diagnosed with right ONP accompanied with anti-GQ1b antibody, and intravenous immunoglobulin (IVIG) therapy for 5 days was administered. The limitation of adduction and vertical gaze improved, and ptosis markedly resolved after IVIG treatment. Given the temporal sequence of disease progression, laboratory findings, and a favorable response to IVIG, a causal relationship cannot be ruled out between the occurrence of ONP and COVID-19 immunization. Since immunomodulatory treatments significantly hasten the recovery and minimize the residual symptoms in anti-GQ1b antibody syndrome, clinicians should be aware of this clinical condition following COVID-19 vaccination.", "keywords": [ "oculomotor nerve palsy", "Miller Fisher syndrome", "anit-GQ1b antibody", "ganglioside", "COVID-19", "vaccination", "IVIG" ], "content": "Introduction\n\nOculomotor nerve palsy (ONP) is a neurological condition that manifests as diplopia, ptosis, and pupillary mydriasis. The various etiologies of ONP include cerebrovascular disease, cerebral aneurysm, diabetes, tumor, infection, collagen disease, hyperthyroidism, and Tolosa-Hunt syndrome1. In some cases, ONP can be caused by an aberrant immune response that develops directly against ganglioside GQ1b, a sialic acid-containing glycosphingolipid enriched in the paranodal region in the III (oculomotor), IV (trochlear), and VI (abducens) cranial nerves2. The para-infectious, immune-mediated ONP, along with ataxia and loss of tendon jerks, was originally described by Charles Miller Fisher as a variant of Guillain-Barré Syndrome (GBS)3. Since there are incomplete or atypical forms of Miller Fisher syndrome (MFS), an umbrella term, “anti-GQ1b antibody syndrome” has emerged to encompass these clinical conditions4.\n\nIn addition to an antecedent infectious illness, vaccine-mediated immunization can trigger GBS and MFS5–8, for example, MFS following influenza6–8, pneumovax8, and DPT (diphtheria, pertussis, tetanus toxoid) vaccination5 has been reported. GBS has been listed as a very rare neurological complication of the COVID-19 vaccine9–15. However, to the best of our knowledge, there have been no case reports of isolated, unilateral ONP with anti-GQ1b antibody following vaccination. Here, we report an adult case of acute-onset right ONP with anti-GQ1b antibody following COVID-19 vaccination with a literature review.\n\n\nCase description\n\nA 65-year-old Asian male office worker began to notice persistent double vision without preceding upper respiratory or gastrointestinal infection. The diplopia worsened in the left gaze, and three days later, he developed right ptosis. He was vaccinated with a second dose of Pfizer-BioNTech COVID-19 (BNT162b2) mRNA vaccine 17 days before his presentation. His medical history included a seven-year history of diabetes, glaucoma, and benign paroxysmal positional vertigo. He did not have diabetic retinopathy or neuropathy in his right eye. His medication included one tablet per day of Canalia® (teneligliptin and canagliflozin), a diabetic combination drug which the patient had been taking for one year and one drop per day of prostaglandin analogue eye drops for glaucoma (time taken for unknown).\n\nThe general medical condition of the patient on admission (day 22) was unremarkable. Neurological examination revealed mild blepharoptosis, limitation of adduction, and vertical gaze on the right side (Figure 1A) with convergence insufficiency. Pupils were slightly asymmetric (right: 3.5 mm, left: 3.0 mm) and the right pupil was slightly slowly reactive to light. The other cranial nerves were preserved normally. These findings were consistent with the diagnosis of right ONP. Gait was normal, with no evidence of muscle weakness, ataxia, or sensory disturbances. Deep-tendon reflexes are normally elicitable.\n\n(A) Mild blepharoptosis, limitation of adduction and vertical gaze on the right side on day 30. (B) The limitation of adduction and vertical gaze improved and ptosis completely resolved after IVIG treatment (day 52).\n\nRoutine hematological and biochemical analyses, including thyroid function, were normal except for the elevation in glucose concentration 162 mg/dL (normal range: 78–109 mg/dL) and HbA1c level 7.8% (normal range: 4.6–6.2%). Serological tests identified the presence of anti-GQ1b IgG antibody (1.82, normal cut-off index <1), a pathognomonic marker for MFS. Other antibodies against glycoconjugates, including ganglioside GM1, antinuclear antibodies, perinuclear antineutrophil cytoplasmic antibody (ANCA), cytoplasmic ANCA, and acetylcholine receptor antibodies were negative. Cerebrospinal fluid showed mild albuminocytologic dissociation with protein levels of 52 mg/dL (normal range: 10–40 mg/dL) and 2 mononuclear cells/mm3 (normal range: 0–5 cells/mm3). High-resolution, constructive interference in steady-state magnetic resonance imaging (CISS-MRI) showed swelling with gadolinium enhancement in the right cavernous segment of the oculomotor nerve (Figure 2), but no signs of aneurysm, tumor, and inflammation in the cavernous sinus and orbital apex were noted. A nerve conduction study in the limbs was normal.\n\nBased on these clinical and laboratory findings, we diagnosed the patient with isolated, unilateral ONP associated with anti-GQ1b antibody and administered intravenous immunoglobulin (IVIG, 400 mg/kg) for consecutive 5 days. On the fourth day of IVIG administration (day 36), the limitation of the vertical gaze and ptosis mildly improved. There were no adverse events during or after the IVIG treatment. The patient was discharged on day 40 and was followed up at an outpatient clinic on day 52. The limitation of the adduction and vertical gaze markedly improved and ptosis completely resolved on day 52 (Figure 1B). The patient also noticed an improvement in his diplopia. The patient was afraid of receiving further vaccinations, including the COVID-19 vaccine. We explained to him that the incidence of GBS and MFS caused by vaccinations are extremely rare and the causal link between neurological complication and COVID-19 vaccinations is still unclear. It is undoubtedly true that benefits of vaccination outweigh the risks and the truth of the matter is that rare side effects shouldn't rule out vaccines. If he is going to be vaccinated in the future, we will carefully watch his condition and seek medical attention as soon as possible if he experiences any complications.\n\n\nDiscussion\n\nWe report the first case of isolated unilateral ONP with anti-GQ1b antibody following COVID-19 vaccination. Clinically, there were many similarities between our case and the previous four cases of unilateral ONP with anti-GQ1b antibody (Table 1)16–18. First, all cases, including ours, had ptosis without ataxia, and three cases showed normal deep tendon reflexes. Second, three patients demonstrated albuminocytologic dissociation in the CSF. Third, all of the cases showed a normal pattern in the nerve conduction studies. Finally, four patients were successfully treated with IVIG and/or steroids, with different recovery periods ranging from 22 days to 6 months16–18. From an etiopathological point of view, two cases of isolated ONP with anti-GQ1b antibody were preceded by acute upper respiratory tract infection or gastroenteritis within two weeks of onset17,18. On the other hand, there have been no case reports of vaccine-induced, isolated ONP with anti-GQ1b antibody. Similar to GBS, the majority of MFS and other anti-GQ1b antibody-associated disorders showed a good response to immunotherapy, such as IVIG and plasmapheresis. However, if ONP is the sole manifestation of anti-GQ1b antibody syndrome, it can be difficult to diagnose, leading to a substantial therapeutic delay. In agreement with our case, cranial MRI demonstrated abnormal swelling, T2 hyperintensity, and gadolinium enhancement of the affected oculomotor nerve in classic forms of MFS, providing a useful tool for early diagnosis19–21.\n\nURI, upper respiratory infection; CSF, cerebrospinal fluid; NCS, Nerve conduction study; MRI, magnetic resonance imaging; NA, Not available; IVIG, intravenous immunoglobulin.\n\nA growing concern among recent coronavirus vaccines is vaccine-related side effects. The most commonly observed adverse events with COVID-19 vaccines are fatigue, headache, muscle and joint pain, fever, pain at the site of injection, and to a much lesser degree, severe allergic reactions including anaphylaxis. The occurrence of these side effects can be predicted based on what is already known about the clinical trials of other vaccines. While not all reported side effects are directly related to vaccine administration, life-threatening side effects such as thromboembolism and neurological complications, including GBS, have also been reported following COVID-19 vaccines. In the United States, as of July 13, 2021 there were 100 preliminary reports of GBS after receiving the Janssen COVID-19 vaccine and 1 death after 12.5 million vaccine doses administered. GBS usually develops 3–22 days after the administration of COVID-19 vaccines9–15. Although MFS following COVID-19 vaccination has not been reported so far, MFS can be observed from 5 to 21 days after immunization with influenza6–8, pneumovax8, and DPT vaccines5. Similarly, our case also presented with unilateral ONP with elevated anti-GQ1b antibody 17 days after Pfizer-BioNTech COVID-19 (BNT162b2) vaccination without any preceding infection. Based on the temporal sequence of disease progression, laboratory findings, and a favorable response to immunotherapy, the possibility that preceding COVID-19 vaccination may provoke unfavorable immune responses, leading to ONP in our patient, cannot be ruled out.\n\nIt should be noted that ONP is the most common cranial neuropathy in patients with diabetes22. Diabetes not only causes ischemic neuropathy, but also induces chronic low-level inflammation in peripheral nerves through the elevation of various inflammatory markers such as C-reactive protein, tumor necrosis factor, and interleukin-622. Thus, one can imagine that the pre-existing diabetes in our case might impair oculomotor nerve function, thereby aggravating demyelinating oculomotor damage by the anti-GQ1b antibody. Indeed, diabetes has been reported as a risk factor for the exacerbation and poor outcomes of GBS23–25.\n\n\nConclusion\n\nUnilateral, isolated ONP with anti-GQ1b antibody was observed following COVID-19 vaccination. Insufficient recognition of this treatable condition often leads to misdiagnosis, which delays the receipt of adequate immunomodulatory therapy. Physicians should consider this rare clinical entity, even when the classical triad of MFS is absent. While the benefits of COVID-19 vaccination substantially outweigh the rare, possible adverse events, healthcare professionals should carefully monitor the hazardous effects of all COVID-19 vaccines and continue to work closely to manage potential risks and to harness science and big data to drive feedback and recommendations.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this case report and any associated 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": "Acknowledgments\n\nWe thank all the neurology medical wards and department staff of Tohoku University Hospital.\n\n\nReferences\n\nRaza HK, Chen H, Chansysouphanthong T, et al.: The aetiologies of the unilateral oculomotor nerve palsy: a review of the literature. Somatosens Mot Res. 2018; 35(3–4): 229–239. PubMed Abstract \| Publisher Full Text\n\nChiba A, Kusunoki S, Shimizu T, et al.: Serum IgG antibody to ganglioside GQ1b is a possible marker of Miller Fisher syndrome. Ann Neurol. 1992; 31(6): 677–679. PubMed Abstract \| Publisher Full Text\n\nFisher M: An unusual variant of acute idiopathic polyneuritis (syndrome of ophthalmoplegia, ataxia and areflexia). N Engl J Med. 1956; 255(2): 57–65. PubMed Abstract \| Publisher Full Text\n\nPanda S, Tripathi M, Odaka M, et al.: Anti-GQ1b IgG antibody syndrome: clinical and immunological range. J Neurol Neurosurg Psychiatry. 2002; 72(3): 418–419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrämer HH, Niemöller U, Döring K, et al.: Postvaccination Miller Fisher syndrome after combined pertussis, diphtheria and tetanus toxoid vaccine. J Infect. 2013; 66(5): 460–461. PubMed Abstract \| Publisher Full Text\n\nShoamanesh A, Chapman K, Traboulsee A: Postvaccination Miller Fisher syndrome. Arch Neurol. 2011; 68(10): 1325–1327. PubMed Abstract \| Publisher Full Text\n\nBlanco-Marchite CI, Buznego-Suárez L, Fagúndez-Vargas MA, et al.: [Miller Fisher syndrome, internal and external ophthalmoplegia after flu vaccination]. Arch Soc Esp Oftalmol. 2008; 83(7): 433–435. PubMed Abstract \| Publisher Full Text\n\nThaler A: Miller Fisher syndrome in a 66-year-old female after flu and pneumovax vaccinations. J Am Med Dir Assoc. 2008; 9(4): 281–283. PubMed Abstract \| Publisher Full Text\n\nTrimboli M, Zoleo P, Gennarina A, et al.: Guillain-Barré syndrome following BNT162b2 COVID-19 vaccine. Neurol Sci. 2021; 42(11): 4401–4402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaheed S, Bayas A, Hindi F, et al.: Neurological Complications of COVID-19: Guillain-Barre Syndrome Following Pfizer COVID-19 Vaccine. Cureus. 2021; 13(2): 2–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRazok A, Shams A, Almeer A, et al.: Post-COVID-19 vaccine Guillain-Barré syndrome; first reported case from Qatar. Ann Med Surg (Lond). 2021; 67: 102540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMin YG, Ju W, Ha YE, et al.: Sensory Guillain-Barre syndrome following the ChAdOx1 nCov-19 vaccine: Report of two cases and review of literature. J Neuroimmunol. 2021; 359: 577691. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIntrona A, Caputo F, Santoro C, et al.: Guillain-Barré syndrome after AstraZeneca COVID-19-vaccination: A causal or casual association? Clin Neurol Neurosurg. 2021; 208: 106887. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMckean N, Chircop C: Guillain-Barré syndrome after COVID-19 vaccination. BMJ Case Rep. 2021; 14(7): e244125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan M, Han SC, Kelly S, et al.: A case series of Guillain-Barré Syndrome following Covid-19 infection in New York. Neurol Clin Pract. 2021; 11(4): e576–e578. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee SH, Lim GH, Kim JS, et al.: Acute ophthalmoplegia (without ataxia) associated with anti-GQ1b antibody. Neurology. 2008; 71(6): 426–429. PubMed Abstract \| Publisher Full Text\n\nIchikawa H, Kamiya Y, Susuki K, et al.: Unilateral oculomotor nerve palsy associated with anti-GQ1b IgG antibody. Neurology. 2002; 59(6): 957–958. PubMed Abstract \| Publisher Full Text\n\nUeno T, Kon T, Kurihara AI, et al.: Unilateral oculomotor nerve palsy following campylobacter infection: A mild form of miller fisher syndrome without ataxia. Intern Med. 2017; 56(21): 2929–2932. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNagaoka U, Kato T, Kurita K, et al.: Cranial nerve enhancement on three-dimensional MRI in Miller Fisher syndrome. Neurology. 1996; 47(6): 1601–1602. PubMed Abstract \| Publisher Full Text\n\nGarcia-Rivera CA, Rozen TD, Zhou D, et al.: Miller Fisher syndrome: MRI findings. Neurology. 2001; 57(10): 1755. PubMed Abstract \| Publisher Full Text\n\nLantos JE, Strauss SB, Lin E: COVID-19-Associated miller fisher syndrome: MRI findings. AJNR Am J Neuroradiol. 2020; 41(7): 1184–1186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHartge MM, Unger T, Kintscher U: The endothelium and vascular inflammation in diabetes. Diab Vasc Dis Res. 2007; 4(2): 84–88. PubMed Abstract \| Publisher Full Text\n\nPeric S, Bozovic I, Bjelica B, et al.: Diabetes mellitus may affect short-term outcome of Guillain-Barré syndrome. J Peripher Nerv Syst. 2017; 22(2): 127–130. PubMed Abstract \| Publisher Full Text\n\nZhang Y, Zhao Y, Wang Y: Prognostic factors of Guillain-Barré syndrome: a 111-case retrospective review. Chin Neurosurg J. 2018; 4: 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBae JS, Kim YJ, Kim JK: Diabetes mellitus exacerbates the clinical and electrophysiological features of Guillain-Barré syndrome. Eur J Neurol. 2016; 23(3): 439–446. PubMed Abstract \| Publisher Full Text" }	[ { "id": "99811", "date": "30 Nov 2021", "name": "Chieko Suzuki", "expertise": [ "Reviewer Expertise 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\nThis report shows unilateral oculomotor nerve palsy associated with anti- GQ1b antibody following COVID-19 vaccination. It is important because there is a growing concern about the adverse effects of COVID-19 vaccines worldwide.\nAnti GQ1b antibody-associated isolated oculomotor nerve palsy is rare. On the other hand, diabetic oculomotor nerve palsy is a common disease. It is necessary to examine whether diabetes has any effect on this condition. In this case, treatment was started more than one month after the onset of the disease.\nIf the condition is related to GQ1b, recovery phase may begin. Were there any signs of recovery before starting treatment?\nElevated CSF protein is often observed in diabetes mellitus. Do you have follow-up data of CSF? If this condition is associated with GQ1b, there may be changes in CSF protein.\n\nContrast-enhanced findings of the oculomotor nerve on MRI have been reported in diabetic oculomotor palsy and idiopathic oculomotor palsy (Zhao et al. 20211; Yang et al. 20202). This finding is not specific to MFS.\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": "8030", "date": "05 Apr 2022", "name": "Takafumi Hasegawa", "role": "Author Response", "response": "Comment #1: If the condition is related to GQ1b, recovery phase may begin. Were there any signs of recovery before starting treatment? Reply: Thank you very much for your comment. There were no obvious signs of recovery before IVIG treatment. He had gradually improved after the therapy. To further clarify the process of recovery, we added the last follow-up when he completely recovered on day 77 in the second paragraph of the diagnostic assessment section as follows: “He completely recovered on day 71 (Figure 1 C).” Comment #2: Elevated CSF protein is often observed in diabetes mellitus. Do you have follow-up data of CSF? If this condition is associated with GQ1b, there may be changes in CSF protein. Reply: Thank you very much for your comment. We are sorry but we did not perform follow-up CSF analysis. As you mentioned, albuminocytologic dissociation in CSF might have improved after the therapy. Comment #3: Contrast-enhanced findings of the oculomotor nerve on MRI have been reported in diabetic oculomotor palsy and idiopathic oculomotor palsy (Zhao et al. 20211; Yang et al. 20202). This finding is not specific to MFS. Reply: We humbly accept this reviewer’s comment. We deleted the following sentence in the 1st paragraph of the discussion: “In agreement with our case, cranial MRI demonstrated abnormal swelling, T2 hyperintensity, and gadolinium enhancement of the affected oculomotor nerve in classic forms of MFS, providing a useful tool for early diagnosis.”" } ] }, { "id": "126605", "date": "22 Mar 2022", "name": "Miguel García-Grimshaw", "expertise": [ "Reviewer Expertise Currently working on acute and long-term neurologic manifestations of COVID-19 and neurologic adverse events following immunization against SARS-CoV-2." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this report, the authors describe the case of a male who developed an isolated unilateral oculomotor palsy following immunization with BNT162b2 and positivity anti-GQ1b antibody.\nReal-world evidence (outside clinical trials) is essential to detect potential adverse events of these newly developed vaccines. However, this case does not fulfill the clinical diagnostic criteria for the Miller Fisher syndrome (MFS) variant of Guillain-Barré syndrome (GBS).\n\nIt would be interesting if the author could discuss the positivity of anti-GQ1b antibody in the general population patients (without clinical suspicion of GBS), including sensitivity and specificity of this antibody for diagnosing MFS.\nIn the discussion section, the author describes the possibility of diabetic cranial neuropathy. It would be interesting to know if the patient was tested for oligoclonal bands in the cerebrospinal fluid and serum anti-AQP-4 antibodies as part of unilateral optic neuritis differential diagnosis as his contrast-enhanced MRI demonstrates swelling and enhancement in the right oculomotor nerve.\n\nLastly, it would be interesting if the author could discuss or speculate on some of the pathophysiological mechanisms on how mRNA-based vaccines may be related to GBS or MFS.\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": "8031", "date": "05 Apr 2022", "name": "Takafumi Hasegawa", "role": "Author Response", "response": "Comment #1: It would be interesting if the author could discuss the positivity of anti-GQ1b antibody in the general population patients (without clinical suspicion of GBS), including sensitivity and specificity of this antibody for diagnosing MFS. Reply: Thank you very much for your valuable comment. From two previous studies, all ten patients with MFS were positive for anti-GQ1b antibody, whereas all 81 control people without GBS were negative. Thus, the sensitivity and specificity of anti-GQ1b antibody in the patients with MFS should be very close to 100% (Chiba A et al. Ann Neurol (1992) 31:677–679. Willison HJ et al. J Neurol Neurosurg Psychiatry (1993) 56:204–206). We added this point in the first paragraph of introduction as follows: “Compared to control subjects without neurological complications, the sensitivity and specificity of anti-GQ1b antibody in the patients with MFS are very close to 100% (2,4).Since there are incomplete or atypical forms of Miller Fisher syndrome (MFS), an umbrella term, “anti-GQ1b antibody syndrome” has emerged to encompass these clinical conditions (5).” Comment #2: In the discussion section, the author describes the possibility of diabetic cranial neuropathy. It would be interesting to know if the patient was tested for oligoclonal bands in the cerebrospinal fluid and serum anti-AQP-4 antibodies as part of unilateral optic neuritis differential diagnosis as his contrast-enhanced MRI demonstrates swelling and enhancement in the right oculomotor nerve. Reply: We appreciate this reviewer’s constructive comment. Oligoclonal bands were negative in this case, but we did not measure anti-AQP-4 antibodies. It is true that anti-AQP-4 antibodies can cause oculomotor nerve palsy in a very rare case (Yasuda K et al. J Clin Neurosci. 2019 Aug;66:271-272.); however, our case did not have any other symptoms and signs of NMOSD. Hence, we think it unlikely that anti-AQP-4 antibodies contribute to the manifestation of oculomotor nerve palsy in our case. Taking the above into consideration, we added the data of oligoclonal bands and myelin basic protein in the diagnostic assessment section as follows: “Oligoclonal bands were negative and myelin basic protein was less than 31.3 pg/mL (normal range: < 102 pg/mL).”. Comment #3: Lastly, it would be interesting if the author could discuss or speculate on some of the pathophysiological mechanisms on how mRNA-based vaccines may be related to GBS or MFS. Reply: Thank you very much for your comment. We add the possible mechanism by which mRNA-based vaccine produce anti-GQ1b antibody and subsequent neurological deficits in the second paragraph of the discussion as follows: “BNT162b2 vaccine of messenger RNA enter the body and export spike proteins on the cell, which provoke the production of antibodies and T cell reactions (19). These immunological alterations may produce neutralizing antibodies as well as anti-GQ1b antibody, thereby leading to unfavorable neurological complications.”" } ] } ]	1	https://f1000research.com/articles/10-1142
https://f1000research.com/articles/10-923/v1	15 Sep 21	{ "type": "Research Article", "title": "Effects of 3% binahong (Anredera cordifolia) leaf extract gel on alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)", "authors": [ "Olivia Avriyanti Hanafiah", "Diana Sofia Hanafiah", "Gostry Aldica Dohude", "Denny Satria", "Livita Livita", "Nindha Siti Moudy", "Rahma Rahma", "Diana Sofia Hanafiah", "Gostry Aldica Dohude", "Denny Satria", "Livita Livita", "Nindha Siti Moudy", "Rahma Rahma" ], "abstract": "Background: Binahong (Anredera cordifolia (Ten.) STEENIS) is a widely available herbal plant in Indonesia and has been intensely researched for its healing abilities due to its biological activities, but few have studied its capability in accelerating hard tissue healing in post-extraction tooth sockets. The purpose of this study was to analyse the effects of 3% binahong leaf extract gel on alveolar bone healing in post-extraction sockets in Wistar rats. Methods: In this study, 48 male Wistar rats were randomly allocated to twelve groups. After the extraction of the left mandibular incisor, sockets in Group I to IV were given 3% binahong leaf extract gel, group V to VIII were given a control gel, and group IX to XII were given Gengigel® for 14 days. The residual socket volume (RSV) and fibroblast proliferation were observed on the 3rd, 7th, and 14th day post-extraction, while the osteoblast and osteocyte proliferation were observed on the 7th, 14th, and 28th day post-extraction. The RSV data were analysed using repeated measure ANOVA and one-way ANOVA, while the histopathological data were analysed using one-way ANOVA. Results: The results showed that the binahong group had the lowest RSV and the highest fibroblast proliferation compared to the other groups on the 7th day (p<0.05) and the highest osteoblast and osteocyte proliferation compared to the other groups on the 14th day (p<0.05). Conclusion: The experiment showed that 3% binahong leaf extract gel could accelerate wound closure, which was characterized by a greater decrease in the RSV value in comparison to the other treatment groups and could enhance alveolar bone healing by increasing the proliferation of fibroblasts, osteoblasts, and osteocytes.", "keywords": [ "Anredera cordifolia", "tooth extraction", "wound healing", "fibroblasts", "osteoblasts", "osteocytes" ], "content": "Introduction\n\nTooth extraction will leave a socket wound and can affect a person's quality of life, especially in terms of the ability to eat and speak.1,2 Socket wound healing involves the healing of soft tissue, which are comprised of the gingiva and connective tissue, as well as the healing of hard tissue, which is the alveolar bone.3 Both soft tissue and hard tissue undergo the same healing phases, namely the inflammatory, proliferative, and remodelling phases.2\n\nSome of the cells that play important roles during the healing of socket wounds are fibroblasts, osteoblasts, and osteocytes. At the beginning of alveolar bone healing, the cells that are more active are fibroblasts because these cells produce collagen, glycosaminoglycans, proteoglycans, and adhesive glycoproteins that will form granulation tissue.4 Granulation tissue is rich in blood vessels and aims to restore tissue unity and prepare for bone formation by osteoblasts, osteoclasts, and osteocytes in the remodelling phase.5,6 In mice, the fibroblasts in alveolar bone begin to proliferate on the 3rd day after tooth extraction and continue to increase until the 7th day.7,8 From the 7th day to day 14th day, there will be a decrease in the number of fibroblasts as new alveolar bone starts to fill the socket.8 A proper proliferation of fibroblasts will trigger good bone formation and accelerate socket wound closure because fibroblasts also take part in wound retraction.9 Osteoblasts are mononuclear cells that synthesize osteoid matrix and play an important role from the proliferative phase to the remodelling phase of healing.2,3,10,11 Based on previous research, osteoblasts in the bone healing process after tooth extraction can be found on the 7th day and will continue to differentiate for more than 21 days.3,7 Osteocytes are the matured form of osteoblasts.12 When osteoblasts form bone, they can be trapped in the matrix formed by osteoblasts and differentiate into osteocytes.11,12 In the bone healing process, osteocytes play a role in bone remodelling by maintaining the integrity and vitality of the new bone.11 Based on previous research, there are more osteocytes on the 28th day post-extraction.13\n\nBinahong (Anredera cordifolia (Ten.) STEENIS) is a plant used in herbal medicine. Binahong is often considered as a wild plant and harmful to other plants because its growth is invasive. However, the characteristics of binahong, which are its abilities to grow easily and rapidly in varied climates, can also be advantages because binahong can be used as an alternative medicine that is also highly-sustainable.14,15 Binahong has been used in traditional medicine by placing its leaves over wounds and it has shown an affinity in accelerating wound healing.16,17 This is due to the presence of the secondary metabolites contained in the binahong leaf extract such as tannins, saponins, flavonoids, alkaloids, anthraquinones, phenolic acids, triterpenes, steroids, and glycosides.18 The flavonoids found are quercetin, rutin, apigenin, apigetrin, morin, myricetin, and vitexin.19–22 The phenolic acid found in binahong leaf extract is P-coumaric acid, and the triterpenes found are ursolic acid and oleanolic acid.19,23 A few studies have been conducted to observe the effects of binahong leaf extract on wound healing. Hanafiah et al showed that binahong leaf extract could increase the proliferation of NIH-3T3 fibroblasts and also proved that 3% binahong leaf extract gel could accelerate palatal mucosal wound healing in rats by increasing the proliferation of fibroblasts.24,25 The study of Khoswanto et al showed that the application of 10% binahong leaf extract gel could increase the expression of BMP-2 (bone morphogenetic protein-2) and osteoblasts in the tooth socket wound of rats on the 7th day post-extraction.3\n\nThe purpose of this study was to determine the effects of 3% binahong leaf extract gel application on the acceleration of socket wound closure and the proliferation of fibroblasts, osteoblasts, and osteocytes in the healing of alveolar bone in tooth socket wounds up to 28 days post-extraction.\n\n\nMethods\n\nAll experimental procedures in this study were carried out in accordance with the Institutional Animal Care and Usage Committee (ARRIVE) guidelines 2.0. Ethical clearance had been approved by the Health Research Ethics Committee (KEPK) at the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia with letter numbers: 0206/KEPH-FMIPA/2021 and 0202/KEPH-FMIPA/2021.\n\nSample size\n\nThe sample size in this study was calculated based on previous research of a similar nature.3 The final sample size will be four animals per group (with a total of 12 groups) or 48 animals in total.\n\nRats\n\nAnimals used in this study were 48 male Wistar rats (Rattus norvegicus) of two to three months old and with an average body weight of 200-250 grams. The rats should also be in good health and had never received any research treatment previously. Rats with abnormalities or those that died before the experiment ended were excluded from the study. The rats were acquired and housed at the Animal House in the Faculty of Mathematics and Natural Sciences in Universitas Sumatera Utara, Medan, North Sumatra, Indonesia. Before administering the treatment, the rats were acclimatized for seven days to assure that the rats could adapt to their surroundings and were allowed ad libitum feeding and drinking.\n\nThe study conducted was an in vivo experiment with a post-test only control design. The rats were randomized by simple random sampling and allocated into twelve groups by the Animal House lab technicians: group I to IV were given 3% binahong leaf extract gel, group V to VIII were given base gel as the negative control, and group IX to XII were given Gengigel® as the positive control. The treatments were given for 14 days as that was the approximate amount of time needed for the socket wound to close completely.2 The residual socket volume (RSV) and fibroblast proliferation were observed on the 3rd, 7th, and 14th day post-extraction, while the osteoblast and osteocyte proliferation were observed on the 7th, 14th, and 28th day post-extraction. All researchers were blinded to the box assignation of the rats, except for the researchers who were in charge of applying the gel to the socket wounds.\n\nWistar rats were anesthetized intraperitoneally using a combination of 91 mg/kgBW ketamine (Agrovet market, Peru) and 9.1 mg/kgBW xylazine (Interchemie, Netherlands) with an anesthetic dose of 0.1 mL/100 g rat weight to alleviate the pain induced by the procedure.26 After that, the mandibular left incisor was extracted using an artery clamp (Wells Spencer, London) with a luxation movement.27 After the extraction, the socket was irrigated with distilled water to clean the socket from debris. The extraction was performed by the same veterinarian who was blinded to the group allocation.\n\nThe binahong leaf extract used in this study was obtained from the Pharmacognosy Laboratory, Faculty of Pharmacy, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia. A total of 400 g fully opened binahong leaves and aged approximately 12 weeks were selected and obtained from Simpang Perdagangan village, Karo district, North Sumatra, Indonesia in April 2017. Binahong leaves were extracted by the maceration method using 80% ethanol (Smart Lab Indonesia, Indonesia) as the solvent. The binahong leaves were initially grinded into powder with an electric blender (Philips HR2115, Indonesia) and then soaked in 80% ethanol in a closed container and distilled for five days at room temperature. After five days, the 80% ethanol solvent was replaced with new solvent and soaked again for two days. Subsequently, a final filtering process was carried out. Then, a water bath was used to evaporate the solvent until the extract dried.28\n\nThe base gel was made by adding 10 mL of hot distilled water to a mortar, then 0.125 g of carbopol (Merck, Germany) was added and the mixture was stirred with a pestle. Next, 1.5 g of triethanolamine (TEA) (Merck, Germany) and 2 g of glycerin (Merck, Germany) were added and the mixture was stirred until it was homogeneous. In a second mortar, a mixture of 10 mL distilled water, 0.125 g of hydroxypropyl methylcellulose (HPMC) (Merck, Germany), nipagin (Merck, Germany), and nipasol (Merck, Germany) were stirred until homogeneous.25 The second mortar mixture was poured into the first mortar and the mixture was stirred until it was homogeneous. To obtain 20 g of 3% binahong leaf extract gel, 0.6 g of binahong leaf extract was added to the base gel and the mixture was stirred until it was homogenous.\n\nThe socket wounds in group I to VI were applied with 3% binahong leaf extract gel; group V to VIII were given base gel; and group IX to XII were given Gengigel® (Ricerfarma, Italy). In every application, 0.1 mL of gel was applied directly to the socket wound using a 1 mL syringe (One Med Health Care, Indonesia) with a bent needle irrigation tip (Ivoclar Vivadent, ∅: 1,2 mm, Liechtenstein) until the gel covered the entire wound surface to make sure that the gel would be directly in contact with the wound. Applications were made twice a day in the morning at 8.00 a.m.-10.00 a.m. and in the afternoon at 4.00 p.m.-6.00 p.m. for 14 days. The time was chosen to ensure consistent schedule for the gel application each day and the application was conducted by a researcher who was aware of the group allocation.\n\nMeasurements of the socket volume were made with a digital caliper (Digital Caliper, China), a pair of compasses (Joyko®, Indonesia), and a periodontal probe (Kohler, NR-3182, Germany) and were performed on the rats in group III, VII, XI, so that repeated measurements could be made on the same samples. The measurements were performed by the same researcher blinded. The average socket volume was calculated for each male Wistar rat. To determine the residual socket volume for each rat, the socket volume obtained from the measurements on the 3rd day, 7th day, and 14th day were each divided by the socket volume on the 1st day. Socket volume = mesiodistal width × buccolingual width × probing depth.29\n\nAfter reaching the 3rd day (group I, V, IX), the 7th day (group II, VI, X), the 14th day (group III, VII, XI), and the 28th day (group IV, VIII, XII) post-extraction, the rats from the respective groups were sacrificed by cervical dislocation. This method was chosen to ensure rapid termination and lower chance of tissue contamination. The mandible of the rats was removed from the skull and the socket wound tissue was excised. Fresh tissue was fixed in 10% Buffered Neutral Formalin (BNF) solution (Milestone Medical, Italy) with a pH of 6.8-7.0 and the ratio of tissue to the BNF solution was 1:10. The tissue containers were labelled and fixation was carried out for 12-48 hours, after which the tissue was immersed in 10% EDTA solution (Merck, Germany) for 10 days for decalcification.30 The tissue was then cut using a scalpel with a thickness of 4 mm and the tissue was placed in tissue cassettes and put into a basket. The baskets were loaded into an automatic processor machine and then transferred to a dehydration machine for tissue dehydration. The next stage was embedding, in which the tissue was placed in a mould and submerged in liquid paraffin. The cooled paraffin blocks were cut with a thickness of 4-5 μm using a microtome machine and the slices were placed in a water bath at 45°C. The tissue slices would be collected on clean glass slides. The slides were labelled and placed in an incubator at 37°C to dry overnight. The slides were then stained with haematoxylin-eosin (Merck, Germany) to see osteoblasts and osteocytes and with Masson's Trichrome (Merck, Germany) to see fibroblasts microscopically.31 The stained tissue slides were then observed under an electric microscope (Primo Star, Carl Zeiss, Germany) with a magnification of 400 times in 10 viewing fields. The calculation of the mean cell count was carried out by two observers blinded to avoid bias and was aided with a tally counter (SXH, 5136, China) and a calculator (Casio, ƒχ-82MS, Japan). The results of the fibroblast, osteoblast, and osteocyte cell counts from 10 viewing fields were averaged.32\n\nThe data were first analysed with Shapiro-Wilk normality test to ascertain the distribution of the data. Data that were normally distributed would be analysed with parametric tests. The RSV data were analysed using repeated measures ANOVA and one-way ANOVA test, while the histopathological data of the mean cell count of fibroblasts, osteoblasts, and osteocytes were analysed using one-way ANOVA test. The multiple comparison tests were performed with Least Significant Difference (LSD) test. The results were considered as significant if the p-value was below 0.05. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS), version 21 (IBM® Inc., USA) by a researcher who was aware of the group allocation.\n\n\nResults\n\nThe residual socket volume (RSV) was measured on the 3rd, 7th, and 14th day post-extraction. The RSV in all treatment groups decreased over time and based on the repeated measures ANOVA test, there were significant differences in the RSV on the 3rd, 7th, and 14th day in the binahong group, the Gengigel® group, and the base gel group (p < 0.05) (Figure 1A). In the binahong group, significant RSV differences were observed between the 3rd and 7th day, between the 3rd and 14th day, and between the 7th and 14th day, while in the Gengigel® group and the base gel group, significant RSV differences were only observed between the 3rd and 14th and 7th and 14th day (p < 0.05). There was no difference in the RSV between the 3rd and 7th day in the Gengigel® group and the base gel group (Table 1).\n\nResults were shown as the means of the residual socket volume (clinical observation) and the number of fibroblasts, osteoblasts, and osteocytes (histopathological observation).\n\nBased on the one-way ANOVA test, there was a significant difference in the RSV among the treatment groups on the 7th and 14th day (p < 0.05). On the 7th day, there were significant RSV differences between the binahong group and the Gengigel® group, between the binahong group and the base gel group, and between the Gengigel® group and the base gel group, while on the 14th day, the significant RSV differences were only between the binahong group and the base gel group and between the Gengigel® group and the base gel group (Table 2).\n\nFibroblast proliferation was examined on the 3rd, 7th, and 14th day post-extraction. There was an increase trend in the number of fibroblasts from the 3rd to 7th day and a decrease from the 7th to 14th day (Figure 1B). Fibroblasts can be seen among the blue-stained collagen fibre (Figure 2). Based on the one-way ANOVA test, there were significant differences in the number of fibroblasts on the 3rd, 7th, and 14th day in the binahong, Gengigel®, and base gel groups (p < 0.05). In the binahong group, there were significant differences in the number of fibroblasts between the 3rd and 7th day and between the 7th and 14th day, while in the Gengigel® group, there were significant differences in the number of fibroblasts between the 3rd and 7th day and between the 3rd and 14th day. In the base gel group, the significant differences were only observed between the 3rd and 7th day (Table 1).\n\nThe observation was conducted on the 3rd, 7th, and 14th day post-extraction socket wounds stained with Masson’s Trichrome. (Abbreviation: Fb: Fibroblasts (×400)).\n\nSignificant differences in the number of fibroblasts among the binahong group, Gengigel® group, and the base gel group were observed on the 3rd, 7th, and 14th day (p < 0.05). On the 3rd day, there were significant differences in the number of fibroblasts between the binahong group and the base gel group and between the Gengigel® group and the base gel group. On the 7th day, there were significant differences between the binahong group and the Gengigel® group, between the binahong group and the base gel group, and between the Gengigel® group and the base gel group. On the 14th day, a significant difference was only observed between the Gengigel® group and the base gel group (Table 2).\n\nThe proliferation of osteoblasts was examined on the 7th, 14th, and 28th day post-extraction. In the binahong group and the Gengigel® group, there was an increase trend in the number of osteoblasts from the 7th to 14th day and a decrease from the 14th to 28th day, although in the base gel group, the number of osteoblasts kept rising over time (Figure 1C). Osteoblasts can be seen along the alveolar bone matrix (Figure 3). Based on the one-way ANOVA test, there were significant differences in the number of osteoblasts among the 7th, 14th, and 28th day in the binahong group and the base gel group (p < 0.05), while the Gengigel® group showed no differences in the number of osteoblasts among those days. In the binahong group, there were significant differences between the 7th and 14th day and between the 7th and 28th day, while in the base gel group, there were significant differences between the 7th and 14th day, between the 7th and 28th day, and between the 14th and 28th day (Table 1).\n\nThe observation was conducted on the 7th, 14th, and 28th day post-extraction socket wounds stained with haematoxylin-eosin. (Abbreviations: Ob: osteoblasts; Oc: osteocytes (×400)).\n\nThe differences in the number of osteoblasts among the binahong group, Gengigel® group, and the base gel group were only observed on the 14th day post-extraction (p < 0.05). On the 14th day, significant differences were observed between the binahong group and the base gel group and between the Gengigel® group and the base gel group (Table 2).\n\nOsteocyte proliferation was examined on the 7th, 14th, and 28th day post-extraction. In the binahong group and the base gel group, there was a trend of increase in the number of osteocytes over time, although in the Gengigel® group, the increase only happened from the 7th to 14th day and was followed by a decrease from the 14th to 28th day (Figure 1D). Osteocytes can be seen in the alveolar bone matrix inside lacunae (Figure 3). Based on the one-way ANOVA test, there were significant differences in the number of osteocytes among the 7th, 14th, and 28th day post-extraction only in the binahong group and the base gel group (p < 0.05). No difference was observed in the Gengigel® group among those days. In the binahong group, there were significant differences between the 7th and 14th day and between the 7th and 28th day, while in the base gel group, significant differences were observed between the 7th and 14th day, between the 7th and 28th day, and between the 14th and 28th day (Table 1).\n\nDifferences in the number of osteocytes among the treatment groups were only observed on the 14th and 28th day post-extraction (p < 0.05). On the 14th day, there were significant differences between the binahong group and the base gel group and between the Gengigel® group and the base gel group. On the 28th day, significant differences were observed only between the binahong group and the base gel group (Table 2).\n\n\nDiscussion\n\nThe concentration of the binahong leaf extract gel in this study was chosen based on a previous research by Hanafiah et al, which examined the effects of binahong leaf extract gel on the healing of palatal mucosal wounds.25 Among the various binahong leaf extract gel concentrations in the research, 3% binahong leaf extract gel showed the best affinity in increasing fibroblast proliferation.25 In this study, the RSV in every treatment group decreased over time and the binahong group showed a lower RSV in comparison with the other treatment groups on the 7th and the 14th day (p < 0.05). A lower RSV implied a decrease in socket wound size, so it can be assumed that the binahong group showed a better affinity in accelerating wound closure compared to the positive and negative control groups. Fibroblast is one of the cells that affects wound retraction and it can be assumed that the acceleration of wound socket closure is influenced by the number of fibroblasts.28\n\nThe number of fibroblasts in every treatment group increased from the 3rd to the 7th day and was followed by a decrease from the 7th to 14th day, except for the Gengigel® group that did not show any significant differences between the 7th and 14th day. According to Vieira et al, who examined the physiologic socket wound healing in mice, the fibroblasts in the alveolar bone would proliferate and would continue to increase until the 7th day post-extraction. The number of fibroblasts would decrease from the 7th day to the 14th day following the formation of new alveolar bone.8 On the 7th day, the highest fibroblast proliferation was in the binahong group. In the Gengigel® group, there was no significant difference in the number of fibroblasts between the 7th day and the 14th day because the hyaluronic acid in Gengigel® had more effects in the early phase of wound healing in fibroblast proliferation compared to alveolar bone formation.33 Fibroblast can secrete collagen and extracellular matrix, which will make up the granulation tissue. Granulation tissue will then be replaced by the provisional matrix, which has fewer inflammatory cells and more matrix and new blood vessels.34\n\nIn the study, the increase in osteoblast proliferation happened between the 7th day and the 14th day and the decrease between the 14th day and the 28th day. Osteoblast proliferation reached its peak on the 14th day and started to decrease until the 28th day, in which the osteoblasts had matured.8 In the base gel group, osteoblast proliferation continued to increase until the 28th day. It could be assumed that in the base gel group, the socket wound healing was still on the early phase of bone formation, which was shown by the high number of undifferentiated osteoblasts. Osteoblasts will produce a matrix, which is called osteoid, and osteoid will be mineralized, forming woven bone.34 Osteoblasts that become trapped in the matrix will become osteocytes. Osteocytes have a role in managing bone remodelling because they can influence the activities of osteoblasts and osteoclasts.4 In the study, there was an increase in the number of osteocytes from the 7th day until the 28th day in the binahong group and the highest osteocyte number was on the 28th day. The osteocytes were found among the alveolar bone matrix. This finding concurred with the experiment of Olaitan et al that showed a higher osteocyte proliferation in the 4th week compared to the 2nd week post-extraction.13\n\nThe binahong plant contains various secondary metabolites that function in the proliferation of fibroblasts, osteoblasts, and osteocytes, optimising wound healing and in turn lowering the value of RSV. In the binahong leaf extract, there is saponin, which can increase the expression of TGF-α (transforming growth factor-alpha) and TGF-β (transforming growth factor-beta).14,35 TGF-β can activate fibroblasts and TGF-α can activate Osterix, which will function in osteoblast differentiation.14,35 Saponin is also antiseptic and it can affect cell membrane integrity and cause the lysis of pathogens, especially fungi.36\n\nApigenin, a flavonoid found in binahong, can increase the expression of TGF-β and PDGF (platelet-derived growth factor) that also function in fibroblast activation so that it can migrate towards the clot.37,38 PDGF is also a protein that affects fibroblast proliferation.38 Apigenin also has anti-inflammatory properties because it can inhibit the activation of NF-κB (nuclear factor kappa B).39 Inhibited NF-κB can prevent the production of inflammatory mediators that can increase inflammation.39 The other flavonoids in the binahong plant that function in socket wound healing are quercetin and vitexin.40–42 Quercetin has the ability to reduce osteoclast formation through inhibiting IL-17 (interleukin-17), which is induced by RANKL (receptor activator of nuclear factor kappa-B ligand), and quercetin can also increase osteogenesis, angiogenesis, and function as an antioxidant.40,41 Vitexin can affect bone formation by increasing osteoblast differentiation through p-Smad (phosphorylation-small mother againts) and Runx2 (runt-related transcription factor 2).42\n\nThe tannin in the binahong plant functions in fibroblast migration because it can increase VEGF (vascular endothelial growth factor) in the early phase of wound healing.43 VEGF is a protein that plays a role in fibroblast migration.38 Tannin is also an antioxidant. Antioxidant is needed to neutralise free radicals that are produced during wound healing. Free radicals can damage cell protein structure, which will prevent cell proliferation.44\n\nThe triterpenes in the binahong leaf extract, such as ursolic acid and oleanolic acid, have anti-inflammatory, antiseptic, and antioxidant properties.14,45 Ursolic acid can also influence the differentiation and proliferation of osteoblasts and improve the activity and mineralisation of ALP (alkaline phosphatase).46\n\n\nConclusion\n\nThe study concluded that the application of 3% binahong leaf extract gel could enhance alveolar bone healing, which could be shown through the decreasing value of residual socket volume and the increasing proliferation of fibroblasts, osteoblasts, and osteocytes.\n\n\nData availability\n\nZenodo: The dataset of ‘The effects of 3% binahong (Anredera cordifolia) leaf extract gel on the alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)’. https://doi.org/10.5281/zenodo.5189362.47\n\nThis project contains the following underlying data:\n\n• Raw data of fibroblast mean cell count.csv (Raw data of fibroblast mean cell count)\n\n• Raw data of osteoblast mean cell count.csv (Raw data of osteoblast mean cell count)\n\n• Raw data of osteocyte mean cell count.csv (Raw data of osteocyte mean cell count)\n\n• Raw data of residual socket volume.csv (Raw data of residual socket volume)\n\n• Readme.txt (Explanations about the raw data in the dataset)\n\nZenodo: Rat tooth extraction and 3% binahong (Anredera cordifolia (Ten.) STEENIS) leaf extract gel. https://doi.org/10.5281/zenodo.5202954.48\n\nThis project contains the following underlying data:\n\n• Rat anesthesia.tiff (Intraperitoneal anesthesia on the rat)\n\n• Rat tooth extraction.tiff (The extraction of the rat’s mandibular left incisor)\n\n• The binahong leaf extract gel.tif (3% binahong leaf extract gel)\n\n• The binahong leaf extract.tif (Binahong leaf extract)\n\nZenodo: ARRIVE checklist for ‘The effects of 3% binahong (Anredera cordifolia) leaf extract gel on the alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)'. https://doi.org/10.5281/zenodo.5203068.49\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAdeyemo W, Taiwo O, Oderinu O, et al.: Oral health-related quality of life following non-surgical (routine) tooth extraction: A pilot study. Contemp Clin Dent. 2012; 3(4): 427–32. 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St.Louis: Elsevier; 2011: 164. .\n\nFunakoshi-Tago M, Nakamura K, Tago K, et al.: Anti-inflammatory activity of structurally related flavonoids, Apigenin, Luteolin and Fisetin. Int Immunopharmacol. 2011; 11(9): 1150–9. PubMed Abstract \| Publisher Full Text\n\nWong SK, Chin KY, Ima-Nirwana S: Quercetin as an agent for protecting the bone: A review of the current evidence. Int J Mol Sci. 2020; 21(17): 1–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim HR, Kim BM, Won JY, et al.: Quercetin, a Plant Polyphenol, Has Potential for the Prevention of Bone Destruction in Rheumatoid Arthritis. J Med Food. 2019; 22(2): 152–61. PubMed Abstract \| Publisher Full Text\n\nKim KM, Son HE, Min HY, et al.: Vitexin enhances osteoblast differentiation through phosphorylation of Smad and expression of Runx2 at in vitro and ex vivo. Mol Biol Rep. 2020; 47(11): 8809–17. PubMed Abstract \| Publisher Full Text\n\nLi K, Diao Y, Zhang H, et al.: Tannin extracts from immature fruits of Terminalia chebula Fructus Retz. promote cutaneous wound healing in rats. BMC Complement Altern Med. 2011; 11: 86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrunicardi FC, Andersen DK, Biliar TR, et al.: Schwartz’s Principles of Surgery. 11th ed. New York: McGraw Hill Education; 2019; 140. : 272–277 86.\n\nSutrisno E, Ketut Adnyana I, Sukandar EY, et al.: Anti-inflammatory study of Anredera cordifolia leaves and Centella asiatica herbs and its combinations using human red blood cell-membrane stabilization method. Asian J Pharm Clin Res. 2016; 9(5): 78–80. Publisher Full Text\n\nLee S: Protective effects of ursolic acid on osteoblastic differentiation via activation of IER3/Nrf2. J Dent Hyg Sci. 2019; 19(3): 198–204. Publisher Full Text\n\nHanafiah OA, Hanafiah DS, Dohude GA, et al.: The dataset of 'The effects of 3% binahong (Anredera cordifolia) leaf extract gel on the alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)' [Data set]. Zenodo. 2021. Publisher Full Text\n\nHanafiah OA, Hanafiah DS, Dohude GA, et al.: Rat tooth extraction and 3% binahong (Anredera cordifolia (Ten.) STEENIS) leaf extract gel. Zenodo. 2021. Publisher Full Text\n\nHanafiah OA, Hanafiah DS, Dohude GA, et al.: The ARRIVE guidelines 2.0 (author checklist) for 'The effects of 3% binahong (Anredera cordifolia) leaf extract gel on the alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)'. Zenodo. 2021. Publisher Full Text" }	[ { "id": "96681", "date": "03 Nov 2021", "name": "Lidia Audrey Rocha Valadas", "expertise": [ "Reviewer Expertise Dentistry", "Pharmacology", "Biofilms", "Natural products" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research is an experimental study on the use of Anredera cordifolia in gel form to be used after extraction.\nThe study is innovative especially in the area, with few works in the literature about it. In addition, the methodology has reproducibility.\nA) Introduction:\n1 - I suggest a paragraph about the medicinal properties reported in the literature and not just isolated constituents.\n2 - There was a gap between the 2nd and 3rd paragraphs, before starting the third, I should start by addressing the use of medicinal plants for this purpose.\n3 - I suggest checking the articles of these authors, it may help (some are from medicinal plants): Alexandre et al. 20181, Guimarães et al. 20162, Lima MDR et al. 20173, Lima V et al. 20204, Ribeiro et al. 20185, and Teixeira et al. 20176.\nB) Methodology:\n4 - If it is a study with natural products, I suggest improving the details of georeferencing, with the addition of coordinates and season.\n5 - I suggest a flowchart for the division of groups and a chart with the constituents of the gel.\nC) Discussion:\n6 - In the discussion, I suggest reviewing the second and third paragraphs so as not to repeat information that is already in the results.\n7 - I also believe it is important to talk about the cytotoxicity and safety of the species, since the intention is that the gel will one day be clinically applicable.\n8 - To further address the importance of investigating properties of natural products in Dentistry, especially in surgery, since most studies are related to cariogenic or periodontal biofilm.\n9 - Finally, add limitations and future perspectives.\n\nIs the work 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": "8021", "date": "05 Apr 2022", "name": "Olivia Avriyanti Hanafiah", "role": "Author Response", "response": "We thank the reviewer for reviewing our manuscript and offering their suggestions. Please find below a point-by-point response to the comments. We hope that the reviewer would find the modifications satisfactory. Comment: I suggest a paragraph about the medicinal properties reported in the literature and not just isolated constituents. Response: We have elaborated on the medicinal properties of Binahong in paragraph three in Introduction. Comment: There was a gap between the 2nd and 3rd paragraphs, before starting the third, I should start by addressing the use of medicinal plants for this purpose. Response: We have followed this suggestion, thank you for the insight. Comment: I suggest checking the articles of these authors, it may help (some are from medicinal plants): Alexandre et al. 20181, Guimarães et al. 20162, Lima MDR et al. 20173, Lima V et al. 20204, Ribeiro et al. 20185, and Teixeira et al. 20176. Response: Thank you for the suggestions by the reviewer. We have read the articles and they have provided us with some helpful insights. Comment: If it is a study with natural products, I suggest improving the details of georeferencing, with the addition of coordinates and season. Response: We have added these details in \"Binahong leaf extract gel and base gel preparation\" section in Methods. Comment: I suggest a flowchart for the division of groups and a chart with the constituents of the gel. Response: Following the suggestion by the reviewer, we have added these details in Figure 1 and Figure 2. Comment: In the discussion, I suggest reviewing the second and third paragraphs so as not to repeat information that is already in the results. Response: Thank you for the suggestion, we have made some changes in paragraph two and three in Discussion. Comment: I also believe it is important to talk about the cytotoxicity and safety of the species, since the intention is that the gel will one day be clinically applicable. Response: Thank you for this insight, we have thus added information about the toxicity of Binahong, which can be found in the fourth paragraph in Discussion Comment: To further address the importance of investigating properties of natural products in Dentistry, especially in surgery, since most studies are related to cariogenic or periodontal biofilm. Response: As suggested by the reviewer, we have added this in the last paragraph in Discussion Comment: Finally, add limitations and future perspectives. Response: As suggested by the reviewer, we have added this in the last paragraph in Discussion" } ] }, { "id": "101384", "date": "17 Jan 2022", "name": "Dalia Hussein El-Rouby", "expertise": [ "Reviewer Expertise Oral tumors", "use of natural products for chemo-prevention and improving oral condition", "use of bone substitutes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEffects of 3% binahong (Anredera cordifolia) leaf extract gel on alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)\nThe points that should be addressed to make the article scientifically sound:\nIntroduction:\n'The healing of soft tissue, which are comprised of the gingiva and connective tissue...' - Please revise the language style\nBinahong: the letter B should always be capitalised\nMethods:\nSample size calculation: 48 male Wistar rats were randomly allocated to twelve groups. Please mention the effect size obtained from the previous study (Khoswanto et al, 2019)\nRats with abnormalities or those that died before the experiment ended were excluded from the study: how did you replace rats who died before the end of the study to maintain the sample size?\nResults\nPlease provide figures showing the 95% confidence interval in error bars\nThe residual socket volume (RSV): Please specify the unit of measurement (mm3 ?) in the graph, table 2 and text\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8022", "date": "05 Apr 2022", "name": "Olivia Avriyanti Hanafiah", "role": "Author Response", "response": "We thank the reviewer for reviewing our manuscript and offering their suggestions. Please find below a point-by-point response to the comments. We hope that the reviewer would find the modifications satisfactory. Comment: 'The healing of soft tissue, which are comprised of the gingiva and connective tissue...' - Please revise the language style Response: Thank you for the suggestion, we have revised the diction of this sentence. Comment: Binahong: the letter B should always be capitalised Response: Thank you for the insight, we have thus made the changes. Comment: Sample size calculation: 48 male Wistar rats were randomly allocated to twelve groups. Please mention the effect size obtained from the previous study (Khoswanto et al, 2019) Response: We have added this detail in \"Sample Size\" section in Methods, thank you. Comment: Rats with abnormalities or those that died before the experiment ended were excluded from the study: how did you replace rats who died before the end of the study to maintain the sample size? Response: The rats were not replaced as long as the minimal sample size were still maintained. Elaboration on this can be found in \"Animals\" section in Methods. Comment: Please provide figures showing the 95% confidence interval in error bars Response: Following the suggestion, we have added these details in the improved Figure 3. Comment: The residual socket volume (RSV): Please specify the unit of measurement (mm3 ?) in the graph, table 2 and text Response: Thank you for the suggestion. We have thus added the unit of measurement in the graph, table, and text for fibroblast, osteoblast, and osteocyte proliferation. Unfortunately, there is no unit of measurement for RSV as it is the ratio of end volume and beginning volume (end volume: beginning volume)." } ] }, { "id": "96680", "date": "17 Jan 2022", "name": "Mohd Farhan Hanif Reduan", "expertise": [ "Reviewer Expertise Histopathology", "natural product development", "animal study", "veterinary" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 and the study is worth for indexing on this platform as it highlights the use of local plants available for the alveolar bone healing.\nFew considerations should be taken to improve the quality of the article:\nIntroduction - to include all pharmacological activities of the plant\n\nMethodology - to cite for the H&E and Masson Trichome procedures. It is good if the authors can explain how to differentiate the various type of cells; fibroblasts, osteocyctes, osteoclasts and osteoblasts\n\nResults - the good quality of histopathology figures must be included in the article, referring to Figure 2: the bluish stain fibroblasts cannot be seen clearly by the reviewers and readers. Same comment for the Figure 3.\n\nDiscussion - to include future direction of the present study, the limitation of the current study can be added and explained.\n\nConclusion - Well concluded\n\nIs the work 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": "8023", "date": "05 Apr 2022", "name": "Olivia Avriyanti Hanafiah", "role": "Author Response", "response": "We thank the reviewer for reviewing our manuscript and offering their suggestions. Please find below a point-by-point response to the comments. We hope that the reviewer would find the modifications satisfactory. Comment: Introduction - to include all pharmacological activities of the plant Response: Thank you for the insight, we have added this in Paragraph 4 of Introduction. Comment: Methodology - to cite for the H&E and Masson Trichome procedures. It is good if the authors can explain how to differentiate the various type of cells; fibroblasts, osteocyctes, osteoclasts and osteoblasts Response: Citations have been added in the \"Histopathological analysis\" section in Methods. Details about cells have also been added in the same section. Thank you for the input. Comment: Results - the good quality of histopathology figures must be included in the article, referring to Figure 2: the bluish stain fibroblasts cannot be seen clearly by the reviewers and readers. Same comment for the Figure 3. Response: Following the suggestion, we have uploaded improved versions of Figure 2 and Figure 3 (Now referred to as Figure 4 and Figure 5 in the new version) Comment: Discussion - to include future direction of the present study, the limitation of the current study can be added and explained. Response: Following the suggestions, we have added these details in the last paragraph in Discussion. Comment: Conclusion - Well concluded Response: We thank you for this input." } ] } ]	1	https://f1000research.com/articles/10-923
https://f1000research.com/articles/11-82/v1	24 Jan 22	{ "type": "Data Note", "title": "Dataset of the adoption of digital tools for research sharing among academics in African Varsities during the COVID-19 pandemic in 2020", "authors": [ "Valentine Joseph Owan", "Jennifer Uzoamaka Duruamaku-Dim", "Francisca Nonyelum Odigwe", "Mercy Valentine Owan", "Jennifer Uzoamaka Duruamaku-Dim", "Francisca Nonyelum Odigwe", "Mercy Valentine Owan" ], "abstract": "This dataset was collected from a total of 1,977 university lecturers across 24 African countries, that were purposively targeted due to their level of exposure to scholarly publications. The dataset was collected through an online survey that was sent to respondents through email, WhatsApp, and the Association of African Universities Telegram group. The questionnaire was designed by the researchers and validated by five experts for face and content validity. The demographic information of the data was analysed and the softcopy of the data uploaded to the Mendeley database for easy retrieval after deidentification (see Data Availability statement). The associated questionnaire can be found in the extended data. In Africa, this appears to be the broadest dataset associated with academics’ perception of utilizing digital platforms for research sharing. This implies that scholars can use this dataset to quantitatively analyse the extent to which different internet-based platforms are being utilized for research communication. Considering the current restrictions on in-person social gatherings due to COVID-19, researchers working on related studies may readily utilize this set of data, saving time and cost. A comprehensive but non-exhaustive number of 20 online platforms were assessed based on academics' awareness and current engagement with them, and the challenges they have faced using them. This offers a wide range of areas for studies to be anchored. Furthermore, researchers interested in specific online platforms can also evaluate the extent to which academic staff in African universities are aware of and willing to utilize them for research dissemination. This data will enable scholars and researchers in Africa and beyond to understand the extent to which academics in varsities are willing to adopt digital repositories for research sharing in the context of Africa.", "keywords": [ "Africa", "data", "digital tools", "higher education", "research", "research dissemination", "staff adoption", "varsities" ], "content": "Introduction\n\nAccording to the most recent statistics gathered in 2021, on the COVID-19 pandemic from Johns Hopkins University and the Africa Center for Disease Control, there are 874,036 active cases of COVID-19, with 18,498 total confirmed cases, 330,981 recoveries, and 524,557 deaths in Africa (Coronavirus Resource Centre, John Hopkins University, 2021). As cases are confirmed daily across the globe, the breakdown remains variable. As of May 13, 2020, every African country had been infected, and instances of COVID-19 have been reported in 213 nations and territories throughout the world; while the whole world grieved and felt the uncertainty and continual fear of losing their loved ones (Cohut, 2020). The COVID-19 pandemic has posed a challenge to the way people and organizations go about their daily lives. As a result, there is an increasing need for individuals and organizations to make changes to their behaviour in order to eliminate the spread of the virus. Research sharing and communication occupy a central position in the world (Odigwe et al., 2020), and has proliferated during the COVID-19 pandemic because of social distancing. Many academics have welcomed the use of digital platforms to share the results of intellectual property or research (Bik & Goldstein, 2013; Bougioukas et al., 2020; Siedlok et al., 2020; Jarreau, 2015; Yammine et al., 2018; Zientek et al., 2018). Currently, the importance of internet technologies for wider dissemination of research results, visibility of researchers, quick file sharing, as well as the uploading and downloading of academic publications cannot be overstated. Furthermore, multimedia technologies have a huge potential for increasing the visibility, reputation, placement, and public value of academics and universities (Anenene et al., 2017).\n\nDespite the importance of digital repositories in the academic world, they have been described as difficult to implement. For example, research has revealed that academics make little use of open-source academic resources (Kodua-Ntim, 2020). Insufficient campaigning, ICT accessibility, facilities, finances, power supplies, lack of technical capabilities, institutional repository regulation, lack of resources, organizational culture/politics, and patent issues have all been identified as major barriers to academic personnel adopting open-access university libraries (Kodua-Ntim, 2020; Owan et al., 2021). This dataset was created because, during the peak of the COVID-19 outbreak, many conferences and other intellectual gatherings were moved to internet platforms to maintain social distance and prevent the virus from spreading. The readiness of academics to accept and use online technologies may be a determining factor in the utilisation of digital platforms for such academic goals. It is critical to know how ready researchers are to use online resources for research communication, particularly from the perspective of developing African countries. This is because their level of readiness to adopt digital tools may have an impact on the utilization of digital platforms for various purposes, which in turn may have an impact on their research dissemination practices.\n\nThis dataset is an excel document showing a person-by-item matrix of the responses to the various aspects of the online questionnaire (Owan, 2021). There are a total of 32 rows and 1,977 columns, with the first row indicating the variables. There is one dummy variable (gender), four ordinal variables (age, educational qualification, rank, and years of work experience), and two nominal variables (research area and country of residence). Columns H to Column AA contain data on the extent staff are willing to adopt 20 unique online platforms for research sharing (see Figure 1). Column AB contains a dichotomous response of participants regarding their perception of classical/traditional versus modern/electronic approaches to research sharing. Each cell in Column AC contains a listing of platforms that respondents indicated the extent they currently utilize them. Each cell in column AD contains the total number of publications each participant has published, while column AE contains the total number of respondents’ scholarly works that are currently on the internet. The data in column AD and AE can be used to compute the ratio of scholars' work that is on the internet as a percentage of their total number of publications. Lastly, column AF contains qualitative data on the challenges scholars face in using online platforms for research sharing.\n\n\nMethods\n\nAn electronic survey, consisting of three main parts, was used for data collection. The survey was created by the researchers, using information from a review of related literature (Owan, ‘Electronic …’, 2021). Google forms was used in designing the survey for financial reasons and because it was easy to use. The virtual snowball approach was used in targeting the respondents using the Association of African Universities’ (AAU) Telegram platform. Some respondents were sent a link to the survey and asked to forward it to other colleagues who are academic staff. The AAU Telegram is composed of academic staff from different universities across Africa. This qualified all of them as participants for the study.\n\nThis study involved human subjects whose consent were sought in the cover letter of the questionnaire. The authors stated clearly to the respondents what the research involved and how the data will be handled. Respondents were made to understand that by completing the survey, they have consented to participate. Ethical approval was not required for this study according to the National Health Research Ethics Committee of Nigeria guidelines (NHREC).\n\nSection one included a lengthy cover letter detailing the purpose of the study, its duration, the planned delivery period, the nature of the responses/data needed and an ethical declaration stating how privacy and confidentiality would be maintained. The respondents were assured that all the information solicited was going to be used for the research and that no personal data was going to be revealed to anyone. The decision to participate was voluntary and respondents were further assured that collected data would be aggregated with all identifying information removed. At the end of the letter in section 1, respondents were informed that responding to the items in subsequent sections of the questionnaire, would indicate that they have consented to participate. Section 2 gathered demographic information from respondents, such as gender, age, qualifications, academic ranks, years of job experience, areas of research and countries of residence. The demographic information was gathered using a list of options for respondents to tick.\n\nThere was no potential bias, since the research was exploratory and the medium used for data collection was transparent, inclusive and open to all African universities. The researchers had no control of snowballing distribution to subsequent participants.\n\nThe third section was divided into six sub-sections. The first sub-section was a five-point rating system for a set of 20 online sites in which participants were supposed to indicate their willingness to use them for research sharing (See Table 1). Table 1 provides information about the extent of staff willingness to utilise internet platforms for research dissemination using mean and standard deviation. The second sub-section consisted of a closed-ended inquiry designed to ascertain academic personnel perceptions of conventional and contemporary approaches to research sharing. The third sub-section was a checklist of 20 websites on which respondents could check the ones they currently use for research sharing. We visualised the resources scholars are interested in using and identified the resources that are more likely to be utilised in the future (Figure 1). Table 2 provides information on the current state of academic staff utilisation of myriad online platforms in the dissemination of research.\n\nThe fourth sub-section was structured to assess respondents’ total number of scholarly publications (including journal articles, theses/dissertations, conference papers, book chapters, and books). Textboxes were provided for the respondents to write the total number of their published works. The fifth sub-section focused on the overall number of respondents’ academic publications available online (including those on the websites of publishers and those that are manually submitted to internet sites). The sixth sub-section was created to allow respondents to share their thoughts on the difficulties they experience while attempting to use online channels for research distribution. The bar chart in Figure 2 highlights the challenges limiting African scholars’ use of online for research dissemination. The various tools were chosen for each sub-section because of their effectiveness in collecting the required information.\n\nFor face and content validity, the survey was reviewed by three instructional technology specialists and two psychometrists from the University of Calabar's Faculty of Education. The face and content validity were carried out to ensure that the contents and arrangements of the questions/items were reasonably clear, eliminating extraneous information and ambiguity. The link to the e-survey was shared on the Telegram forum of the Association of African Universities, which has 1,622 participants from various African countries and regions. Members of the group, who were university academic employees, were invited to complete the survey and post it on their institutions' internet-based forums. The researchers warned the participants intending to share the link to the questionnaire, to distribute it to only academics who meet the selection criteria. To be qualified as a respondent for the study, an academic staff must have obtained a doctorate degree and must have published an article in a peer reviewed journal.\n\nThe survey was open from July 2020 to January 2021, reflecting a seven-month data collection period. The accompanying data was imported, translated to an Excel file (.xlsx), reviewed, cleaned, and re-coded after the survey was closed (where necessary). The data obtained was deidentified in line with the privacy statement given to participants following the Safe Harbour method. All information pertaining to dates such as age and years of experience, were grouped into a range (e.g., 20 – 29 years). The data was cleaned in sections where respondents were given the freedom to provide short answers. Cleaning was done through fixing spelling errors, matching cases for responses and grouping responses. The survey received feedback from 1,977 academics in African universities. The survey was stopped not because of saturation but because no further response was obtained two months after the last response was obtained. At this point it became clear that responses were no longer forthcoming.\n\n\nData availability\n\nMendeley: Electronic Cross-Sectional Data of Academic Staff Preparedness to Adopt Digital Tools for Research Sharing in African Varsities. http://dx.doi.org/10.17632/69k939yr4n.1 (Owan, ‘Electronic …’, 2021).\n\nThe project contains the following underlying data:\n\n- Utilisation of online platforms for research dissemination further analysis 2.xlsx\n\n- Utilisation of online platforms for research dissemination further analysis.xlsx\n\n- Utilisation of online platforms for research disseminato=.sav\n\n- Utilization of Online Platforms for Research Dissemination Questionnaire (UOPRDQ) (Responses).xlsx\n\nThis project contains the following extended data:\n\n- Utilization of Online Platforms for Research Dissemination Questionnaire (UOPRDQ)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe researchers are very grateful to the participants of this research who after receiving the survey link, worked actively to network with their colleagues. We are also grateful to the Association of African Universities (AAU) resource persons for offering us the platform to disseminate the instrument and engage respondents across different countries in Africa.\n\n\nReferences\n\nAnenene EE, Alegbeleye GB, Oyewole O: Factors contributing to the adoption of institutional repositories in Universities in South-West Nigeria: Perspectives of Library Staff. Library Philosophy and Practice (e-journal). 2017.Reference Source\n\nBik HM, Goldstein MC: An introduction to social media for scientists. PLoS Biol. 2013; 11(4): e1001535. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBougioukas KI, Bouras EC, Avgerinos KI, et al.: How to keep up to date with medical information using web-based resources: A systematised review and narrative synthesis. Health Inf. Libr. J. 2020; 37: 254–292. PubMed Abstract \| Publisher Full Text\n\nCohut M: Covid-19 global impact: How the coronavirus is affecting the world.2020.Reference Source\n\nJarreau PB: All the science that is fit to blog: An analysis of science blogging practices. 2015. Unpublished Doctor of Philosophy, (Louisiana State University), Baton Rouge, LA.Reference Source\n\nKodua-Ntim K: Sage of open access institutional repositories in University Libraries in Ghana. 2020. Unpublished PhD. Dissertation, (University of South Africa).Reference Source\n\nOdigwe FN, Bassey BA, Owan VJ: Data management practices and educational research effectiveness of university lecturers in South-South Nigeria. J. Educ. Soc. Res. 2020; 10(3): 24–34. Publisher Full Text\n\nOwan VJ, Asuquo ME, Makuku V, et al.: The Extent of Online Platforms Utilization for Scholarly Research Work Dissemination: A Survey of Academic Staff in African Universities. SocArXiv Preprint. 2021. Publisher Full Text\n\nOwan VJ: Electronic Cross-Sectional Data of Academic Staff Preparedness to Adopt Digital Tools for Research Sharing in African Varsities. Mendeley Data. 2021; V1. Publisher Full Text\n\nSiedlok F, Hibbert P, Whitehurst F: Social network influences employee responses to organizational withdrawals. Org. Manage. J. 2020; 17(1): 15–35. Publisher Full Text\n\nYammine SZ, Liu C, Jarreau PB, et al.: Social media for social change in science. Science. 2018; 360(6385): 162–163. PubMed Abstract \| Publisher Full Text\n\nZientek LR, Werner JM, Campuzano MV, et al.: The use of Google Scholar for Research and Research Dissemination. New Horizons in Adult Education and Human Resource Development. 2018; 30: 39–46. Publisher Full Text" }	[ { "id": "120882", "date": "18 Feb 2022", "name": "Neha Lata", "expertise": [ "Reviewer Expertise ICT applications to Library and Information Services", "Digital Library and Information Retrieval System", "Library Automation and Networking", "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\nThe article presents a broad dataset from 24 African countries about their intention to utilise internet-based outlets for research dissemination during the COVID-19 pandemic. The paper's general quality is good: it is well-written, and some important considerations are highlighted. The research study is well-structured, with clear ideas and concise, persuasive language. The introduction is relevant and theoretically sound. The literature evaluation is thorough, and authors successfully discussed the significance of the research from both a theoretical and practical standpoint.\nThe methodology of the paper is rich and commendable because it was stated in a manner that allows for replication, and the methodologies and data analysis have no flaws in my opinion. Secondly, the authors addressed the issue of potential bias by ensuring that the link to the survey was posted only on platforms hosting academics from African universities, who in turn shared it with their colleagues.\nThere was also a clear statement of ethical consideration, with evidence of a national guideline exempting the need for ethical clearance because of no associated risk of participation.\nGenerally, the authors were detailed in their methodology with the safe harbour methods followed for data privacy. As a data article, the information provided is sufficient for re-use.\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": "121430", "date": "25 Feb 2022", "name": "Noorhidawati Abdullah", "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 abstract provides clear information.\nThe introduction is good and cited recent references. But please check again the following statement: “There are a total of 32 rows and 1,977 columns, with the first row indicating the variables.” It should be 1,977 rows (indicating number of respondents) and 32 columns (the variables).\nFigure 1 should include label for the data on the bar chart, either percentage or frequency. Table 1 and Table 2 should include the N (number of responses).\nMethod\nIn the abstract it was mentioned - “This dataset was collected from a total of 1,977 university lecturers across 24 African countries”. But in the Introduction it was reported - ”The link to the e-survey was shared on the Telegram forum of the Association of African Universities, which has 1,622 participants from various African countries and regions.” Please check again the figure, as the responses gathered (1,977) were more that the total Telegram participants (1,622).\nThe method section is quite clear, but I have some comments for improvement:\nIt would be good to have a separate sub-section on instrument development, where information on each section of the survey instrument is discussed, currently it is all under Ethics and Consent sub-section. It is a good practice to include Ethics and Consent sub-section. Include information on the scale used to measure “willingness” as shown in Table 1. Provide explanation on the Average Score = 5839 as indicated in Table 1. Report on the pilot study and the changes made after it was conducted. A total of 1,9777 responses were gathered, all of them are usable data/completed responses? It is good to report the incomplete survey (that was excluded from the analysis) if that is the case. If none please inform so.\n\nThe title used the term “digital tools” but throughout the article various term were used such as internet platform, digital platform, online platform, internet-based platform – one consistent term should be used throughout the article and survey instrument to avoid confusion.\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": [ { "c_id": "7875", "date": "02 Mar 2022", "name": "Valentine Owan", "role": "Author Response", "response": "Response to reviewer Comment #1: The abstract provides clear information. Response#1: Thanks Comment #2: The introduction is good and cited recent references. But please check again the following statement: “There are a total of 32 rows and 1,977 columns, with the first row indicating the variables.” It should be 1,977 rows (indicating number of respondents) and 32 columns (the variables). Response #2: The researchers are grateful to the reviewer for the inputs. However, we tend to disagree with the reviewer regarding the statement made about rows and columns. There are 1,977 columns (representing the number of respondents) and 32 rows (representing the number of variables). Columns are vertically arranged, while rows flow horizontally. The reviewer should kindly recheck that. However, we have modified the statement to make it clearer. Comment #3: Figure 1 should include a label for the data on the bar chart, either percentage or frequency. Table 1 and Table 2 should include the N (number of responses). Response #3: We are grateful to the reviewer for this insightful observation. We have indicated the percentage for Figure 1 for clarity. We have also provided the number of responses (N) to the titles of Tables 1 and 2. Method Comment #4: In the abstract, it was mentioned - “This dataset was collected from a total of 1,977 university lecturers across 24 African countries”. But in the Introduction, it was reported -” The link to the e-survey was shared on the Telegram forum of the Association of African Universities, which has 1,622 participants from various African countries and regions.” Please check again the figure, as the responses gathered (1,977) were more than the total Telegram participants (1,622). Response #4: We thank the reviewer for bringing this point out. However, we are clarifying that the virtual snowball was used. Although there were 1,622 members in the Telegram group, the respondents further distributed the link to the questionnaire to other institution-specific online groups. Thus, the responses gotten was beyond the number of participants in the group. It was not an error. Comment #5: The method section is quite clear, but I have some comments for improvement: It would be good to have a separate sub-section on instrument development, where information on each section of the survey instrument is discussed, currently it is all under Ethics and Consent sub-section. It is a good practice to include Ethics and Consent sub-section. Response #5: The authors are very impressed with this comment. The original draft of the article had this section, but it was recommended for removal during the initial editorial check. Nevertheless, we have added the section “instrument development” to the revised article because we agree with the reviewer that it will make it clearer to readers. We are grateful. Comment #6: Include information on the scale used to measure “willingness” as shown in Table 1. Provide an explanation on the Average Score = 5839 as indicated in Table 1. Response #6: The researchers wish to draw the attention of the reviewer to the description of the third section of the survey, where it was stated that a five-point rating scale was used. However, in the revision, we have stated that a scale with response options ranging from 0 (no willingness) to 5 (very willing) was used. The average score of 5839 in Table 1 has also been explained in the revised version. Comment #7: Report on the pilot study and the changes made after it was conducted. Response #7: Thanks to the reviewer. Although we felt it was not necessary to document the pilot study procedure in a Data Note. However, following your suggestion, we can confirm that details of the pilot study including reliability are now provided. Comment #8: A total of 1,9777 responses were gathered, all of them are usable data/completed responses? It is good to report the incomplete survey (that was excluded from the analysis) if that is the case. If none please inform so. Response #8: We are grateful to the reviewer for raising it. It is important information that should be added. As you will see in the revised version of the article, a statement has been made that complete data were collected from the 1,977 respondents without any missing data. Comment #9: The title used the term “digital tools” but throughout the article, various terms were used such as internet platform, digital platform, online platform, internet-based platform – one consistent term should be used throughout the article and survey instrument to avoid confusion. Response #9: The authors can confirm that the term \"digital tools\" is now consistently used in the revised version of the article." } ] } ]	1	https://f1000research.com/articles/11-82
https://f1000research.com/articles/11-390/v1	04 Apr 22	{ "type": "Research Article", "title": "Machine learning techniques for predicting depression and anxiety in pregnant and postpartum women during the COVID-19 pandemic: a cross-sectional regional study", "authors": [ "Radwan Qasrawi", "Malak Amro", "Stephanny VicunaPolo", "Diala Abu Al-Halawa", "Hazem Agha", "Rania Abu Seir", "Maha Hoteit", "Reem Hoteit", "Sabika Allehdan", "Nouf Behzad", "Khlood Bookari", "Majid AlKhalaf", "Haleama Al-Sabbah", "Eman Badran", "Reema Tayyem", "Malak Amro", "Stephanny VicunaPolo", "Diala Abu Al-Halawa", "Hazem Agha", "Rania Abu Seir", "Maha Hoteit", "Reem Hoteit", "Sabika Allehdan", "Nouf Behzad", "Khlood Bookari", "Majid AlKhalaf", "Haleama Al-Sabbah", "Eman Badran", "Reema Tayyem" ], "abstract": "Background: Maternal depression and anxiety are significant public health concerns that play an important role in the health and well-being of mothers and children. The COVID-19 pandemic, the consequential lockdowns and related safety restrictions worldwide negatively affected the mental health of pregnant and postpartum women. Methods: This regional study aimed to develop a machine learning (ML) model for the prediction of maternal depression and anxiety. The study used a dataset collected from five Arab countries during the COVID-19 pandemic between July to December 2020. The population sample included 3569 women (1939 pregnant and 1630 postpartum) from five countries (Jordan, Palestine, Lebanon, Saudi Arabia, and Bahrain). The performance of seven machine learning algorithms was assessed for the prediction of depression and anxiety symptoms. Results: The Gradient Boosting (GB) and Random Forest (RF) models outperformed other studied ML algorithms with accuracy values of 83.3% and 83.2% for depression, respectively, and values of 82.9% and 81.3% for anxiety, respectively. The Mathew’s Correlation Coefficient was evaluated for the ML models; the Naïve Bayes (NB) and GB models presented the highest performance measures (0.63 and 0.59) for depression and (0.74 and 0.73) for anxiety, respectively. The features’ importance ranking was evaluated, the results showed that stress during pregnancy, family support, financial issues, income, and social support were the most significant values in predicting anxiety and depression. Conclusion: Overall, the study evidenced the power of ML models in predicting maternal depression and anxiety and proved to be an efficient tool for identifying and predicting the associated risk factors that influence maternal mental health. The deployment of machine learning models for screening and early detection of depression and anxiety among pregnant and postpartum women might facilitate the development of health prevention and intervention programs that will enhance maternal and child health in low- and middle-income countries.", "keywords": [ "Machine Learning", "Anxiety", "Depression", "Pregnancy", "COVID-19", "Random Forest" ], "content": "Introduction\n\nThe emergence of the Coronavirus disease (COVID-19) in late 2019 and early 2020 has severely impacted the global population. Being characterized as an infectious disease primarily spreading through droplets of saliva or nasal discharge1, the infection rate is significant and its consequences can be lethal2–4. The disease is particularly dangerous to vulnerable populations, such as the elderly and those with underlying medical conditions including cardiovascular disease, diabetes, respiratory disease, and cancer3–5. Nonetheless, the specific implications of COVID-19 infection on pregnancy and childbirth have remained unidentified throughout the pandemic6–9.\n\nThe uncertainty about the nature, transmission, and mortality of the virus, together with its rapid spread and the consequential social and mobility restrictions (quarantines, lockdowns, and social distancing) have impacted the mental health of pregnant women worldwide5,10. In fact, the psychological effects of COVID-19 on pregnant women may lead to the appearance or increment of stress, anxiety, and depression symptoms as indicated in Broche-Perez et al. study11. In a 2020 study by Tokgoz et al.12, the authors demonstrated that pregnant women during the COVID-19 pandemic presented higher rates of depression, stress, and anxiety than pregnant women before the pandemic. The study further evidenced that mental health disorders during pregnancy can result in pre-term labour, low birth weight, delayed neuropsychiatric development in children, preeclampsia, and unscheduled caesarean delivery11,13.\n\nHowever, mental health disorders among pregnant women are widely undiagnosed and could result in worse consequences for mother and child14,15. Furthermore, the traditionally applied screening programs for psychological conditions rely on self-reporting and are for the most part designed to detect the population with pre-existing symptoms6,16. Contrary to the available traditional assessment of psychological disorders, artificial intelligence (AI) models can predict potential incidence of depression and anxiety among pregnant women, which would facilitate pre-emptive action, treatment, and early diagnosis16,17. As a matter of fact, one subarea of AI, machine learning (ML), has previously been used in the field of mental health for the prediction of psychological conditions such as anxiety, depression, obsessive-compulsive disorder (OCD), and post-traumatic stress disorder (PTSD), both prior to and during the onset of the COVID-19 pandemic18,19.\n\nIn a 2020 study by Seah et al.20, five ML algorithms where applied for the prediction of anxiety, depression, and stress on individuals around the world using the Depression, Anxiety and Stress Scale questionnaire (DASS 21). The Random Forest classifier, a machine learning algorithm used for data classification, had the best performance accuracy in predicting psychological conditions. A study by Priya et al.21, utilized ML tools for the creation of a new diagnostic methodology for anxiety and depression to replace traditional diagnosis through self-reported symptoms. The tool represented an improvement in diagnosis and treatment. Similarly, a study by Richter et al.22 applied eight ML algorithms, including a hybrid model, for the prediction of psychological problems such as anxiety, depression, and stress. The study found that the hybrid model presented higher accuracy rates than the single algorithms used. In relation to maternal health, only a few studies have been found to use ML models for the prediction of psychological disorders16,19,23,24. Among the available studies in this field, a study by Shin et al.24, developed a predictive model for postpartum depression using nine different ML approaches, the results showed that the Random Forest model achieved the highest accuracy rates. In addition, the study of Hochman et al.16, provided evidence that ML models are able to accurately screen and identify populations at high risk of postpartum depression for preventive intervention.\n\nThus, the use of ML techniques in mental health prediction and diagnosis might yield positive results in the reduction of self-harm and the provision of timely treatment for at-risk patients. However, very limited studies have used ML in maternal mental health, especially in relation to COVID-1911,16,19,24. This study aims to enrich the literature by assessing the performance of ML techniques in studying the effect of the COVID-19 lockdown on maternal mental health in low- and middle-income countries. The study used ML for predicting depression and anxiety symptoms from different features during the COVID-19 lockdown. To date, this is the first international study using population-based datasets from five countries (Palestine, Lebanon, Jordan, Saudi Arabia, and Bahrain) that accounts for multiple maternal and mental health variables.\n\n\nMethods\n\nThe study obtained written approval of the Ethics Committee in Scientific Research of University of Jordan, Jordan (19/2020/585), as well as universities from all participating countries. Written informed consent was obtained from all participants.\n\nData set. This study is the first of its kind as it utilized a regional dataset for evaluating the performance of ML algorithms in predicting depression and anxiety among pregnant and postpartum women during the COVID-19 lockdown in five Arab countries. The stratification of participants into different sets of data according to country of residence and the overall prediction for the total participants provide important and interesting information about the effect of the COVID-19 lockdown on pregnant women. A total of 3,569 women (1,939 pregnant and 1,630 postpartum) from five countries (Jordan, Palestine, Lebanon, Saudi Arabia, and Bahrain) participated in the study. Data were collected during the period of lockdown from July to December 2020.\n\nThe data set was extracted from a regional study conducted by the authors for assessing the impact of the COVID-19 pandemic on pregnant and postpartum women's physical and mental health. The study collected data from five Arab countries including: Lebanon, Palestine, Jordan, Bahrain, and Saudi Arabia. A total of 3569 women (currently pregnant or were pregnant during the COVID-19 pandemic lockdown) were selected in this study. The data set including socio-demographic variables and risk factors related to depression, anxiety, and physical and mental health among pregnant women is shown in Table 1.\n\nA cross-sectional study design was used for collecting the study data during the COVID-19 pandemic from August to November 2020, in the five listed countries: Lebanon, Palestine, Jordan, Bahrain, and Saudi Arabia) in the Arab region. The snowball sampling method was used to recruit pregnant women. The initial participants were contacted through the research team’s professional network, and the obstetric and maternity clinics in the participating countries. Data was collected through a web-based questionnaire, which was previously validated in two published studies25,26, and the software was designed by Palestinian National Nutrition Platform. The questionnaire was disseminated by researchers through their social media networks (Facebook, WhatsApp, and Instagram), and the participating universities network. Furthermore, hard copies of the questionnaire were distributed to women in some areas with limited internet access through obstetric and maternity clinics. The survey considered several sociodemographic features of pregnant women, medical history, nutrition patterns, physical activity, smoking, education, residency, economic situation, anxiety indicators, and depression indicators. Questions regarding the pre-pregnancy period were not included in the survey. For the full questionnaire, see Extended data27.\n\nThe following criteria guided the data collection process: (i) pregnancy during the COVID-19 pandemic period; (ii) normal pregnancy (i.e., no complications); (iii) aged over 18 (iv) place of residence (the five study countries); (v) having answered all questions in the questionnaire. Moreover, the exclusion criteria included conception during the intra-COVID19 pandemic period, as well as risk factors such as miscarriage and chronic health complications.\n\nOutcome variables. The outcome variables included pregnant women's depression and anxiety levels. Participants were assessed for depression and anxiety using the Patient Health Questionnaire (PHQ-9) and Generalized Anxiety Disorder (GAD-7) scales.\n\nDepression: The depression data was collected using the Patient Health Questionnaire (PHQ), a self-reported scale designed by 28 to screen for symptoms of depression. The PHQ items are composed of four answer categories (Never =0; several days =1, more than half of the days =2, and nearly every day =3). The total score was calculated by summing the scale items responses. The PHQ total score was classified into the following groups: Low=0; Moderate=1; High =2.\n\nAnxiety: The GAD-7 scale28 was used for measuring generalized anxiety disorder. The anxiety score was estimated by assigning scores of 0, 1, 2, and 3 to the response categories of (Never =0; several days =1, more than half of the days =2, and nearly every day =3). The total score was calculated by summing the scale items responses. The GAD-7 total score was classified into the following groups: Low=0; Moderate=1; High =2.\n\nFeatures. All potential features (predictors), including associated risk factors and socio-demographic variables were considered in the ML models for the assessment of pregnant women before and during the COVID-19 lockdown. The socio-demographic features included women’s age, age at marriage, country of residence, education level, work status, family income, and locality.\n\nAssociated risk factors included pre- and post-COVID-19 pandemic food consumption patterns, smoking status, body mass index (BMI), physical activity, healthy food consumption (fruits, vegetables, meat, grains, and dairy products), unhealthy food consumption (sweets, soft drinks, energy drinks, and fast food), physical activity level, technology-related activities, COVID-19 diagnosis, relatives diagnosed with COVID-19, underlying health conditions (diabetes, gestational diabetes, hypertension, gestational hypertension, heart and arterial diseases, liver diseases, high cholesterol, high triglycerides, thyroid disorders, or respiratory problems), cancellation of follow up appointment due to COVID-19, number of pregnancies, number of abortions, family problems, social problems, psychological stress, and work-related stress.\n\nData analysis. General descriptive analysis and ANOVA tests were used for describing the distribution of women based on risk factors, while prediction and classifications were measured using ML techniques. The classification accuracy, confusion matrix, precision, sensitivity, and specificity were used for evaluating the ML prediction performance. The ML algorithms were applied in the Python AI development platform to predict the incidence and severity level of depression and anxiety during COVID-19 among pregnant women. The data set was divided into a 70:20:10 ratio for training, testing and validation.\n\nTo evaluate whether the ML algorithms can predict pregnant women's depression and anxiety, the outcome variables and features were included in the ML models. The performance of ML models was first evaluated for depression and then for anxiety separately. The performance metrics for Gradient Boosting Machines (GB), Distributed Random Forests (RF), Extreme Randomized Forests (XRT), Naïve Bayes (NB), Support Vector Machine (SVM), Multilayers Neural Network (MNN), and Decision Tree (DT) are presented in the results. The accuracy, precision, Area Under the Curve (AUC), Matthew's Correlation Coefficient (MCC) and Receiver Operating Characteristic Curve (ROC) were used for measuring the performance accuracy.\n\n\nResults\n\nTable 2 and Table 3 show the descriptive analysis of the participants’ data by anxiety and depression levels. Results indicated that the women’s mean age was 28.5 (±5.3) years. Among participants, 11.6% and 8.7% had moderate and high levels of depression, respectively while 22.4% and 7.7% had moderate and high levels of anxiety, respectively.\n\nThe rates of anxiety and depression were found to differ by the country of residence, education level, family income, work stress, social problems, health problems, family problems, financial problems, psychological problems, sleeping hours per day, fear of COVID-19 infection, and unhealthy food consumption. A greater percentage of women with high levels of depression and anxiety symptoms were found in Palestine (19.9%, 22.5%) and Jordan (18.6%, 21.0%), respectively. Furthermore, the results in Table 2 indicated that the highest percentages of depression were among women with self-reported family problems (35.3%), sleep deprivation (<6 hours per night) (31.6%), psychological problems (30.5%), financial problems (29.7%), COVID-19 diagnosis (27.4%), social (25.7%), and work stress (23.1%). Furthermore, high levels of anxiety symptoms were found among women with self-reported family problems (33.2%), financial (23.6%), social (21.6%), and psychological problems (22.5%) as shown in Table 3.\n\nDifferent performance measures were considered in our study to evaluate whether the ML models can predict women’s depression and anxiety symptoms during the COVID-19 lockdown. Seven ML classification algorithms were tested on our dataset, including SVM, K-nearest neighbour (KNN), NB, Random Forest (RF), Neural Network (NN), DT, and GB. The performance was evaluated using several assessments measures such as accuracy, precision, Area Under the Curve (AUC), Matthew's Correlation Coefficient (MCC) and Receiver Operating Characteristic Curve (ROC). The performance measures were calculated using the following equations:\n\n1. Specificity\n\n\n\n2. Precision\n\n\n\n3. Recall\n\n\n\n4. F-measure\n\n\n\n5. Matthew’s Correlation Coefficient\n\n\n\n6. Accuracy\n\n\n\nFigure 1 represents the comparison of accuracy rates among the selected machine learning algorithms in predicting women's depression and anxiety symptoms. All tested models reported a high level of accuracy (ranging from 80.0–83.3%) for predicting depression among pregnant women except for NB. On the other hand, various levels of accuracy were reported for the ML models when predicting anxiety. The GB model presented the highest accuracy rate (82.9%) followed by RF and NB (81.3%). Nonetheless, all the ML models reported an acceptable rate of accuracy for both depression and anxiety symptoms.\n\nAdditional performance measures were used for evaluating the ML prediction performance of depression and anxiety symptoms, including AUC, sensitivity, specificity, F-Measure, and MCC. Figure 2 illustrates the different performance measures of depression prediction models. Balanced accuracy, sensitivity, and F measures were observed across the ML models. The AUC varied across models; DT reported the lowest AUC rate (68.8%), while other models ranged from 82.6% to 91.9%. The MCC performance measure showed high variability across models being relatively low among the different ML models; the NB model reported the highest MCC value of 63%. Overall, GB reported the highest AUC, ACC, sensitivity, and F1 measures among all other ML models.\n\nFigure 3 shows the different performance measures of ML models for the anxiety prediction. Performance analysis for the anxiety prediction reported quite similar AUC, ACC, sensitivity, and F1 measures for the RF, NN, KNN, and GB models. SVM and DT models had the lowest accuracy measures. The sensitivity and accuracy were highest at the GB and NB models. The MCC performance measure varied among the ML models, the highest of which were found in the NB and GB models (74.3%, 72.8%, respectively). The SVM had the lowest MCC performance measure (52.4%). Overall, GB achieved the best accuracy and sensitivity, and F1-measures of 82.9%, and a balanced MCC measure of 72.8%.\n\nThe GB and RF receiver operating characteristics (ROC) for the moderate and high depression and anxiety classes is presented in Figure 4 (A and B) and Figure 5 (A and B) respectively. Three numerical categories of student depression and anxiety classes were used: low, moderate, and high. The ROC resides in the upper left corner; thus, the gradient boosting algorithm showed a better prediction of positive value than the other studied algorithms (AUC of 91.9% and 93.5% for depression and anxiety, respectively).\n\nGradient Boosting and Random Forest ROC sensitivity and specificity analysis: (A) Moderate depression symptoms analysis; (B) High depression symptoms analysis.\n\nGradient Boosting and Random Forest ROC sensitivity and specificity analysis: (A) Moderate anxiety symptoms analysis; (B) High anxiety symptoms analysis.\n\nThe 23 variables used for predicting depression and anxiety symptoms in the ML models were classified and ranked from 0 to 100%. The variables with importance level greater than 60% were considered. The participants reported different levels of variables’ importance for depression and anxiety. The distribution of most important variables for depression and anxiety can be found in Figure 6 and Figure 7, respectively. The most significant variables in predicting depression symptoms were stress during lockdown, psychological factors, family problems, and country of residence. While the most significant variables in predicting anxiety were stress during lockdown, financial problems, family problems, social problems, and COVID-19 diagnosis.\n\n\nDiscussion\n\nIn this study, we used machine learning techniques for the prediction of depression and anxiety among pregnant and postpartum women from five Middle Eastern countries (Lebanon, Palestine, Jordan, Bahrain, and Saudi Arabia) during the COVID-19 lockdown. We found that 20.3% of women had moderate to severe maternal depression while 30.1% of them had moderate to severe anxiety, the highest rates being among Palestinian and Jordanian women. The findings of this study are consistent with other studies that indicated high levels of anxiety and depression symptoms among pregnant women during COVID-19 lockdown3. Women reported a significant concern and were found at high risk of developing post-traumatic stress disorder, which requires direct intervention from health care providers for caring of pregnant and postpartum women mental health during COVID-19 pandemic.\n\nThe performance of different ML models in predicting maternal depression and anxiety was evaluated through measuring accuracy, specificity, precision, recall, F-measure, and Matthew's Correlation Coefficient (MCC). The accuracy performance of the studied models was similar and did not indicate a significant difference across models. GB and RF reported the best accuracy, sensitivity, and F1 measures for depression prediction. The MCC has been measured for the selected models as an alternative performance measure which is not affected by an unbalanced dataset. The MCC measure showed acceptable scores for both depression and anxiety symptoms. The NB had the highest MCC values followed by GB and RF. Thus, the results in this study are consistent with other studies that assessed the performance of ML classifiers in predicting depression among pregnant and postpartum women, where the RF model showed the highest accuracy and AUC values16,24.\n\nThe ML prediction models of postpartum depression developed by Shin et al., (2020) achieved an AUC of 0.79. On the other hand29, utilized a multilayer perceptron approach using several risk factors for depression prediction among Spanish pregnant and postpartum women. The model accomplished an AUC value of 0.82, sensitivity value of 0.84, and specificity of 0.81. Furthermore, in a study, Logistic Regression (LR) classifier was used for depression prediction and achieved an accuracy value of 83.3%30 while employing multiple ML algorithms including KNN, LR, Linear Discriminant Analysis (LDA), and B improved the overall accuracy values to 90%28,29. Additionally, our study was consistent with 24 study in which the ML classifiers were used in predicting postpartum depression and found that depression before pregnancy, stress during pregnancy, and smoking were the most significant risk factors for depression. On the contrary of our findings31, reported that women’s age, marital status, and education were the most significant factors relating to postpartum depression.\n\nFurthermore, our study reported a higher AUC performance measure than other similar ML prediction studies, whose AUC measures were 80%32, 79%5, and 78%33. The results in our study showed an accuracy of 83.3%, which is comparable to the 84% accuracy rate reported in other studies26,30. Nevertheless, the study sample used in this research was collected from diverse population groups across countries, thus diverging background and environmental factors were expected to affect the homogeneity of the dataset.\n\nSignificant risk factors for pregnant and postpartum depression and anxiety were found, including country of residence, family income, smoking, COVID-19 diagnosis, number of hours of sleep, stress during the COVID-19 lockdown, family support, social support, financial situation, psychological problems, and work stress. Additionally, risk factors particularly significant for anxiety included education level, locality, and work status. We found the rates of anxiety symptoms to be higher than those of depression among pregnant and postpartum women during the pandemic lockdown. The results showed that Jordanian, Palestinian and Lebanese women had higher anxiety and depression than Saudi and Bahraini women. The increased risk for depression and anxiety among women could be explained by low family income, financial problems, and poor healthcare systems available in these countries34. The study also reported significant differences in anxiety because of locality and education levels. Women with lower education levels reported higher anxiety; similarly, women living in urban areas presented higher anxiety levels. These findings could be explained by the stricter lockdown in cities and the lack of knowledge about the disease among women with lower education levels.\n\nThe machine learning models returned the five highest ranking features affecting women’s depression symptoms: stress during pregnancy, psychological problems, family support, country of residence, and number of hours of sleep-in descending order. The highest-ranking features for anxiety were stress during pregnancy, financial problems, family problems, social problems, and COVID-19 diagnosis. Our findings are consistent with similar studies indicating that stress during pregnancy negatively affects women's mental health and might influence incidence of postpartum depression9,14,32. Furthermore, the results were consistent with other studies indicating that family income, and social and psychological problems had significant impact on maternal mental health3,6,8.\n\nThe study provides an interesting finding that the accuracy performance measures is relatively high and remains stable between the selected ML models, especially for AUC, accuracy, and sensitivity even at reduced number of variables. This finding is consistent with other studies17,31,34–36 that indicated the high correlation between anxiety and depression symptoms and other socio-demographic risk factors. Thus, stress, family support, financial situation, psychological problems, and country of residence were among the most important variables associated with depression and anxiety during the pandemic lockdown. This is important to consider when developing intervention strategies and programs. The stability in performance measures reflects that the self-reported survey methods can be used as a good assessment tool for anxiety and depression. Moreover, pregnant women had more anxiety symptoms than depression during lockdown, which might affect maternal and child health.\n\nOur findings suggest that deploying machine learning techniques for the screening of pregnant and postpartum women will help in identifying those at highest risk of anxiety and depression through clustering and classification, which will in turn aid in the development of effective preventive interventions. Thus, this research not only addresses the integration of innovative technology for the prediction and diagnosis of depression and anxiety among pregnant and postpartum women in low- and middle-income countries, but given the international dataset used, it assesses the prediction power of several ML algorithms across diverging population groups with distinct risk factors. Additionally, the study included variables specific to the COVID-19 lockdown period, which differentiates it from similar studies.\n\nNevertheless, some limitations are found in this study, including the extent of the study sample. Having a smaller dataset limits the power of predictions to train a robust range of algorithms, as well as limits the number of clusters and classifications produced by the ML predictive models. In addition, the study used the online self-reported assessment, which was not fully completed by all study participants. Nonetheless, the incomplete and missing data were excluded from out dataset. Finally, a more comprehensive study with a larger and more representative dataset including clinical data is recommended for future research among low- and middle-income countries.\n\n\nConclusion\n\nThe study assessed the performance measures of machine learning algorithms in predicting depression and anxiety among pregnant and postpartum women in low- and middle-income countries during the COVID-19 pandemic lockdown. Based on the results presented, this research concludes that ML algorithms, particularly (yet not exclusively) Gradient Boosting and Random Forest, are effective predictive models for maternal mental health. These models could be integrated into clinical medical information systems for the automatic prediction of pregnant women’s depression and anxiety based on the identified key variables. The deployment of ML models will provide effective clinical applications for the development of prevention and intervention programs. Likewise, by making use of accurate machine learning techniques such as Random Forest, public health professionals, healthcare providers, and decision-makers will be able to predict rising issues and implement relevant intervention programs to enhance maternal and child health in their respective countries.\n\n\nData availability\n\nHarvard Dataverse: Pregnancy and Mental Health Data during COVID-19\n\nhttps://doi.org/10.7910/DVN/FCDGEB27\n\nThis project contains the following underlying data:\n\nML-DataSet.xlsx\n\nVariables_Descriptions.xlsx\n\nDataverse: Pregnancy and Mental Health Data during COVID-19\n\nhttps://doi.org/10.7910/DVN/FCDGEB27\n\nThis project contains the following extended data:\n\nEnglish questionnaire.xlsx\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to thank the study participants for their time and effort in responding to our study. Furthermore, The authors would like to thank the following for assisting in data collection: Elissa Naim, Manal Fardon (Lebanon); Narmeen Al-Awwad (The Hashemite University, Jordan); Asma Bash (The University of Jordan); Nahla Al-Bayyari (Al-Balqa Applied University, Jordan).; Shreen Sulten, Nada Omar Abdul jawad, and Mahmoud Sami (King Hamad University Hospital, Kingdom of Bahrain); Rana Ghabbash, Asma Imam (Al-Quds University, Palestine); Firas Abdel Jawad (Makassed Hospital); Nabil Thawabteh (Makassed Hospital); Areej Alamery (Ministry of Health, Saudi Arabia).\n\nAuthor Disclaimer: The views expressed in this article do not necessarily represent the views, decisions or policies of WHO, Saudi FDA or the other institutions with which the authors are affiliated.\n\n\nReferences\n\nHatmal MM, Al-Hatamleh MAI, Olaimat AN, et al.: Side effects and perceptions following covid-19 vaccination in jordan: A randomized, cross-sectional study implementing machine learning for predicting severity of side effects. 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PubMed Abstract \| Publisher Full Text\n\nEffati-Daryani F, Zarei S, Mohammadi A, et al.: Depression, stress, anxiety and their predictors in Iranian pregnant women during the outbreak of COVID-19. BMC Psychol. 2020; 8(1): 99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEffati-daryani F, Zarei S, Mohammadi A, et al.: Depression, stress, anxiety and their predictors in Iranian pregnant women during the outbreak of COVID-19. BMC Psychol. 2020; 8(1): 99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeng JS, Sperlich M, Low LK, et al.: Childhood Abuse History, Posttraumatic Stress Disorder, Postpartum Mental Health, and Bonding: A Prospective Cohort Study. J Midwifery Womens Health. 2013; 58(1): 57–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdollahi F, Etemadinezhad S, Lye MS: Postpartum mental health in relation to sociocultural practices. Taiwan J Obstet Gynecol. 2016; 55(1): 76–80. PubMed Abstract \| Publisher Full Text\n\nHochman E, Feldman B, Weizman A, et al.: Development and validation of a machine learning-based postpartum depression prediction model: A nationwide cohort study. Depress Anxiety. 2021; 38(4): 400–411. PubMed Abstract \| Publisher Full Text\n\nŞahan E, Ünal SM, Kırpınar İ: Can we predict who will be more anxious and depressed in the COVID-19 ward? J Psychosom Res. 2021; 140: 110302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUmanandhini D: Survey on Stress Types Using Data Mining Algorithms. Int J Innov Res Adv Eng. 2017; 4(4): 2014–2018. Reference Source\n\nSeah JHK, Jin Shim K: Data Mining Approach to the Detection of Suicide in Social Media: A Case Study of Singapore. 2018 IEEE International Conference on Big Data (Big Data).. 2019; 5442–5444. Publisher Full Text\n\nPriya A, Garg S, Tigga NP: Predicting Anxiety, Depression and Stress in Modern Life using Machine Learning Algorithms. Procedia Comput Sci. 2020; 167(2019): 1258–1267. Publisher Full Text\n\nRichter T, Fishbain B, Markus A, et al.: Using machine learning-based analysis for behavioral differentiation between anxiety and depression. Sci Rep. 2020; 10(1): 16381. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar P, Garg S, Garg A: Assessment of Anxiety, Depression and Stress using Machine Learning Models. Procedia Comput Sci. 2020; 171(2019): 1989–1998. Publisher Full Text\n\nKessler RC, van Loo HM, Wardenaar KJ, et al.: Testing a machine-learning algorithm to predict the persistence and severity of major depressive disorder from baseline self-reports. Mol Psychiatry. 2016; 21(10): 1366–1371. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShin D, Lee KJ, Adeluwa T, et al.: Machine Learning-Based Predictive Modeling of Postpartum Depression. J Clin Med. 2020; 9(9): 2899. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTayyem RF, Allehdan SS, Alatrash RM, et al.: Adequacy of nutrients intake among jordanian pregnant women in comparison to dietary reference intakes. Int J Environ Res Public Health. 2019; 16(18): 3440. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDra AFR: Food Group Intake of Pregnant Jordanian Women Based on the Three Pregnancy Trimesters. Angew. Chemie Int. Ed. 6(11), 951–952. 2017; 25: 1–77.\n\nQasrawi R, Hoteit M, Allehdan S, et al.: Pregnancy and Mental Health Data during COVID19. Harvard Dataverse, V1, 2022. http://www.doi.org/10.7910/DVN/FCDGEB\n\nSpitzer RL, Kroenke K, Williams JB, et al.: A brief measure for assessing generalized anxiety disorder: The GAD-7. Arch Intern Med. 2006; 166(10): 1092–1097. PubMed Abstract \| Publisher Full Text\n\nTortajada S, García-Gomez JM, Vicente J, et al.: Prediction of postpartum depression using multilayer perceptrons and pruning. Methods Inf Med. 2009; 48(3): 291–298. PubMed Abstract \| Publisher Full Text\n\nHosseinifard B, Moradi MH, Rostami R: Classifying depression patients and normal subjects using machine learning techniques and nonlinear features from EEG signal. Comput Methods Programs Biomed. 2013; 109(3): 339–345. PubMed Abstract \| Publisher Full Text\n\nJiménez-Serrano S, Tortajada S, García-Gómez JM: A mobile health application to predict postpartum depression based on machine learning. Telemed J E Health. 2015; 21(7): 567–574. PubMed Abstract \| Publisher Full Text\n\nAndersson S, Bathula DR, Iliadis SI, et al.: Predicting women with depressive symptoms postpartum with machine learning methods. Sci Rep. 2021; 11(1): 7877. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang D, Shen D, Alzheimer's Disease Neuroimaging Initiative: Multi-modal multi-task learning for joint prediction of multiple regression and classification variables in Alzheimer's disease. NeuroImage. 2012; 59(2): 895–907. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGjerdingen DK, Chaloner KM: The relationship of women’s postpartum mental health to employment, childbirth, and social support. J Fam Pract. 1994; 38(5): 465–472. PubMed Abstract\n\nLebel C, MacKinnon A, Bagshawe M, et al.: Elevated depression and anxiety symptoms among pregnant individuals during the COVID-19 pandemic. J Affect Disord. 2020; 277: 5–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOnoye JM, Goebert D, Morland L, et al.: PTSD and postpartum mental health in a sample of Caucasian, Asian, and Pacific Islander women. Arch Womens Ment Health. 2009; 12(6): 393–400. PubMed Abstract \| Publisher Full Text" }	[ { "id": "136365", "date": "08 Jun 2022", "name": "Chadi Ibrahim Fakih", "expertise": [ "Reviewer Expertise Human reproduction", "endocrinology", "ART" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe context of the work proposed in this article is to use machine learning techniques to predict maternal depression and anxiety levels during the COVID-19 pandemic. The data are collected from five Arab countries and composed of 3569 samples (1939 pregnant women and 1630 postpartum). Several machine learning techniques are applied such as Random Forest, Gradient Boosting and Naïve Bayes. As a result, the gradient boosting algorithm outperformed the other methods used in this research.\nRegarding the form, the article is well organized and written. The addressed problem is quite important and the results obtained are promising. However, the article suffers from several weak points:\nHow are features’ importance evaluated? (filtering method) Do authors focus on filter-based, wrapper-based or embedded-based methods? It seems they have based on embedded one, but they should be precise.\n\nWhy feature extraction methods are not used such as LDA, ICA and t-SNE?\n\nArticle contains a lot of figures and tables per evaluation measure. I think these figures and tables can be put in the appendix and focus on one evaluation measure such as accuracy.\n\nYou may provide a matrix that shows the correlation between each couple of features and between each feature and the different classes.\n\nWhat is the bCA mentioned under the figures 2 and 3? (Component analysis or classification accuracy).\n\nAuthors should show a comparison of their work with other works even they are not applied on the same dataset. It is important to see how the efficiency of each machine learning varies between the work and other works.\n\nAuthors stated that they used 3569 samples in their study while when we refer to the table 2 and table 3 there is something missing. For example, if we count in table 2 all the samples in the row that corresponds to the country of residence regardless the depression level, they sum to 1601. In contrast, the sum of the samples in the row smoking during pandemic is 1000 and others sums to 1600. Are there missing features’ values?\n\nThere is something missing in table 2 (table 3 also). Table 2 shows that the total samples belonging to class “No depression” is 878 over 1601, “Moderate depression” is 413/1601 and “High depression” is 310/1601. Is the total number of samples are 1601? I thought they are 3569.\n\nIn page 5 – section “Results”, you mention “Among participants, 11.6% and 8.7% had moderate and high levels of depression, respectively while 22.4% and 7.7% had moderate and high levels of anxiety, respectively”. It means, the class “No depression” constitute the 80% of the dataset and “No anxiety” 70% of the dataset. How is this class-imbalance handled during experimentations?\n\nPut the confusion matrix for depression-prediction and the one for anxiety-prediction to see how the misclassified samples are distributed.\n\nThe dataset is split into 70:20:10 (training:testing:validation). What type of parameters did you use that need validation? Did you split them randomly into three groups? You should use techniques other than random sampling such as cross-validation.\n\nAuthors should show the training error to compare it with the testing error in order to show if there is overfitting.\n\nNo need to put the formulas of the evaluations measures (Precision, recall, AUC,…). They are known.\n\nGradient-boosting and Random-forest are decision-tree based classifiers. It is important to derive the rules that constitute the model showing the set of rules of each class.\n\nIs the work 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": "144638", "date": "05 Sep 2022", "name": "Iyad Tumar", "expertise": [ "Reviewer Expertise My research area is in the fields of Artificial Intelligence (AI). Within AI", "I am interested in problems related to health or education modeling", "machine learning", "and data mining", "and their interdisciplinary applications to real-life problems. Furthermore", "I’m interested in computer networks and cybersecurity", "in which I worked on the development of models and methods of network management and protection." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 used the machine learning techniques in predicting the effect of COVID 19 on the women depression and anxiety. The machine learning models used original data set collected from Arab countries during COVID19-pandemic lockdown. The study sample composed of 3569 women (1939 pregnant and 1630 postpartum). The study indicated that the gradient boosting algorithm reported the highest performance compared to other algorithms.\nThe study addressed an important problem in developing countries, and the result of the study is very encouraging, mainly in the deployment of machine learning in the fields of Mental health and public health.\n\nFurthermore, the study is well-written and organized. However, a few issues need more clarifications:\nThe study contains many figures and tables, will be much better to add some of them in the Appendix.\n\nI recommend double checking the samples and features of Table 2.\n\nAre there any significant differences between pregnant women and postpartum?\n\nDoes the authors use a weighted dataset to analyze and run the ML models.\n\nIt is important to show the overfitting problem during the training and testing.\n\nIs the work 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/11-390
https://f1000research.com/articles/9-251/v1	09 Apr 20	{ "type": "Research Article", "title": "Evaluation of the rapid, multi-country, parallel process, multi-tasking approach to startup of short-term technical assistance to improve service delivery in newborn and child health in the context of USAID’s Zika response in four Eastern and Southern Caribbean countries", "authors": [ "Bulbul Aumakhan", "Astou Coly", "Salwan Hager", "Tamar Chitashvili", "M. Rashad Massoud", "Bulbul Aumakhan", "Astou Coly", "Salwan Hager", "Tamar Chitashvili" ], "abstract": "Background: In 2018, the USAID Applying Science to Strengthen and Improve Systems (ASSIST) Project started a new partnership with four Eastern and Southern Caribbean countries impacted by the Zika virus: Antigua and Barbuda, Dominica, St. Kitts and Nevis, and St. Vincent and the Grenadines. The goal of the project was to provide short-term technical assistance (STTA) to strengthen the health systems’ capacity to detect newborns and young children potentially affected by Zika and to address their health needs. To meet these objectives, ASSIST developed an innovative approach based on its existing model for service delivery improvement. This novel approach is known as Rapid, Multi-country, Parallel Process, Multi-tasking Approach for a Project Startup (RMPP-MAPS). An evaluation was conducted to document the STTA startup activities, to identify enabling and constraining factors, and to capture lessons learned. Methods: An external consultant conducted remote in-depth interviews with individuals involved in the startup using semi-structured interview guides and retrieved data from the review of project documents. Results: Using RMPP-MAPS, the ASSIST Project successfully implemented the startup for complex STTA in four countries within less than four months, spanning mid-May to early September 2018. Project milestones included achieving buy-in from stakeholders, co-developing the technical scope and materials, and rapidly executing critical operational functions. Dedicated project teams, country leaderships, and local champions were essential to overcoming the main challenges, which included a condensed timeframe, lack of in-country offices, and country-level factors such as a shortage of health care workers and a weak health infrastructure. Conclusions: The RMPP-MAPS is a feasible and resource-efficient mechanism of interest to implementers, donors, and low and middle-income countries facing temporal and financial limitations to rapidly addressing public health priorities.", "keywords": [ "Rapid startup", "Zika", "emergency response", "quality improvement", "Eastern and Southern Caribbean", "Antigua and Barbuda", "Dominica", "St. Kitts and Nevis", "St. Vincent and the Grenadines" ], "content": "Introduction\n\nThe rapid spread of the Zika virus (ZIKV) infection to countries in the Latin American and Caribbean (LAC) region in 2015 and 2016 and its association with serious health consequences such as microcephaly prompted the World Health Organization (WHO) to declare the ZIKV infection a public health emergency of international concern (PHEIC) on February 1, 20161. Although the WHO declared an end to the PHEIC in November of the same year, the ongoing Zika transmission in the region as well as the potential for future outbreaks required continuous vigilance to improve health systems preparedness and capacity in prevention, surveillance, and management of emerging infectious diseases2–4.\n\nIn 2016, the United States Agency for International Development (USAID), the international community, and country governments began responding to the ZIKV epidemic in the LAC region5. In 2017, the USAID Applying Science to Strengthen and Improve Systems (ASSIST) Project implemented by University Research Co., LLC (URC) in Chevy Chase, MD, started activities in the Dominican Republic, Ecuador, El Salvador, Guatemala, Honduras, Jamaica, Nicaragua, and Paraguay to strengthen Zika-related health services to deliver evidence-based, person-centered quality care with a focus on pregnant women, newborns, and women of reproductive age6. In May 2018, USAID requested that ASSIST expand its efforts to four more countries in the Eastern and Southern Caribbean (ESC): Antigua and Barbuda, Dominica, St. Kitts and Nevis, and St. Vincent and the Grenadines. The request came in the last year of a three-year USAID Zika response plan in the LAC region. During this time, there was also a substantial decline in the number of Zika cases observed in the region. Therefore, the decision was made to design the effort as a one-year short-term technical assistance (STTA) activity aimed at improving detection and addressing the unique health needs of children potentially affected by Zika in the four countries. The STTA planned to achieve these objectives by strengthening newborn and well-baby care systems and early childhood development (ECD) programs in all functional health facilities providing childbirth services and well-baby care. These efforts were in line with national and USAID goals to improve public health emergency response and to strengthen the resilience of health systems to address future emergencies.\n\nGiven the short timeline and overarching objectives, ASSIST developed and applied the Rapid, Multi-country, Parallel Process, Multi-tasking Approach to Project Startup (RMPP-MAPS) in the four countries. The RMPP-MAPS required simultaneous initiation and execution of multiple planning processes; including but not limited to, achieving buy-in from Ministries of Health and participating facilities, co-developing the workplan and sustainability strategy, developing technical content and operational approach, identifying essential needs of facility teams to implement the activity, and initiating the procurement processes to address the logistical needs of the activity.\n\nThe fundamental concept underlying ASSIST’s health systems improvement approach is understanding that “every system is perfectly designed to achieve exactly the results it achieves7.” ASSIST views health systems as complex yet adaptive systems designed to undergo continuous reiterative processes of change and innovation. That process forms the basis of a six-component “Country Integrated Design” model (1-Improvement Design; 2-Implementation; 3-Sustainability; 4-Scale-up; 5-Institutionalization, and 6- Learning) that has been proven to effect positive outcomes over the three decades of USAID work (through the ASSIST and preceding projects) in the field of health system strengthening and quality improvement in health care services delivery8. To achieve desired outcomes, all six components of the model must be integrated in planning improvement interventions. Thus, the process necessitates the engagement of all stakeholders throughout implementation. Depending on the scale of the problem and/or context the process can take up to several years. A typical model of ASSIST and other global health partners includes setting up, staffing country offices, and a step-by-step implementation approach. For the ESC countries, ASSIST’s task was to implement the improvement activity in multiple countries within a single year. Registering URC field offices within the countries and establishing project teams to locally support the operation was not feasible in the given timeframe. This constraint posed a significant challenge for logistics and implementation. To overcome the challenge, ASSIST undertook an innovative RMPP-MAPS which allowed simultaneous planning and execution of multiple startup processes in all four countries (instead of in linear, sequential fashion) and using real-time data or “feedback loops” to track progress. Given the common language, geographic proximity, and similar socio-economic status, URC was able to apply a universal approach. In documenting and evaluating this novel approach, URC has published observations and recommendations for similar rapid startup of donor-funded short-term technical assistance activities.\n\n\nMethods\n\nA process evaluation design and a qualitative study methodology were employed to address the evaluation objectives9. The evaluation was retrospective with primary data collected through in-depth interviews with persons or key informants (KI) involved in the startup and secondary data retrieved from the review of project documents. Documents reviewed included: (1) startup documents obtained from the ASSIST Project (e.g., meeting records, activity plans, timelines, presentations, trip reports, scoping survey, etc.), (2) online public resources (e.g., USAID Zika program overviews, press-releases, ESC country-specific information, literature related to Zika outbreak in the region, summary of relevant work by implementing partners, etc.), and (3) documents obtained from program managers (e.g., Maternal and Child Survival Program reports and documents). Primary and secondary data were triangulated as necessary to document and develop a cohesive picture of the activity. See Extended Data10 for the documents reviewed.\n\nKIs were defined as individuals who had direct or indirect knowledge, experience, and/or involvement with any stage of the project development or startup activities. The USAID ASSIST Project provided a list of recommended KIs to the independent research consultant (B.A.) who conducted the interviews. The list included technical partners, country program planners, project coordinators, chairs and members of community and hospital nursing divisions, and maternal and child health committees. An initial email was sent to all prospective participants by the USAID ASSIST Project to introduce the consultant and inform the eligible KI about the evaluation. The consultant then contacted each KI via email, provided details about the evaluation purpose and rationale, and invited him or her to participate in an interview via Zoom (or in some cases, over the telephone). When necessary, the USAID ASSIST Project team facilitated contact with stakeholders to confirm their participation as a KI.\n\nA semi-structured interview guide (Extended data10) was developed to cover key topics including identifying startup activities in chronological order, elucidating views or understanding of the approach and methods employed, perceptions related to timeline and speed of the process, identifying facilitating and hindering factors, and capturing lessons learned and future recommendations. Questions were modified according to the type of respondent (national or regional partner, implementing partner, or leading STTA provider) and to fit the extent of participants’ involvement in the startup. Probes were used to elicit more details on specific responses. ASSIST team members were interviewed last to allow for clarification of any issues identified by previous respondents and/or obtain additional information as deemed necessary. All interviews were conducted in English and lasted approximately 45 minutes. The interviews were conducted by an independent, external research consultant (B.A.), who was not part of USAID ASSIST Project and was not involved in a project startup or implementation. The consultant holds a Ph.D. in Epidemiology and has expertise in public health research and program evaluation.\n\nAll interviews were audio-recorded. Audio files were uploaded into password-protected computer files. Automated transcripts were then generated from interviews and transcribed using transcription software (https://otter.ai). Transcripts were then manually checked and edited to ensure accuracy. They were not sent back to interviewees. Data were systematically grouped according to recurrent patterns and themes as related to central topics being evaluated (by B.A.), and codes were applied to identify segments of the interviews where these themes were discussed. The information obtained from the desk review was triangulated with the data retrieved from in-depth interviews to validate the information collected and more precisely map startup activities.\n\nInformed consent was obtained from each KI prior to the interview via email (consent form available as Extended data1). In addition, consent was obtained orally prior to recording each interview. Participants were informed of the voluntary nature of participation and the right to withdraw at any time or decline questions they did not wish to answer. Participants were also informed that their responses would be confidential (i.e., specific quotes or answers not linked to any specific respondent in the report). Interview audio, voice, and transcript records were stored securely in password-protected electronic files.\n\nThis evaluation was performed as part of the learning component of the USAID ASSIST Project and was not considered a research study subject to IRB review.\n\nRecall bias is a potential study limitation as interviews took place four to six months after the startup in the middle of the implementation phase. Participants sometimes had difficulties recalling details related to activities, dates, and sequences of events, and could not always distinguish startup from implementation activities. There is also a possibility for social desirability bias in response to sensitive questions (e.g., what the participant did not like about startup). The unavailability of some respondents due to scheduling or technical challenges is also a potential limitation.\n\n\nResults\n\nInvitations to participate in the startup evaluation interviews were sent out to a total of 34 individuals, out of whom 28 (82.4%) were successfully interviewed. Interviews took place between January 29, 2019 and April 7, 2019. Interviewed participants comprised of key stakeholders involved in the startup, including technical assistance providers, implementers, and partners based in USA and the Caribbean region. The distribution of respondents included: four USAID Washington staff; four URC staff working on the USAID ASSIST Project; one Jhpiego staff member who worked on the USAID Maternal and Child Survival Program (MCSP); three staff of the American Academy of Pediatrics (AAP); one representative of the Caribbean Regional Midwives Association (CRMA); one representative of USAID/Guyana (overseeing USAID’s regional activities in ESC); as well as stakeholders from the four technical assistance recipient countries. Respondent distribution is further illustrated in Table 1.\n\nNotes: Six individuals did not participate, citing “family emergency”, “not being knowledgeable enough about startup activities”, and “no longer being attached to the program” as the reason for declining; three responded to invitation but were not interviewed due to scheduling conflicts or internet access/technical difficulties with Zoom.\n\nCRMA, Caribbean Regional Midwives Association; MCSP, USAID Maternal and Child Survival Program; AAP, American Academy of Pediatrics.\n\nKey stakeholders and their roles and responsibilities are shown in Figure 1. The ASSIST team in collaboration with country partners played a key role in the development of the overall scope, approach, activity plan, and implementation of the STTA. Jhpiego MCSP, a collaborating partner, worked closely with the ASSIST team to coordinate approaches and activities in the region and share lessons and experiences, as well as documents and training materials for adaptation and use in STTA. AAP, the project sub-awardee, was represented by Global Health Program Officers (management team) and pediatric consultants (technical team) and was engaged to bring in the subject matter expertise, specifically, to deliver training curricula on essential care for every baby (ECEB), and developmental pediatrics within the context of Zika. CRMA, the project sub-awardee, is a regional organization with members throughout the Caribbean region and was represented by an executive team. CRMA supported the selection and recruitment of quality improvement (QI) coaches. USAID’s Zika Program guided the overall direction and scope of the STTA.\n\nNotes: The central part of the chart shows the country Ministry of Health as the principal technical assistance recipient and stakeholder and the USAID ASSIST Project as the leading provider and implementing partner of USAID. Key supporting implementing partners and other national stakeholders are arranged around the above two main stakeholders. Arrows reflect the relationship and communication flows between different stakeholders. Sidebars display the key roles each stakeholder played in the startup. USAID Guyana and CRMA are in the middle between the main stakeholders as regional liaisons and partners. MCSP, USAID Maternal and Child Survival Program; AAP, American Academy of Pediatrics; CRMA, Caribbean Regional Midwives Association; MOH, Ministry of Health; PAHO, Pan American Health Organization; CMO, Chief Medical Officer ; WI-HER LLC, Women Influencing Health, Education, and Rule of Law; NGO, non-governmental organization; ECD, early childhood development; ECEB, essential care for every baby; QI, quality improvement.\n\nThe startup consisted of multiple processes implemented in parallel in the four countries between May 11, 2018 (when the assignment was given to the USAID ASSIST Project) and September 3, 2018. In general, all activities fall under four main categories: (1) conception and initiation activities, (2) workplan and timeline development, (3) technical content development, and (4) operations. A detailed summary list of activities under each category is shown in Table 2.\n\nThe timeline of key activities is shown in Figure 2. In less than four months, the USAID ASSIST Project was able to achieve buy-in from all countries, develop a work plan with approval from the USAID and the four Ministries of Health, develop technical content and capacity building materials for the first TA visit, initiate agreements with three sub-awardees and event planners, establish facility-based improvement teams in each country, determine indicators to track improvements in service delivery, and finalize financial, organizational and logistical arrangements for both the scoping and the first technical assistance visits.\n\nAAP, American Academy of Pediatrics; CRMA, Caribbean Regional Midwives Association; MOH, Ministry of Health; SOW, Scope of Work; IID, improvement indicator database.\n\nIn this section, we describe and discuss some of the common themes and issues identified from interviews as related to key processes, challenges encountered, and any enabling and hindering factors.\n\nAchieving buy-in. Given the limited timeframe, it was critical to secure buy-in for USAID Zika expansion efforts from key stakeholders in the new ESC countries from the very beginning. Achieving buy-in involved: (1) describing the problem and providing a convincing rationale for why it needs to be addressed; (2) informing stakeholders of the availability of funds and technical expertise that can be leveraged by countries to co-develop and implement a solution; and (3) mapping out the project’s direct outputs/outcomes and extended benefits. Involving country stakeholders at each step was critical to achieving buy-in both at the decision making and implementation levels. However, there were several constraining factors when it came to engaging country stakeholders. First, ASSIST did not have a prior presence in these four countries. As explained by one respondent, there were \"reservations\" on the part of countries since they were not familiar with ASSIST or its work. Second, by the time USAID approached countries, the Zika epidemic and the heightened urgency around Zika was essentially over. Finally, the countries faced competing priorities such as dealing with the aftermath of devastating hurricanes that ravaged the region in 2017. In order to address the countries’ needs, the activity focused on detection and support of babies potentially affected by Zika through strengthening essential newborn and well-baby care systems and early childhood development programs.\n\nAccording to USAID and ASSIST respondents, initially, there were delays in establishing regular communications with the countries. Different “levels of engagement” were noted, with some country partners being more responsive compared to others. These challenges were mitigated through ASSIST’s continuous engagement with the USAID representative from the regional office, who facilitated direct contact with country stakeholders via email and phone. ASSIST also coordinated face-to-face meetings at a regional conference in Trinidad and during country scoping visits to ensure full commitment from country partners.\n\nFrom the countries’ perspectives, the demonstrated experience of the USAID ASSIST Project and the proposal laid out during the initial discussions were noted as influential in deciding to get on board with the activity. As one country senior Ministry of Health (MOH) officer described:\n\n“First of all, I found (USAID ASSIST) extremely knowledgeable. They have a wealth of experience and have done extensive work in quality all over the world. So, that for us was really important to know. Second, I think that they were quite sensitive to the specificities of all cultural nuances and …were quite responsive to suggestions we made and how to go forward. It was not just cordial… after a couple of weeks, we were talking like we've known each other for years. So, I think, that speaks to the commitment both parties had to this as well as the experience that USAID ASSIST has”.\n\n(senior MOH officer)\n\nAdditional contributing factor for accepting the USAID Zika STTA was the recognition of the need to have ongoing Zika surveillance and up-to-date national policy documents and guidelines for developmental surveillance, screening, referral and support of children potentially affected by Zika. Country pediatricians have noted that the existing policy documents and guidelines on Zika were developed during the height of the epidemic and did not reflect the new evidence. For example, in Antigua and Barbuda, the guidelines for infants exposed in utero to ZIKV were developed in 2016 with technical assistance from Pan American Health Organization (PAHO). This was useful for clinical providers in responding to the outbreak at the time; however, the knowledge and evidence about Zika has since expanded. Additionally, terminology, including the definition of congenital syndrome associated with Zika (CSaZ), was not reflected.Therefore, the USAID Zika technical assistance was viewed by leading pediatricians in these countries as a “fantastic” opportunity to update relevant policy documents with the help of ASSIST and AAP.\n\n\"First of all, we recognize what we should follow in terms of surveillance, that we need to ensure we have the same case definition, that we have national policies, and we recognize that there is a limitation in terms of human resources. We also recognize that there needs to be someone external, who is looking into the weaknesses within the system, and that's one of the reasons that we were happy to have technical assistance. This is how we could improve because we're good on paper, we have many things in place, but the system needed to be improved and (made) more robust.\"\n\n(Country team member)\n\nDefining STTA focus, scope and approach. Interviews with USAID respondents revealed that USAID designed a response framework around the four major lines of effort, each addressing a specific factor in preventing and controlling the spread of the Zika epidemic. The four lines of effort were: (1) vector control; (2) social and behavior change, communication, and community engagement; (3) service delivery; and (4) research, development, and innovation. Depending on the stage of the epidemic and the needs of the countries, implementing partners were guided by USAID to focus their efforts on one or more particular lines of effort to strategically align response activities amongst the various partners. Since ASSIST started its efforts in ESC countries, nearly two to three years after the outbreak had peaked, the country priorities shifted towards improving service delivery around identification of cases of CSaZ and other developmental malformations among infants and children exposed to ZIKV in utero and strengthening newborn and well-baby care systems to address other similar emergencies.\n\n“The disease epidemiology shifted from when the first outbreak started, …trying to minimize another outbreak …to care and support for those who have been affected. Now, we are …trying to answer the question about the transition from emergency response to long-term development.”\n\n(USAID Zika team member)\n\nTherefore, the focus at this stage of the response was to improve and strengthen national health systems to enhance national capacities to address public health emergencies. Based on responses from USAID and country participants, the following factors have played a role in shaping the focus, scope and approach of the STTA: (1) timing or the current phase of the Zika epidemic; (2) timeframe of the USAID Zika response contract; (3) financial and human resources; and (4) country-level factors (Figure 3).\n\nTA, technical assistance; CSaZ, congenital syndrome associated with Zika; STTA, short-term technical assistance; MCSP, USAID Maternal and Child Survival Program; AAP, American Academy of Pediatrics; CRMA, Caribbean Regional Midwives Association; PAHO, Pan American Health Organization; WI-HER LLC, Women Influencing Health, Education, and Rule of Law; URC, University Research Co., LLC; WPP, World Pediatric Project.\n\nAll MOH focal persons and relevant decision-makers were engaged in the process of co-developing the work plan and finalizing the technical and geographic scope of the work.\n\nTechnical partners with specific areas of expertise were contracted after the scope and the types of technical expertise that would be needed had been defined. However, some partners wished they were involved in initial discussions. According to technical partners, earlier engagement would have helped identify clear expectations, roles, and responsibilities; while giving the partners time to plan their part of work according to the country contexts. The time left after achieving country buy-ins and initial discussions with countries to define TA focus and scope was less than a month.\n\nPerspectives on the RMPP-MAPS. The approach was based on the ASSIST’s “model for improvement” and “adaptive management” strategies. These strategies emphasize identification of gaps and testing of local solutions to problems in real time, adjusting plans throughout the process, and implementing the results of regular monitoring of improvement interventions. Among the advantages of a rapid approach, respondents noted faster implementation, early achievement of results, expedited scaling and delivery of patient-favorable outcomes into the health services, and the efficient use of resources. Disadvantages included a possible loss of specificity and the potential for missing subtle details in the design during the early stages of response. Only a few respondents raised concerns about the possibility of diminished quality of response. Respondents emphasized that a continuous feedback mechanism and real-time adaptive management and learning strategies helped to address quality or other concerns promptly. While constant feedback and inputs from all partners were actively sought and incorporated at each step, the limited time frame made it impossible to wait until all components and specific details were in place or agreed upon by all stakeholders. Rather, the strategy adopted by ASSIST was to proceed with all necessary startup activities simultaneously to avoid delays while refining and/or making adjustments along the way as needed.\n\nThe only way to improve care is to change what is happening at facilities. So, our goal was to start facility-level improvement activities at the earliest point as possible. Knowing from previous experiences that planning is most effective when it is informed by implementation, we tried to start as fast as possible and use robust continuous feedback loops at every level to learn and adapt.”\n\n(ASSIST team member)\n\nScale up and engagement of local stakeholders. The small population and geographic size of the four countries facilitated the full scale of the improvement activity from the start. Except for the few community-based clinics in Barbuda and Dominica that were damaged by hurricanes, all facilities were included in the improvement intervention. To promote early adoption and institutionalization of changes and cross-sectoral collaboration ASSIST engaged health workers at all levels of the health system and encouraged the involvement of early childhood developmental programs under the Ministry of Education, and other community-based organizations (e.g. Roving Caregivers Program in Dominica). Both scaling up and institutionalization are essential components of ASSIST’s health service improvement model. These features of the STTA were noted by many country respondents as unique when compared to other technical assistance projects they had experience within their countries. According to country representatives, often in such projects, information \"stays at the top\" while \"people at the bottom\" are left poorly informed and tasks are \"thrown\" at them. Many pointed out that frontline and mid-level health workers (and, in some cases, community health aides and nursing assistants) had benefited greatly from training on clinical care of Zika-affected newborns and children. The science of QI empowered health workers to improve processes within the newborn and well-baby care system and equipped them with the skills and knowledge to apply improvement principles to other priority clinical-content areas.\n\nEnsuring the participation of all stakeholders was not without challenges. The health systems in three of the four countries (except St. Vincent and the Grenadines) were suffering from severe shortages of health care workers, particularly nurses. Many nurses and other health care workers had migrated out to other countries following recent hurricanes, exacerbating pre-existing shortages. Hence, some health facilities were concerned additional project-related responsibilities would overwhelm staff members. Due to this shortage of local health workers and heavy workload, country partners and ASSIST used retired nurse midwives who were already familiar with the health system. St. Vincent and the Grenadines was the only country that did not request external help to provide on-site coaching support to health facilities. According to a MOH officer, there was no shortage of nurses in the country. St. Vincent and the Grenadines relied on the existing nurse supervisors to take on coaching responsibilities. St. Vincent and the Grenadines did, however, experience the same challenge as the other countries when it came to \"pulling the staff\" for a week-long technical session while ensuring no interruption in health services.\n\nCountry teams and facilities had to shuffle various schedules and calendars around clinic days or public holidays to prevent disruption of health services and accommodate project activities.\n\n“Well, the most stressful part of it was getting the persons together…, that was kind of a tedious job getting into different entities. The time that we had in order to prepare for that first visit and the categories of individuals that they were requesting to meet with, the timeframe was a bit short in order to get all those persons together. Persons need prior notice in order to be released. We weren't giving them that prior notice. We are basically giving them “a meeting is tomorrow and I need X number of staff”. They don't have that time to prepare…”\n\n(Country team member)\n\nCountry team leaders noted that promoting a better understanding of the improvement activity, the benefits of adopting changes, and support from ASSIST helped to overcome the initial resistance. As one country focal point put it, they had to “sell the idea” to staff. A dedicated leadership team and local champions were crucial in creating a “positive competitive environment” to motivate increased participation and involvement. The result was increased interest and demand for participation in technical learning sessions observed from the second TA visit, with more health providers attending the workshops than initially planned.\n\nTechnical content development. Technical content development focused on two major areas: (1) the science of QI led by the ASSIST team; and ECEB training with a focus on ZIKV led by AAP. One of the challenges identified by respondents was the lack of technical experts with expertise in both ECEB and ZIKV. According to AAP, the pool of experts trained in ECEB was limited and finding available experts to work on developing the curriculum on “a very short turnaround time” presented significant challenges. Nevertheless, the AAP team was instrumental in “pulling together” their existing materials and information resources, as well as a team of consultants to develop high-level technical tools. In mitigating short timeframe challenges, AAP and other implementing partners emphasized the importance of early involvement of technical partners in the rapid startup. Additionally, providing technical consultants with as much background or context data on local health systems and related issues in advance, was highlighted as an essential best practice.\n\nOperations. Operations included functions related to defining administrative support (documentation, reporting, contracting, etc.), financial management (budgeting, accounting, payments, etc.), streamlining communications between multiple stakeholders, and logistical needs (procurement/supply, event organization, etc.). Given the lack of field offices and URC staff within the countries, administrative and financial management was centralized from ASSIST headquarters in Chevy Chase, MD, USA with country-specific adjustments made where necessary.\n\nLogistical activities were outsourced to external organizations. The main challenge was identifying reliable event planning or logistics services on the ground who have intimate knowledge of local markets, suppliers, distributors, or experience with procurement management and competitive purchasing processes. For example, no reputable event planning organizations meeting the minimum required qualifications were identified in St. Vincent and the Grenadines. A unique logistical challenge for St. Vincent and the Grenadines were the delays arranging transportation for the \"nurses from the Grenadines to come to the mainland\" (some of the Grenadines’ nine islands are small and remote). Therefore, ASSIST contracted two independent consultants to take on the role of event planners. The ASSIST team worked with MOH focal points in all four countries to help identify and provide recommendations on potential vendors and co-implement activity to help \"build local ownership and capacity.\"\n\nFinancial management followed USAID accounting procedures and standards according to the terms of the ASSIST cooperative agreement between USAID and URC. Planning and budgeting took place at the same time as workplan, staffing, and operational needs were being defined. Difficulties were reported in prompt costing of local activities.\n\nFigure 1 illustrates the flow of communication between all stakeholders and partners. The ASSIST team and the focal points from the country teams were the two parties in primary contact through whom all messages from other partners were communicated. This was established to ensure consistency of messages across multiple partners. According to ASSIST, the sub-awardees were not in direct communication with country stakeholders during the early start-up phase to facilitate focused conversations about buy-in or the technical scope. However, relevant updates and decisions were shared in a timely manner. Communication was open and regular, informing all relevant stakeholders, including local stakeholders and partners. There were also regular conference calls involving stakeholders from all islands. Emails to relevant stakeholders and partners were answered within 24 hours, and urgent issues were handled through telephone, Zoom calls, and WhatsApp.\n\nThe speed or pace of the startup. Participants reported that given the substantial amount of work in preparation for the first TA session in early September and very short timeframe, the startup had to be rapid and fast-paced, which had both advantages and disadvantages. The rapid startup galvanized stakeholders to brainstorm and make decisions quickly. However, the rapid speed may not have allowed enough time for detailed or thorough follow-up of some tasks (e.g., not all materials were printed out in time for the first technical session). When asked about how the pace of the startup compared to other projects, most respondents indicated that they are not aware of any other projects of the scale, depth, or the speed and pace that occurred with the USAID ASSIST Project. Both ASSIST team members and partners reported exerting a great level of effort to successfully and efficiently manage multiple tasks at the same time within a short timeframe.\n\n\nLessons learned and recommendations\n\nThis evaluation revealed that a rapid startup of complex service delivery technical assistance with activities in multiple countries without field offices was feasible. Although there were several unexpected implementation bottlenecks, by the end of the startup there was a collective realization among project teams and partners that what seemed at first a “mission impossible” was, in fact, possible, and had been accomplished. In addition, the evaluation proved that in the context of a short timeframe and urgent nature of the task, defining essential startup steps and processes early in the course of activity while at the same time identifying and accounting for specific contextual challenges is critical to avoid delays and facilitate smooth execution.\n\nAdditional lessons gleaned from respondents’ comments are summarized and listed below. While some of these lessons are not unique to the experience of the USAID ASSIST Project, they were distinctly reinforced by the course of startup and proved to be valuable for future startups.\n\nAt technical assistance/implementing partner level:\n\nTo achieve buy-in:\n\n◦ Maintain regular dialogue with senior country-level decision-makers to achieve prompt buy-in\n\nWhen developing response/designing TA:\n\n◦ Involve implementing partners and collaborators early\n\n◦ Establish efficient but flexible communication principles\n\nWhen setting up operations:\n\n◦ Learn as much as possible about host country context and determine optimal operational mechanisms, including:\n\n◾ Identify knowledgeable individuals and/or entities locally to take on or share logistical responsibilities\n\n◾ Determine context-appropriate contracting and financial management strategies\n\nAt the country/beneficiary level:\n\nWhen co-developing and co-implementing TA:\n\n◦ Conduct rapid assessment of the health system and service delivery needs jointly with key national stakeholders and subsequently with implementing partners\n\n◦ Co-develop interventions with a focus on sustaining the impact at scale, tailoring TA to country context, capacity development needs, and achieving local ownership of the activity\n\nWhen implementing TA:\n\n◦ Ensure activities are naturally integrated within the existing system, structures, and functions and not run or perceived by staff as a “parallel” task. Create a positive environment among health workers and avoid or mitigate resistance to change\n\n◦ Establish routine communication with key country stakeholders, partners, and donors to update them about progress, respond to their needs, promptly communicate challenges, and seek joint solutions\n\n◦ Identify and mitigate implementation barriers\n\n◾ Gather intelligence on logistical challenges from key local stakeholders\n\n◾ Identify and engage reliable or experienced local vendors early (before initial scoping visit) to support logistics of field activities, avoid delays, and ensure rapid start-up of TA activities\n\nBoth TA providers and beneficiary countries should aim to find an optimal balance between country needs and/or expectations and what technical partners and donors can offer, and to identify and acknowledge various limitations (programmatic content, financial, human resources, etc.) in advance. This will facilitate successful collaboration and ensure the achievement of project goals and outcomes.\n\nFinally, the presence of committed leadership, dedicated project teams, and local champions with the ability to collaborate on project strategy and adaptation was repeatedly emphasized as a critical process. The latter point consistently comes up as an essential factor in all projects ASSIST has implemented to date globally11.\n\n\nConclusions\n\nIn less than four months, the USAID ASSIST Project developed and carried out an innovative RMPP-MAPS to lay the groundwork for the implementation of a complex STTA activity in the four Caribbean countries. The RMPP-MAPS model proved to be a feasible and resource-efficient mechanism for delivering STTA within a short timeframe in middle-income countries facing numerous human, material, and financial resource challenges, including weak communication and health infrastructure. Startup activities were executed at full scale in all four countries. In doing so, the startup engaged health workers at all levels of the newborn and well-baby service delivery system, including front line providers, teachers, educators, and caregivers of ECD programs. Thus, promoting early institutionalization and long-term sustainability of the planned intervention.\n\nThe conventional process of developing and introducing into clinical practice evidence-based health service delivery and care changes lasts years, if not decades, due to the time it takes to generate the evidence through research, and efforts needed to close the “gap between knowledge and action”. The National Academy of Medicine’s concept of establishing a learning health care system (LHCS) as a “change machine” aims to overcome the above obstacles and expedite the process of uptake of evidence-based interventions into clinical practice12–14. The RMPP-MAPS is in line with the LCHS principle and a step towards achieving this goal.\n\nChallenges encountered in the startup stemmed from the tight timeline, the lack of in-country offices, and the shortage of healthcare staff in the ESC countries. Despite these challenges, the RMMP-MAPS model facilitated the successful implementation of all significant operational and technical aspects of the project while ensuring a timely launch of intervention within a rapid timeframe. Dedicated project teams, country leadership, local champions, and a strong sense of partnership and country ownership were identified as critical factors in facilitating a successful startup.\n\nThe RMPP-MAPS is a feasible, resource-efficient, and useful model to consider by implementers, donors, and low-and middle-income countries facing temporal and financial constraints when responding to public health emergencies.\n\n\nData availability\n\nIn order to encourage free and open sharing of opinions and perceptions, we assured stakeholders that their responses would be confidential. Since interview questions were shaped around a participant’s specific role and involvement level in the startup, their responses were correspondingly specific to their particular experiences and views. Many organizations were represented by no more than 1–2 respondents. Hence, interview files (transcripts and audio recordings) will contain potentially identifying information and thus, cannot be made openly available. Those interested in accessing this data may contact the corresponding author, at rmassoud@urc-chs.com. Requests will be considered on a case-by-case basis and must include contact information, type of data requested, and justification (e.g. proposal) for how the data will be used and for what purposes. Any information that could potentially identify respondents or organizations will be removed from the data shared.\n\nOpen Science Framework: Zika Startup Evaluation Manuscript Extended Data, https://doi.org/10.17605/OSF.IO/B7QGS10\n\nThis project contains the following extended data:\n\n- Short-Term Technical Assistance startup review documents\n\n- Semi-structured interview guides\n\n- Consent form\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWHO: WHO statement on the first meeting of the International Health Regulations (2005) (IHR 2005) Emergency Committee on Zika virus and observed increase in neurological disorders and neonatal malformations. 2016. Reference Source\n\nWHO: Fifth meeting of the Emergency Committee under the International Health Regulations (2005) regarding microcephaly, other neurological disorders and Zika virus. Reference Source\n\nWHO: Zika Situation Report. Reference Source\n\nLeta S, Beyene TJ, De Clercq EM, et al.: Global risk mapping for major diseases transmitted by Aedes aegypti and Aedes albopictus. Int J Infect Dis. 2018; 67: 25–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarrino CA, Minichiello AN, Amaya-Burns A, et al.: Performance evaluation of USAID’s Zika response in the Latin America and Caribbean (LAC) region. 2019. Reference Source\n\nUSAID Applying Science to Strengthen and Improve Systems Project. Eastern and Southern Caribbean FY19 Activity Plan. Submitted by the USAID ASSIST Project. Chevy Chase, MD, University Research Co., LLC (URC). 2018. Reference Source\n\nBatalden PB, Stoltz PK: A framework for the continual improvement of health care: Building and applying professional and improvement knowledge to test changes in daily work. Jt Comm J Qual Improv. 1993; 19(10): 424–447. PubMed Abstract \| Publisher Full Text\n\nMassoud MR, Kimble L: The USAID ASSIST Project’s approach to improving health care in low-middle-income countries. Technical Report. Published by the USAID ASSIST Project. Chevy Chase, MD: University Research Co., LLC (URC). 2018. Reference Source\n\nCrowe S, Cresswell K, Robertson A, et al.: The case study approach. BMC Med Res Methodol. 2011; 11: 100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAumakhan B, Massoud R, Coly A, et al.: Zika Startup Evaluation Manuscript Extended Data. 2020. http://www.doi.org/10.17605/OSF.IO/B7QGS\n\nMassoud MR, Kimble LE, Boguslavsky V, et al.: Managing hundreds of improvement teams [version 1; peer review: 2 approved]. F1000Research. 2018; 7: 1722. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIOM (Institute of Medicine): The learning health system and its innovation collaboratives: Update report. Washington, DC: The National Academies Press. 2011b.\n\nNAM (National Academy of Medicine): The learning health system series: Continuous improvement and innovation in health and health care. 2018. Reference Source\n\nMassoud MR, Barry D, Murphy A, et al.: How do we learn about improving health care: a call for a new epistemological paradigm. Int J Qual Health Care. 2016; 28(3): 420–424. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "62795", "date": "22 Jun 2020", "name": "Lisa Hirschhorn", "expertise": [ "Reviewer Expertise Implementation science", "QI", "MNCH." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 on a rapid approach to provide the start-up across 4 countries in the Eastern and Southern Caribbean to begin work to improve detection of and care for children affected by Zika virus. The work is embedded in the ASSIST project, which brings deep experience in establishing quality improvement work across a range of settings, expertise which is clearly reflected in the development of this rapid approach (RMPP-MAPS) to provide short term TA reflective of the timeline of overall funding.\n\nThe report is important in providing some important strategies for how to rapidly implement the key steps and operations needed to move rapidly, although this is relative to usual times for non-emergency interventions which would require even more rapid work (about 4 months). The strengths of the work include using an external evaluator, and the range of KIIs used. However the report could be significantly strengthened in a number of areas which need clarification.\n\nMethods: What, if any, frameworks were used for the qualitative coding (and KII guide development)? How were contextual factors captured and compared across the countries for similarities and differences which may have influenced implementation success of the start-up? This is described in a few areas (nurses coming from the Grenadines, coaches from existing resources versus retired nurses for example), but would be very helpful for others to be described and any association with variability of RMPP-MAPS implementation. In addition, how did ASSIST decide who to suggest for KIIs? Was there any chance of bias (should include in your limitations-which also typically go into discussion sections).\n\nResults and Discussion The scope of the evaluation is a bit confusing if this is just about start-up (as described in the results and abstract) or overall program implementation, which is described in the discussion. The authors should include information on how this approach was successful in rapid start and the outcomes of the STTA, or modify the language about the definitions of success.\n\nFor example, in the conclusions, I do not see the relevance of the decades from research to implementation, as this was more about implementing and strengthening established EBIs? Also some data on if the EBIs were indeed implemented would strengthen the paper. Particularly given the sentence in the preceding paragraph that the broad engagement of stakeholders resulted in “promoting early institutionalization and long-term sustainability”.\n\nFor the knowledge to be more transferable, more details of how in addition to what was done would be helpful particularly in getting leadership ownership, developing a culture for change and other areas to which ASSIST brought expertise. How did this expertise influence the success and what would another organization need to have (or not need to have) to be able to adopt the RMPP-MAPS. For example, highlighting that this was done without country offices is important for replication. What else? What partners should they choose?\n\nThe focus on engaging a broad range of stakeholders is admirable and important. A bit more detail in how the stakeholders were identified and if the calls involving stakeholders were with all of them or more specific groups (page 11).\n\nThe approach is described as \"resource-efficient”, but no data on cost is included. That would be important as this is indeed an important factors on which TA is not always evaluated.\n\nFinally, the report meets many of the Consolidated criteria for reporting qualitative research (COREQ) and with a small effort would help strengthen the paper.\n\nMinor: Page 8; The authors write the approach was based on the ASSIST “model for improvement” and “adaptive management”. This needs a reference.\n\nTypo: Experience top of page 10 should be experienced.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7664", "date": "27 Jan 2022", "name": "M. Rashad Massoud", "role": "Author Response F1000Research Advisory Board Member", "response": "The authors report on a rapid approach to provide the start-up across 4 countries in the Eastern and Southern Caribbean to begin work to improve detection of and care for children affected by Zika virus. The work is embedded in the ASSIST project, which brings deep experience in establishing quality improvement work across a range of settings, expertise which is clearly reflected in the development of this rapid approach (RMPP-MAPS) to provide short term TA reflective of the timeline of overall funding. The report is important in providing some important strategies for how to rapidly implement the key steps and operations needed to move rapidly, although this is relative to usual times for non-emergency interventions which would require even more rapid work (about 4 months). The strengths of the work include using an external evaluator, and the range of KIIs used. However the report could be significantly strengthened in a number of areas which need clarification. Methods: What, if any, frameworks were used for the qualitative coding (and KII guide development)? Response: The case study approach and associated evaluation or learning framework guided the development of KII tools. In particular, based on initial discussions with ASSIST program officers, the evaluation focus, objectives, main topics of interest were preliminarily defined followed by identifying all stakeholders and key informants involved at each step of the startup. The interview guide was then structured to have sections around identified topics with questions and probes to facilitate detailed responses as necessary to answer evaluation questions of interest. Thematic or pattern-based coding was applied to the data manually. How were contextual factors captured and compared across the countries for similarities and differences which may have influenced implementation success of the start-up? This is described in a few areas (nurses coming from the Grenadines, coaches from existing resources versus retired nurses for example), but would be very helpful for others to be described and any association with variability of RMPP-MAPS implementation. Response: Contextual similarities and differences were systematically identified and documented via scoping survey by the TA team during initial visits to countries. Specifically, similarities across countries in terms of socio-economic or health infrastructure development levels allowed the simultaneous and universal application of the approach. As relevant to the startup, the differences observed were more pronounced in the levels of availability of local human resources (e.g., shortage of nurses) or qualified vendors on the ground for outsourcing operational and logistical tasks. In addition, some differences were observed in the IT infrastructure readiness level, such as availability of computers, internet connectivity, or even availability of electricity in certain facilities due to differences in the destruction level caused by hurricanes that passed through the region the year before (e.g., Dominica’s health infrastructure was severely damaged compared to other countries). Therefore, appropriate specific adjustments were made to the startup planning and implementation to address any unique to specific country challenges. As indicated in the paper, to deal with these contextual differences, ASSIST integrated into its RMPP-MAPS real-time feedback mechanisms, joint brainstorming, and adaptive implementation to address challenges promptly and initiate facility-level QI activities in all four countries at the same time. In addition, how did ASSIST decide who to suggest for KIIs? Was there any chance of bias (should include in your limitations-which also typically go into discussion sections). Response: All individuals who were involved at any stage in the start-up were identified as KI, contacted and invited to participate in the study. The response rate was over 80%. Therefore, we believe the risk for respondent selection bias is minimal. Thank you and we included this information to the limitations which were also moved to the discussion section as suggested. Results and Discussion The scope of the evaluation is a bit confusing if this is just about start-up (as described in the results and abstract) or overall program implementation, which is described in the discussion. The authors should include information on how this approach was successful in rapid start and the outcomes of the STTA, or modify the language about the definitions of success. For example, in the conclusions, I do not see the relevance of the decades from research to implementation, as this was more about implementing and strengthening established EBIs? Also, some data on if the EBIs were indeed implemented would strengthen the paper. Particularly given the sentence in the preceding paragraph that the broad engagement of stakeholders resulted in “promoting early institutionalization and long-term sustainability”. Response: The scope of the evaluation was limited to startup only. However, since the success of the startup phase is inevitably linked to the success of the implementation phase, we tried to mention in some form the effect on the implementation phase or the STTA itself. Regarding EBI, we removed the corresponding part on “decades of research to implementation” to avoid any confusion. We modified the language in the manuscript as shown below: “The RMPP-MAPS model for startup facilitated a rapid launch of the implementation phase of STTA. The STTA was implemented in full scale in all four countries and engaged health workers, educators, and caregivers at all levels of the newborn and well-baby service delivery system promoting early institutionalization and long-term sustainability of the evidence-based interventions”. For the knowledge to be more transferable, more details of how in addition to what was done would be helpful particularly in getting leadership ownership, developing a culture for change and other areas to which ASSIST brought expertise. How did this expertise influence the success and what would another organization need to have (or not need to have) to be able to adopt the RMPP-MAPS? For example, highlighting that this was done without country offices is important for replication. What else? What partners should they choose? Response: Operating under the USAID “Journey to Self-Reliance” agenda, the ASSIST Project worked closely with government leaders to achieve locally sustained results and strengthen local capacities, with the end goal being that the work would fully transition to the government by the end of the project. This was reiterated in conversations over the life of the activity, and plans for sustainability and transition were discussed regularly. The combination of improvement expertise and upfront clarity on roles, responsibilities, and transition expectations facilitated teaming up with host country nationals and transferring competencies to them. The focus on engaging a broad range of stakeholders is admirable and important. A bit more detail in how the stakeholders were identified and if the calls involving stakeholders were with all of them or more specific groups (page 11). Response: Key contacts and stakeholders in the country and the region were identified in conjunction with USAID and then in conjunction with the key contacts in the country for other partners within the country. The approach is described as \"resource-efficient”, but no data on cost is included. That would be important as this is indeed an important factors on which TA is not always evaluated. Response: No specific cost analysis was done as part of this evaluation. The conclusion was based on the previous experience of ASSIST program leaders and TA implementers, other stakeholders, who had decades of experience in implementing similar health systems strengthening TA in multiple countries in different regions. Similar TA under normal circumstances typically involved setting up country offices, hiring local staff, external and internal consultants with startup activities taking much longer, often over a year incurring significantly higher costs before TA is ready to launch. Finally, the report meets many of the Consolidated criteria for reporting qualitative research (COREQ) and with a small effort would help strengthen the paper. Minor: Page 8; The authors write the approach was based on the ASSIST “model for improvement” and “adaptive management”. This needs a reference. Response: We added references. Typo: Experience top of page 10 should be experienced. Response: Corrected." } ] }, { "id": "99249", "date": "22 Nov 2021", "name": "Manoja Das", "expertise": [ "Reviewer Expertise Public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report presents the process and experience of the technical assistance to the four LAC countries for addressing the Zika challenge. The documentation and dissemination are important for lessons and potential application in the future.\nThe manuscript/article can be improved by considering the following points:\n1. Data collection and analysis 1.1. How were the KI IDI guides drafted/developed? It should be mentioned.\n\n1.2. Which method was used for qualitative data analysis? It should be mentioned.\n\n1.3. Was the data analysis done by only one person (B.A.)? Usually, qualitative analysis is done by more than one person.\n\n1.4. What was the degree of error with the use of the transcription software?\n\n2. Results 2.1. How did the status of preparedness, responses, perceptions, practices, and response components vary across these countries? These should have been mentioned.\n\n2.2. Triangulation with the record review/desk review has been mentioned in methods. The observations have not been mentioned clearly in the results section.\n2.3. Some narration about the results of the start-up activities should be given - whether the countries carried on the activities as planned or not.\n2.4. The conclusion mentions the TA as a resource-efficient model. Some information about the resource-efficiency should be mentioned.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7567", "date": "27 Jan 2022", "name": "M. Rashad Massoud", "role": "Author Response F1000Research Advisory Board Member", "response": "Response to Reviewer #2: The manuscript/article can be improved by considering the following points: 1. Data collection and analysis 1.1. How were the KI IDI guides drafted/developed? It should be mentioned. Author response: To draft KI interview guides initial list of topics of interest to be evaluated around the startup components was developed based on preliminary discussions with ASSIST program officers. These initial conversations helped define the focus and objectives of the evaluation, develop a comprehensive list of stakeholders and key informants involved at any stage of the startup, precisely define the startup period and outline specific processes or activities that took place within the given timeframe. Additionally, project documentation, including meeting minutes, presentations, trip reports, work plans, USAID and partners' program documents, etc., were reviewed to more precisely construct activities and identify additional topics of interest for inclusion in the interview guide. The interview guide was structured to have sections built around identified topics and startup components with specific questions and probes drafted to facilitate getting answers for evaluation objectives. Prepared draft tools were reviewed by ASSIST, and received feedback was used to finalize the tools. We have described the above development process in the manuscript, in brief, to keep the length of the manuscript within the recommended guidelines. 1.2. Which method was used for qualitative data analysis? It should be mentioned. Author response: The number of respondents was not large, under 30, but respondents varied widely by which stakeholders or countries they represented. Additionally, interviews were conducted in a free-flow conversation style which allowed a diverse range and at the same time often unique answers or specific issues raised by respondents. Therefore, to best capture the range, the grouping or coding of emerging common and unique response themes was done manually (i.e., no automated qualitative analysis software or tools were used). 1.3. Was the data analysis done by only one person (B.A.)? Usually, qualitative analysis is done by more than one person. Author response: Data analysis was done by B.A., who was not involved in the project and contracted to perform an independent evaluation of the activity and avoid potential bias if the evaluation was to be done by ASSIST people themselves. The evaluation was performed as part of the learning process of the project. It was not considered a research subject to IRB review, which would have, perhaps, required more rigorous data analysis or verification strategies. However, all collected data (audio records, transcripts of interviews) were submitted to ASSIST and independently verified by supervising program officer who was not involved in the startup activities for completeness, data integrity, and record-keeping purposes according to program rules. 1.4. What was the degree of error with the use of the transcription software? Author response: The accuracy of the transcription software varied depending on the respondent’s English speaking style and accent (native English speakers in the US vs. English of respondents from Caribbean countries). Also, technology factors such as whether the interview was via phone, or Zoom platform, in-person, recorded sound quality, presence of background noise, etc., played a role. On average, the accuracy was between 80 and over 90%. 2. Results 2.1. How did the status of preparedness, responses, perceptions, practices, and response components vary across these countries? These should have been mentioned. Author response: The four countries were similar in terms of economic development level or health infrastructure state. As relevant to the startup, most differences were observed in the levels of availability or the shortage of local health care providers, particularly nurses; availability of qualified local vendors for outsourcing operational and logistical tasks; as well as relatively poor technological infrastructure in facilities, in terms of availability of computers, internet connectivity, even electricity in some cases. The latter was partly due to the differing severity of damages caused by hurricanes that passed through the region the prior year. Hence, priorities or promptness in responsiveness varied somewhat between countries but consistent follow-up and identifying bottlenecks in a timely manner helped the team address the challenges and needs without significant delay and allow simultaneous initiation of the technical phase of the TA. 2.2. Triangulation with the record review/desk review has been mentioned in methods. The observations have not been mentioned clearly in the results section. Author response: Because KI typically were few in numbers representing different stakeholders and each played a unique role in the project, no one informant could provide a comprehensive picture “from the start to the end.” Therefore, triangulation or tracing and comparing data obtained from multiple sources (preliminary discussions, in-depth interviews, project documents, etc.) was done to corroborate information, construct a cohesive and accurate picture of the startup, deduce the timeline and sequence of events, or elucidate enabling and limiting factors in order to reach evaluation objectives. 2.3. Some narration about the results of the start-up activities should be given - whether the countries carried on the activities as planned or not. Author response: The start-up activities were implemented in all four countries as described. 2.4. The conclusion mentions the TA as a resource-efficient model. Some information about the resource-efficiency should be mentioned. Author response: No specific cost analysis was done as part of this evaluation. The conclusion was based on the previous experience of ASSIST program leaders and TA implementers, other stakeholders, who had decades of experience in implementing similar health systems strengthening TA in multiple countries in different regions. Similar TA under normal circumstances typically involved setting up country offices, hiring local staff, external and internal consultants with startup activities taking much longer, often over a year incurring significantly higher costs before TA is ready to launch." }, { "c_id": "7665", "date": "27 Jan 2022", "name": "M. Rashad Massoud", "role": "Author Response F1000Research Advisory Board Member", "response": "The manuscript/article can be improved by considering the following points: 1. Data collection and analysis 1.1. How were the KI IDI guides drafted/developed? It should be mentioned. Response: To draft KI interview guides, an initial list of topics of interest to be evaluated around the startup components was developed based on preliminary discussions with ASSIST program officers. These initial conversations helped define the focus and objectives of the evaluation, develop a comprehensive list of stakeholders and key informants involved at any stage of the startup, precisely define the startup period and outline specific processes or activities that took place within the given timeframe. Additionally, project documentation, including meeting minutes, presentations, trip reports, work plans, USAID and partners' program documents, etc., were reviewed to more precisely construct activities and identify additional topics of interest for inclusion in the interview guide. The interview guide was structured to have sections built around identified topics and startup components with specific questions and probes drafted to facilitate getting answers for evaluation objectives. Prepared draft tools were reviewed by ASSIST, and received feedback was used to finalize the tools. We added the above additional details to the revised version. 1.2. Which method was used for qualitative data analysis? It should be mentioned. Response: The number of respondents was not large, under 30, but respondents varied widely by which stakeholders or countries they represented. Additionally, interviews were conducted in a free-flow conversation style which allowed a diverse range and at the same time often unique answers or specific issues raised by respondents. Therefore, to best capture the range, the grouping or coding of emerging common and unique response themes was done manually (i.e., no automated qualitative analysis software or tools were used). 1.3. Was the data analysis done by only one person (B.A.)? Usually, qualitative analysis is done by more than one person. Response: Data analysis was done by B.A., who was not involved in the project and contracted to perform an independent evaluation of the activity and avoid potential bias if the evaluation was to be done by ASSIST people themselves. The evaluation was performed as part of the learning process of the project. It was not considered a research subject to IRB review which would have, perhaps, required more rigorous data analysis or verification strategies. However, all collected data (audio records, transcripts of interviews) were submitted to ASSIST and independently verified by supervising program officer who was not involved in the startup activities for completeness, data integrity, and record-keeping purposes according to program rules. 1.4. What was the degree of error with the use of the transcription software? Response: The accuracy of the transcription software varied depending on the respondent’s English-speaking style and accent (native English speakers in the US vs. English of respondents from Caribbean countries). Also, technology factors, such as whether the interview was via phone, or Zoom platform, in-person, recorded sound quality, presence of background noise, etc., played a role. On average, the accuracy was between 80 and over 90%. We added this information to the manuscript. 2. Results 2.1. How did the status of preparedness, responses, perceptions, practices, and response components vary across these countries? These should have been mentioned. Response: The four countries were similar in terms of economic development level or health infrastructure state. As relevant to the startup, most differences were observed in the levels of availability or the shortage of local health care providers, particularly nurses, availability of qualified local vendors for outsourcing operational and logistical tasks, as well as relatively poor technological infrastructure in facilities in terms of availability of computers, internet connectivity, even electricity in some cases. The latter was partly due to the differing severity of damages caused by hurricanes that passed through the region the prior year. Hence, priorities or promptness in responsiveness varied somewhat between countries but consistent follow-up and identifying bottlenecks in a timely manner helped the team address the challenges and needs without significant delay and allow simultaneous initiation of the technical phase of the TA. 2.2. Triangulation with the record review/desk review has been mentioned in methods. The observations have not been mentioned clearly in the results section. Response: Because KI typically were few in numbers representing different stakeholders and each played a unique role in the project, no one informant could provide a comprehensive picture “from the start to the end”. Therefore, triangulation or tracing and comparing data obtained from multiple sources (preliminary discussions, in-depth interviews, project documents, etc.) was done to corroborate information, construct a cohesive and accurate picture of the startup, deduce the timeline and sequence of events, or elucidate enabling and limiting factors in order to reach evaluation objectives. 2.3. Some narration about the results of the start-up activities should be given - whether the countries carried on the activities as planned or not. Response: Results of the start-up phase The start-up activities in all four countries were conducted simultaneously except for the country visits, which were conducted sequentially by the start-up team visiting one country after the other. All four countries committed to taking on the work and to allocating the human resources needed for it. In the start-up discussions, differences between the resource availability in different countries became clear. For example, some countries had shortages in staff required to act as coaches, while others did not. These were factored into the workplan through arrangements with the Caribbean Regional Nurses Association (CRNA). By the end of the start-up phase all four countries embarked on implementing the workplan. The above paragraph was added to the article to address this point 2.4. The conclusion mentions the TA as a resource-efficient model. Some information about the resource-efficiency should be mentioned. Response: No specific cost analysis was done as part of this evaluation. The conclusion was based on the previous experience of ASSIST program leaders and TA implementers, other stakeholders, who had decades of experience in implementing similar health systems strengthening TA in multiple countries in different regions. Similar TA under normal circumstances typically involved setting up country offices, hiring local staff, and external and internal consultants, with startup activities taking much longer, often over a year, incurring significantly higher costs before TA is ready to launch." } ] } ]	1	https://f1000research.com/articles/9-251
https://f1000research.com/articles/11-235/v1	25 Feb 22	{ "type": "Case Study", "title": "Using social annotation to construct knowledge with others: A case study across undergraduate courses", "authors": [ "Esteban Morales", "Jeremiah H. Kalir", "Alice Fleerackers", "Juan Pablo Alperin", "Jeremiah H. Kalir", "Alice Fleerackers" ], "abstract": "Background: Social annotation (SA) is a genre of learning technology that enables the addition of digital notes to shared texts and affords contextualized peer-to-peer online discussion. A small body of literature examines how SA, as asynchronous online discussion, can contribute to students’ knowledge construction (KC)—or a process whereby learners collaborate through shared socio-cognitive practices. This case study analyzed how SA enabled student participation in seven KC activities, such as interpretation and elaboration. Methods: We analyzed 2,121 annotations written by 59 students in three undergraduate courses at a Canadian University in the Winter 2019 semester. Using a method of open coding and constant comparison, we coded each annotation for evidence of KC activities. Results: Results showed a range of KC activities in students’ SA. Across courses, interpretation was the most common KC activity (40%), followed by elaboration (20%). Annotations that were part of peer-to-peer discussion included all seven types of KC activities, but some activities, such as consensus building, support, and conflict, were almost exclusively found in replies to others. Conclusions: This study suggests that SA is a productive form of online learning through which undergraduate students in multiple disciplinary contexts can interact with peers, make sense of academic content, and construct knowledge by reading and writing together.", "keywords": [ "social annotation", "knowledge construction", "computer-supported collaborative learning", "case study" ], "content": "Introduction\n\nOnline conversation—as with chat rooms or discussion forums—is a ubiquitous practice with societal implications (Paulus and Wise 2019). In educational contexts, online discussion emerged alongside popular online bulletin boards in the mid-1990s, and written digital discourse is now an essential feature of contemporary educational technologies, such as learning management systems (LMS), discussion boards, and blogs (Weller 2020). Online discussion allows students to converse about topics pertinent to course content (Loncar, Barrett, and Liu 2014), is often asynchronous (Sheail 2018), and encourages learners’ participation at their own pace. Asynchronous online discussion is a tenet of social learning in digital environments (Hill, Song, and West 2009) with research indicating such discourse enables social and collaborative learning (Chan and Pow 2020; Hambacher, Ginn, and Slater 2018; McMahon 1997).\n\nThe growth of asynchronous online discussion in digital education presents both opportunities and challenges. For learners, the benefits of online discussion include social knowledge construction (KC) (Eryilmaz et al. 2013; Kent, Laslo, and Rafaeli 2016), meaningful dialogue with information shared and negotiated (Gao, Zhang, and Franklin 2013; Wise, Hausknecht, and Zhao 2014), collaboration with peers (Pratt and Back 2013), and reflection (Truhlar, Walter, and Williams 2018). Online discussion can improve student learning processes and outcomes (Hambacher, Ginn, and Slater 2018; Kent, Laslo, and Rafaeli 2016). Alternatively, online discussion has been associated with low levels of student participation (Aloni and Harrington 2018), instructor bias (Baker et al. 2018), and non-substantive learner interaction (Hambacher, Ginn, and Slater 2018), perhaps due to the imposition of discussion structure and order (Gao, Zhang, and Franklin 2013). Indeed, the “dreaded threaded” discussion has been a trope of unsatisfying and unproductive online learning for at least two decades (Chabon, Cain, and Lee-Wilkerson 2001). The respective benefits and challenges of online discussion motivates additional research on how text-based, asynchronous online discourse can productively promote learners’ interaction, collaboration, and reflection in digital learning environments.\n\nGiven the ubiquity and timeliness of asynchronous online discussion, particularly in higher education (Bettinger et al. 2017), our study examines undergraduate student participation in social annotation (SA) as a form of online discussion. Specifically, we studied how peer-to-peer dialogue via SA contributed to KC activities in multiple courses from different disciplines. We first reviewed literature about SA and KC, highlighting the relevance of this relationship to online discussion and learning. We then present a case study of KC activities and patterns in 2,121 annotations written by students from three undergraduate courses at a Canadian university. We present findings about: a) The discursive, or peer-to-peer, characteristics of student SA; b) the prevalence of KC activities evidenced in student SA; and c) patterns of KC activities in student SA, including a comparison among courses. Our discussion considers the strengths and limitations of this study, the social qualities and value of student participation in SA, and implications for the use of SA as asynchronous online discussion.\n\n\nSA, collaboration, and KC\n\nIn this study, we embrace a sociocultural stance toward learning (Gutiérrez and Rogoff 2003; John-Steiner and Holdbrook 1996). We extend computer-supported collaborative learning (CSCL) research that considers cognition a socially-situated, group accomplishment (Enyedy and Hoadley 2006; Stahl 2017). We recognize communication, whether spoken or written, as central to the social construction of reality (McMahon 1997); consequently, social and discursive activities like thinking aloud, asking questions, and providing explanation promote meaningful learning (King 2007). We understand collaboration as a “social contract” (Dillenbourg 1999) reflecting shared situations and interactions, as well as orchestrated participation in online learning (i.e., Chen et al. 2018). Our orientation to cognition, communication, and collaboration positions us to study how SA as online discussion can encourage engagement in structured dialogue and shared epistemic practices (Kalir 2020a; Eryilmaz et al. 2013).\n\nAnnotation—or the addition of notes to texts (Kalir and Garcia 2021)—has, for centuries, informed how people read, write, and interact with texts and other readers (Jackson 2001). Today, the proliferation of digital annotation tools (Wolfe and Neuwirth 2001), particularly in education as with SA technology (i.e., Paradis and Fendt 2016; Seatter 2019; Zhu et al. 2020), has enabled readers to annotate online documents using text, links, and multimedia while engaging in dialogue. SA affords contextualized discussion as peer-to-peer dialogue is “anchored” to a source text (Gao, Zhang, and Franklin 2013), in contrast to conventional online discussion forums, which are distal from learners’ texts. Anchored online discussion helps learners acquire discipline-specific terminology and methods (Kararo and McCartney 2019), collaborate with peers (Chan and Pow 2020), and engage in public discourse (Kalir and Garcia 2019; Marshall and Brush 2004). SA is an alternative approach to online discussion forums as anchored dialogue enables learners to engage in proximal, meaningful conversation with texts and peers (Plevinski, Weible, and Deschryver 2017).\n\nIn higher education—the context of our study—a growing body of research indicates that SA promotes productive online discussion and student learning. Novak et al. (2012) reviewed SA use across seven higher education disciplines and found that student reading comprehension, peer review, motivation, and attitudes toward technology use were all positively influenced by SA activities. A more recent systematic review, conducted by Zhu and colleagues (2020), highlighted how SA can help students process domain-specific knowledge, support argumentation and inquiry, improve literacy skills, and can aid instructor and peer assessment. As online discussion, SA promotes critical thinking via peer-to-peer dialogue (Mendenhall and Johnson 2010), builds collaborative sensemaking (Chen 2019), and can offer students social, linguistic, and cultural learning opportunities (i.e., Brown and Croft 2020; Thoms and Poole 2017). Because SA affords dialogic, collaborative learning in digital learning environments (i.e., Allred, Hochstetler, and Goering 2020; Sprouse 2018; Wranovix and Isbell 2020), it is appropriate to further investigate how SA—as asynchronous online discussion—supports peer-to-peer activity like KC.\n\nWith roots in cognitive psychology and constructivism, KC is defined in the CSCL literature as a social process “by which students solve problems and construct understanding of concepts, phenomena, and situations” (van Aalst 2009, 261). In online learning, KC emphasizes the social processes whereby divergence of ideas lead to the convergence of negotiated meanings (Onrubia and Engel 2009). KC differs conceptually and processually from the transmission of information (knowledge sharing) or the innovative use of ideas and tools (knowledge creation). KC relies on peer-to-peer dialogue as an instrument for learning (Pena-Shaff and Nicholls 2004), group participation in shared problem-solving environments and opportunities (Hmelo-Silver and Chernobilsky 2004) and concerns how different perspectives are assimilated among groups and incorporated into individual thinking and metacognition (Yu and Wu 2016; Luo and Clifton 2017). KC activities—such as learners’ collaborative engagement in elaboration, argumentation, question-asking, and explanation (i.e., Fu, van Aalst, and Chan 2016)—are understood as situated, reflexive, and related to deep learning (De Wever et al. 2009; van Aalst 2009).\n\nNot every online interaction among students leads to KC or learning. Nonetheless, CSCL research has shown that learners’ technology-mediated, dialogic interaction (i.e., Enyedy and Hoadley 2006; Hambacher et al. 2018) can lead to the meaningful co-construction of knowledge (Heo, Lim, and Kim 2010) and collaborative learning (Eryilmaz et al. 2013). KC activities are frequently associated with student digital dialogue as such conversation has the potential to “increase the level of participation and interaction among students and … has the capacity to provide a meaningful supplement to regular class discussions” (Pena-Shaff and Nicholls 2004, 264). Online discussion presents an ideal scenario within which to research KC as students’ interaction patterns may be easily orchestrated (Hmelo-Silver and Chernobilsky 2004), accessed (Schrire 2006), and analyzed to differentiate among cognitive tasks and accomplishments (Luo and Clifton 2017). For example, when a group of students discuss project management, they may engage in socio-cognitive KC processes like questioning, summarizing, and elaborating (Onrubia and Engel 2009). Whereas most studies of student KC in the CSCL literature examine activity in more conventional discussion forums, SA may also productively promote KC activities through anchored discourse that encourages collaborative and meaningful learner dialogue (van der Pol, Admiraal, and Simons 2006).\n\nThere are but a handful of studies that examine how SA, as online discussion, can enable KC activities. Eryilmaz et al. (2013) describe how SA reduced coordination activities among learners, which consequently lead to greater individual learning gains and increased some group KC activities like elaboration and conflict. Plevinski, Weible, and Deschryver (2017) found that SA can support multiple KC activities, primarily learner elaboration and interpretation, determining that “coordination activities were relatively minimal and that [anchored annotation systems] supported KC activities closely aligned with the cognitive processes of remembering and understanding” (117). Zarzour and Sellami (2017) concluded that SA effectively encouraged learner engagement with multiple perspectives, with students “gaining ideas, seeing others’ different viewpoints, linking more external data, and building knowledge about the annotated content” (394). Kalir (2020b) detailed how SA discussion supported a repertoire of group-level epistemic expressions including critical inquiry, associative connections across contexts, and discernment among multiple perspectives.\n\nOur study is motivated by opportunities for further scholarly inquiry at the intersection of KC and SA. There is little discussion in the literature about how different SA practices lead to specific KC activities and patterns of students’ social interaction. There is also a need to further understand how KC promoted by SA may vary across instructional settings and disciplinary contexts. Our case study therefore focuses on social practices afforded by SA as online discussion and analyzes how SA enables student participation in KC activities. We do so by studying student SA in three undergraduate courses at a Canadian university. Specifically, our study addresses three research questions (RQs):\n\n(1) How does student SA and the prevalence of discursive threads differ across courses from multiple disciplines?\n\n(2) What specific KC activities are most frequently observed within student SA, and how do KC activities differ across courses?\n\n(3) What patterns of KC activities are most frequently observed within students’ discursive SA, and how do patterns of discursive KC activities differ across courses?\n\n\nMethods\n\nThis study was conducted using data collected for the assessment of students as part of their regular academic work and, as such, was exempt from ethics board review according to Article 2.5 of the Canadian Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans.\n\nThis research employed a single-case study design (Yin 2009) at a Canadian university throughout the winter 2019 semester. Seven faculty from multiple disciplines at the undergraduate and graduate levels were invited to integrate SA as online discussion into their courses using Hypothesis (Hypothes.is, RRID:SCR_000430) (https://web.hypothes.is/), an open-source technology that enables SA and which has been widely studied in higher education (e.g., Chen 2019; Goller et al. 2021; Sprouse 2018; Seatter 2019). Faculty were provided with technical and pedagogical support by the research team.\n\nAll seven courses used SA for asynchronous online discussion in some capacity. Three were selected for this study: Publishing Studies (PUB); Gerontology (GERO); and Gender, Sexuality and Women’s Studies (GSWS). These courses were selected using the following inclusion criteria to assure the quality and consistency of data: 1) Similar course-level (upper-level undergraduate); 2) High student engagement with SA as evidenced by number of annotations; and 3) Close contact between the instructor of each course and the research team.\n\nAs detailed in our related research about student perceptions of SA (Kalir et al. 2020), these three courses differed in terms of instructor pedagogy, enrolled students, and the use of SA for online discussion. PUB organized class sessions into two components—a lecture followed by face-to-face discussion—and required that students make at least two annotations on each reading before class to receive a 15% participation grade. GERO sessions typically started with student face-to-face discussion and then transitioned to a traditional lecture. SA represented 15% of students’ final grades and was assessed using a rubric that included engagement with peers and annotation consistency, quantity, and originality. GSWS sessions relied on a combination of lectures and seminars; participation accounted for 20% of students’ final grade and included SA. Table 1 summarizes student engagement with SA in the three courses.\n\nA total of 59 students from PUB, GERO, and GSWS authored 2,121 annotations during online discussion (instructors’ 24 annotations were removed from analysis). When the term ended, SA was collected using the Hypothesis API to provide detail of annotation data and metadata. This enabled our team to describe basic characteristics of student SA—including descriptive statistics of total annotations and replies, discursive threads per course, and SA mean length—as well as to analyze student annotation content for evidence of KC activities.\n\nThe content of student SA was analyzed using open coding and constant comparison (Strauss and Corbin 1990), with all 2,121 annotations coded for evidence of KC. A single annotation could be coded as exhibiting more than one KC activity. To do so, we adapted the codebook developed by Plevinski, Weible and Deschryver (2017) which draws on relevant CSCL literature to identify seven KC activities: Clarification, conflict, consensus building, elaboration, interpretation, question, and support. Definitions and examples for these codes are provided in Table 2 (Morales, Fleerackers, Alperin 2022).\n\nNumbers in square brackets (e.g. [SA100]) represent anonymized identification for each annotation.\n\nTo assess the reliability of Plevinski et al.’ (2017) codebook in the context of our multi-course analysis, two authors (EM and AF) independently coded a set of student annotations from all three courses and compared results. The authors’ coding was measured and achieved a Kappa value of 0.86, which is considered to be a strong level of rater agreement (Mchugh 2012). EM subsequently coded the remaining annotations for evidence of KC activities. The coding process used NVivo version 12 (NVivo, RRID:SCR_014802) (http://www.qsrinternational.com/nvivo-product) (freely available alternative software: Taguette, https://www.taguette.org/). Annotations that lacked evidence of KC activity were often associated with coordination, such as “This relates to what I highlighted earlier in the paper” [631], or with informal social interactions, like “Hello world!” [739].\n\n\nResults\n\nOur study examined the use of SA for online discussion in three different undergraduate courses and features three complementary sets of findings. To address RQ1, we report characteristics associated with student SA by categorizing the types of annotation written by students and identifying the prevalence of discursive—or peer-to-peer—threads composed via SA. In response to RQ2, we present quantitative summaries of KC activities evidenced in student SA. A third set of findings addressing RQ3 details the patterns of KC activities most frequently observed within students’ SA and identifies how patterns of KC activities within discursive threads differed across courses.\n\nWe first report three types of annotation written by students to identify the basic discursive characteristics of SA for online discussion. This first set of findings distinguishes individual annotations from those that were part of discursive threads. A thread is an instance in which one students’ annotation elicited at least one reply from a peer (Figure 1). As reported in Table 3, all student SAs were categorized as: a) an individual annotation that did not appear in a thread and elicited no peer response, henceforth referred to as no-thread; b) an annotation that elicited at least one peer response, referred to as top-of-thread; and c) an annotation reply that developed discursive interaction among peers. In reporting our findings, it is important to recall that even no-thread SA had social qualities; this category of annotation was written for group-level participation in each course and was visible to peers throughout online discussion activities.\n\nSA, social annotation.\n\nAcross all three courses, 76% (1,619 annotations) of student SA received no peer response and were not part of any threaded discussion (no-thread). A total of 24% (502) of all SA appeared in a thread, with 10% (202) of annotations as top-of-thread and 14% (300) as a reply. In GERO, nearly half (46%) of student SA appeared in threads, including 135 top-of-thread annotations and 212 replies. In GSWS, just over one third (36%) of student SA appeared in threads, with 37 top-of-thread annotations and 56 replies. And in PUB, only 6% of student SA were in threads, with 30 top-of-thread annotations and 32 replies.\n\nWhether or not student SA appeared in a discursive thread, we found that 71% (1,511) of all annotations included a single KC activity, 18% (380) included two KC activities, and 5% (105) included three or more KC activities. A total of 6% of all annotations (125) did not include evidence of any KC activity. Across courses, the most common KC activity was interpretation: 40% of all annotations (1,051) included interpretation in the form of an inference, conclusion, or summary of the text. Examples of interpretation included a GERO student who wrote “[i] t seems that this is a big problem with categorizing but is it possible to avoid ‘othering’? to me, it seems we can not avoid it in research because that is how we can compare and contrast findings to present conclusions” [SA725], as well as a GSWS student who observed, “I think this is a really great point for illustrating how media coverage isn’t just about representation, but about a lack of representation” [SA050].\n\nAs summarized in Figure 2 (Morales, Fleerackers, Alperin 2022), elaboration was the second most common KC activity featured in all student SA, appearing in 20% of all annotations (532). For example, a student in PUB wrote “[t] his is similar to what we see with the Internet and the use of tools like Facebook or Twitter or blogging. The Internet is fairly accessible to a large percentage of the world where anyone can post anything they want to” [SA917]. Across our study sample, other KC activities present in students’ SA included clarification (13% of all annotations), asking a question (12%), consensus building (8%) and providing support (6%). Conflict was the least common KC activity across courses and occurred in less than 1% of annotations, indicating that students may have avoided contrasting points of views when writing SA for online discussion.\n\nKC, knowledge construction.\n\nThe distribution of KC activities within online discussion further demonstrated the prevalence of interpretation and elaboration in student SA. As indicated in Figure 3, interpretation accounted for 58% of the KC activities within PUB and over one third of KC activities in GERO and GSWS. Elaboration occurred in 26% of the KC activities of GERO and GSWS, and 24% of KC activities in PUB. The prevalence of other KC activities differed among the three courses. Students in GERO, for example, more frequently included consensus building in their SA during online discussion (“I agree, this question is very subjective but I wonder if that is the point for this study” [SA293]), whereas few annotations from PUB included consensus building or support. Results further suggest that the KC activities of asking a question and providing clarification appeared in students’ online discussion with about the same frequency across all three courses.\n\nKC, knowledge construction; PUB, Publishing Studies; GERO, Gerontology; GSWS, Gender, Sexuality and Women’s Studies.\n\nOur third set of findings detail patterns of KC activities within discursive, or peer-to-peer, SA and includes a comparison of differences among courses. To address RQ3, we first recall that over three-quarters of student SA in this study (76%) received no peer response and were not part of threaded discussion (no-thread annotation). Figure 4 illustrates the distribution and prevalence of seven KC activities (as detailed in RQ2) by SA category (as described in RQ1).\n\nKC, knowledge construction; SA, social annotation.\n\nAcross courses, four KC activities were predominantly concentrated in annotations that were not part of a thread: Interpretation (with 79% of the 1,051 annotations evidencing interpretation found in no-thread annotations); elaboration (76% of 532 annotations); clarification (89% of 338 annotations); and asking a question (70% of 320 annotations). Among these four most prominent KC activities, less than one-third of student SA that included a question was discursive, only about one-quarter evidencing interpretation or elaboration was discursive, and just one in ten annotations that provided clarification were discursive. In PUB, for example, in which 94% of student SAs were not in threads, prominent KC activities were interpretation (52% of 1,056 no-thread annotations), elaboration (21%), clarification (15%), and asking a question (12%). In GERO, the course with the lowest percentage of no-thread annotation (54%), prominent KC activities within this category included interpretation (36% of 402 no-thread annotations), elaboration (26%), and clarification (25%). Similarly, in GSWS—in which approximately two-thirds of student SA were no-thread—the most frequent KC activities in this subset were interpretation (40% of 161 no-thread annotations) and elaboration (29%).\n\nStudents’ KC activities also occurred in the 24% of SA that were discursive and located within threads as either top-of-thread or a reply. Four of the same five KC activities found among no-thread annotation were also identified, across all three courses, and with approximately the same frequency, in annotations that began threads: Interpretation, in 8% of 1,051 annotations evidencing interpretation; elaboration (11% of 532 annotations); clarification (9% of 338 annotations); and asking a question (14% of 320 annotations).\n\nAs previously shown in Figure 4, all seven KC activities were found in students’ replies to peers. Three KC activities were almost exclusively discursive, appearing only in student replies: consensus building (with 100% of the 198 annotations coded for consensus occurring in replies); support (99% of 148 annotations); and conflict (100% of 18 annotations). Some KC activities were infrequently evident in replies, such as clarification (2% of 338 annotations).\n\nA further analysis of peer-to-peer online discussion in GERO, GSWS, and PUB reveals course-level patterns of KC activity in students’ discursive SA. Table 4 reports the percentage of annotations that contained evidence of each KC activity among annotations in threads—either as top-of-thread or as a reply—for each of the three courses. In both GERO and GSWS, prominent KC activities that appeared in top-of-thread SA included interpretation and elaboration. Interpretation was also the most common KC activity among top-of-thread SA in PUB. Across courses, interpretation, consensus building, and support were the three KC activities that more frequently appeared in replies. Overall, the three courses showed relatively similar patterns of KC activities among SA threads.\n\nTwo examples illustrate course-level patterns of KC present in students’ discursive SA, specifically interpretation and elaboration appearing atop threads followed by consensus building and additional interpretation in replies. One GERO student’s top-of-thread annotation (coded for both interpretation and elaboration) noted: “I was surprised that so many people disagree with improving the quality of life for immigrant seniors. The commenters are ‘othering’ and blaming the immigrants for not adapting to ‘Canadian culture.’ It is a sad reality that people believe this, especially since Canada was colonized by immigrants” [SA624]. A peer’s reply included consensus building and interpretation: “Canadian culture is very diverse - agreed! I feel this is also the reason that everyone needs to compromise to come up with a sustainable solution for all because it’s near impossible to cater to all the specific/individualized needs of such a diverse society” [SA587]. In PUB, one top-of-thread annotation that demonstrated interpretation stated: “Mentors and role models for diversity are so important in the workplace. When people trying to break into the publishing world see others succeeding that they can identify with, they may be inspired and more confident to chase after their goals” [SA274]. In response, a peer annotation featured consensus building and interpretation: “I agree. To add to your point, connections are so important in the job market now. To get into an industry, it seems like you need to know someone. If the industry is dominated by white people, and those people only are connected to other white people, then it would be hard for people of other backgrounds to get a foot in the door” [SA171].\n\n\nDiscussion\n\nThis descriptive study of three undergraduate courses from different disciplines at one university examined how student participation in asynchronous online discussion via SA contributed to KC activities. Our study builds on research about online discussion in higher education (i.e., Chen et al. 2018; Sun and Gao 2017) and extends insight from CSCL literature regarding the role of social learning technologies to enable learner cognition, communication, and collaboration (i.e., Chan and Pow 2020; King 2007). From this stance, SA—as a popular approach to online discussion (Allred, Hochstetler, and Goering 2020; Zhu et al. 2020)—was studied because it allowed students to add interactive notes to shared digital texts, anchor discussion in meaningful social contexts (Gao, Zhang, and Franklin 2013), and make their thinking visible and responsive to peers (Kalir and Garcia 2019; Marshall and Brush 2004). It has been nearly a decade since Novak et al. (2012) encouraged investigation about the promises and limitations of SA across varied higher education learning environments and among diverse groups of learners. While subsequent research has documented SA as productively mediating collaborative dialogue and learning (Brown and Croft 2020; Wranovix and Isbell 2020), only a small subset of CSCL literature details the intersection of SA practices and KC activities. In this discussion we: Address the contributions of this exploratory case study, with attention to the strengths and limitations of our research design and context; consider the discursive qualities and social value of student SA as participation in KC activities; and present methodological and instructional implications for SA as asynchronous online discussion.\n\nThe design of our study included multiple features that strengthened the relevance of this case for researchers interested in online discussion and CSCL constructs like KC. First, we expanded on a small but important literature that examines KC activities as enabled by SA (Eryilmaz et al. 2013; Plevinski et al. 2017; Zarzour and Sellami 2017). Whereas previous studies examined student KC within a single discipline, our case is the first instance to document undergraduate students’ KC via SA across three disciplines and does so with an SA corpus larger than that of prior studies. Second, we studied KC activities made visible by the SA technology Hypothesis, which strengthens our study by making use of a SA technology that has been both widely studied and widely adopted by global educational institutions (https://web.hypothes.is/blog/our-view-from-20-million-annotations/). Third, our approach to data analysis borrowed from Plevinski et al.’ (2017) codebook to deductively identify seven KC activities in student SA. We hope our findings further establish these particular activities and definitions as the future benchmark when investigating SA for evidence of KC. A fourth strength of this case is its comprehensive account of students’ collaborative learning as aided by SA. Having previously reported how this sample of students perceived SA as a valuable contribution to their learning (Kalir et al. 2020), we can now pair prior insight with these findings about student participation in SA for online discussion. As research suggests asynchronous online discussion can be unsatisfying and unproductive (i.e., Aloni and Harrington 2018), our two studies jointly indicate that students find SA satisfying and can use it as a productive means to construct knowledge.\n\nThere are two limitations of our study design that other researchers of SA and collaborative learning should work to mitigate. Both concern the extent to which we were able to comprehensively document students’ discursive activities in context, a methodological challenge noted in the CSCL literature that reflects complex social, cultural, and cognitive qualities of group discourse as situated across meaning-making contexts (i.e., Arvaja 2011). First, despite frequent coordination with participating faculty we had limited access to on-the-ground and online learning environments. Accordingly, we were unable to document via direct observation how SA was introduced to students as an approach to online discussion, nor were we able to observe how SA activities were orchestrated in coordination with other synchronous course activities (i.e., Zhu, Shui, and Chen 2020). A second limitation concerned unanticipated technical challenges that constrained our ability to document with nuance the online discursive context within which KC activities occurred. While Hypothesis SA has been extensively studied across various open and group-based online learning arrangements (i.e., Allred, Hochstetler, and Goering 2020; Kalir 2020a; Goller et al. 2021), the use of this technology within a university LMS is a recent development. Unexpected difficulties in mapping annotations to readings, caused by time-bound URLs given to students, curtailed our efforts to trace how KC activities progressed through a given text. We recommend future SA studies of Hypothesis establish technical workflows to contextualize discussion at multiple scales (i.e., thread, text, course), and examine threads as a unit of analysis to better understand the social sense-making processes of students as KC activities occur over time (i.e., Eryilmaz et al. 2013).\n\nIn light of our study’s strengths and limitations, this case described undergraduate student participation in asynchronous, group-based online discussion by detailing the extent to which their SA was discursive, and by analyzing the presence and prevalence of KC activities in SA. Notably, only a quarter (24%) of student SA across all three courses was discursive, although this varied substantially between courses. Threads accounted for almost half the SA written in GERO, over a third of the SA in GSWS, and less than one-tenth of the SA in PUB (annotations in PUB comprised more than half the total corpus, see Table 3). Nonetheless, online discussion did evidence all seven KC activities when students’ SA was discursive, albeit to differing degrees. Across the annotation corpus, interpretation was by far the most common KC activity (Figure 2), a finding consistent with previous research (Eryilmaz et al. 2013; Plevinski et al. 2017). Yet unlike Eryilmaz et al.’ (2013) analysis of KC sequences within SA threads, we found that interpretation seldom elicited peer response (Figure 4).\n\nOur exploratory study is pertinent to long-standing interest in the “social life” of texts (Brown and Duguid 1996) and the value of collaborative annotation (i.e., van der Pol, Admiraal, and Simons 2006) during asynchronous online discussion. Moreover, our findings about KC activities may be useful for researchers interested in the cognitive and social qualities of student annotation. For example, with respect to Gao et al.’ (2013) model of productive online discussion, we found student participation in SA primarily demonstrated discussion for comprehension (as evidenced by the KC activities of interpretation and elaboration) as well as discussion for improved understanding. Yet our analysis of the distribution of KC activities by course and category (Figure 3) revealed that students less frequently utilized SA to critique or actively negotiate meanings, reconsider assertions, or revise their thinking. KC activities analogous to engagement with diverse perspectives—like elaboration, questioning, consensus building, and conflict—were identified within discursive SA though in varying degrees (Table 4). We found noteworthy course-level variation in how students wrote and shared annotation as a participatory social process through which divergent ideas were subsequently negotiated and synthesized (i.e., Onrubia and Engel 2009). In this respect, we speculate that discursive SA productively mediated the ongoing and social negotiation of meaning-making in GERO, perhaps also in GSWS, though probably not in PUB where only 6% of course SA were discursive. SA can, in some circumstances, make visible complex group-level cognitive processes (like conflict and consensus building) through online discourse. Moreover, our prior research indicated that students perceived social value in reading and writing annotation to clarify confusion, confirm ideas, and engage diverse perspectives (Kalir et al. 2020). Nonetheless, further research should clarify the processual ways in which discursive SA aids student groups in sharing conflicting ideas and synthesizing among divergent perspectives.\n\nNotably, student participation in SA may also reflect course-specific factors, including instructor expectations about online discussion. In PUB—the largest course in our sample—students were required to write at least two annotations per reading, perhaps explaining why every student in the class annotated and wrote annotations of the greatest length (Table 1). However, the PUB assessment rubric did not emphasize peer interaction, which may explain, in part, both the overwhelming quantity of no-thread SA (94% of the course annotation) and interpretation as that course’s prominent KC activity (Figure 3). In GERO, alternatively, an assessment rubric incentivized discursive SA. Students in this course were evaluated, partly, on “engaging with other students (responding to others’ comments).” It may not be surprising that GERO featured the highest percentage of top-of-thread SA and peer replies among courses. This may also explain other characteristics of GERO annotations, such as the relatively high prevalence of consensus building, and the fact that less frequent KC activities—like consensus building, support, and conflict—comprised over one-third of course SA (the highest percentage of such KC activities in the study). Across all courses, instructors approached their involvement in SA from a “more is not always better” (Zhu et al. 2020, 267) stance, collectively writing just 1% of the corpus. Given difference and similarity among instructional contexts and practices, future SA research—and, in particular, design-oriented rather than descriptive studies—should carefully consider how to scaffold students’ writing of SA so that it is both discursive and encouraging of particular KC activities.\n\nThis study provides further evidence that SA is a productive form of online learning enabling students’ collaborative KC. As such, we conclude our discussion by noting methodological and instructional implications that should be useful for other researchers and educators interested in SA. First, with only a handful of SA studies examining KC activity, we made an intentional decision to borrow Plevinski et al.’ (2017) codebook rather than create a bespoke analytic scheme. We encourage other SA researchers to take up this common method when studying KC in online learning with larger samples of students, more courses, and other disciplines. Second, future studies might also combine thread-level analysis of student annotation with additional data sources like educator interviews, student focus groups, and direct course observations (as with the methods in Schneider et al. 2016) to better contextualize how SA enables KC progressions within the broader social life of a course. Third, a range of instructional opportunities are also possible if instructors can, in real-time, be made aware of students’ SA use, emerging participation patterns, and the ongoing development of KC activities. The design of complementary technologies, such as learning analytics dashboards that dynamically report student SA (Kalir 2020a), could be attuned to KC patterns so that instructors might better support group-level discourse across course texts and orchestrate sequential collaborative activities. Aiding instructor knowledge of student SA through efficient feedback processes would also help inform the ways in which instructors participate in online discussion to clarify student misunderstanding and build connections to relevant disciplinary literature and methods. New instructional methods could also encourage students to write SA evidencing a wider range of KC activities. Our final instructional recommendation is that instructors model and transparently assess how SA enables productive, discipline-specific online discussion in accordance with course learning objectives (Zhu, Shui, and Chen 2020).\n\n\nConclusions\n\nThis study examined the affordances of SA as asynchronous online discussion and analyzed how SA enabled students’ KC activities. Our case provides further evidence that SA, as mediated by the Hypothesis technology, is a productive form of online discussion through which undergraduate students in multiple disciplinary contexts interacted with peers (i.e., Kalir et al. 2020), made sense of academic content (i.e., Kararo and McCartney 2019), and constructed knowledge by reading and writing together (i.e., Sprouse 2018). As the first descriptive cross-disciplinary account of students using Hypothesis SA to construct knowledge together, this case details the ways in which SA made cognition visible and collaborative activity possible (i.e., Kalir 2020a; Chan and Pow 2020). Undergraduate student SA as online discussion was a socio-cognitive context within which we identified the predominance of textual interpretation, a supporting set of KC activities that included elaboration, clarification, and asking questions, as well as a less frequent group of KC activities that included consensus building, support, and conflict. Given long-standing interest in SA and learning (i.e., Novak et al. 2012) and recent changes in digital education, this study offers a valuable and timely contribution to the literature. While our broader study was designed and conducted prior to the coronavirus disease 2019 (COVID-19) pandemic, we analyzed data for this case and wrote this article throughout the disrupted 2020-21 academic year. Amid the pandemic, hybrid and online courses became the primary mode of learning in higher education. Changes to digital learning arrangements exacerbated by the pandemic may, in part, benefit from light-touch and highly-collaborative learning technologies—like SA—that promote online discussion and aid student KC about discipline-specific concepts, methods, and content. In this respect, our study extends an established line of inquiry about the educational benefits of SA, provides practical and relevant insight about SA as asynchronous online discussion, and can help shape future research about the social practices and cognitive qualities of collaborative learning in higher education.\n\n\nData availability\n\nDataverse: Knowledge Construction Activities in the Online Social Annotations of Three Classes, https://doi.org/10.7910/DVN/SVDFCF (Morales, Fleerackers, Alperin 2022).\n\nThis project contains the following underlying data:\n\n- AnnotationsCoded_Course.tab (raw coded knowledge construction activities)\n\n- Codebook.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nvan Aalst J: Distinguishing Knowledge-Sharing, Knowledge-Construction, and Knowledge-Creation Discourses. Int. J. Comput.-Support. Collab. 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Educ. 2004; 42(3): 243–265. Publisher Full Text\n\nPlevinski J, Weible J, Deschryver M: Anchored Annotations to Support Collaborative Knowledge Construction Introduction. Making a Difference: Prioritizing Equity and Access in CSCL, 12th International Conference on Computer Supported Collaborative Learning (CSCL) 2017. 2017; no. 2013: 111–18.\n\nvan der Pol, J, Admiraal W, Simons PRJ: The Affordance of Anchored Discussion for the Collaborative Processing of Academic Texts. Int. J. Comput.-Support. Collab. Learn. 2006; 1(3): 339–357. Publisher Full Text\n\nPratt N, Back J: Using Communities of Practice as a Tool to Analyse Developing Identity in Online Discussion. Learn. Media Technol. 2013; 38(3): 284–300. Publisher Full Text\n\nSchneider E, Hartman S, Eshel A, et al.: Making Reading Visible: Social Annotation with Lacuna in the Humanities Classroom. The Journal of Interactive Technology and Pedagogy. 2016; (9). Reference Source\n\nSchrire S: Knowledge Building in Asynchronous Discussion Groups: Going beyond Quantitative Analysis. Comp. Educ. 2006; 46(1): 49–70. Publisher Full Text\n\nSeatter L: Towards Open Annotation: Examples and Experiments. KULA: Knowledge Creation, Dissemination, and Preservation Studies. 2019; 3(1): 1–10. Publisher Full Text\n\nSheail P: The Digital University and the Shifting Time–Space of the Campus. Learn. Media Technol. 2018; 43(1): 56–69. Publisher Full Text\n\nSprouse ML: Social Annotation and Layered Readings in Composition. 2018 Computers & Writing Conference. 2018; 39–52.\n\nStahl G: Group Practices: A New Way of Viewing CSCL. Int. J. Comput.-Support. Collab. Learn. 2017; 12(1): 113–126. Publisher Full Text\n\nStrauss A, Corbin J: Basics of Qualitative Research. Basics of Qualitative Research 2nd Edition. 1990. Publisher Full Text\n\nSun Y, Gao F: Comparing the Use of a Social Annotation Tool and a Threaded Discussion Forum to Support Online Discussions. Internet High. Educ. 2017; 32: 72–79. Publisher Full Text\n\nThoms JJ, Poole F: Investigating Linguistic, Literary, and Social Affordances of L2 Collaborative Reading. Lang. Learn. Technol. 2017; 21(2): 139–156.\n\nTruhlar AM, Walter MT, Williams KM: Student Engagement with Course Content and Peers in Synchronous Online Discussions. Online Learn. J. 2018; 22(4): 289–312. Publisher Full Text\n\nWeller M: History of Educational Technology – Editorial. J. Interact. Media Educ. 2020; 2020(1): 1–2. Publisher Full Text\n\nWever B, Van Keer H, Schellens T, et al.: Structuring Asynchronous Discussion Groups: The Impact of Role Assignment and Self-Assessment on Students’ Levels of Knowledge Construction through Social Negotiation. J. Comput. Assist. Learn. 2009; 25(2): 177–188. Publisher Full Text\n\nWise AF, Hausknecht SN, Zhao Y: Attending to Others’ Posts in Asynchronous Discussions: Learners’ Online ‘Listening’ and Its Relationship to Speaking. Int. J. Comput.-Support. Collab. Learn. 2014; 9(2): 185–209. Publisher Full Text\n\nWolfe JL, Neuwirth CM: From the Margins to the Center: The Future of Annotation. J. Bus. Tech. Commun. 2001; 15(3): 333–371. Publisher Full Text\n\nWranovix M, Isbell M: The Digital Common Read: Creating a Space for Authentic Engagement with Social Annotation. Journal of the European Honors Council. 2020; 4(1): 1–10. Publisher Full Text\n\nYin R: Case Study Research and Applications: Design and Methods. SAGE Publications; 2009.\n\nYu FY, Wu CP: The Effects of an Online Student-Constructed Test Strategy on Knowledge Construction. Comp. Educ. 2016; 94: 89–101. Publisher Full Text\n\nZarzour H, Sellami M: A Linked Data-Based Collaborative Annotation System for Increasing Learning Achievements. Educ. Technol. Res. Dev. 2017; 65(2): 381–397. Publisher Full Text\n\nZhu X, Chen B, Avadhanam RM, et al.: Reading and Connecting: Using Social Annotation in Online Classes. Inf. Learn. Sci. 2020a; 121(5): 261–271. Publisher Full Text\n\nZhu X, Shui H, Chen B: A Scaffolding Framework for Social Annotation in Online Classes.2020b. Publisher Full Text" }	[ { "id": "125500", "date": "17 Mar 2022", "name": "Emily J. Ragan", "expertise": [ "Reviewer Expertise Science education", "biochemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMorales et al. explore knowledge construction and its intersection with social annotation, where students collaboratively annotate a text. They analyzed a full term worth of student annotations from three different upper division undergraduate courses, comparing the number of annotations that stood alone (no-thread) to those that became part of a thread (with one or more student(s) responding to an initial post). They also coded student annotations of course readings based on seven established types of knowledge construction and compared patterns across the three courses. Then, combining the first two analyses, they looked at how knowledge construction activities related to no-thread posts or discourses (a thread) between students. Student survey data on student perceptions of social annotation from same three courses has been shared in a previous paper (Kalir et al. 2020).\nThe percent of annotations that became threads varied between courses, from 6% to 46%. A vast majority of annotations (94%) were found to be related to at least one of the seven knowledge construction categories. The most common categories across all three courses were interpretation and elaboration. Certain types of knowledge construction were much more likely to occur in responses to threads: consensus building, support, and conflict. This paper offers a strategy for cataloging the knowledge construction activities within social annotation, which may be helpful for faculty seeking to better understand how their students are using social annotation. It also offers a way faculty could measure potential impacts of changes to assignment instructions and/or assessment rubrics on student knowledge construction activities as documented through social annotation.\nIntroduction\nI recommend the authors acknowledge that knowledge creation can be individual as well as social. In the section “Knowledge construction” they state: With roots in cognitive psychology and constructivism, KC is defined in the CSCL literature as a social process \"by which students solve problems and construct understanding of concepts, phenomena, and situations\" (van Aalst 2009, 261). However, I note that van Aalst’s next sentence is “It is effortful, situated, and reflective, and can be individual or social (Sullivan Palincsar 1998).” I think it is a mistake to imply that all knowledge construction is inherently social. Social annotation allows individual effort and reflection to be made visible to the group, thus blurring the boundary between individual and social, but the fact that individuals construct knowledge cannot be ignored.\nA brief introduction of coordination activities in the section “SA enabling KC” would be helpful. I also personally would prefer to have social annotation and knowledge construction written out, avoiding SA and KC abbreviations.\nMethods\nThe format that I assume is indicating a specific annotation in the last sentence of the Methods, [631] and [739], is slightly different that that used in other portions of the paper, where the number in square brackets is preceded by SA.\nNo statistical analyses were performed. While I believe that is reasonable for the scope of this descriptive project, future work on this topic would be strengthened by greater statistical rigor.\nDiscussion\nThere is a sentence in the discussion that I found unclear. “Unexpected difficulties in mapping annotations to readings, caused by time-bound URLs given to students, curtailed our efforts to trace how KC activities progressed through a given text.” I assume that the Hypothesis API, which provided detail about annotations, lacked information relating threads to specific readings. Clearly stating what you would have liked to do (compare knowledge creation activities across different texts, or within different sections of the same text, etc.) would help the reader better understand this specific limitation. More detail in the Methods section about the annotation data and metadata available from the Hypothesis API may also be helpful.\nData availability\nThe data after coding into knowledge construction categories is available but lacks the original annotation text.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [ { "c_id": "8009", "date": "01 Apr 2022", "name": "Juan Pablo Alperin", "role": "Author Response", "response": "Thank you for taking the time to provide us with this thoughtful review. We are uploading a revised manuscript that addresses these comments in addition to this response. Introduction Dr. Ragan helpful suggests that we \"acknowledge that knowledge creation can be individual as well as social\". We very much agree with this suggestion and appreciate the opportunity to better clarify KC (in general) and the particular stance toward KC that we are extending in our paper. We have revised the \"Knowledge Construction\" subsection to better reflect that KC is both an individual and social phenomena, and that we are highlighting the social qualities and affordances of KC within our study (and related CSCL literature). The new text can be found in the first few sentences of this section. Dr. Ragan also suggests that we add a \"brief introduction of coordination activities in the section 'SA enabling KC'\". We have now edited the opening paragraph of this section to do this, including the addition of a new second sentence in this section. Finally, in the introduction, Dr. Ragan suggests we avoid using the SA and KC abbreviations. We discussed this, but feel that the terms too frequent in the manuscript and that writing them out would only encumber the reader. We see this as a matter of style and have opted to retain the abbreviations. Methods We have now standardized the way in which we identify participants by adding \"SA\" inside the square brackets. Thank you for catching this discrepancy. Discussion Dr. Ragan noted a sentence that was unclear. We agree that it was so and have re-written the sentence. The new sentence can be found near the end of the third paragraph of the Discussion. Data Availability Both reviewers noted that we did not publish the text of the annotation with our open dataset. This was intentional, as we did not feel like we could sufficiently anonymize the texts to guarantee the privacy of the students and the instructors." } ] }, { "id": "125503", "date": "21 Mar 2022", "name": "Federico Pianzola", "expertise": [ "Reviewer Expertise digital social reading", "computational literary studies" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMorales et al. provide a robust investigation of social annotation in the context of higher education by looking at 3 different undergraduate courses from 3 different disciplines. The large number of annotations analysed suggests that the results may be valid in many other educational contexts. The contextualisation of the research within an existing scholarly tradition is very accurate and the choice of using an established protocol (codebook) for qualitative research is excellent for the comparability of the results. The discussion and interpretation are appropriate and rightfully acknowledge limitations that can occur in field studies and prevented the researchers to gain more detailed insight.\nAn important issue that is not clear from the article is whether informed consent has been obtained from the students. Moreover, it is not possible to fully reproduce the results of the study because the full text of the annotations is missing.\nA few smaller remarks that could improve the presentation of the research:\nGiven the inconsistencies between academic calendars in various countries and the differences between Northern and Southern hemisphere, it would be better to express the time range of the data collection in number of weeks and months of the year. Now it is only stated: winter 2019 semester.\n\nIn place of Table 4, Figure 3 could be modified by using stacked bars to show the proportion of annotations for each course within each bar (e.g. 3 different shades of orange for no-thread)\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [ { "c_id": "8010", "date": "01 Apr 2022", "name": "Juan Pablo Alperin", "role": "Author Response", "response": "Thank you for taking the time to provide us with this review. We have now uploaded a revised manuscript that addresses many of your comments and provide a response to all here. In his review, Dr. Pianzola expressed concern regarding whether informed consent was obtained from the students. We take research ethics seriously and sincerely appreciate his concern. The short answer is that we did not obtain consent because the data were collected for the assessment of students as part of their regular course work. We then followed the Canadian Tri-Council Policy Statement (as is required at Canadian Universities) and affirm our understanding that our study is exempt from ethics board review according to Article 2.5 of this policy. A statement to this effect appears at the beginning of the Methods section. Dr. Pianzola also noted that different academic calendars, especially between the Northern and Southern hemispheres, would not be clear of when \"Winter Semester\" has taken hold. Again, we deeply appreciate this small, but significant suggestion. This was an oversight on our part, as we do attempt to avoid centering the Global North in so much of our other work. We have now changed this in the abstract and in the text to indicate it was between January and April 2019. Finally, Dr. Pianzola suggests that the results presented in Table 4 could be incorporated into Figure 3. We have not done this for two reasons: 1) we believe figures can be less effective if they present too much information and become confusing or overwhelming for readers; but more importantly, 2) it would be difficult to read from the stacked-bar graph suggested the information being highlighted by Table 4." } ] } ]	1	https://f1000research.com/articles/11-235
https://f1000research.com/articles/11-387/v1	01 Apr 22	{ "type": "Research Article", "title": "What energy infrastructure will be needed by 2050 in the EU to support 1.5°C scenarios?", "authors": [ "Igor Arduin", "Christopher Andrey", "Tobias Bossmann", "Christopher Andrey", "Tobias Bossmann" ], "abstract": "Background: The European Commission has settled ambitious objectives in order to reach climate neutrality by 2050. This will imply the shift from fossil fuel to low carbon energy supply and an adaptation of the energy system according to it. Electrification and production of green hydrogen are seen as structural pillars. The objective of the study is to quantify energy infrastructure needs required in various climate neutral scenarios at the 2050 time horizon. Methods: The work was based on Artelys Crystal Super Grid, a tool developed at Artelys for modelling and simulating energy markets on a continental or national scale. In this study, we apply a multi-energy (i.e., power, hydrogen, methane) capacity expansion and dispatch optimisation methodology, featuring hourly and national granularity, covering the European Union plus major neighbouring countries. Several investments options are considered: storage assets, electrolysers, cross-border electricity, hydrogen and CH4 interconnections (including repurposing of CH4 infrastructures), and gas-to-power capacities. Results: Important needs for cross-border electricity infrastructure appear in all the considered scenarios. Cross-border hydrogen infrastructure needs strongly depend on the geographic allocation of renewable energy sources across Europe. Security of supply in Europe can be maintained without investing in additional cross-border methane pipelines. Existing methane pipelines will be repurposed or characterized by low utilisation rates at the 2050 horizon. Conclusions: The multi-energy optimization approach developed is well suited to assess electricity, methane and hydrogen infrastructure projects and their interdependencies considering various scenarios. While electricity and methane infrastructure needs are quite robust across several sensitivities on a climate neutral scenario, hydrogen infrastructure needs are more uncertain and depend on various factors such as the level of hydrogen demand, its competition with biomethane and the level of colocation between RES generation and hydrogen demand.", "keywords": [ "infrastructure", "networks", "2050", "decarbonisation", "methane", "repurposing", "EU", "multi-energy", "renewables", "power-to-gas" ], "content": "Introduction\n\nThe European Green Deal sets out the European Commission’s ambitions in tackling climate and environmental-related challenges. The Green Deal targets a 55% reduction in greenhouse gases emissions at the 2030 horizon compared to 1990 levels, and aims at achieving climate neutrality by 2050.\n\nReaching these targets will require colossal efforts in energy efficiency to reduce the energy demand, in the deployment of decarbonised energy sources, and in infrastructure to enable dynamic interlinkages between sectors and energy vectors.\n\nThe Energy System Integration Strategy and Hydrogen Strategy that have been presented by the European Commission during the summer 2020 aim at transforming the currently still siloed design of the European energy sector into a much more integrated system, allowing electrons and molecules to play a complementary role. One of the technologies that interlinks the electricity and gas systems is electrolysis, i.e. the conversion of electricity into hydrogen, estimated to reach up to 500 GW by 2050 in the policy scenarios of the European Commission1 in order to meet the demand for decarbonised gases and fuels.\n\nThe European Commission has performed multiple modelling exercises aiming at designing transition pathways, based on different technological options and behavioural assumptions. The Long-Term Strategy, published in November 2018, analyses the role of different technological options. In particular, the so-called 1.5TECH scenario reaches carbon neutrality at the 2050 horizon and foresees a dramatic increase in Renewable Energy Sources (RES) generation capacities compared to current levels, to allow for direct and indirect electrification.\n\nThe important deployment of variable renewable energy sources, the interlinkage of methane, hydrogen, electricity and heat, and the flexibility services that can be offered by end-uses will need to be supported by the right type of infrastructure (grids, pipeline, storage), to facilitate full integration and minimize costs. Yet, several infrastructure configurations can be considered (e.g., electrolysis located close to RES generation or close to hydrogen consumption centres, repurposing of existing methane infrastructure to make it compatible with 100% hydrogen, etc.), leading to vastly different investment needs. In all cases, a system-wide, integrated and forward-looking approach is required to identify interdependencies and synergies between vectors and sectors, and provide insights into the optimal level of energy infrastructure to support a 1.5°C-compatible economy.\n\n\nObjective of the analysis\n\nThis analysis applies a multi-energy modelling framework to evaluate the needs for infrastructure in a 2050 1.5°C-compatible scenario. It builds upon framework assumptions from the European Commission’s 1.5TECH scenario as well as on variations of this scenario based on the Paris Agreement Compatible (PAC) scenarios2 and 1.5LIFE3 scenario of the EU’s Long-Term Strategy (LTS). Many hypotheses are characterised by an important level of uncertainty (e.g., cost of repurposing pipelines, economic case for hydrogen distribution networks, cost of electrolysers, etc.). Therefore, we have focused the analysis on four high-level questions:\n\n1. Cross-border methane – Is there a need to reinforce the European methane infrastructure beyond its current level? Due to the change of the structure of gas flows that can be expected at the 2050 horizon, are there pipelines with very low utilisation rates?\n\n2. Cross-border electricity – How important is the need for cross-border electricity interconnectors, considering the impacts of electrolysers and their geographical allocation?\n\n3. Cross-border hydrogen – Is there a need for cross-border transport of hydrogen? If yes, could part of the existing methane infrastructure be repurposed?\n\n4. Robustness – How does the need for infrastructure depend on key assumptions, and how does the (non-)colocation of renewables and hydrogen demand affects infrastructure needs?\n\nIn order to provide insights into these questions, we perform a modelling exercise where we jointly optimise the capacity of hydrogen, methane and electricity infrastructure and their use, for a given 1.5°C-compatible scenario at the 2050 horizon, using the Artelys Crystal Super Grid software. We assess the robustness of the conclusions to several key assumptions by performing sensitivity analyses with respect to hydrogen demand levels, bio-methane supply, and wider use of direct electrification to supply low-temperature heat.\n\nThis study does not aim at identifying the precise set of infrastructure projects that should be built at the 2050-time horizon, but rather to identify key lessons that can be learned from this exercise, and to translate them into policy recommendations.\n\nThis study significantly extends the state-of-the-art of multi-energy simulations. As far as the authors are aware, this exercise is the first one to explore the needs for energy infrastructure with a joint electricity, hydrogen and methane model that maintains an hourly time resolution over the entire year. The use of an hourly time resolution is of primary importance to ensure one captures the impacts of the variability of RES that can heavily impact the operational management of electrolysers and hence the deployment of electrolysers, their location and the infrastructure to transport energy (via electrons or molecules).\n\n\nMethods\n\nThe modelling exercise carried out uses the multi-energy system modelling platform Artelys Crystal Super Grid (ACSG). A detailed description of the underlying mathematical system, incl. objective functions, constraints and equations describing asset behaviours used is openly available for download from the METIS website here. METIS was likewise developed by Artelys and relies on the same methodological principles. The approach we apply could be reproduced with an open optimisation model such as PyPSA-Eur-Sec.\n\nIn this analysis, a joint model of the European electricity, hydrogen and methane systems was used.The model allows for a joint optimisation of investments and operations (with cost minimisation as its objective function) for a given year using an hourly time resolution and a national granularity. All generic data sourced from the METIS website and the data specific to the present analysis is available in Underlying data1.\n\nThe model is able to simultaneously optimise the operations of and investments in all categories of assets, including different generation technologies, flexible consumption technologies, storage assets, cross-border interconnections and pipelines between areas. The model allows for global constraints to be introduced, such as CO2 budget or maximum output by a given technology over the year (e.g., maximum running hours of coal assets may be constrained).\n\nThe costs that are considered in the optimisation include operational costs, i.e. fuel and CO2 costs, variable O&M costs and loss of load penalties (if any), as well as investment costs for all technologies subject to capacity optimisation. The model has to ensure that electricity, hydrogen and methane demands are met at any moment in time4 in the considered area (EU27 + Norway, Switzerland, UK, Macedonia, Montenegro, Serbia and Bosnia-Herzegovina).\n\nThe catalogue of investment options considered in the present analysis is composed of the following assets:\n\n□ Cross-border infrastructure for electricity, hydrogen (incl. via repurposing) and methane\n\n□ Hydrogen storage assets\n\n□ Electrolysis, steam methane reformers (SMR) and methanation assets\n\n□ Batteries and pumped-hydro storage assets\n\n□ Gas power plants (combined cycle gas turbines, CCGTs, and open-cycle gas turbines, OCGTs)\n\nSeveral demand-side flexibilities are included in the modelling of the European power system:\n\n□ The charging patterns of electric vehicles are optimised, depending on the user profiles (home charging/work-charging).\n\n□ The operation of residential and tertiary heat pumps is simulated by optimising the heat production from heat pumps (considering the impacts of the temperature on the effective coefficient of performance, COP, of heat-pumps), the operation of thermal storage and the heat production by electric back-up heaters5.\n\nFigure 1 provides an overview of all the types of assets that are represented in the model and indicates whether they are subject to dispatch optimisation only (highlighted by a dotted circle) or capacity and dispatch optimisation (highlighted by a shaded circle).\n\nH2, hydrogen; CH4, methane; SMR, steam methane reformers.\n\nThe results of the optimisation carried out with Artelys Crystal Super Grid include the investments in the previously mentioned investment options and the hourly dispatch of the different system components of the system (power generation, storage, interconnections, flexible consumers etc.). The Artelys Crystal solution further delivers a set of pre-defined key performance indicators (KPIs), such as total investment and production costs, CO2 emissions, RES curtailment, and security of supply indicators. The full list of KPIs is available in our online documentation.\n\nThe reference scenario is largely inspired by the 1.5TECH scenario for 2050 developed by the European Commission in its 2018 Long-Term Strategy6. The main hypotheses adopted from this scenario include (all figures corresponding to EU27+UK geographic scope):\n\n□ Demand levels: circa 4000 TWh of final electricity demand (including 500 TWh for electric vehicles and 250 TWh for heat pumps), 1600 TWh of final hydrogen demand and 1200 TWh of final methane demand. Additional losses from the system of 6% is added to the electricity overall consumption.\n\n□ Electricity supply: variable renewable power generation (vRES) as main source with circa 1000 GW of solar PV, 750 GW of onshore wind and 450 GW of offshore. The thermal fleets complete the capacity mix with around 120 GW of nuclear capacity, 50 GW of bioenergy capacities with carbon capture and storage (BECCS) and 135 GW of fossil fuel-based capacities (including 90 GW of gas-fired capacities and 45 GW of coal and oil-fired capacities, mainly used as reserve).\n\n□ Biomethane supply: 825 TWh\n\nSince the country-level assumptions of the LTS pathways have not been made publicly available by the European Commission, we have developed a disaggregation methodology to generate a country-level scenario. The disaggregation methodology is based on the following principles:\n\n□ Adopt the EU-wide assumptions of the LTS 1.5TECH scenario (e.g. total demand by fuel, total installed capacity for each technology, etc.).\n\n□ Disaggregate these assumptions at country level using distribution keys (cf. Figure 2 for the breakdown of final energy demand as an example). In practice, most of the distribution keys are based on the use of country-level assumptions published in the Ten-Year Network Development Plan (TYNDP) 2020 of the European network of transmission system operators for electricity and gas (ENTSO-E and ENTSOG, respectively). The plausibility of the disaggregated figures is then analysed via a literature review (e.g. compatibility with RES potentials, order of magnitude of hydrogen demand compared with other scenarios, etc.).\n\nElectric interconnections are optimized starting from the net transfer capacities (NTCs) provided in the 2020 Best Estimate scenario of the TYNDP 2018, representing the current European power grid. The installable capacities are limited to 20 GW per border, as costs and impacts on internal networks become very uncertain for high levels of additional interconnection capacity. Investments costs are based on line-by-line transmission projects included in TYNDP 2018: for each cross-border interconnector, an aggregated cost per MW of additional NTC are calculated (for CAPEX and OPEX).\n\nCross-border pipelines are optimized starting from the “Low” scenario of the TYNDP 2018. Data includes the existing infrastructures in 2018 and projects with ‘Final Investment Decision’ status representing the minimum level of infrastructure development considered for the identification of infrastructure gaps. It has been assumed that both directions of gas interconnectors are able to transport the same capacity by 2050 (the additional costs to enable reverse flows are not taken into account). Investment costs in additional pipelines are based on the transmission project list provided by ENTSOG in the 2018 edition of the TYNDP.\n\nThe model used for this study optimises the repurposing of methane pipelines and investments in new hydrogen pipelines. The costs used were extracted from European Hydrogen Backbone and Hydrogen Generation in Europe. In order to consider refurbishment, the number of pipes was estimated on the basis of GIE’s map for each interconnection. We start from a situation without hydrogen storage, letting the model decide whether to invest in such a technology considering a cost of 334 € / MWh of stored hydrogen (value of salt cavern storage from Hydrogen Generation in Europe).\n\nThe commodity prices are based on different sources, including the ENTSOs’ TYNPD, the World Energy Outlook (WEO) of the International Energy Agency (IEA) and the 2016 Reference scenario from the European Commission. Since the CO2 price is a key assumption for the modelling, the figure we have adopted originates from the LTS 1.5TECH scenario and equals 350 €/tCO2.\n\nThree sensitivity analyses have been carried out to test the robustness of the evaluation of the infrastructure needs of the European energy systems, by modifying structural assumptions of the reference scenario. They are governed by the willingness to reach higher ambitions on different aspects of the scenario, with updated assumptions based on the Paris Agreement Compatible (PAC) scenarios, and LTS 1.5LIFE scenario.\n\nA lower hydrogen demand and a smarter allocation of vRES capacities. The objective of this sensitivity is to assess the impacts of two important factors: the level of hydrogen demand in the system and the ability to produce it closer to hydrogen demand centres.\n\nThis sensitivity analysis assumes a hydrogen consumption that is around 30% lower compared to the one of the reference scenarios, inspired by the PAC scenario’s lower level of hydrogen demand, which is around 1100 TWh across the EU and the UK. The hydrogen demand decrease is shared homogeneously between all countries and associated with a decrease of 600 TWh of renewable power generation (considering an efficiency of 85% for electrolysers). Also, variable RES capacities are reallocated across countries to obtain a better alignment between load centres (for electricity and H2) and power generation volumes, instead of allocating RES to least-cost RES potentials, cf. Figure 3.\n\n(Belgium [BE], Germany [DE], Denmark [DK], Spain [ES], France [FR], United Kingdom [GB], Italy [IT], Netherlands [NL], Romania [RO], Sweden [SE]).\n\nA lower biomethane potential. The objective of this sensitivity is to assess the impact of a lower biomethane supply and analyse the impacts of a more local use of biomethane.\n\nThe second sensitivity analysis assumes a lower biomethane potential at EU level, by reducing the capacity supply to reach the level of the LTS 1.5LIFE scenario: around 600 TWh of biomethane production are assumed in this sensitivity (cf. Figure 4) compared to 825 TWh in the reference scenario.\n\nThe biomethane reduction has been performed with the objective of building a more consistent alignment between biomethane supply and methane demand. The reduced biomethane supply potential is compensated by increasing synthetic gas production via electrolysers and methanation plants. Thus, additional renewable capacities are added in the sensitivity in order to cope with the additional demand for carbon neutral power generation induced by the synthetic gas needs. With a respective efficiency of 85% and 79% for electrolyser and methanation plants, around 300 TWh of renewable power generation (wind and solar) are added in this sensitivity.\n\nA higher energy efficiency and a deeper electrification. The objective of this sensitivity is to assess the impact of a deeper electrification of the heat sector on energy infrastructure needs.\n\nIt assumes a deeper electrification of the heating end-uses in the residential and tertiary sectors as the remaining gas boilers are replaced by heat pumps, reflecting the PAC scenario assumption of a gas boiler phase out. Since the overall efficiency of producing heat from synthetic-gas-fired boilers is lower than the heat pump efficiency, this sensitivity induces a reduction in electricity demand compared to the reference scenario. In the reference scenario, the gas demand for boilers reaches around 250 TWh. With an 85% efficiency assumption for boilers, the heat demand covered by gas boilers in the reference scenario would reach a little more than 200 TWh. In order to meet this heat demand via the installation of heat pumps, around 50 TWh of additional electricity demand is required. This figure is based on using following assumptions:\n\n□ 95% of this heat demand is covered by heat pumps\n\n□ The remaining heat is provided by an electric back-up heater (during the coldest hours of the year)\n\n□ An average COP of 3.6 is assumed for heat pumps (simulations represent the impact of temperatures on the effective capacity and COP of heat pumps, see e.g. METIS study S6)\n\n□ Electrical heater’s efficiency of 100%\n\nThe 200 TWh of heat demand would thus be met with a little more than 50 TWh of electricity consumption from heat pumps. Figure 5 depicts the electricity demand repartition from heat pumps and relative increase compared in the sensitivity compared to the reference scenario.\n\nAs with the other sensitivities, the vRES capacities have been updated in order to adapt to the lower electricity consumption (since part of the boilers were using electricity-derived gases). Total RES capacities were reduced by 5% or 100GW compared to the 2 240 GW of the reference scenario.\n\n\nResults\n\nIn this section, we provide the key findings of the study. These findings have been identified by evaluating the required investments in electricity, methane and hydrogen infrastructure in several 2050 scenarios: a central scenario based on the assumptions of the European Commission’s LTS 1.5TECH scenario and three sensitivity analyses (see previous section for definitions).\n\nThe reference scenario as well as all the sensitivities see major investment needs in the electricity infrastructure appear (from 220 GW up to 280 GW additional electricity interconnectors, cf. Table 1 for the result per scenario).\n\nInvestments would be concentrated around countries that structurally need to import electricity (Italy, Germany, Belgium, Poland, Romania) but also for some countries located on major transit routes (Netherlands, Spain, Switzerland or Austria), as shown in Figure 6.\n\nThe key outcome of the hydrogen sensitivity is that the level of required investments in power interconnections can be mitigated by around 10% by a smart distribution of RES capacities in a way that is consistent with hydrogen demand centres.\n\nGiven the magnitude of the required investments in electricity infrastructure, a recommendation resulting from this study is that procedures (e.g. related to permitting) should be streamlined and simplified to facilitate investment in cross-border electricity infrastructure.\n\nInvestments in cross-border hydrogen infrastructure will be required in specific areas, notably by repurposing part of the existing methane pipelines in addition to investing in new H2 pipelines. In the reference scenario, we estimate that 320 GW of gas pipeline are repurposed (representing circa 260 GW of hydrogen transmission capacities). Additionally, 310 GW of new hydrogen pipelines are also found to be necessary in the reference scenario. Figure 7 shows the geographical distribution of hydrogen infrastructures.\n\nThe hydrogen infrastructure enables to connect main hydrogen exporters (the Netherlands, Spain and France) to the main hydrogen importers (Germany, Italy and Poland).\n\nHowever, the cross-border hydrogen infrastructure requirements are found to be highly dependent on the geographical allocation of renewables in Europe. Our simulations show that the combination of a 30% decrease of hydrogen demand (representing a limitation of the role of hydrogen to hard-to-abate sectors, in line with the PAC scenario) and the reallocation of renewable production sites in proximity to power and H2 demand centres lead to a 60% decrease of the required hydrogen infrastructure (see Figure 8).\n\nTwo phenomena are impacting the trade-off between repurposing gas pipelines and building new hydrogen pipes:\n\n□ A lower demand for hydrogen means that one is more likely not to repurpose a given pipeline.\n\n□ A lower demand for methane leaves more room for repurposing as was highlighted in the biogas and electrification sensitivities (see Figure 8).\n\nThe key policy recommendation that emerges from this analysis is that scenarios and cost-benefit analyses, CBAs, (especially for power-to-x projects) should examine the impacts of a consistent deployment of RES and electrolysers, in order to avoid unnecessary investments in wires and pipelines.\n\nOur optimisation results show that there is no need for additional investments in additional methane infrastructure. Indeed, no additional investments are found to be required to ensure security of supply in any of the considered scenarios. As can be seen in Figure 9, part of the existing infrastructure is found to be characterised by low utilisation rates at the 2050 horizon (up to 10 cross-border pipelines with a use rate below 1% over the year) due to the structural evolution of gas flows (a local production combined with the competition between biomethane and hydrogen). Some corridors still remain relevant connecting the north and south to central Europe. The repurposing of part of the existing gas infrastructure is found to be relevant to support the cross-border transport of hydrogen.\n\nThe key policy conclusion that comes from this finding is that new methane projects that do not demonstrate benefits in terms of hydrogen transport if repurposed should be excluded. Thus, the evaluation of projects should consider the entire lifetime of the project, considering the possibility to use methane at first stage, and hydrogen in a second phase). Indeed, our results show that new methane infrastructure are unnecessary from a security of supply point of view. Therefore, any additional investments in such infrastructure assets should only be made if they can also serve as hydrogen infrastructure. This transition from transporting methane to transporting hydrogen should be captured when assessing infrastructure projects.\n\n\nConclusions and outlook\n\nWe apply a European multi-energy model to assess the required level of cross-border electricity, hydrogen and methane infrastructure in 2050 to ensure the energy demand of a carbon-neutral EU energy system can be met at the lowest cost. The assessment of the reference scenario and the different sensitivities leads to the conclusion cross-border electricity interconnections will be required in any case, whereas there is no need for additional cross-border methane pipelines. The required degree of establishing cross-border hydrogen infrastructure depends on various factors:\n\n□ The level of hydrogen demand, which is highly uncertain. This impacts two aspects: the level of infrastructure and the ratio between new hydrogen and repurposed gas pipelines. Repurposing is a binary process: the lower the hydrogen demand, the less likely it is that repurposing is favoured as the existing gas pipelines have capacities that may be too high compared to the need to transport hydrogen. Investing in a dedicated hydrogen solution might be cheaper in some cases.\n\n□ The level of colocation between RES capacities and hydrogen demand centers. Scenarios and guidelines for cost-benefit analysis should carefully examine the impacts of a consistent deployment of renewable technologies and hydrogen consumption centres in order to avoid unnecessary investments in pipes and wires. Our sensitivities tend to show that hydrogen infrastructure levels can be mitigated by enhancing the degree of colocation between RES production and H2 consumption.\n\n□ The potential competition between biomethane and hydrogen for the use of existing gas pipelines. While there is no need for additional investments in methane infrastructure to ensure security of gas (i.e. methane) supply within the EU, the use of existing gas pipelines will have to be balanced between transportation of methane or hydrogen. If the methane flows remain important, the system needs to keep a sufficient level of methane infrastructure. Reducing the biomethane injection or methane demand and considering biomethane as a local supply - as in the two last sensitivities - will leave more room to repurposing.\n\nFrom a policy point of view, this analysis demonstrates that the assessment of system needs at the 2050 horizon should be conducted jointly for the electricity, hydrogen and methane systems. For this to be the case, simulation guidelines and tools have to represent all interlinkages within a single framework via an integrated model. Additionally, the repurposing of part of the existing gas infrastructure is found to be a cost-effective way to develop the hydrogen infrastructure and should be considered in the planning of hydrogen infrastructure. Depending on the considered scenarios, the repurposing of gas pipelines could represent between 35% and 50% of the global hydrogen infrastructure. CBA methodology used to assess infrastructure projects should set forward the repurposing of existing pipelines. This advocates for a joint approach to the planning of all infrastructure projects, as decisions related to one vector can heavily impact the value brought by projects transporting another of the energy vectors.\n\nThe analysis framework that has been developed could support further analysis of infrastructure needs, and in particular the construction of entirely new transition pathways.\n\n\nData availability\n\nA detailed description of the underlying mathematical system used, including objective functions, constraints and equations describing asset behaviours is openly available for download from the METIS website here: https://energy.ec.europa.eu/document/download/de87d30a-1915-4ba9-b900-50456ea213b1_en\n\nAll generic data (which applies to all scenarios and is independent from the present study context), such as techno-economic data or time series for demand profiles, the availability of power plants or temperature data is contained in the data package available here: https://energy.ec.europa.eu/metis-scripts-and-data_en.\n\nZenodo: What energy infrastructure to support 1.5°C scenarios? - Scenario data. https://doi.org/10.5281/zenodo.63590011.\n\nThis project contains the following underlying data:\n\n- EMP-E_Data_Arduin et al_v1.0.xlsx (the assumptions relating to the present analysis, including energy demand volumes and installed capacities).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank Amaury Salauze, Laurent Cornaggia and Luc Humberset (all working at Artelys at the time this analysis has been produced) for their contribution.\n\nThe related project report on behalf of the European Climate Foundation is available online (https://www.artelys.com/wp-content/uploads/2020/12/Artelys-2050EnergyInfrastructureNeeds.pdf). However, it reflects the views of the authors only.\n\n\nFootnotes\n\n1 SWD (2020) 176 final Stepping up Europe’s 2030 climate ambition - Investing in a climate-neutral future for the benefit of our people - Figure 48\n\n2 CAN Europe/European Environmental Bureau: Building a Paris Agreement Compatible (PAC) energy scenario, June 2020,\n\n3 The reader is referred to the LTS reference document for further details about the LTS 1.5LIFE scenario, cf. https://ec.europa.eu/clima/eu-action/climate-strategies-targets/2050-long-term-strategy_en.\n\n4 The model accepts loss of load (LoL), yet it comes at high costs, implying that LoL is restricted to 3 hours per year on average.\n\n5 Because the coefficient of performance of heat pumps degrades with low outside temperatures, an electric back-up heater is associated to the heat pump, to ensure the heat demand is met cost-efficiently.\n\n6 In-depth analysis in support of the Commission Communication COM(2018) 773\n\n\nReferences\n\nArduin I: What energy infrastructure to support 1.5°C scenarios? - Scenario data (Version v1) [Data set]. Zenodo. 2022. http://www.doi.org/10.5281/zenodo.6359001" }	[ { "id": "129675", "date": "06 Apr 2022", "name": "Sonja Wogrin", "expertise": [ "Reviewer Expertise mathematical modeling and optimization of energy systems", "techno-economic analyses of energy systems." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 makes an ambitious attempt to depict the European energy system and optimize the required infrastructure to achieve 1.5°C scenarios by 2050. Using the METIS model, every country is represented in an aggregated form (by one node) and sectors gas (hydrogen and methane are considered separately) and power are represented explicitly. Cross-border power lines and pipelines are assessed and conclusions about the EU’s energy infrastructure are presented. The article addresses the important challenge of achieving climate neutrality in Europe by 2050 and is a valuable foundation for future discussions.\nPlease find some more detailed comments and questions below:\nIt would be interesting to include a brief description of the METIS model formulation. What are the most prominent constraints that have been accounted for by sector? For example, is the power flow considered, or is it a transport problem, what about gas flow equations, etc?\nWith respect to the model, the main simplifications should be clearly stated, and it would be interesting to have your opinion on the impact of such simplifications on model outcomes. Are there plans to make an open-source version of METIS available to the public?\nIt would improve the paper if you have a summary of the main data, and in what exact granularity you found the data in the source. And most importantly, if data is only available in an aggregated format, how did you specify it. You mention this for the regionalization of energy demand for example, but I am particularly interested in how you obtained hourly granularity for such data. Could you please expand on that? Because METIS runs on an hourly basis but public data sources might now have the same granularity.\nWith respect to your findings. In Finding 1, it seems intuitive that a massive expansion of the electric power grid is the cheapest option to achieve climate neutrality. However, there are some bottlenecks here, which should be mentioned. You have discussed permits, but what about political and economic considerations. Often, when cross-country transmission lines are not built, it is not (only) a permit issue. There might be winners/losers of this transmission line, so while I agree that this is technically a great option, there are political complications that should be discussed.\nYou mention “variable RES capacities are reallocated .. to obtain a better alignment between load centers”. It seems artificial to re-allocate RES closer to load centers. I think it is fair to say that generated power has the highest value for the system (and also for the investors). I am doubtful that this would happen in reality.\n\nIs the work 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": "135614", "date": "03 May 2022", "name": "Ilija Batas-Bjelić", "expertise": [ "Reviewer Expertise Simulation based optimization of national energy systems with policy constraints" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTo my knowledge, this is only constrained multi-energy interconnector joint capacity and operation optimization study at EU former/future member states level, and therefore all conclusions should be carried carefully. The electricity interconnection increase (for 250-280 GW) clearly illustrates the electrification of all sectors as the first step of decarbonisation. Although the approach is directed toward identifying interdependencies and synergies between energy vectors and sectors, the presented conclusions regarding optimal levels of energy infrastructure are rather particular and not applicable to regions other than Europe. The conclusions are somewhat robust, which at least lead to the identification of key lessons for policy recommendations at EU level. There are studies e.g. covering operation optimization at the higher renewable energy penetrations in more details (Pfeifer et al., 20211), but they lack proven effect of interconnection on the relaxation of the total system costs as goal function. Maybe the importance of interconnection contribution should be stressed stronger. The loss of load maximum hours and maximum interconnection are bounded smartly to narrow the optimization search space. One of the weak spots of this research might be the utilization of some cross-border pipelines at a rate of less than 1% per year. Thus, this has to be added to constraints. The most interesting direction of future research will be the competition between three interconnector energy vectors. The article is rich in illustration and open data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-387
https://f1000research.com/articles/11-386/v1	01 Apr 22	{ "type": "Research Article", "title": "The management of tubo-ovarian abscess - A retrospective analysis of a centre offering outpatient intravenous antibiotic therapy", "authors": [ "Jhia Jiat Teh", "Sarah Wali", "Josephine Mollier", "Mark Gilchrist", "Tariq Miskry", "Josephine Mollier", "Mark Gilchrist", "Tariq Miskry" ], "abstract": "Background Tubo-ovarian abscess (TOA) carries long-term sequale in women of reproductive age. Consensus of the optimal treatment of tubo-ovarian abscess remains lacking. The aims of this study are to identify risk factors predicting the need for early drainage and compare clinical outcomes of current management practices of TOA.\n\nMethods From 2015 to 2019, a retrospective cohort study of 92 women admitted to a tertiary centre for gynaecological surgery was performed. Patients with diagnosed TOA were classified into two groups: treatment with antibiotics only, and those receiving additional drainage. Primary outcomes included length of hospital stay (LoS), length of antibiotic treatment (LoA) and need for re-intervention.\n\nResults In this study, 52 women (56.5%) were successfully treated with first line intravenous antibiotics; 40 (43.5%) received surgical drainage. Significant predictors for successful medical treatment only include age < 35 (OR: 0.89, 95% CI: 0.82-0.97) and abscess size < 6cm (OR: 0.17, 95% CI: 0.04-0.64), using multivariate analysis. Pyrexia ≥ 38°C predicted a need for drainage (OR: 3.82, 95% CI: 1.01-8.12). Patients who received additional drainage had significantly longer LoA, LoS and higher rates of re-intervention. Within this group, drainage within 72 hours of admission resulted in a trend towards shorter LoA and LoS than drainage after 72 hours, albeit not statistically significant.\n\nConclusions Parameters include age > 35 years, pyrexia ≥ 38°C and a TOA size > 6cm may independently predict the need for drainage of TOA. Early identification of these patients is imperative for timely surgical intervention to avoid prolonged hospitalisation, antibiotic usage, and patient morbidity. More work is required to identify whether early drainage may reduce length of hospital stay and antibiotic treatment, including identifying certain patient groups who most likely to benefit from outpatient antibiotic intravenous therapy.", "keywords": [ "Tubo-ovarian abscess", "Pelvic inflammatory disease", "outpatient intravenous antibiotic therapy", "malignancy", "infection" ], "content": "Abbreviations\n\nAD: additional drainage\n\nAO: antibiotics only\n\nBASHH: British association of sexual health and HIV\n\nCI: confidence interval\n\nCRP: C- reactive protein\n\nHIV: Human immunodeficiency virus\n\nLoA: length of antibiotics\n\nLoS: length of stay\n\nOPAT: outpatient intravenous antibiotic therapy\n\nOR: odds ratio\n\nPID: Pelvic inflammatory disease\n\nTOA: tubo-ovarian abscess\n\nWCC: white cell count\n\n\nIntroduction\n\nPelvic inflammatory disease (PID) is a polymicrobial infection of the female upper genital tract caused by ascending lower genital tract organisms, both sexually transmitted and normal microbiome. Its lifetime prevalence of 4.4% in sexually active women of reproductive age (18–44 years)1 may be an underestimate as PID can be subclinical and is increasingly managed in the outpatient setting on clinical suspicion only.\n\nTubo-ovarian abscess (TOA) is a severe complication of PID which affects approximately one third of women hospitalised with PID.2 Rupture of a TOA is a rare surgical emergency, presenting with acute peritonitis and sepsis requiring emergency laparotomy with a mortality of 65-100% if treated with antibiotics only.3 The majority of TOA, however, are unruptured and their management has changed over the years from radical surgery, often involving unilateral or bilateral adnexectomy and pelvic clearance, to more medical management involving enteral and parenteral broad-spectrum antibiotics, with or without drainage (radiological or simple surgical). This conservative approach has a documented efficacy of 16-95%, with recent studies showing success of 70% or greater.4 The high success rate of medical management has meant that there is often a reluctance to manage these cases surgically with the associated risks of hemorrhage, visceral injury, and consequences of reduced ovarian tissue. There is published guidance on the use of outpatient parenteral antimicrobial therapy (OPAT) for intra-abdominal abscess although this does not address TOA specifically.5\n\nSurgical drainage has been reserved by some for patients with diagnostic or therapeutic uncertainty.6 The surgical approach is now more commonly laparoscopic drainage and washout. Alternatively, radiological drainage is a less invasive option and has additional advantages of being done under local anaesthesia and is repeatable with less risks than surgical drainage. It has also been suggested that this approach may also lead to faster recovery compared to antibiotics alone.7\n\nPrimary prevention of pelvic infection as well as early identification and treatment of women with suspected PID is pivotal to reduce the incidence of TOA and associated acute and chronic morbidity. The UK guidelines on the management of PID are available from the British Association of Sexual Health and HIV (BASHH) and it recommends various combinations of enteral and parenteral antibiotic regimens.8 For TOA, the BASHH guidelines suggest consideration of drainage either laparoscopically or radiologically to help early resolution of disease. However, these guidelines do not provide explicit indications for drainage. In contrast, French guidelines strongly recommend that the first line approach should be radiological drainage for TOA > 3 cm.9\n\nThese differences have contributed to the ongoing debate on optimal management. As a result, currently, in the UK there is an ad hoc approach to the management of TOA with variation between clinicians and drainage generally only being considered when initial antibiotic therapy is unsuccessful (e.g. persistent pyrexia, pain, or rising C-reactive protein (CRP)). This study assesses women with TOA at our tertiary referral centre, St Mary’s Hospital, in West London, investigating their demographics, management approach and outcomes. We aim to firstly compare risk factors present in patients who required antibiotics alone compared to those who required additional radiological or surgical drainage. Secondly, we aim to describe outcomes in these two groups to determine clinical factors that influence the selection of a particular treatment approach, including those treated with outpatient parenteral antimicrobial therapy.\n\n\nMethods\n\nFrom January 2015 to June 2019, a historical cohort study was performed on a total of 92 patients (n = 92) admitted to a tertiary west London hospital for TOA. Patients were identified through a search of the hospital’s electronic database, using diagnostic codes for TOA. Patients were included if a TOA was confirmed either radiologically or surgically. A total of 370 cases were reviewed to confirm eligibility, of which 278 patients were excluded as they had past medical history of TOA rather than an active encounter or did not have radiological or surgical confirmation of the diagnosis. Patients who had secondary TOA due to pre-existing intra-abdominal pathology were included in our analysis and discussion.\n\nFollowing institutional approval from the audit team (Imperial NHS Trust service evaluation project reference number 404), data on demographics, clinical management, investigations, and patient outcomes were extracted from the hospital’s electronic database. As the data was retrospectively collected and did not affect the included patients care no further ethical approval was required. Additionally, consent was waived by the ethics committee as the data collected from the hospital records were anonymized.\n\nDemographic data collected included: age, body mass index (BMI), ethnicity, parity, and presence of comorbidities including endometriosis, intrauterine contraceptive device (IUCD) use, previous PID, immunocompromised state and smoking status (See Underlying data).10 TOA data included its size, laterality, high vaginal swab (HSV) cultures and presence of any other intra-abdominal pathology.\n\nPatients’ records were reviewed for signs and symptoms of PID on presentation including lower abdominal pain, abnormal vaginal discharge, fever (defined as a temperature ≥ 38°C), white cell count (WCC) and CRP at admission (See Underlying data).10 Microbiology results of any microscopy, culture and sensitivity were also noted.\n\nAfter confirmation of TOA, all patients were started on the hospital’s broad spectrum intravenous antibiotic regime for PID. Those that did not show improvement or who grew organisms with specific sensitivities were discussed with microbiology and had their antibiotic regimen tailored.\n\nBased upon the management approach, all patients were classified into two groups. Group one consisted of patients treated successfully with intravenous antibiotics only (either short course or long course including OPAT). Successful treatment was defined as good clinical and biochemical response to antibiotics with down trending inflammatory markers. Conversely, Group two consisted of patients requiring drainage in addition to intravenous antibiotics due to lack of improvement or worsening of symptoms or inflammatory markers or recommendation on radiology report. Decision for the route of drainage was made on an individual patient basis by a multidisciplinary team involving gynecologists, radiologists, and general surgeons. The drainage approach included: (i) radiologically guided drainage, or (ii) laparotomy/laparoscopy. Laparotomy or laparoscopy involved a combination of washout, salpingectomy, adnexectomy, hysterectomy with bilateral salpingo-oophorectomy or bowel resection.\n\nThe main outcome measures were length of hospital stay (LoS), length of antibiotic treatment (LoA) and need for reintervention, including further antibiotics or drainage. Successful treatment was defined as clinical and biochemical improvement without recurrence of symptoms, further intervention, or hospital readmission.\n\nData was analysed using IBM SPSS Statistics version 25 (SPSS Inc, Chicago, IL, USA). Kolmogorov-Smirnov tests were used to assess for normality. Univariate analysis involved Mann-Whitney-U tests for comparison of medians of continuous variables and Chi-squared tests of independence for comparison of categorical variables to establish predictive risk factors for requiring additional drainage. Multivariate analysis involved a binary logistic regression model for any predictive factors found to be significant on univariate analysis. Statistical significance was defined as p < 0.05.\n\n\nResults\n\nAt the time of this study, 92 women were diagnosed with TOA. 52 (57%) patients were treated successfully with antibiotics alone and 40 (44%) patients required surgical or radiological drainage in addition to antibiotics (Figure 1). 15% of patients had a short course of antibiotics of no more than 14 days and 85% had a longer course of antibiotics ranging 15-64 (average 26 days). For patients requiring additional drainage, the decision made to perform drainage ranged between 0 to 196 days from presentation. 26 (65%) patients underwent image-guided drainage and 14 (35%) had surgical drainage. Of those 14 patients, 7 (50%) patients had immediate surgery on admission under the care of general surgeons due to clinical suspicion of other intra-abdominal pathologies.\n\nDemographic characteristics, clinical and biochemical laboratory values on admission between the two groups were compared and presented in Table 1a and 1b. Univariate analysis found that patients who required drainage (Group 2) were more likely to be febrile (>38°C) on admission (p = 0.038) compared to patients treated with antibiotics only (Group 1). Patients in Group 2 had a significantly higher median age than those in Group 1 (42 vs 44 years, p = 0.041). Size was also found to be a significant predictor of requiring drainage, with the median TOA size of 8 cm in Group 2 compared to 6.2 cm in Group 1 (p = 0.0001). Positive high vaginal swab (HVS) cultures were also more likely in patients who required drainage (p = 0.027). Although not statistically significant, all three positive blood cultures were in Group 2. There are no significant differences in parity, BMI, ethnicity, PID risk factors and admission inflammatory markers between the two groups. All patients received imaging with half receiving multiple imaging modalities. 74 (80%) patients underwent transvaginal/abdominal ultrasounds, 58 (63%) underwent CT, and 10 (11%) underwent MRI.\n\n† Values are given as number (percentages), unless indicated otherwise.\n\n‡ Drainage performed in either radiologically or surgically.\n\n§ Positive HVS growths: Chlamydia, Gonorrhoea, E coli, Enterococcus faecalis, Group B Streptococcus, mixed anaerobes, normal flora including coliforms, Prevotella bivia, Staph aureus yeasts.\n\n¶ Positive blood culture growths: Gram positive cocci, Burkholderia multivorans, E. coli.\n\n† Appendicitis, small bowel obstruction, diverticulitis, enteritis.\n\n‡ Complete miscarriage, post-partum, necrotic fibroid.\n\n§ Hysteroscopy, laparoscopy treatment of endometriosis, Mirena coil insertion, ovarian cystectomy, hysteroscopy and coil insertion, hysterosalpingogram, hysteroscope, colposcopy.\n\nA binary logistic regression model adjusting for age, fever, high vaginal swab, and TOA size for multivariate analysis is shown in Table 2. Variables found to be significant independent predictors of successful treatment with antibiotics only on multivariable analysis were age < 35 (OR: 0.89, CI: 0.82-0.97) and TOA size < 6 cm (OR: 0.17 CI: 0.04-0.64). A pyrexia ≥ 38°C on admission significantly predicted the need for additional drainage (OR: 3.82, CI: 1.01-8.12). Although HVS was significant on univariate analysis, this was not significant on multivariate analysis.\n\nComparison of clinical outcomes between the two groups is presented in Table 3. Patients in Group 2 had a significantly prolonged inpatient stay, length of antibiotics and need for re-intervention compared to Group 1 (p <0.001). In Group 2, the median length of time between admission and decision for surgical drainage was two days (IQR 1-5 days).\n\nThose that had drainage within 72 hours of admission had a trend towards shorter LoA and LoS compared to patients receiving drainage after 72 hours, although this did not reach statistical significance. There was no difference in need for re-intervention between the two subgroups. 3 (3.26%) patients had malignant histology at surgery (all colorectal or intestinal). 69 patients received post-treatment follow-up imaging (Table 4). Patients in Group 2 were more likely to show no improvement or worsening of the TOA than those in group 1 (p = 0.0251)\n\nIn this cohort, 23 (25%) patients received OPAT ranging between 5-50 days (Table 5). In Group 1, OPAT was used to successfully manage 11 (21%) patients, with a median total antibiotics’ duration of 47 days (IQR: 35-56 days) and a median OPAT length of 25 days (IQR: 16-35.5) The average age of this group was 40 years (± 9 years), with an average TOA size of 7.0 cm (±1.3 cm), and 45% had bilateral abscess. Among those patients who required invasive intervention, 7 (58%) patients had pre drainage OPAT, and 5 (42%) had post drainage OPAT. There is no significant difference in the length of OPAT days between Group 1 and Group 2 (25 vs 26 days, p = 0.853). There was no significant difference in OPAT days between those that received OPAT pre and post drainage (p = 0.749), although numbers are small.\n\n\nDiscussion\n\nOur study described a cohort of patients with confirmed tubo-ovarian abscess (TOA) in a tertiary institute with detailed outcome measures. Our main findings were: age > 35 years, pyrexia ≥ 38°C and a TOA size >6 cm may independently predict the need for drainage. Our studies also compared the outcomes in patients who received short term inpatient antibiotics with OPAT.\n\nIncreased age was the only demographic risk factor between the treatment groups in our study. Other studies have investigated possible risk factors for needing early drainage, but the literature is heterogeneous and often sample sizes used are too small to reach statistical significance. Chan et al. found that BMI ≥ 24.9 had an increased risk of needing drainage.11 Fouks et al. created a risk assessment tool to predict patients that will need drainage based on four variables: age > 35 years, TOA size ≥ 7 cm, bilateral abscess and WCC > 16 cells/mm3.12 Similarly, we found that women older than 35 years are more likely to need drainage. This could be due to associated comorbidities, more aggressive microorganisms, delay in diagnosis and treatment, and increased likelihood of a secondary TOA in older patients.\n\nAlthough TOA size has been well-recognised as a risk factor for failing antibiotic management requiring early drainage, the recommended size threshold varies between 3-10 cm across multiple studies and international guidelines.7,9,11,13–17 For example, French guidelines recommend all TOA ≥ 3 cm must be drained either radiologically as a first-line treatment or surgically due to higher risk of treatment failure and serious complications with antibiotics alone.9 In our cohort, we found a TOA of 6 cm to be the maximum size of TOA for successful management with antibiotics only. The ability of antibiotics to penetrate large abscesses effectively is known to be limited because of reduced vascular supply, as well as the effects of encapsulation and acidity.18 Benefits of early drainage also include the ability to identify causative organisms and sensitivities to better target antibiotic regimes. In our study group, a variety of organisms were grown from vaginal swabs, blood and aspirate cultures (Table 6). Many patients required discussion with microbiologists to guide antibiotic treatment in the event of unsuccessful empirical management of PID.\n\nStudies have also found a raised WCC15 or leucocyte to neutrophil ratio19 or CRP15 to be predictors for the need for drainage. We found no correlation between WCC or CRP and the need for drainage but found pyrexia ≥ 38°C to be significantly associated with the need for surgical drainage, which corroborates with other groups.11,15,17\n\nOur study observed a significantly longer duration of inpatient stay and antibiotics use for patients that received invasive intervention (Group 2) compared to those who had medical treatment only (Group 1). This is consistent with findings by Chan et al. and Habboub et al.,11,20 which may be reflected by increased clinical severity in patients in Group 2. On the contrary, Perez et al. found that patients who had drainage had a significantly shorter LoS compared to the antibiotic only group,13 however their group was small and only included unilateral abscess and decision for drainage was within 6-12 hours of antibiotics.\n\nFurthermore, we found that patients who received additional drainage were more likely to require readmission or further reintervention. This is consistent with the increased antibiotic duration and length of stay observed in these patients. In terms of methods of drainage, we found no difference in management outcomes when comparing surgical and radiological drainage. Differentiating a TOA from other intra-abdominal pathologies may pose a diagnostic challenge for radiologists. Nonclinical resolution of TOA despite broad spectrum intravenous antibiotics with or without drainage should prompt suspicion of alternate pathology causing a secondary TOA, especially non-gynecological malignancy. In our cohort, 3 patients (3%) were found to have colorectal or caecal malignancy. We could not find related incidences of colorectal or gastrointestinal malignancy presenting as TOA in the literature and it is possible that this is an under-recognised underlying pathology.\n\nJaiyeoba et al. suggested that failure of response to antibiotics within 48-72 hours (as characterised by persistent fever and increasing leukocytosis) should be considered for surgical drainage21 and this is also highlighted in the BASHH guidance.8 Our study found that patients who failed medical treatment received a median of two days (IQR 1-5 days) of antibiotics before a decision for invasive intervention was made, which is similar to the duration of 4.0±2.1 days by Chan et al.11 In our study, 58% of patients who received invasive intervention within 72 hours of admission, justified by either poor clinical response to antibiotics or radiologists’ recommendation. We found a trend towards reduction in length of antibiotics (24 vs 40 days) and length of stay (11 vs 17 days) in the subgroup that was drained within 72 hours compared to those that were drained >72 hours from admission, but this did not reach statistical significance. Our work suggests that there may be scope to recommend early intervention in specific groups of patients who have clinically severe disease whilst recognising there may be delays in accessing radiological drainage. In addition, surgical concerns about operating on friable tissues in the presence of sepsis can result in inappropriate perseverance with medical management with changing antibiotic regimens despite lack of initial clinical or biochemical improvement.\n\nAlthough the general fear of operating in the acute phase of a TOA is the friability of tissues and bleeding, it is easier to operate on acute adhesions than on dense and vascular chronic adhesions, and Reich et al. describes the recommended technique of careful blunt dissection using a probe or aqua dissection.22 Reich et al. also argues that patients with fertility desires should be managed with early surgical drainage to reduce the risks to fertility, as evidenced by Elmoghazy et al. who found that on second look laparoscopy the incidence of extensive adhesions with bilateral tubal block was significantly higher in the radiologically compared to the surgically drainage group.4 A review on the fertility outcomes of different management of TOA concluded that with medical management alone the reported pregnancy rates were 4-15% and with the addition of laparoscopic drainage within 24 hours of antibiotics the reported pregnancy rates were 32-63%.23\n\nOPAT is a safe, evidence-based, and cost-effective regimen for the management of patients with a wide range of infections including complex deep-seated infection5 and can be a valuable management option in patients not fit for surgery. In our cohort of patients, OPAT was used in the management of about a quarter of our patients. To our knowledge, our study is the first published study looking at the use of OPAT specifically in the management of TOA. The decision for the use of OPAT was made by an MDT involving gynecologists, infectious disease specialists, interventional radiologists and our established regional OPAT service.\n\nIn the literature, the role of OPAT specifically for the management of TOA is unclear, and further research should aim to compare the efficacy of inpatient vs outpatient antibiotic therapy, as well as identify a subset of patients who may benefit most from OPAT. Interestingly, we identified three patients in our cohort who had TOA as the first presentation of colorectal or caecal malignancy. Two out of three patients had received OPAT for their presumed primary TOA. Although numbers are small, this suggests that all patients considered for OPAT should be managed with a high index of suspicion, whilst considering further investigations to exclude non-gynaecological malignancy, especially in those who did not respond to long-term antibiotics therapy. Hatcher found an 88% success rate of OPAT in the management of intraabdominal infection, they do not comment on the reason for failure for the 12% of patients and it would be interesting to know if they identified underlying malignancies as a cause.24\n\nThe study has several limitations. Firstly, data was collected retrospectively using hospital electronic records and may reflect retrospective case ascertainment. The relatively limited sample size captured the clinically unwell patients admitted with tubo-ovarian abscess. Secondly, potential selection bias may occur with the decision for surgical intervention in patients with TOA driven by decision of the clinical team based on personal clinical experience and judgement rather than based on hospital protocols. However, we believe the effect of heterogeneity was limited since the study is based on a tertiary hospital and decision making was largely confined to a number of experienced consultant gynaecologists. We did not make any specific conclusions on the use of OPAT in TOA since the number of cases were limited.\n\n\nConclusion\n\nThere is a plethora of literature over more than 60 years on the management and outcomes of TOA yet there is to be consensus on the optimal management and timing of drainage for these women. Our study adds to this literature and helps in the understanding of our patient population’s response to the varying management approaches. Our results suggest that older, febrile patients with larger TOA are less likely to respond to antibiotic only management and may benefit from early drainage in combination with standard antibiotics. We await with interest the results from a French randomised control trial to shed more light on whether surgical drainage is more beneficial than radiological drainage in terms of long-term outcomes.25\n\nOur future work includes developing a local policy to streamline the management of women with TOA in an attempt to reduce patient morbidity and duration of antibiotics and hospital stay. Importantly, such a policy should help identify women who may have secondary etiology for their TOA sooner to better manage these women. Given the associated morbidity in patients who fail medical management, we recommend early identification of this patients group for prompt and timely drainage with advantages such as early identification of causative organisms, improved antibiotic penetration, shorter hospital stay, as well as mitigating potential long-term sequelae including infertility.\n\n\nData availability\n\nOpen Science Framework: Tubovarian abscess, https://doi.org/10.17605/OSF.IO/JYA8D10\n\nThis project contains the following underlying data: TOA data file: Excel Sheet containing anonymized data of patients\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAuthor contributions\n\nJJT contributed to data curation, investigation, methodology, project administration, resources, validation, visualization, writing – original draft, writing – review and editing.\n\nSW contributed to conceptualisation, data curation, investigation, methodology, project administration, resources, supervision, validation, visualization, writing – original draft, writing – review and editing.\n\nJM contributed to data curation, data curation, formal analysis, software, investigation, methodology, resources, validation, visualization, writing – original draft, writing – review and editing.\n\nMG contributed to conceptualisation, investigation, supervision, writing – review and editing.\n\nTM contributed to conceptualisation, investigation, methodology, project administration, resources, supervision, validation, visualization, writing – review and editing.", "appendix": "Acknowledgements\n\nThe study protocol was approved by the ethics (Imperial NHS Trust service evaluation project reference number 404).\n\n\nReferences\n\nKreisel K, Torrone E, Bernstein K, et al.: Prevalence of Pelvic Inflammatory Disease in Sexually Experienced Women of Reproductive Age - United States, 2013-2014. MMWR Morb. Mortal. Wkly Rep. 2017; 66(3): 80–83. PubMed Abstract \| Publisher Full Text\n\nWiesenfeld HC, Sweet RL: Progress in the management of tuboovarian abscesses. Clin. Obstet. Gynecol. 1993; 36(2): 433–444. PubMed Abstract \| Publisher Full Text\n\nCollins CG, Nix FG, Cerha HT: Ruptured tuboovarian abscess. Am. J. Obstet. Gynecol. 1956; 72(4): 820–829. Publisher Full Text\n\nElmoghazy DA Sr.: Operative Laparoscopy versus trans-vaginal ultrasound-guided aspiration in the management of acute tubo-ovarian abscess: A comparative clinical trial. Fertil. Steril. 2004; 82: S138. Publisher Full Text\n\nChapman AL, Seaton RA, Cooper MA, et al.: Good practice recommendations for outpatient parenteral antimicrobial therapy (OPAT) in adults in the UK: a consensus statement. J. Antimicrob. Chemother. 2012; 67(5): 1053–1062. PubMed Abstract \| Publisher Full Text\n\nGjelland K, Ekerhovd E, Granberg S: Transvaginal ultrasound-guided aspiration for treatment of tubo-ovarian abscess: A study of 302 cases. Am. J. Obstet. Gynecol. 2005; 193(4): 1323–1330. PubMed Abstract \| Publisher Full Text Reference Source\n\nDoganay M, Iskender C, Kilic S, et al.: Treatment approaches in tubo-ovarian abscesses according to scoring system. Bratislavske lekarske listy. 2011; 112(4): 200–203. PubMed Abstract\n\nRoss J, Cole M, Evans C, et al.: United Kingdom National Guideline for the Management of Pelvic Inflammatory Disease (2019 Interim Update). British Association for Sexual Health and HIV Clinical Effectiveness Group (BASHH); 2019.\n\nBrun JL, Castan B, de Barbeyrac B , et al.: Pelvic inflammatory diseases: Updated French guidelines. J. Gynecol. Obstet. Hum. Reprod. 2020; 49(5): 101714. S2468-7847(20)30044-1 [pii]. PubMed Abstract \| Publisher Full Text\n\nTeh J: Tuboovarian abscess.2022, March 21. Publisher Full Text\n\nChan GMF, Fong YF, Ng KL: Tubo-Ovarian Abscesses: Epidemiology and Predictors for Failed Response to Medical Management in an Asian Population. Infect. Dis. Obstet. Gynecol. 2019; 2019: 4161394–4161398. PubMed Abstract \| Publisher Full Text\n\nFouks Y, Cohen A, Shapira U, et al.: Surgical Intervention in Patients with Tubo-Ovarian Abscess: Clinical Predictors and a Simple Risk Score. J. Minim. Invasive Gynecol. 2019; 26(3): 535–543. S1553-4650(18)30335-2 [pii]. PubMed Abstract \| Publisher Full Text\n\nPerez-Medina T, Huertas MA, Bajo JM: Early ultrasound-guided transvaginal drainage of tubo-ovarian abscesses: a randomized study. Ultrasound in Obstetrics & Gynecology: The Official Journal of the International Society of Ultrasound in Obstetrics and Gynecology. 1996; 7(6): 435–438. PubMed Abstract \| Publisher Full Text\n\nGreenstein Y, Shah AJ, Vragovic O, et al.: Tuboovarian abscess. Factors associated with operative intervention after failed antibiotic therapy. J. Reprod. Med. 2013; 58(3-4): 101–106. PubMed Abstract\n\nGüngördük K, Guzel E, Asicioğlu O, et al.: Experience of tubo-ovarian abscess in western Turkey. Int. J. Gynaecol. Obstet. 2014; 124(1): 45–50. S0020-7292(13)00504-3 [pii] Publisher Full Text\n\nFarid H, Lau TC, Karmon AE, et al.: Clinical Characteristics Associated with Antibiotic Treatment Failure for Tuboovarian Abscesses. Infect. Dis. Obstet. Gynecol. 2016; 2016: 5120293–5120297. PubMed Abstract \| Publisher Full Text\n\nKinay T, Unlubilgin E, Cirik DA, et al.: The value of ultrasonographic tubo-ovarian abscess morphology in predicting whether patients will require surgical treatment. Int. J. Gynaecol. Obstet. 2016; 135(1): 77–81. S0020-7292(16)30204-1 [pii]. Publisher Full Text\n\nWagner C, Sauermann R, Joukhadar C: Principles of antibiotic penetration into abscess fluid. Pharmacology. 2006; 78(1): 1–10. 94668 [pii]. PubMed Abstract \| Publisher Full Text\n\nAlay I, Kaya C, Karaca I, et al.: The effectiveness of neutrophil to lymphocyte ratio in prediction of medical treatment failure for tubo-ovarian abscess. J. Obstet. Gynaecol. Res. 2019; 45(6): 1183–1189. Publisher Full Text\n\nHabboub AY: Middlemore Hospital experience with tubo-ovarian abscesses: an observational retrospective study. Int. J. Women's Health. 2016; 8: 325–340. PubMed Abstract \| Publisher Full Text\n\nJaiyeoba O, Lazenby G, Soper DE: Recommendations and rationale for the treatment of pelvic inflammatory disease. Expert Rev. Anti-Infect. Ther. 2011; 9(1): 61–70. PubMed Abstract \| Publisher Full Text\n\nReich H: Laparoscopic Treatment of Tubo-ovarian and Pelvic Abscess. Azziz R, Murphy AA, editors. Practical Manual of Operative Laparoscopy and Hysteroscopy. New York, NY: Springer; 1997.\n\nRosen M, Breitkopf D, Waud K: Tubo-ovarian abscess management options for women who desire fertility. Obstet. Gynecol. Surv. 2009; 64(10): 681–689. PubMed Abstract \| Publisher Full Text\n\nHatcher J, Costelloe C, Cele R, et al.: Factors associated with successful completion of outpatient parenteral antibiotic therapy (OPAT): A 10-year review from a large West London service. Int. J. Antimicrob. Agents. 2019; 54(2): 207–214. S0924-8579(19)30094-9 [pii]. PubMed Abstract \| Publisher Full Text\n\nPatrick L, Michel C, Jean-Luc P, et al.: Drainage of Tubo - Ovarian Abscess: DTOA. Reference Source" }	[ { "id": "282007", "date": "12 Jun 2024", "name": "Toni Darville", "expertise": [ "Reviewer Expertise Infectious disease specialist who studies chlamydia pathogenesis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written paper describing results of medical only versus medical and surgical treatment of TOA. The data acquisition and statistical methods are appropriate and the results are well presented. This is an important question in clinical medicine and the authors present non-biased results. Patient numbers are sufficient to make some conclusions based on the data despite it being a retrospective study.\n\nThe tables are easy to follow. Since multiple organisms were obtained from patients it would be helpful to include the numbers of patients that grew specific microorganisms so that the reader can appreciate whether specific pathogens were more or less common - to potentially aid in determining antibiotic choices. Data on specific antibiotics used in the patients should also be included.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-386
https://f1000research.com/articles/10-1046/v1	15 Oct 21	{ "type": "Research Article", "title": "Stacked deep analytic model for human activity recognition on a UCI HAR database", "authors": [ "Ying Han Pang", "Liew Yee Ping", "Goh Fan Ling", "Ooi Shih Yin", "Khoh Wee How", "Liew Yee Ping", "Goh Fan Ling", "Ooi Shih Yin", "Khoh Wee How" ], "abstract": "Background Owing to low cost and ubiquity, human activity recognition using smartphones is emerging as a trendy mobile application in diverse appliances such as assisted living, healthcare monitoring, etc. Analysing this one-dimensional time-series signal is rather challenging due to its spatial and temporal variances. Numerous deep neural networks (DNNs) are conducted to unveil deep features of complex real-world data. However, the drawback of DNNs is the un-interpretation of the network's internal logic to achieve the output. Furthermore, a huge training sample size (i.e. millions of samples) is required to ensure great performance. Methods In this work, a simpler yet effective stacked deep network, known as Stacked Discriminant Feature Learning (SDFL), is proposed to analyse inertial motion data for activity recognition. Contrary to DNNs, this deep model extracts rich features without the prerequisite of a gigantic training sample set and tenuous hyper-parameter tuning. SDFL is a stacking deep network with multiple learning modules, appearing in a serialized layout for multi-level feature learning from shallow to deeper features. In each learning module, Rayleigh coefficient optimized learning is accomplished to extort discriminant features. A subject-independent protocol is implemented where the system model (trained by data from a group of users) is used to recognize data from another group of users. Results Empirical results demonstrate that SDFL surpasses state-of-the-art methods, including DNNs like Convolutional Neural Network, Deep Belief Network, etc., with ~97% accuracy from the UCI HAR database with thousands of training samples. Additionally, the model training time of SDFL is merely a few minutes, compared with DNNs, which require hours for model training. Conclusions The supremacy of SDFL is corroborated in analysing motion data for human activity recognition requiring no GPU but only a CPU with a fast- learning rate.", "keywords": [ "smartphone", "one-dimensional motion signal", "activity recognition", "stacking deep network", "discriminant learning" ], "content": "Introduction\n\nHuman activity recognition (HAR) can be categorized into vision-based and sensor-based. In vision-based HAR, an image sequence, in the form of video, recording the human activity is captured by a camera.1 This sequence will be analysed to recognize the nature of an action. This system is applied for surveillance, human-computer interaction and healthcare monitoring. For sensor-based HAR, human activities are captured by inertial sensors, such as accelerometers, gyroscopes or magnetometers. Among these approaches, sensors are more favourable due to their lightweight nature, portability and low energy usage.2 With the advancement of mobile technology, smartphones are equipped with high-end components. Accelerometer and gyroscope sensors embedded in the smartphone make it feasible as an acquisition device for HAR. Smartphone-based HAR has been an area of contemporary research in recent years.3–9 In this work, we categorize the smartphone-based HAR as part of the sensor-based HAR. Activity inertial signals are collected through smartphone sensors.\n\nHand-crafted approaches using manually computed statistical features have been proposed.10–12 These authors applied various machine learning techniques such as decision tree, logistic regression, multilayer perceptron, naïve Bayes, Support Vector Machine etc. to classify the detected activities. The performance of the handcrafted approaches might be affected when dealing with complex scenarios due to their feature representation incapability. The algorithms could easily plummet into the local minimum despite the global optimal.\n\nHence, various deep neural networks (DNNs) were explored in HAR owing to the capability of extracting informative features. DNN is a machine learner that can automatically unearth the data characteristics hierarchically from lower to higher levels.13 The work of Ronao and Cho (2016),14 Lee et al. (2017)15 and Ignatov (2018)16 explored the deep convolutional neural networks by exploiting the activity characteristics in the one-dimensional time-series signals captured by the smartphone inertial sensors. The empirical results substantiated that the extracted deep features were crucial for data representation with promising recognition performance.\n\nZeng et al. (2014) proposed a modified convolutional neural network to extract scale-invariant characteristics and local dependency of the acceleration time-series signal.17 The weight sharing mechanism in the convolutional layer was modified. Unlike in the vanilla model where the local filter weights were shared by all positions within the input space, the authors incorporated a more relax weight sharing strategy (partial weight sharing) to enhance the performance.\n\nRecurrent Neural Network (RNN) was proposed to process sequential data by analysing previously inputted data and processing it linearly. Due to the vanishing gradient problem, RNN was enhanced and Long Short-Term Memory – LSTM was introduced. Chen et al. (2016) explored the feasibility of LSTM in predicting human activities.18 Empirical results demonstrated an encouraging performance of LSTM in HAR. Further, an enhanced version of LSTM, known as bidirectional LSTM, was proposed.19 Unlike LSTM, bidirectional LSTM tackles both past and future information during the feature analysis. With this, a richer description of features could be extracted for classification.\n\nA cascade ensemble learning (CELearning) model was proposed for smartphone-based HAR.20 There are multiple layers in this aggregation network and the model goes deeper layer by layer. Each layer contains Extremely Gradient Boosting Trees, Random Forest, Extremely Randomized Trees and Softmax Regression. The CELearning model gains higher performance, and the training process is rather simple and efficient. Besides, Hierarchical Multi-View Aggregation Network (HMVAN) is also one of the aggregation models.21 This model integrates features from various feature spaces in a hierarchical context. In this network, three aggregation modules from the aspect of feature, position and modality levels are designed.\n\nIn DNNs, there are learning modular components in multiple processing layers for multiple-level feature abstraction. These layers are trained based on a versatile learning principle, which does not require any manual design by experts.22 These DNNs accomplish excellent performances in pattern recognition. However, these networks are not well trained if they have limited training samples, leading to performance degradation. Furthermore, there is a lack of theoretical ground on how to fine-tune the gigantic hyper-parameter series.21 The outstanding accomplishment of DNNs can only be achieved if and only if sufficient training data is accessible for fine-tuning the large parameter set. A high specification of GPU is needed to train the network from gargantuan datasets.\n\nThus, a stacking-based deep learning model for smartphone-based HAR is proposed. Inspired by the hierarchical learning in the DNNs, the proposed stacked learning network is aggregated with multiple learning modules, one after another, in a hierarchical framework. Specifically, a discriminant learning function is implemented in each module for discriminant mapping to generate discriminative features, level by level. The lower (generic) to higher level (deeper) features are input to a classifier for activity identification. This proposed approach is termed Stacked Discriminant Feature Learning, coined as SDLF.\n\nThe contributions of this work are summarized in three-fold:\n\n1. A deep analytic model is proposed for smartphone based HAR to extract deep features without the need of a gigantic training set and tenuous hyper-parameter tuning.\n\n2. An adaptable modular model is developed with a discriminant learning function in each module to extract discriminant features from lower to higher levels demanding no graphics processing unit (GPU) but only a central processing unit (CPU) with a fast-learning rate.\n\n3. An experimental analysis using various performance evaluation metrics (i.e. recall, precision, the area under the curve, computational time, etc.) with subject-independent protocol implementation in which there is no overlap in subjects between training and testing sets.\n\n\nMethods\n\nSmartphone inertial sensors were used to capture 3-axial linear (total) acceleration and 3-axial angular velocity signals. These signals were pre-processed into time- and frequency-domain features, as listed in Table 1. Next, the pre-processed data was inputted into the Stacked Discriminant Feature Learning (SDFL) for feature learning. The extracted feature template was fed into the nearest-neighbour (NN) classifier for classification. The overview of the system is illustrated in Figure 1.\n\nSDFL is a pile of multiple discriminant learning layers interleaved with a nonlinear activation unit, as illustrated in Figure 2. By cascading multiple discriminant learning modules, each layer of SDFL learns based on the input data and the learned nonlinear features of the preceding module. The depth of the stacking layer is determined using the database subset. If the performance is not improving but showing degradation, the depth of the stacking layer is determined. In this case, the depth of three showed the optimal performance, so we adopted this architecture with three layers. To be detailed, the first discriminant learning module learns based on the input data and the second learning module learns based on an input vector (concatenating the input data and the learned features of the first learning module). This is similar to the third learning process where the third learning module learns based on an input vector (comprising the input data and the learned features of the second learning module).\n\nLet xiyii=1N be a set of N transformed data, yi is the class label of xi, C is the number of training classes, each of C classes has a mean μj and total mean vector μ=1N∑i=1Nmjμj with mj denotes the number of training samples for jth class. In the first learning layer, the input vector is the transformed data xi. The computation of the intrapersonal scatter matrix Σintra and interpersonal scatter matrix Σinter are defined as:\n\nwhere T denotes a transpose operation. Next, a linear transformation Φ is computed by maximizing the Rayleigh coefficient. With this optimization, the data from the same person could be projected close to each other, while data from different people is projected as far apart as possible. This optimization function is termed as Fisher’s criterion,23\n\nThe mapping Φ is constructed through solving the generalized eigenvalue problem,\n\nThe learned features are produced through the projection of the input data xi onto the mapping subspace,\n\nx̂ is transformed to C−1 dimensions. We denote l for the index of modular layer in SDFL. The learned feature vector of the first modular unit is notated as x̂il=1=x̂i1. A nonlinear input-output mapping is applied to x̂i1 via a nonlinear activation function. In this study, we adopt a sigmoid function, xˇi=Sx̂i=11+e−x̂i for the nonlinear projection. To be specific, xˇi1=11+e−x̂i1 is the nonlinear learned features of the first modular unit.\n\nFor deeper modules, the input vector of the respective module is a stacking vector containing the input data and the learned features, i.e. zil=xixˇil−1 where l=2 and 3. The intrapersonal scatter matrix Σintral and interpersonal scatter matrix Σinterl are formulated,\n\nIn this case, μjl is the jth class mean computed from the input vectors of jth class, zil∈Cj and the total mean vector μl=1N∑i=1Nmjμjl at lth modular unit. The final feature vector is the nonlinear learned features of each modular layer,\n\n\nResults\n\nWe scrutinized how well SDFL could analyse the inertial data and correctly classify those activities. The experimental hardware platform was constructed on a desktop with an Intel® Core™ i7-7700 processor with 4.20 GHz and 48.0 GB main memory; whereas the experimental software platform was a 64-bit operating system of Windows 10 with Matlab R2018a (MATLAB, RRID:SCR_001622) software (An open-access alternative that provides an equivalent function is GNU Octave (GNU Octave, RRID:SCR_014398)).\n\nWe used the UCI HAR dataset12: There were 30 subjects with 7352 training samples and 2947 testing samples. Each subject was required to carry a smartphone (Samsung Galaxy SII) on the waist and perform six different activities. The activities were “walking”, “walking_upstairs”, “walking_downstairs”, “sitting”, “standing” and “laying”.\n\nThe generalization level of SDFL was evaluated in a user-independent scenario. SDFL was trained using training samples from a group of users. Then, the model was applied to new users without the necessity of collecting additional samples of these new users to retrain the model. In this experiment, the UCI HAR dataset was partitioned into two sets: 70% of the volunteers were selected to generate the training data and the remaining 30% of the volunteers’ data was used as the testing data. There was no subject overlapping between the training and test data sets. Table 2 records the performance of SDFL and Table 3 records the performance comparison with other approaches.\n\nTable 4 tabulates the computational time. The computational time of SDFL is benchmarked with that of the ordinary methodology, which is directly performing classification on the pre-processed data. Instead of using a multiclass support vector machine as in,12 we adopt Nearest Neighbour (NN) classifier for classification because the focus of this work is the feature extraction capability and the classification is standardized with the simplest classifier, i.e. NN.\n\nClassifier = Nearest Neighbour (NN) classifier.\n\n\nDiscussion\n\nFrom the empirical results, we observed that the proposed SDFL was able to demonstrate superior classification performance compared to most of the existing techniques, even though a simple classifier was adopted in the system. The exceptional performance of SDFL explains the capability of SDFL in capturing the essence of the inertial data without heavily depending on the classifier. Furthermore, SDFL also exhibited its superiority to most of the existing approaches, including deep learning models. To be specific, SDFL obtained an accuracy of 96.3%, whilst Deep Belief Network’s accuracy was 95.8%,4 CNN achieved 95.75% accuracy14 and ANN’s accuracy was 91.08%.\n\nLast but not least, it was discerned that the performance of SDFL is on a par with the Cascade Ensemble Learning model (CELearning).20 Both approaches are ensemble learning methods with multiple layers for data learning. The key difference between these approaches is the analysis algorithms in each layer. CELearning is comprised of four different classifiers, i.e. Random Forest, Extremely Gradient Boosting Trees, Softmax Regression and Extremely Randomized Trees and the final classification result is obtained through the last layer via the score-level fusion of the four complex classifiers. On the other hand, in SDFL, merely Rayleigh coefficient optimization is implemented to extract the low-to-higher level of discriminant features. Further, a simple classifier, i.e. NN classifier, is adopted in SDFL. This deduces that the discrimination capability of SDFL primarily depends on the SDFL modular model to extract discriminant features demanding no complex classifier.\n\nFrom Table 4, we can notice that the overall training and testing time of SDFL are much lesser than those of the benchmark method. On average, SDFL just needs ~4.3×10−4seconds per sample (sps) for the training phase and ~4.2×10−4 sps for the testing phase. The fast feature learning of SDFL and the dimensionality reduction in SDFL to project the data onto a lower-dimensional subspace are the main reasons for having such an efficient computation.\n\n\nConclusions\n\nA cascading learning network for human activity recognition using smartphones is proposed. In this network, a chain of independent discriminant learning modules is aggregated, layer by layer in a stackable framework. Each layer is constituted by a discriminant analysis function and a nonlinear activation function to effectively extract the rich features from the inertial data. This proposed SDFL network possesses characteristics of good performance even on small-scale training sample sets, as well as less hyper-parameter fine-tuning, and fast computation compared with the other deep learning networks. Despite showing computational efficiency, the proposed network also demonstrated its classification superiority to most of the state-of-the-art approaches with an accuracy score of ~97% in differentiating human activity classes.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nPoppe R: A survey on vision-based human action recognition. Image Vis. Comput. 2010; 28(6): 976–990. Publisher Full Text\n\nAhmed N, Rafiq JI, Islam MR: Enhanced Human Activity Recognition Based on Smartphone Sensor Data Using Hybrid Feature Selection Model. Sensors . Jan. 2020; 20(1): 317. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCao L, Wang Y, Zhang B, et al.: GCHAR: An efficient Group-based Context—aware human activity recognition on smartphone. J. Parallel Distrib. Comput. Aug. 2018; 118: 67–80. Publisher Full Text\n\nLi H, Trocan M: Deep learning of smartphone sensor data for personal health assistance. Microelectronics J. Jun. 2019; 88: 164–172. Publisher Full Text\n\nHernández F, Suárez LF, Villamizar J, et al.: Human Activity Recognition on Smartphones Using a Bidirectional LSTM Network. 2019 22nd Symp. Image, Signal Process. Artif. Vision, STSIVA 2019 - Conf. Proc. 2019: 1–5. Publisher Full Text\n\nYang Z, Raymond OI, Zhang C, et al.: DFTerNet: Towards 2-bit Dynamic Fusion Networks for Accurate Human Activity Recognition. IEEE Access . Jul. 2018; 6: 56750–56764. Publisher Full Text\n\nYang J, MN N, PP S, et al.: Deep Convolutional Neural Networks on Multichannel Time Series for Human Activity Recognition. IJCAI. 2015.\n\nSun J, Fu Y, Li S, et al.: Sequential Human Activity Recognition Based on Deep Convolutional Network and Extreme Learning Machine Using Wearable Sensors. J. Sensors . 2018; 2018: 8580959. Publisher Full Text\n\nNweke HF, Teh YW, Al-garadi MA, et al.: Deep learning algorithms for human activity recognition using mobile and wearable sensor networks: State of the art and research challenges. Expert Systems with Applications. Elsevier Ltd; Sep. 01, 2018; vol. 105. . pp. 233–261. Publisher Full Text\n\nKwapisz JR, Weiss GM, Moore SA: Activity recognition using cell phone accelerometers. ACM SIGKDD Explor. Newsl. 2011; 12(2): 74–82. Publisher Full Text\n\nWu W, Dasgupta S, Ramirez EE, et al.: Classification accuracies of physical activities using smartphone motion sensors. J. Med. Internet Res. Sep. 2012; 14(5): e130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnguita D, Ghio A, Oneto L, et al.: A public domain dataset for human activity recognition using smartphones.2013. Publisher Full Text\n\nTemitope Yekeen S, Balogun AL, Wan Yusof KB: A novel deep learning instance segmentation model for automated marine oil spill detection. ISPRS J. Photogramm. Remote Sens. Sep. 2020; 167: 190–200. Publisher Full Text\n\nRonao CA, Cho SB: Human activity recognition with smartphone sensors using deep learning neural networks. Expert Syst. Appl. 2016; 59: 235–244. Publisher Full Text\n\nLee SM, S M, Cho H, et al.: Human Activity Recognition From Accelerometer Data Using Convolutional Neural Network. IEEE Int. Conf. Big Data Smart Comput. (BigComp). 2017; vol. 62, pp. 131–134. Publisher Full Text\n\nIgnatov A: Real-time human activity recognition from accelerometer data using Convolutional Neural Networks. Appl. Soft Comput. J. 2018; 62: 915–922. Publisher Full Text\n\nZeng M, et al.: Convolutional Neural Networks for human activity recognition using mobile sensors Article.2014: 381–388. Publisher Full Text\n\nChen Y, Zhong K, Zhang J, et al.: LSTM Networks for Mobile Human Activity Recognition. no. Icaita. 2016; pp. 50–53. Publisher Full Text\n\nYu S, Qin L: Human activity recognition with smartphone inertial sensors using bidir-LSTM networks. Proc. - 2018 3rd Int. Conf. Mech. Control Comput. Eng. ICMCCE 2018 . 2018: 219–224. Publisher Full Text\n\nXu S, Tang Q, Jin L, et al.: A cascade ensemble learning model for human activity recognition with smartphones. Sensors (Switzerland) . May 2019; 19(10). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang X, Wong Y, Kankanhalli MS, et al.: Hierarchical multi-view aggregation network for sensor-based human activity recognition. PLoS One . 2019; 14(9): e0221390. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLecun Y, Bengio Y, Hinton G: Deep learning. Nature . 2015; 521: 436–444.\n\nFukunaga K: Introduction to Statistical Pattern Recognition. Elsevier; 1990.\n\nSeto S, Zhang W, Zhou Y: Multivariate time series classification using dynamic time warping template selection for human activity recognition. Proc - 2015 IEEE Symposium Series on Computational Intelligence, SSCI 2015. 2015: 1399–1406. Publisher Full Text\n\nRonao CA, Cho SB: Recognizing human activities from smartphone sensors using hierarchical continuous hidden Markov models. Int. J. Distrib. Sens. Networks . 2017; 13(1). Publisher Full Text" }	[ { "id": "97058", "date": "25 Nov 2021", "name": "Cheng-Yaw Low", "expertise": [ "Reviewer Expertise Stacked neural networks", "deep neural 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\nThis manuscript proposes a stacked deep analytic model, dubbed stacked discriminant feature learning (SDFL), for mobile-platform human activity recognition (HAR). Based on the reported experimental results, the layer-wise SDFL trained with small-scale training data outperforms the SOTAs, including the conventional (end-to-end-trained) deep neural networks.\n\nMy concerns are as follows:\nObjective 2: The authors claim that SDFL learns low-level to higher-level feature representation. This hypothesis should be demonstrated, if possible. Otherwise, I think this objective should be revised accordingly.\n\nObjective 3: The authors should elaborate the subject-independent protocol from the motivation section as a problem statement to be resolved. In addition to that, the advantages of the subject-independent protocol should also be revealed.\n\nThe methodology section is ambiguous.\na) For clarity, the data/feature dimensionalities for all mathematical equations should be indicated accordingly.\nb) The SDFL hyper-parameters are unknown?\n\nThe experiment section may not be convincing as it is reliant on only a single dataset, and there is no comprehensive analysis conducted.\na) For reproducibility, the parameter configurations should be disclosed.\nb) It would be better if an ablation study is presented, e.g., exploration of the number of SDFL layers, etc. (refer to [141]). Furthermore, the baseline performance, the accuracy for the preprocessed data prior to SDFL learning, is not presented.\nc) I suggest the authors include additional small-scale HAR datasets for more extensive experiments.\n\nd) I think cross-validation should also be performed.\ne) I suggest the authors double-check the results reported for precision, recall, f-score, and accuracy. It is rare that these varying matrices give the same value, approximately 96.3%. Alternatively, the confusion matrices for these evaluation matrices should be disclosed.\n(f) In Table 4, I think the inference time should be computed for the SDFL and other conventional models, in place of the pre-processed data? Otherwise, this comparison may not be meaningful.\n(g) In Table 3, the authors compare SDFL with [24], [25], [4], [3], etc., however, not all are reviewed in the related work section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "7813", "date": "18 Feb 2022", "name": "Ying Han Pang", "role": "Author Response", "response": "First of all, we would like to express our heartiest thanks to the Editor-in-Chief and Reviewers who have provided us with many insightful comments and a chance to improve our works. Objective 2: The authors claim that SDFL learns low-level to higher-level feature representation. This hypothesis should be demonstrated, if possible. Otherwise, I think this objective should be revised accordingly. Response: Thanks for the feedback. Authors have revised Objective 2 for better clarification. Objective 3: The authors should elaborate the subject-independent protocol from the motivation section as a problem statement to be resolved. In addition to that, the advantages of the subject-independent protocol should also be revealed. Response: Authors have included the preference of subject-independent solution in real-time applications in the Motivation section for better relating the motivation and the Objective 3. a) For clarity, the data/feature dimensionalities for all mathematical equations should be indicated accordingly. b) The SDFL hyper-parameters are unknown? Response: Authors have included dimensionalities of data/feature, number of training classes C and the generated final feature vector in the Methods section for better clarification. Parameters of SDFL are the number of stacking layers, nonlinear activation function, dimensions of intermediate feature vectors and dimensions of the final feature vector. The Method section has been revised to include the information of these parameters for better clarification. a) For reproducibility, the parameter configurations should be disclosed. b) It would be better if an ablation study is presented, e.g., exploration of the number of SDFL layers, etc. (refer to [141]). Furthermore, the baseline performance, the accuracy for the preprocessed data prior to SDFL learning, is not presented. c) I suggest the authors include additional small-scale HAR datasets for more extensive experiments. d) I think cross-validation should also be performed. e) I suggest the authors double-check the results reported for precision, recall, f-score, and accuracy. It is rare that these varying matrices give the same value, approximately 96.3%. Alternatively, the confusion matrices for these evaluation matrices should be disclosed. (f) In Table 4, I think the inference time should be computed for the SDFL and other conventional models, in place of the pre-processed data? Otherwise, this comparison may not be meaningful. (g) In Table 3, the authors compare SDFL with [24], [25], [4], [3], etc., however, not all are reviewed in the related work section. Response: The information of these parameters has been included in the Methods section for better clarification. Table 3 has been revised to include the benchmark method, that is Multiclass Support Vector Machine, proposed by the original author of the UCI database. This baseline performance which is the accuracy of the preprocessed data with Support Vector Machine has been included in the table. In order to have a better performance comparison with the existing methods, the testing protocol of this study follows the train-test split protocol defined by the database provider, i.e. this database has a version that is already split into training and test sets that contain data from different participants. This paper is using this version of the database. The results have been double-checked and the confusion matrix of SDFL has been included. Table 4 has been revised by just presenting the computational time of SDFL. With this, readers can have a picture of the training and inference time of the proposed SDFL. The Related Work section has been revised to include the reference in Table 3." } ] }, { "id": "99185", "date": "30 Nov 2021", "name": "Andrews Samraj", "expertise": [ "Reviewer Expertise AI", "Patterns", "Bionics", "ML and DEEP Learning", "signals", "sensors" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe motivation and contributions may be rewritten to make it simpler to understand the purpose and achievements of the work (purpose of deep features have to be mentioned).\n\nAny chances of an Intra personal scatter matrix with any other inter personal scattermatrix? How was the threshold or borderline decided? There needs to be explanations with sample values.\n\nThe used hardware and software details, and details about dataset should be shifted from result to any other section in methodology.\n\nSlight corrections on technical writings need to be carried out. (eg: Table 4 tabulates: can be changed as either: Table4 presents or it is tabulated in table 4)\n\nA sample subject wise (Personnel) data table would be nice if presented to know the inter personnel differences.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7814", "date": "18 Feb 2022", "name": "Ying Han Pang", "role": "Author Response", "response": "First of all, we would like to express our heartiest thanks to the Editor-in-Chief and Reviewers who have provided us with many insightful comments and a chance to improve our works. The motivation and contributions may be rewritten to make it simpler to understand the purpose and achievements of the work (purpose of deep features have to be mentioned). Response: The motivation and contribution have been revised for better clarification. Any chances of an Intra personal scatter matrix with any other inter personal scattermatrix? How was the threshold or borderline decided? There needs to be explanations with sample values. Response: The Method section has been revised for better clarification. The optimal projection in SDFL is computed by optimizing the Rayleigh coefficient for modelling the difference between the classes of data through maximizing the ratio of the inter-personal scatter matrix and intra-personal scatter matrix. The variability between data is contained in a subspace spanned by the eigenvectors corresponding to the number of class -1 largest eigenvalues. Hence, the threshold is C-1 in this work. The used hardware and software details, and details about dataset should be shifted from result to any other section in methodology. Response: The amendment has been done and the details have been shifted to the Method section. Slight corrections on technical writings need to be carried out. (eg: Table 4 tabulates: can be changed as either: Table4 presents or it is tabulated in table 4) Response: The correction has been done. A sample subject wise (Personnel) data table would be nice if presented to know the inter personnel differences. Response: Figure 3 is added to illustrate the inter-personnel inertial signal differences of standing activity" } ] } ]	1	https://f1000research.com/articles/10-1046
https://f1000research.com/articles/11-384/v1	01 Apr 22	{ "type": "Brief Report", "title": "Exploring pandemic-related stress and resilience among digital workers: A basic interpretive qualitative study", "authors": [ "Hawa Rahmat", "Alexius Weng-Onn Cheang", "Choon-Hong Tan", "Ah-Choo Koo", "Wei-Fern Siew", "Elyna Amir Sarji", "Kin-Meng Cheng", "Alexius Weng-Onn Cheang", "Choon-Hong Tan", "Ah-Choo Koo", "Wei-Fern Siew", "Elyna Amir Sarji", "Kin-Meng Cheng" ], "abstract": "Background: Following the COVID-19 pandemic, the world's socio-economic structure must adjust to operate within the new normal for each country for its citizens to survive. Many jobs are now operating online, which has changed regular workers into “digital workers”. The abrupt shift in the nature of work has caused pandemic-related stress among workers and raises the question - can these digital workers thrive during the pandemic? The study aims to evaluate pandemic-related stress faced by digital workers and their resilience and ability to thrive during the pandemic.\n\nMethods: A qualitative research methodology in the form of a thematic approach was used to gauge digital workers’ pandemic-related stressors and resilience strategies during the COVID-19 pandemic. 15 digital workers from various backgrounds were interviewed using a semi-structured questionnaire.\n\nResults: Five themes were identified as the pandemic-related stressors among digital workers. These include anxiety about COVID-19 infections, mental exhaustion, physical exhaustion, feeling insecure and uncertain about financial concerns. The resilience factors include an active approach towards problem-solving, a positive outlook even during suffering, staying focused on responsibility for others, managing one’s well-being and a strong reliance on faith.\n\nConclusions: The outcomes of the study were derived from the culmination of a qualitative approach that comprehended the feelings, ideas, and experiences of digital workers. Therefore, there is a need to conduct a quantitative analysis in order to test the hypotheses and assumptions of the study.", "keywords": [ "Digital workers", "resilience", "pandemic-related stress", "covid-19", "thematic approach" ], "content": "Introduction\n\nThe study aims to explore the pandemic-related stress experienced by digital workers and their resilience and ability to thrive during the pandemic.\n\nDigital workers are virtual workers responsible for enhancing and augmenting human work by combining artificial intelligence, machine learning, robotic process automation and analytics to automate business functions from end to end.1 Although this is the way of the future, the COVID-19 pandemic has accelerated this transformation by increasing the number of professions moving towards the digital domain since early 2020. This has shifted the nature of employment for most occupations around the world. As a result, most who have to work virtually (online) from home can be classified as a “digital worker”. These workers are mostly employed in high-skilled occupations that allow for flexibility in working from home.2 The Department of Statistics (DOS) recently released the data from a study on the impact of COVID-19 which illustrates that a large number of workers (44%) work from home.\n\nFor companies to ensure their long-term viability and survivability during the pandemic, workers must work from home (WFH) and adhere to strict standard operating procedures (SOP). Working from home often involves frequent online meetings (or sometimes a few meetings at once), sitting for long hours, increased screen time, communication challenges and distress due to virtual environments. Those working from home often also work more than the standard nine to five office hours without having clear boundaries for their time. This abrupt shift in the nature of work has caused anxiety and distress among digital workers. Their resiliency despite these challenges is a very interesting issue to be explored.\n\nA study by Lai, Ma, and Wang indicated a significant number of healthcare workers experienced pandemic-related stress.3 A total of 1257 healthcare workers from 34 hospitals in multiple regions of China participated in their study. The hospitals were equipped with fever clinics and wards to treat COVID-19 patients. It was found that women, nurses, and those in Wuhan experienced symptoms of depression, anxiety, insomnia and distress. Mosheva et al. specifically studied pandemic-related stress and resilience among physicians during the COVID-19 pandemic.5 Their study surveyed 1106 Israeli physicians during the COVID-19 outbreak. The results of the study showed an inverse relationship between resilience and anxiety. Mental exhaustion, anxiety about being infected, anxiety about COVID-19 affecting family members and sleep difficulties were all found to have a positive relationship with anxiety scores.\n\nThe studies by Lai et al. and Mosheva et al. demonstrated the reality faced by healthcare personnel.3,5 The current study, on the other hand, is focused on “digital workers” who have experienced a rapid change in the digitalization of work and education and a shift to work-from-home (WFH) approaches.17 Therefore, the study aims to explore digital workers’ pandemic-related stress, due to the high percentage of jobs that have gone digital.\n\nResilience is defined by the presence of protective personal, societal, and family factors and institutional safety nets which enable people to withstand adversity.4,6,7 However, the concept of resiliency also includes dangerous, adverse and life-threatening conditions which affect individual vulnerability. Therefore, the resiliency of a person at any moment is determined by the ratio between the presence of protective factors and the presence of hazardous circumstances. Some people exhibit a remarkable ability to withstand great stress, torture, trauma or disaster, known as “resiliency.” In other words, resilience can be understood as possessing a set of adaptive characteristics which allows an individual to cope and to recover from (or even thrive after) experiencing stress or trauma.\n\nA qualitative descriptive study was conducted by Ching, Cheung, Hegney, and Rees to explore the stressors and coping strategies of nursing students with differing levels of resilience and burnout during their clinical placement.13 The results showed that students with high resiliency scores in the quantitative phase of the research tended to display self-consciousness while students who had low scores of resilience tended to use self-blame. This study demonstrated that when faced with the stressors from their clinical placement, students with different burnout and resilience scores used various coping strategies. This poses the question whether a similar pattern appears for digital workers, as well as whether these workers can thrive despite the uncertainty and stress around their current state.\n\n\nMethods\n\nThis qualitative research project used a thematic approach to gauge pandemic-related stressors and resilience in the face of the COVID-19 pandemic. 15 participants (digital workers) from various backgrounds were interviewed using a -structured interview.16 In the qualitative approach, convenient sampling is used for data collection. The criteria of the participants were they must currently be home workers, who were normal full-time workers before the pandemic. They have to be working from home due to the COVID-19 pandemic. Based on the criteria, two digital workers were identified and recruited through the contact of one of our research team’s researchers in HR. Then the rest of the samples were selected through snowball sampling in which research participants were asked to assist the researchers in identifying other potential participants with the said criteria. 15 home workers participated in the study after the researchers reach feedback saturation.19,22\n\nData collection in qualitative research allows the researcher to answer pertinent questions, assess results, and forecast future probability and trends. Important demographic background information was also collected to gain insight into different points of view regarding pandemic-related stress and resilience among home workers. The method of data collection was a structured interview. The structured interview questions were developed based on the existing literature review and the pandemic situation. A structured interview is a kind of interviewing method in which all candidates are asked the same preset questions in the same sequence and these are then assessed using a standardized scoring system. The effectiveness of this strategy is nearly double that of a standard interview. Two sets of structured questions were asked. The first question was on pandemic-related stress: “Could you tell me what terrified you the most during the outbreak and why?” The second question was on the respondents’ resilience: “How do you encounter with this emotional disturbance?” Respondents had the option of answering in English or Malay, which would be translated following transcribing. The interviews lasted between 30 and 45 minutes. Data collection started on March 3, 2021 and ended on April 15, 2021. The interviews took place through telephone conversation which were recorded using pen and paper. Before each interview, informed consent was obtained from each participant. Participants were free to withdraw from the interview session if they did not want to be interviewed.\n\nTo validate the transcribed data, thorough verification was performed beginning with data input, data coding, and anonymization of excerpts. The transcribed data was also shown to the respondents to ensure the accuracy of the data. The study’s validity was further assessed via triangulation, which entails many people analysing the same data. The researcher selects moderators to ensure that the data is not skewed by what the researcher wants to see or hear in order to help overcome personal bias. Three individuals reviewed the interview questions and data to check its validity.\n\nThe respondents’ interviews were analyzed according to Braun and Clarke’s (2006) six steps for thematic analysis.18 Adapted from Braun and Clarke’s thematic analysis method, the first step involved transcription of the recorded interviews, reading through the text, and making initial notes in order to become familiar with the data. Second, sections of texts were highlighted and assigned codes to describe the content. Third, the data was then collated together into groups as identified by the codes. Fourth, patterns among the codes were identified and themes were formed. Any vague, and/or irrelevant codes were discarded. Those steps were conducted by HR with help of a research assistant. Fifth, the themes were reviewed by all the researchers to make sure that they accurately represented the data by comparing the themes with the data set. Sixth, the list of themes were named and defined to produce a coherent and substantial argument which applied to the facts obtained from the respondents’ own experiences. As a result, there were five themes extracted from the interview around pandemic-related stress: anxiety about COVID-19 infections, mental exhaustion, physical exhaustion, feeling insecure and uncertain about financial concerns. Meanwhile, four themes were extracted for resilience, which was based on Werner and Smith11: the active approach towards problem-solving, the tendency to maintain a positive outlook, staying focused on responsibility for others to manage one’s well-being and a strong reliance on faith to maintain a positive life view.\n\nEthical approval was granted by the Research Ethics Committee (REC) of Technology Transfer Office (TTO) (approval number: EA0752021).\n\nInformed oral consent was obtained from the respondents for the study and usage of data. The interviews took place over the phone and the informants were told that their data would be kept private and used solely for research purposes. The informants were also assured that they could stop the interview session at any point. The participants were sent a copy of the manuscript and approved the excerpts that were included for publication. The participants consented to the excerpts of their interviews being shared publicly, but not the full transcripts.\n\n\nResults\n\nAbout 40% (6) males and 60% (9) females participated in the study. Table 1 shows the detailed demographic background of the participants. The full dataset can be found in the Underlying data.16\n\nThe findings of the study provide valuable input on the pandemic-related stressors among these digital workers and their resiliency during the pandemic. The five themes extracted from the interviews included anxiety about COVID-19 infections, mental exhaustion, physical exhaustion, feeling insecure and uncertain about financial concerns as shown in Table 2. Excerpts from the interview transcripts can be found in the Underlying data.16\n\nThe first theme was anxiety about being infected with COVID-19. The respondents felt anxious not only about being infected with the virus but also had the fear of infecting family members after they returned home from work. At the same time, they also developed anxiety about losing their job, losing a loved one, and losing family members as confessed by one participant #3, who said “I was afraid of losing family due to COVID-19. Knowing some of my relatives and close friend died due to COVID-19 make me felt anxiety and worried”. One of the respondents was a recovered COVID-19 patient. These experiences of anxiety were seen even after the recovery, as mentioned by participant #7, “I developed post COVID-19 anxiety after being infected with it last 3 months. I called it as fear of long COVID symptoms. At that time, I was pregnant and now I still have a bit of asthmatic symptom. My worries are more towards my yet to be born baby”. The second theme was mental exhaustion. The respondents experienced sleep difficulties, especially working mothers with young children. Since home-based teaching and learning was implemented, most parents experienced issues with childcare as schools and childcare facilities were closed. The implementation of working from home coupled with home-based teaching and learning turned out to be very challenging for most parents and has affected their work efficiency and life satisfaction. Participant #10 claimed “I felt like I neglected my children’s needs and wants because I was focusing more on my work rather than looking after them. I felt like they were already at home, I forgot that they still need my attention and affection. Actually, I have a problem with my own time management. I am always unable to finish work on time. This situation makes me stressed and I felt I have neglected my children. I feel sad, guilty, and bad towards my children.” In short, parents had to take on the role of teachers at home while concurrently struggling with their own work meetings and deadlines. They experienced emotional distress from multitasking their responsibilities.\n\nThe third theme was physical exhaustion. The participants expressed that they perceived their employers had increased the workload because they thought workers had extra time since they were WFH. This created pandemic-related stress among the workers because there is no clear boundary regarding their work hours. The participants found themselves frequently working longer than the typical nine to five office hours. The effects of multitasking also led to physical cramps or exhaustion, and in one participant’s case, obesity. As highlighted by participant #10, “I felt physically tired and cramped. Even though I work from home, I felt like I was working 24 hours a day, with the kids playing tumble and the noises from their gadgets made me collapse physically and emotionally. I also felt that I have gain weight because I ate a lot when I am under pressure. I just can’t stop my cravings. So now I am scared of obesity I have mixed feelings of inability to finish work on time, improper mealtime, and never-ending household chores.” Moreover, participant #5 added that “I am facing physical exhaustion over reaction or misjudgment due to COVID-19 cases I developed a fear syndrome every time I heard ambulance sirens. I am asking myself, what to do next. This has made me feel not only mentally but physically exhausted. I am also afraid and nervous all the time about the pandemic situation, not able to ignore or accept news related to the pandemic because lack of awareness can cause panic if I suddenly received news about our close friends or relatives that were infected with COVID-19. The research has shown that experiencing stress may lead to obesity due to overeating, as food cravings was part of the body’s reaction to stress.9\n\nThe fourth theme was feeling insecure about the future. This includes concern about the future and whether the vaccine will work and businesses will return to normal. In addition, several major organizations have had to incorporate pay cuts or have had to close down and lay off employees. Therefore, participants tended to develop mixed feelings about losing their work skills, experiencing job insecurity, feeling abandoned and insecure about the new normal, as mentioned by participant #12 “I have a special needs son and I am the source of income in the family. If something happens to me, either I am infected with COVID-19 or lose my job due to COVID-19, who is going to feed my family and especially my son. I also worry if something happens to him if he is infected with COVID-19, how can he deal with it. Thinking about it make me feel insecure, worried, and anxious about my son’s future”. Interestingly, government employees tended to not share as much concern about losing their job compared to employees from the private sector, participant #2, a working mother married to a government servant confessed “I am lucky my husband works with the government, so he still has a steady income.” But I am just thinking, now we have to stay at home, I feel bored, there is nowhere to go now. That is only my worry now.”\n\nThe final theme was financial concern. Participant #11, a lecturer in a private college expressed concern about her salary deduction “salary deduction (due to less students registered in the college - I have a PhD with very low salary) Now, it is hard to find job - still looking for another job. Remain there until got new better offer”. The respondents feared losing their job or having salary deductions because they had to support their families and pay monthly mortgages (bank loans; car/housing/personal). Younger respondents tended to be very concerned about paying off their study loan. They expressed that, if they did not have a steady income, they would not be able to settle their debts and this would increase their stress and anxiety.\n\nResearch on birds, rodents, and humans suggests that developmental exposures to stress can improve components of attention, perception, learning, memory and problem-solving which are ecologically relevant in harsh-unpredictable situations.10 After several implementations of the Movement Control Orders (MCO) and observing events around the world, the exposure to such changes has developed resiliency in each of the digital workers participants to persevere in the face of the situation that befalls them now. In psychological adaptation, resilience is defined as an individual’s ability to recover quickly from transitions, diseases, injuries, or adversity.6\n\nIn this study, the resilience of the participants during the pandemic seemed to align with the compensatory model of resilience. In this compensatory model, resilience is viewed as a factor that neutralizes risk exposure. The result of the prediction is influenced by both risk and compensating factors.11 Similarly, the themes of resilience extracted from the interviews included an active approach towards problem-solving, the tendency to maintain a positive outlook even when they were suffering, staying focused on responsibility for others to manage one’s well-being and a strong reliance on faith to maintain a positive life view as shown in Table 3.\n\nThe themes for resilience are as follows:\n\n1) Active approach towards problem-solving\n\nThe respondents expressed that staying positive and being prudent in their spending helped them to remain calm. They also believed that having health insurance coverage was very crucial especially with the high rates of infection and hospitalizations. They felt assured that if they were to die from COVID-19 infection, at least there was support left behind for their family.\n\n2) Tendency to maintain a positive outlook\n\nMaintaining a positive outlook was among the effective ways to neutralize pandemic-related stress.8 The participants were very aware that all countries were currently working hard to fight the virus and, with the availability of the vaccines, there was hope that the situation would improve. They expressed these hopes despite the daily uncertainties they faced. Some respondents were also grateful for the stability of their spouse’s government job which made them feel secure as there was a stable income. However, private sector employees did not share this belief.\n\n3) Staying focused on responsibility for others to manage one’s well-being\n\nMany participants expressed that it was very important to stay focused during the pandemic by thinking about the responsibility they had for their family members. Some of the respondents reported that they had to stay healthy for their children. They did not want their children to see that they were weak, stressed, or worried. Many participants also stayed focused by thinking about their parents who were in their hometowns.7 Thus, they tended to be compliant to the SOPs so that they could live a normal life again. Doing whatever they could in order to ensure a good future for their family helped them to maintain their well-being. A positive outlook may influence the health of the whole family physically and mentally, which can indirectly spread positivity to others.8,9,12\n\n4) Strong reliance on faith to maintain a positive life view\n\nParticipants with a strong reliance on faith13,14 such as tawakkal to Allah (“trusting in God’s plan” for Muslims) and a hopeful view throughout the pandemic helped them to maintain a positive outlook on life which is still worthwhile and meaningful. In addition, participants also expressed their faith and confidence in the government as well as the Ministry of Health (MOH) in leading the fight against COVID-19.\n\n\nConclusion\n\nThis study explored the pandemic-related stressors among digital workers as well as their ability to manage their well-being during the pandemic. By using the thematic approach,15 five themes have been identified in order to understand the pandemic related stress among these digital workers, while the level of resilience of digital workers is seen to be similar to the existing level in the study conducted by Werner and Smith.11 The themes are anxiety about COVID-19 infection, mental exhaustion, physical exhaustion, feeling insecure about the future, and financial concerns. Meanwhile, the participants’ resilience throughout the pandemic found in this study seems to fit with the compensating model of resilience, i.e., active approach toward problem-solving, tendency to maintain a positive outlook, staying focused on responsibility for others to manage one’s well-being, and strong reliance on faith. Nonetheless, the study cannot be generalized to all digital workers in Malaysia because the goal of a qualitative method is to understand digital workers’ feelings, thoughts, and experiences, with a focus on pandemic-related stress and elements that encourage them to be resilient as a result of the pandemic. According to Carminati20 and Delmar,21 the goal of qualitative research is to provide in-depth explanations and interpretations, as well as unique contributions, rather than to generalize results. Thus, to test the hypotheses and assumptions, a quantitative analysis should be conducted. Future studies should also further solidify the criteria for “digital workers” during the pandemic.\n\n\nAuthor contributions\n\nMain author is responsible for the project planning and all authors contributed to the findings and discussion.\n\n\nData availability\n\nThe full transcripts cannot be publicly shared due to ethical restrictions. They are available upon request from the corresponding author.\n\nDANS: Exploring pandemic-related stress and resilience among digital workers. https://doi.org/10.17026/dans-zgt-tbpc16\n\nThis project contains the following underlying data:\n\n- Data_Interview.ods (contains participant demographic information and interview responses)\n\n- Interview sessions_Verbatim.pdf (contains deidentified transcript excerpts)\n\nThis project contains the following extended data:\n\n- Blank copy of interview questions.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThank you to the “digital workers” who participated in the study.\n\n\nReferences\n\nMaselli I, Fabo B: Digital workers by design? An example from the on-demand economy (No. 11030). Centre for European Policy Studies; 2015.\n\nTumin SA: How common is working from home. KRI Discussion paper.2020; 1–13. Reference Source\n\nLai J, Ma S, Wang Y, et al.: Factors associated with mental health outcomes among health care workers exposed to Coronavirus Disease 2019. JAMA Netw. Open. 2020; 3(3): e203976. PubMed Abstract \| Publisher Full Text\n\nRosenberg AR: Cultivating deliberate resilience during the coronavirus disease 2019 pandemic. JAMA Pediatr. 2020; 174(9): 817–818. PubMed Abstract \| Publisher Full Text\n\nMosheva M, Hertz-Palmor N, Dorman Ilan S, et al.: Anxiety, pandemic-related stress and resilience among physicians during the COVID-19 pandemic. Depress. Anxiety. 2020; 37(10): 965–971. PubMed Abstract \| Publisher Full Text\n\nKaplan CP, Turner S, Norman E, et al.: Promoting resilience strategies: A modified consultation model. Child. Sch. 1996; 18(3): 158–168.\n\nSamuelsson K, Barthel S, Colding J, et al.: Urban nature as a source of resilience during social distancing amidst the coronavirus pandemic.2020.\n\nVinkers CH, van Amelsvoort T , Bisson JI, et al.: Stress resilience during the coronavirus pandemic. Eur. Neuropsychopharmacol. 2020; 35: 12–16. PubMed Abstract \| Publisher Full Text\n\nTomiyama AJ: Stress and obesity. Annual review of psychology.2019; 70: 703–718.\n\nEllis BJ, Bianchi J, Griskevicius V, et al.: Beyond risk and protective factors: An adaptation-based approach to resilience. Perspect. Psychol. Sci. 2017; 12(4): 561–587. PubMed Abstract \| Publisher Full Text\n\nWerner EE, Smith RS: Journeys from childhood to midlife: Risk, resilience, and recovery. Cornell University Press; 2001.\n\nPrime H, Wade M, Browne DT: Risk and resilience in family well-being during the COVID-19 pandemic. Am. Psychol. 2020; 75(5): 631.\n\nChing SSY, Cheung K, Hegney DG, et al.: Resilience among nursing students in clinical placement.2020.\n\nChang MC, Chen PF, Lee TH, et al.: The Effect of Religion on Psychological Resilience in Healthcare Workers During the Coronavirus Disease 2019 Pandemic. Front. Psychol. 2021; 12.\n\nCoelho M, Menezes I: University Social Responsibility, Service Learning, and Students’ Personal, Professional, and Civic Education. Front. Psychol. 2021; 12: 436.\n\nRahmat, Dr Hawa (Multimedia university): Exploring Pandemic-related Stress and Resilience among Digital Workers. DANS. 2021. Publisher Full Text\n\nRahul D, Neena P, Abhipsa P: Impact of digital surge during Covid-19 pandemic: A viewpoint on research and practice. Int. J. Inf. Manag. 2020; 55: 102171. Published online 2020 Jun 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBraun V, Clarke V: Using thematic analysis in psychology. Qual. Res. Psychol. 2006; 3(2): 77–101. Publisher Full Text\n\nGuest G, Bunce A, Johnson L: How many interviews are enough? An experiment with data saturation and variability. Field Methods. 2006; 18(1): 59–82.\n\nCarminati L: Generalizability in qualitative research: A tale of two traditions. Qual. Health Res. 2018; 28(13): 2094–2101. PubMed Abstract \| Publisher Full Text\n\nDelmar C: “Generalizability” as recognition: Reflections on a foundational problem in qualitative research. Qualitative Studies. 2010; 1: 115–128.\n\nMason M: Sample size and saturation in PhD studies using qualitative interviews. Forum Qualitative Sozialforschung/Forum: Qualitative Social Research. 2010, August; 11(3)." }	[ { "id": "153596", "date": "01 Nov 2022", "name": "Nor Diana Mohd Mahudin", "expertise": [ "Reviewer Expertise Applied psychology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe goal of this paper is relatively straightforward: applying thematic analysis to 15 semi-structured interviews and drawing on the psychological resilience concept to understand how workers who previously worked from the office (WFO) but then had to change to work from home (WFH) due to the COVID-19 pandemic, see and overcome pandemic-related stress. Clearly, this is a timely issue in light of the recent changes that COVID has wrought. Some studies have attempted to investigate similar issues with teachers, students, parents, healthcare workers, and patients, among others. Naturally, extending the investigation to WFH workers is an understandable attempt. Given this background and my general curiosity about the findings, I have some serious concerns about the paper, as outlined below:\nWork cited and literature:\nThe first concern relates to the usage of the term “digital workers” in the title and throughout the manuscript to refer to the informants/participants in the study. The authors stated that digital workers are “virtual workers responsible for enhancing and augmenting human work by combining artificial intelligence, machine learning, robotic process automation and analytics to automate business functions from end to end” and then proceeded by stating that “most who have to work virtually (online) from home can be classified as a digital worker” (p. 3). I believe that the definition and criteria of the informants/participants in the study need to be elaborated. This is because working from home does not automatically turn individuals into digital workers. It is best to explain in what way the informants/participants in the study (those depicted on p. 5, e.g., QC assistant, admin executives, HR admin, teachers, lecturers, physiotherapist, etc.) fit the definition of a digital worker.\n\nStudies on pandemic-related stress and resilience cited in the Introduction are not aligned with the WFH workers or issues that the authors are attributing. I would also like to question how those studies are relevant to the current samples which the authors are investigating. Another bizarre example can be seen on p. 6 in the sentence, “Research on birds, rodents, and humans suggests that developmental exposures to stress can improve components of attention, perception, learning, memory and problem-solving which are ecologically relevant in harsh-unpredictable situations”. The authors need to show how this literature relates to the discussion of the findings. There are other examples too throughout the manuscript where the readers are confronted with a large number of claims and assertions that are ungrounded.\n\nIn light of these observations, I suggest doing the following steps as they can add value to the manuscript: (i) review, synthesise, and cite studies that are relevant and directly related to the target sample, i.e., WFH workers and issues of interest only; (ii) include a critical analysis of the relevant concepts and theories, and situate the study in the theoretical context of the literature; and (iii) since this is a qualitative study, make explicit the philosophical underpinning of the study.\nStudy design, method, and analysis:\nWhile the study design seems appropriate, the work is not technically sound in general.\n\nThe description of the method used is relatively brief, with some points requiring rechecking. For example, a semi-structured questionnaire was stated in the Abstract, but in the Method and Data Collection, structured interviews were stated.\n\nThe authors also stated, “….. all candidates are asked the same pre-set questions in the same sequence, and these are then assessed using a standardized scoring system” (p. 4), but no discussion regarding the results of this standardised scoring assessment is presented.\n\n“How do you encounter with this emotional disturbance?” is stated on p. 4. This sentence is incorrect.\n\n“The interviews took place through telephone conversation which were recorded using pen and paper” on p. 4 requires elaboration. What do the authors mean by “recorded using pen and paper”?\n\nIn the Analysis section, the authors stated, “The researcher selects moderators to ensure that the data is not skewed by what the researcher wants to see or hear in order to help overcome personal bias. Three individuals reviewed the interview questions and data to check its validity”. For a clearer description of this process, it is best that the authors describe (i) the researcher(s) who selected the moderators, (ii) who the moderators are and on what basis they were selected, as well as (iii) who the three individuals are and on what basis they were selected.\n\nSentences starting from \"...the first step involved transcription of the.....Fourth, patterns among the codes were identified and themes were formed. Any vague, and/or irrelevant codes were discarded. Those steps were conducted by HR with help of a research assistant\" are vague. Are the authors implying that it was the HR (best to write in full to avoid readers guessing its meaning) who conducted all these steps (together with a research assistant)? Are the HR personnel equipped with qualitative data analysis skills? All these aspects need to be clarified.\nResults and reproducibility:\nFull reproducibility is not always possible in qualitative studies, and in fact, it is not one of the objectives of conducting qualitative studies. However, looking at the excerpts provided in the manuscript and supplementary documents, there is a tendency to reproduce the excerpts in a very reduced form, with minimal or no interpretation of those data.\n\nIn particular, it is observed that the authors (i) summarised the themes of what the informants/participants said without analysing the discourse within the interaction; and (ii) treated the findings as reflective of all members of a given category in which the informants/participants are cast. These two aspects affect the conclusions and worth of the manuscript because only providing brief excerpts or quotes with little or no detailed analysis does not reflect a high-quality study.\nConclusion:\nIt is not at all clear as to why the authors assert the sentence, “Therefore, there is a need to conduct a quantitative analysis in order to test the hypotheses and assumptions of the study” in the Abstract and repeat it in the Conclusion section. It is best to describe explicitly the hypotheses and assumptions arising from the findings of the qualitative study.\n\nThe applied implications from the study, though cursorily explained, do not give readers a clear take-home message.\nOverall comment: While the manuscript addresses a worthy topic, it is hard to discern whether the study has been well executed from this reporting. If, on fuller reporting, the method and instruments used turn out to be blunt, then it is not clear how much interest will be aroused by short, structured interviews with no probes of not-so-well-defined samples of “digital workers”. On the other hand, if the study is sharp, with good analysis, then there may be a case for encouraging re-submission. However, if so, much more must be said about the method, participants, data, and analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "175132", "date": "14 Jun 2023", "name": "Nabisah Ibrahim", "expertise": [ "Reviewer Expertise Mental Health", "Educational Psychology", "Counseling", "Group Work and Intervention", "Gerontology", "Women 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\nThe objectives, procedures, and findings of the study are succinctly summarized in the abstract. It effectively drives home the need for more research into the stress that the COVID-19 epidemic caused for digital employees as well as their ability to remain resilient. However, there is a lack of specific information regarding the sample size and key findings, which might be fixed.\nThe study design and data collection procedures are adequately covered in the methods section, which is generally acceptable for addressing the research question. The use of qualitative interviews enables a thorough investigation of the experiences of digital employees.\nHowever, it is challenging to fully evaluate the study's methodological rigor due to the lack of information regarding participant characteristics, specific interview questions, intercoder reliability, and data saturation. A list of the precise themes covered would be beneficial in addition to the quick description of the structured interview questions. The analysis process is described, but there is a paucity of data on intercoder reliability and data saturation.\nTo maintain replicability and openness, it is essential to provide thorough information about the methodology and analysis. The entire research process is described in full, from participant recruitment to data gathering and analysis. Without these details, it might be difficult for other researchers to replicate the study or to compare the results to their own.\nIt is therefore recommended to provide a thorough explanation of the study's design, methodology, and analysis procedures. This will increase the transparency of the study, enable others to replicate it, and make it simpler to assess the study's technical soundness.\nThe provided text lacks the discussion part, which is a significant omission. Interpreting the results, comparing them to the body of literature, and providing insight into the study's implications and limits all depend on the discussion. The inclusion of the discussion section would significantly improve the research article's overall quality and thoroughness.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-384
https://f1000research.com/articles/11-383/v1	31 Mar 22	{ "type": "Brief Report", "title": "A Sanger sequencing-based method for a rapid and economic generation of SARS-CoV-2 epidemiological data: A proof of concept study to assess the prevalence of the A23403G SNP (D614G) mutation in Quito, Ecuador.", "authors": [ "Paula Leoro-Garzón", "Galo Leoro-Monroy", "David Ortega-Paredes", "Marco Larrea-Álvarez", "Gabriel Molina-Cuasapaz", "Pablo González-Andrade", "Gabriela Sevillano", "Camilo Zurita-Salinas", "Jeannete Zurita", "Paula Leoro-Garzón", "Galo Leoro-Monroy", "David Ortega-Paredes", "Marco Larrea-Álvarez", "Gabriel Molina-Cuasapaz", "Pablo González-Andrade", "Gabriela Sevillano", "Camilo Zurita-Salinas" ], "abstract": "The Single Nucleotide Polymorphism (SNP) A23403G associated with the D to G change in position 614 of the SARS-CoV-2 spike protein has recently become dominant. The most utilized and robust approach is the study of whole genome sequences, generally available at public databases. However, this technology is not suited for massive testing as it requires expensive reagents, equipment, and infrastructure. Consequently, developing rapid and accessible protocols will be fundamental for producing epidemiological data linked to this SNP, especially in countries with limited resources. This report has evaluated an easy cost-effective approach, based on sanger sequencing, for detection of the A23403G (D614G) mutation. This strategy was tested in SARS-CoV-2 positive samples collected in Quito during March and October of 2020. In March, a total of 264 out of 1319 samples yielded positive results (20%), while 777 out of 5032 (15%) did so in October. From these cases, almost all samples were associated with the G23403 (G614) variant (>98%). This technique proved to be reliable, reproducible, and might be expandable to study other mutations without major protocol amendments. The application of this method allowed the production of epidemiological data regarding the A23403G (D614G) mutation in Quito, where no previous reports were available. This approach will be crucial for producing relevant information for public health management, especially during the ongoing pandemic.", "keywords": [ "SARS-CoV-2 infection", "D614G", "epidemiology", "SNP", "Sanger sequencing", "low-cost method", "Quito-Ecuador" ], "content": "Introduction\n\nSARS-CoV-2 is the agent responsible for the COVID-19 pandemic that has caused almost 4 million deaths worldwide (World Health Organization (WHO) 2021). The genome of this virus is approximately 30-kb long (Bar-On et al. 2020). Two Open Reading Frames (ORFs) (1a and 1b), that cover most of its genome, encode non-structural proteins needed for viral replication as well as an RNA-dependent RNA polymerase (RdRp). Structural proteins, such as the Spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein are encoded by short sub-genomic RNAs (sgRNAs) (Kim et al. 2020). The S protein recognizes and binds to the angiotensin-converting enzyme 2 (ACE2) receptor to gain entry to hosts cells.\n\nEvidently, mutations in the gene encoding the S protein alter the affinity to the ACE2 receptor enhances infectivity (Watanabe et al. 2020), and increased infection capacity. The aspartate (D) to glycine (G) substitution related to the non-synonymous A23403G SNP (single nucleotide polymorphism) has been associated with the rapid spread of the virus (Yuan et al. 2020).\n\nThe A23403G (D614G) mutation was first described in early 2020, and rapidly detected in various regions around the globe (Isabel et al. 2020; Raghav et al. 2020; Dao et al. 2021; Elizondo et al. 2021; Molina-Mora et al. 2021; Pandey et al. 2021). Studies related to this mutation have used, in the majority, whole genome sequences (WGS). However, this approach is too expensive to be used in massive testing campaigns, especially in countries with limited resources. Moreover, the number of cases studied by WGS may not necessarily be representative of the number of cases detected by quantitative Polymerase Chain Reaction (qPCR) at these locations.\n\nAs a result, several methodologies have been developed as affordable alternatives that provide rapid detection of not only relevant SNPs in the S protein, but also SNPs in variants of concern (Hashemi et al. 2020; Bezerra et al. 2021; Chakraborty et al. 2021; Vogels et al. 2021). Low-cost rapid technologies have been put forward to investigate the A23403G (D614G) mutation. These technologies rely on: Probe-based real-time reverse transcriptase PCR (rRT-PCR) (Chan et al. 2022), an amplification refractory mutation system (ARMS) (Islam et al. 2021), and restriction fragment length polymorphism (RFLP) (Niranji & Al-Jaf 2021). Arguably, it is urgent to establish and standardize as many molecular tools as possible in order to expand the screening capacity of SARS-CoV-2 mutations. Consequently, and as an effort to scale up molecular diagnosis, we have developed and tested a cost-effective method based on Sanger sequencing (Jørgensen et al. 2021; Park et al. 2021) to detect the A23403G (D614G) mutation.\n\nThis investigation was carried out in Quito, the capital city of Ecuador, during March and October of 2020. The first SARS-CoV-2 case detected in the country was registered in March 2020, and from that point the virus has been reported in all 24 provinces. Until the 7th of July 2021, the number of positive cases in Ecuador was 467,878 (Johns Hopkins University & Medicine 2021). However, to this date, there are no studies reporting the prevalence of this mutation in the city or in the country, despite its predominance in various regions (Loureiro et al. 2021; Pandey et al. 2021; Viedma et al. 2021).\n\n\nMethods\n\nIn the present study, we evaluated 1319 samples collected during March and 5032 gathered during October of 2020 (all nasopharyngeal swabs) by Zurita & Zurita Laboratorios and Laboratorio Clínico Inmunolab from both symptomatic and asymptomatic patients (see Underlying data) (Sevillano 2022). The two laboratories carried out detection independently. These clinical laboratories do not perform quantification in routine samples; results were reported as positive or negative. Samples from the first time point (March 2020) were extracted using the High Pure Viral Nucleic Acid kit (Product No. 11858874001, Roche, Germany), and analyzed using the LightMix® Modular SARS and Wuhan CoV E-gene kits (Product No. 09155368001, Roche, Germany). Both genes were analyzed using a LightCycler® 480 Real-Time PCR. Samples collected in October 2020 were extracted using CommaPrep® RNA Extraction Columns (Product No. RP20, Biocomma, China), following the provided guidelines, and analyzed using STANDARD M nCoV Real-Time Detection kit (Product No. M-NCOV-01, SB Biosensor, Korea) on a Stratagene MX3005P Real-Time PCR System or Allplex™ SARS-CoV-2 Assay (Product No. RV10248X, Seegene Inc, Korea) on CFX96 Touch Real-Time PCR Detection System. Interpretation of results (positive/negative) were carried out based on the guidelines provided by the manufacturers. Both kits are currently approved by the ARCSA (National Agency for Health Regulation, Control and Surveillance of Ecuador) for their use in routine diagnostics.\n\nTo estimate the prevalence of the A23403G (D614G) mutation, the sample size was first calculated based on the prevalence of SARS-CoV-2 positive cases, 0.2 for March and October (see Underlying data) (Sevillano 2022). The following equation, with a 10% margin of error and a confidence level of 95%, was used for calculating such value:\n\nAccording to equation (1), where z is the statistic corresponding to the level of confidence, P is the expected prevalence, and d is the precision (Pourhoseingholi et al. 2013). The estimated number was 61, although we analyzed a total of 96 samples per month. Selection of samples was carried out using a random number generator.\n\nInitially, the sequence encoding for the SARS-CoV-2 S protein (Gene Bank accession number: MT252819.1) was utilized for designing primers using the Geneious Prime software Version 2019.2.3 (https://www.geneious.com/prime/). Primers flanked the region between nucleotide positions 23,301 and 23,511 of the of WH-Human 1 coronavirus (MN908947) reference genome. Primers were aligned to genomes reported in Latin American countries in order to identify potential catastrophic mutations within the annealing sites of the primers. Forward sequence (D614G_F2): 5’-GATGCTGTCCGTGATCCACA-3’; reverse sequence (D614G_R2): 5’-AAACAGCCTGCACGTGTTTG-3’. The resulting amplicon was expected to be 230bp long. In the A23403 (D614) variant, adenine is the primary nucleotide that encodes the amino acid aspartate at position 614 of the polypeptide. Glycine has been observed to replace this aspartate if guanine is present instead of adenine, this variant is known as G23403 (G614).\n\nAt the next step, PCR analyses were performed on the positive samples with the following conditions: 45 °C for 15 min, 95 °C for 2 min with 45 cycles of 95 °C for 10 sec and 60 °C for 50 sec, followed by the reading of dissociation curves at temperatures from 55 °C to 95 °C. The reaction used 10 μL GoTaq G2 Hot Start Colorless Master Mix (Product No. M7422, Promega, USA), 50 U of RocketScript RTase H Minus (Product No. BQ-042-101-04, Bioneer, Korea), 1 μL of EvaGreen 20x (Product No. 31000-31000-T, Biotium, USA), 0.75 μl of primers D614G_F2 and D614G_R2 10 mM, 10ng RNAse Inhibitor (Product No. RB0478, BioBasic, Canada) and template up to a final volume of 15 μL. Samples were analyzed on a Stratagene MX3005P Real-Time PCR system, using EvaGreen dye, and melting curves (dissociation curves) were analyzed using the MxPro application. This analysis permits the measurement of the temperature at which DNA strands separate into single strands, which provides a reference of the melting temperature at which 50% of DNA is denatured.\n\nFinally, all samples that produced an amplicon with a melting temperature around 81 °C were sent to be sequenced commercially using Sanger technology (Macrogen, Korea). The resulting sequences were aligned to the reference WH-Human 1 coronavirus (MN908947) genome using the Geneious Prime software Version 2019.2.3 (https://www.geneious.com/prime/).\n\n\nResults\n\nAt the first sampling point, 20% of the samples (264 out of 1319) tested positive for SARS-CoV-2. Likewise, at a later stage, 15% of the samples (777 out of 5032) were infected by the virus (Table 1).\n\nPer month, 96 samples were evaluated and, after sequencing, were assigned to their corresponding type (Table 2). From these samples 99% possessed a guanine at position 23403 (G614), while 1% contained an adenine at this position (D614). In March and October, the G23403 (G614) variant represented more than 98% of the samples (see Underlying data) (Sevillano 2022). However, the A23403 (D614) variant were registered only in the samples collected in March.\n\nThe sequence of 230 bp (Figure 1A) was sufficient to effectively identify the A23403G (D614G) mutation (Figure 1B). Moreover, high resolution melting curve analysis (HRM) was helpful to confirm the amplicon identity before Sanger sequencing, as the Tm value of the expected amplicon ranged from 78 °C to 82 °C. However, this approach was not suitable for genotype differentiation since no marked differences were observed between the wildtype version and the A23403G (D614G) mutation (Figure 1C).\n\nA) Adenine to guanine modification and its consequences for the S protein. Primers flanking the area of interest between nucleotide positions 23,301 and 23,511 of the WH-Human 1 coronavirus genome (MN908947), used as reference in the sequencing analysis. B) Segments of the electropherograms by Sanger sequencing indicating the A23403G mutation. C) Melting temperature analysis of selected samples for the missense mutation. All samples had a similar Tm of around 81 °C indicating the correct amplicon size, although not sufficient to discriminate between the G and D types.\n\n\nDiscussion\n\nSince its appearance in late 2019, SARS-CoV-2 has overwhelmed the entire global population with its geographical distribution and mutational rate. The virus has been constantly evolving into new variants with high genetic diversity. To study the G23403 (G614) variant, researchers commonly analyze whole-genome sequences, available in public databases e.g., GISAID (https://www.gisaid.org/), as well as sequences of their own.\n\nWGS is the most reliable and utilized method, notwithstanding its cost and inadequacy to work with a large number of samples. WGS is high-priced to be employed as a method for massive testing, predominantly in low-income countries. Consequently, several techniques have been put forward as alternatives for an economic and rapid detection of not only spike mutations, but also variants of concern (Rhee et al. 2019; Hashemi et al. 2020; Bezerra et al. 2021; Chakraborty et al. 2021; Islam et al. 2021; Park et al. 2021; Vogels et al. 2021; Chan et al. 2022).\n\nIn this investigation, the efficacy of a low-cost method, based on sanger sequencing, to detect the A23403G (D614G) mutation was assessed. This approach proved to be affordable, user-friendly and consistent, which simplifies its implementation in common laboratory facilities worldwide; especially in regions with limited health systems such as Sub-Saharan Africa and Latin America (Kirby 2020; Okoi & Bwawa 2020).\n\nThe primers employed to produce the amplicon were straightforwardly designed and can be easily adjusted to detect novel variants without major protocol amendments. Arguably, other mutations of relevance could be studied in the same way. For instance, the evaluated technique might prove useful for detecting the N439K mutation, associated with an increased affinity to ACE2 and reduced sensitivity to SARS-CoV-2 antibodies (Thomson et al. 2021).\n\nOther approaches have been proposed to study the A23403G (D614G) mutation. For example, one study successfully tested a combined point-of-care nucleic acid and antibody assay (Mlcochova et al. 2020). Similarly, another method effectively detected the mutation, although based on an engineered Cas12a guide RNA (Meng et al. 2021). Despite encouraging results, these techniques appear particularly laborious and expensive for laboratories with limited resources. Rapid and economic approaches have also been developed, which are based on (i) probe-based real-time reverse transcriptase PCR (qRT-PCR) (Chan et al. 2022), (ii) an amplification refractory mutation system (ARMS) RT (Islam et al. 2021) and (iii) restriction fragment length polymorphism (RFLP) (Niranji & Al-Jaf 2021), the latter method has proved particularly intricate to optimize as original results (Hashemi et al. 2020) had to be revised (Niranji & Al-Jaf 2021).\n\nIn the present research, a simple straightforward technique consisting of a simple PCR reaction and further sequencing was tested, thus reducing the steps required for sample processing. This study aimed to use melting curve analysis to identify the screened mutation. This approach has been utilized successfully to detect different viruses (Liu et al. 2018) and recently applied to detect SARS-CoV-2 mutations (Barua et al. 2021; Sarkar et al. 2021).\n\nUnfortunately, the melting curves obtained during amplification were not suitable to discriminate between variants. However, they did provide key information about the size of the amplicon, which is fundamental for sequencing the right samples. Clearly, this step must be optimized. Another limitation is that Sanger sequencing could be regarded as time consuming, although it brings several benefits as it is useful to detect developing mutations and even to differentiate variants of concern (Bezerra et al. 2021).\n\nData from the Emergency Operations Center of Pichincha (COE-Pichincha) showed that around 579 positive cases were registered in Quito by March 2020. In October 2020, the official number reported was 15,890 (CEO Provincial Pichincha 2021). However, no reports have been published with regards to the prevalence of the A23403G (D614G) mutation in Quito. In this investigation determined that 190 out of 192 samples (99%) belong to the G23403 (G614) variant, which was dominant at both time points. Other studies have similarly reported a predominance of this SNP in different regions (Loureiro et al. 2021; Pandey et al. 2021; Viedma et al. 2021); these outcomes show the rapid spread of this variant which might relate to a higher transmission rate, as suggested by certain authors (Arora et al. 2021; Ozono et al. 2021).\n\nAs aforementioned, in Ecuador, there are no studies reporting the prevalence of this missense mutation. Hence, to estimate its prevalence in the months studied, this research referred to GISAID (https://www.gisaid.org/) to retrieve WGS and metadata of SARS-CoV-2 from the period of study. Our in-silico study revealed that from the total of sequences registered in March, five belonged to the G23403 (G614) variant and three to A23403 (D614) variant. Likewise, in October, G23403 (G614) was predominant with four sequences out of five being this type (Table 3). Evidently, a major limitation of studying datasets is that they represent a small fraction of the cases detected by RT-PCR, especially in the periods of study.\n\nThe results of this study corroborate what can be inferred from WGS analysis, the G23403 (G614) proved to be the most common in samples from Quito during March and October of 2020. Arguably therefore, using this affordable method permits us to expand the information drawn from databases.\n\nThis scheme was not intended as a replacement of WGS or other PCR-based approaches. Instead, it was devised as a supplementary technique that could be used, in combination with the aforementioned methods, to rapidly generate ample data on the prevalence and epidemiology of relevant SNPs around the globe, mainly in regions with limited facilities and equipment for detection.\n\nThe importance of the present approach lies in the speed and easiness with which results can be obtained. This simple technique might help a large group of laboratories to report results quicker and more abundantly, which appears fundamental in the context of public health management during the ongoing pandemic.\n\nThe present outcomes demonstrate that the evaluated methodology produced reliable and reproducible information with regards to the A23403G (D614G) mutation. Using this approach, we showed that the G23403 (G614) variant was the most common, during the months of March and October of 2020, among samples from Quito, the capital city of Ecuador. This information contributes to expanding the results obtained by WGS, which will undoubtedly benefit public health decisions during novel outbreaks. Future work must place emphasis on testing a larger number of samples to accurately estimate the prevalence of this mutation in the area, and to assess its relationship with the transmissibility and severity of the disease.\n\n\nData availability statement\n\nRepository: A Sanger sequencing-based method for a rapid and economic generation of SARS-CoV-2 epidemiological data: A proof of concept study to assess the prevalence of the A23403G SNP (D614G) mutation in Quito, Ecuador.\n\nDOI: 10.17605/OSF.IO/PMVGJ (Sevillano 2022).\n\nThis project contains the following underlying data:\n\nData file 1. March 2020 Positive SARS-CoV-2 Tests\n\nData file 2. October 2020 Positive SARS-CoV-2 Tests\n\nData file 3. Samples distribution by period\n\nData file 4. March SARS-CoV-2 Sanger sequencing results of samples\n\nData file 5. October SARS-CoV-2 Sanger sequencing results of samples\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\nEthical approval\n\nThe ethical Committee of the General Coordination for Strategic Development in Health, as stipulated by the Ecuadorian Ministry of Public Health, approved the research described herein (Reference number: 074-2020). Data were anonymized for the purpose of protecting private information.\n\n\nAuthor contributions\n\nP.L-G.: methodology, editing, funding acquisition, methodology first draft; G.L-M.: conceptualization, editing, first draft; D.O-P.: conceptualization, methodology, supervision, final draft preparation, writing, editing; M.L-A.: conceptualization, final draft preparation, writing, editing; G.M-C.: methodology, editing, first draft, P.G-A.: methodology; G.S.: methodology, first draft; J.Z. and C.Z-S.: methodology, editing, funding acquisition.", "appendix": "References\n\nArora P, Pöhlmann S, Hoffmann M: Mutation D614G increases SARS-CoV-2 transmission. Signal Transduct Target Ther. 2021; 6: 1–2. Publisher Full Text\n\nBar-On YM, Flamholz A, Phillips R, et al.: SARS-CoV-2 (COVID-19) by the numbers. Elife. 2020; 9: e57309. PubMed Abstract \| Publisher Full Text\n\nBarua S, Hoque M, Kelly PJ, et al.: High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations. J Med Virol. 2021; 93: 5588–5593. Publisher Full Text\n\nBezerra MF, Machado LC, Carvalho V, et al.: A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern. Infect Genet Evol. 2021; 92: 101348–104910. Publisher Full Text\n\nCEO Provincial Pichincha: Situación cantonal por COVID-19. [WWW Document].2021. Reference Source\n\nChakraborty D, Agrawal A, Maiti S: Rapid identification and tracking of SARS-CoV-2 variants of concern. Lancet (London, England). 2021; 397: 1346–1347. PubMed Abstract \| Publisher Full Text\n\nChan CTM, Leung JSL, Lee LK, et al.: A low-cost TaqMan minor groove binder probe-based one-step RT-qPCR assay for rapid identification of N501Y variants of SARS-CoV-2. J Virol Methods. 2022; 299: 114333. PubMed Abstract \| Publisher Full Text\n\nDao MH, Phan LT, Cao TM, et al.: Genome-wide analysis of SARS-CoV-2 strains circulating in Vietnam: Understanding the nature of the epidemic and role of the D614G mutation. J Med Virol. 2021; 93: 5660–5665. Publisher Full Text\n\nElizondo V, Harkins GW, Mabvakure B, et al.: SARS-CoV-2 genomic characterization and clinical manifestation of the COVID-19 outbreak in Uruguay. Emerg Microbes Infect. 2021; 10: 51–65. PubMed Abstract \| Publisher Full Text\n\nHashemi SA, Khoshi A, Ghasemzadeh-moghaddam H, et al.: Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2. Infect Genet Evol. 2020; 86: 104625. PubMed Abstract \| Publisher Full Text\n\nIsabel S, Graña-Miraglia L, Gutierrez JM, et al.: Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide. Sci Rep. 2020; 10: 14031. PubMed Abstract \| Publisher Full Text\n\nIslam MT, Alam ARU, Sakib N, et al.: A rapid and cost-effective multiplex ARMS-PCR method for the simultaneous genotyping of the circulating SARS-CoV-2 phylogenetic clades. J Med Virol. 2021; 93: 2962–2970. PubMed Abstract \| Publisher Full Text\n\nJohns Hopkins University & Medicine: COVID-19 Map [WWW Document].2021. Reference Source\n\nJørgensen TS, Blin K, Kuntke F, et al.: A rapid, cost efficient and simple method to identify current SARS-CoV-2 variants of concern by Sanger sequencing part of the spike protein gene. medRxiv. 2021. 2021.03.27.21252266.\n\nKim D, Lee J-Y, Yang J-S, et al.: The Architecture of SARS-CoV-2 Transcriptome. Cell. 2020; 181: 914–921.e10. PubMed Abstract \| Publisher Full Text\n\nKirby T: South America prepares for the impact of COVID-19. Lancet Respir Med. 2020; 8: 551–552. PubMed Abstract \| Publisher Full Text\n\nLiu P, Lu L, Xu M, et al.: A novel multiplex PCR for virus detection by melting curve analysis. J Virol Methods. 2018; 262: 56–60. PubMed Abstract \| Publisher Full Text\n\nLoureiro CL, Jaspe RC, D´Angelo P, et al.: SARS-CoV-2 genetic diversity in Venezuela: Predominance of D614G variants and analysis of one outbreak. PLoS One. 2021; 16: e0247196. PubMed Abstract \| Publisher Full Text\n\nMeng Q, Wang X, Wang Y, et al.: Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA. Biotechnol J. 2021; 16: 2100040. Publisher Full Text\n\nMlcochova P, Collier D, Ritchie A, et al.: Combined Point-of-Care Nucleic Acid and Antibody Testing for SARS-CoV-2 following Emergence of D614G Spike Variant. Cell Reports Med. 2020; 1: 100099. PubMed Abstract \| Publisher Full Text\n\nMolina-Mora JA, Cordero-Laurent E, Godínez A, et al.: SARS-CoV-2 genomic surveillance in Costa Rica: Evidence of a divergent population and an increased detection of a spike T1117I mutation. Infect Genet Evol. 2021; 92: 104872. PubMed Abstract \| Publisher Full Text\n\nNiranji SS, Al-Jaf SMA: Comments on ‘Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2.’. Infect Genet Evol. 2021; 87: 104661. PubMed Abstract \| Publisher Full Text\n\nOkoi O, Bwawa T: How health inequality affect responses to the COVID-19 pandemic in Sub-Saharan Africa. World Dev. 2020; 135: 105067. PubMed Abstract \| Publisher Full Text\n\nOzono S, Zhang Y, Ode H, et al.: SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity. Nat Commun 2021. 2021; 12(12): 1–9. Publisher Full Text\n\nPandey U, Yee R, Shen L, et al.: High Prevalence of SARS-CoV-2 Genetic Variation and D614G Mutation in Pediatric Patients With COVID-19. Open Forum Infect Dis. 2021; 8: 1–9. Publisher Full Text\n\nPark SY, Faraci G, Ward PM, et al.: High-precision and cost-efficient sequencing for real-time COVID-19 surveillance. Sci Rep. 2021; 11: 1–10. Publisher Full Text\n\nPourhoseingholi MA, Vahedi M, Rahimzadeh M: Sample size calculation in medical studies. Gastroenterol Hepatol From Bed to Bench. 2013; 6: 14.\n\nRaghav S, Ghosh A, Turuk J, et al.: Analysis of Indian SARS-CoV-2 Genomes Reveals Prevalence of D614G Mutation in Spike Protein Predicting an Increase in Interaction With TMPRSS2 and Virus Infectivity. Front Microbiol. 2020; 11: 2847. Publisher Full Text\n\nRhee C, Jones T, Hamad Y, et al.: Prevalence, Underlying Causes, and Preventability of Sepsis-Associated Mortality in US Acute Care Hospitals. JAMA Netw open. 2019; 2: e187571. PubMed Abstract \| Publisher Full Text\n\nSarkar SL, Alam ASMRU, Das PK, et al.: Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis. medRxiv. 2021; 165, 1–13.\n\nSevillano G: A Sanger sequencing-based method for a rapid and economic generation of SARS-CoV-2 epidemiological data: A proof of concept study to assess the prevalence of the A23403G SNP (D614G) mutation in Quito, Ecuador.2022. Reference Source\n\nThomson EC, Rosen LE, Shepherd JG, et al.: Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity. Cell. 2021; 184: 1171–1187.e20. PubMed Abstract \| Publisher Full Text\n\nViedma E, Dahdouh E, González-Alba JM, et al.: Genomic epidemiology of SARS-CoV-2 in Madrid, Spain, during the first wave of the pandemic: Fast spread and early dominance by D614G variants. Microorganisms. 2021; 9: 1–11. Publisher Full Text\n\nVogels CBF, Breban MI, Ott IM, et al.: Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2. PLOS Biol. 2021; 19: e3001236. PubMed Abstract \| Publisher Full Text\n\nWatanabe Y, Berndsen ZT, Raghwani J, et al.: Vulnerabilities in coronavirus glycan shields despite extensive glycosylation. Nat Commun. 2020; 11: 1–10. Publisher Full Text\n\nWorld Health Organization (WHO): Coronavirus Disease (COVID-19) [WWW Document].2021. Reference Source\n\nYuan F, Wang L, Fang Y, et al.: Global SNP analysis of 11,183 SARS-CoV-2 strains reveals high genetic diversity. Transbound Emerg Dis. 2020; 68: 3288–3304." }	[ { "id": "178950", "date": "16 Aug 2023", "name": "Fernanda de-Paris", "expertise": [ "Reviewer Expertise Molecular laboratory diagnosis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript offers an alternative method to genomic surveillance based on melting curves and Sanger sequencing methods. The rapid spread of SARS-CoV-2 has shown us the importance of having methodologies capable of rapidly generating epidemiological and genomic data. The subject of the paper could contribute to the surveillance of SARS-CoV-2 variants but needs some changes to clarify certain issues and make this approach more useful.\nIssues pointed on the review form (comments for questions considered partially or unanswered):\n\nIs the work clearly and accurately presented and does it cite the current literature?\nPartly. The authors discuss the genomic organization of SARS-CoV-2 and the A23403G mutation but do not fully elaborate on the significance of this mutation in the virus's phylogenetic evolution. The emergence of the A23403G mutation facilitated the rapid global spread and is found in all SARS-CoV-2 lineage B, including the variants of concern and the omicron sublineages. It is expected that all circulating variants at the moment would possess this mutation. It is crucial to include this information to emphasize the importance of genomic surveillance targeting this specific mutation. A reference must be inserted in the final text about this.\nAre sufficient details of the methods and analysis provided to allow replication by others?\nPartly. The authors discuss the RT-qPCR method utilizing the melting curve approach as a screening method to select positive samples for Sanger sequencing. However, they do not provide a description of the Sanger sequencing method itself, including the fragment amplification and purification procedures. This information is crucial for reproducibility, as the amplification reaction and PCR cycling conditions are specific to each primer pair used. The study mentions a large number of samples included in order to generate epidemiological surveillance data. However, in the results section, the authors mention that not all samples tested positive. This should be clarified, or alternatively, only the number of samples that were successfully sequenced should be indicated.\nFurthermore, the study primarily focuses on samples that may not fully represent the current scenario of SARS-CoV-2. It is important to include samples from other periods of the pandemic to provide a more comprehensive analysis and data. It is crucial to include these samples in the study since the dynamics of SARS-CoV-2 demonstrate that the virus is constantly mutating and adapting. The current subvariants exhibit genomic profiles that significantly differ from the previous variants circulating at the time of the study.\nAre all the source data underlying the results available ensure full reproducibility?\nThe authors do not describe reagents and procedures to obtain cDNA viral. SARS-CoV-2 is a RNA genome virus, so it is crucial a transcriptase reverse reaction to study viral genome by PCR and Sanger Sequencing.\nAre the conclusions drawn adequately supported by the results?\nThe conclusion should be based on the findings of Sanger sequencing. It was Sanger sequencing that correctly discriminated between mutated and non-mutated samples for the targeted viral SNP. The authors need to rewrite the conclusion to emphasize Sanger's results.\nOthers points to observe:\n\nPoint 1: The genomic position should be verified in the methods section. Based on the forward primer and the reference sequence MN908947, the initial position of the amplicon is 23,282. Please ensure to check this information for accuracy.\nPoint 2: Considering the melting curve approach used as a screening method and the low variability of the analyzed region (when compared to regions such as the RBD region), it may be more appropriate to focus on evaluating samples that exhibit a melting temperature different from 81 ºC. This approach would allow for the sequencing of samples that have a nucleotide sequence distinct from those containing the D614G mutation. Techniques such as High-Resolution Melting (HRM) have the potential to differentiate single nucleotide differences by a melting curve. This is important since the D614G mutation is what facilitated the rapid global spread and is present in all SARS-CoV-2 lineage B, including variants of concern and omicron sublineages. Therefore, it is expected that all samples would contain this mutation.\nPoint 3: The discussion section should address why the utilization of the Sanger sequencing methodology in this case serves as an alternative approach, highlighting the benefits it offers compared to whole-genome sequencing (WGS) technologies. These benefits of a targeted approach to analyze a single nucleotide polymorphism (SNP) using Sanger sequencing may include factors such as process time, cost-effectiveness, simplified analysis, and the ability to specifically identify and monitor the A23403G SNP, and this could be highlighted in the discussion section.\nPoint 4: The prevalence equation in the results section is unnecessary and can be removed.\nPoint 5: The English needs to be revised. Some words like 'gathered' or 'arguably' do not appear to be commonly used in scientific writing.\nPoint 6: In the methods section, the authors state that 'both genes were analyzed using a LightCycler® 480 Real-Time PCR', but they only cite the E gene. Please verify this information.\"\nPoint 7: The Table 1 in the results section presents the percentages of positive cases in both periods included in the study. This data appears to be unnecessary in table format and could be described in the text.\nPoint 8: The Table 3 in the results section presents the prevalence of the A23403G mutation using WGS data obtained from GISAID during the same period as the study. This information should be described more comprehensively. The data highlights the limited number of Ecuador sample sequences deposited in GISAID, underscoring the urgent need for genomic surveillance, which can be facilitated through the methodology proposed in this study. This information should be reinforced in the authors' discussion to support the importance of searching for the A23403G mutation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "264014", "date": "06 May 2024", "name": "Anissa Chouikha", "expertise": [ "Reviewer Expertise Virology", "Molecular Biology", "Sanger Sequencing", "Next generation sequencing", "PCR", "Loop-Lamp amplification", "Measles/Rubella Viruses", "Polioviruses", "Rotaviruses", "SARS-CoV-2", "Hepatites viruses (HAV", "HEV", "HBV and (HCV)" ], "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\nReviewer’s report for F1000Research Manuscript title: A Sanger sequencing-based method for a rapid and economic generation of SARS-CoV-2 epidemiological data: A proof of concept study to assess the prevalence of the A23403G SNP (D614G) mutation in Quito, Ecuador.\n\nIs the work clearly and accurately presented and does it cite the current literature? NO The authors of the study detail a Sanger sequencing approach to identify the D619G mutation of the SARS-CoV-2 virus. This method aims to aid in the detection of this mutation, particularly in regions lacking Next Generation sequencing capabilities. This mutation was initially identified during the early stages of the COVID-19 pandemic and exhibited a significant impact on the virus's transmissibility. Subsequently, this mutation became prevalent in all circulating SARS-CoV-2 viruses, alongside other mutations of concern within the spike protein. These mutations gave rise to variants of concern such as alpha, delta, and omicron, as well as other variants under surveillance.\nPrevious studies have already documented a partial Sanger sequencing approach targeting regions within the spike protein. However, in the current context, this study may be considered outdated. The references provided may not reflect the most current literature, as the authors still discuss the D614G mutation as if it were a recent development. As of mid-2024, many real-time PCR assays have been developed for the detection of mutations of concern. Additionally, numerous laboratories have been trained in and have adopted Next Generation sequencing technologies.\nIs the study design appropriate and is the work technically sound? Partially While it's true that Sanger technology offers simplicity and the rapid detection of variants is crucial, the current study's sequencing region and primary objective appear outdated. A similar approach could be applied to detect new variants within the current circulating Omicron variants.\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-383
https://f1000research.com/articles/11-382/v1	31 Mar 22	{ "type": "Software Tool Article", "title": "IsoAligner: dynamic mapping of amino acid positions across protein isoforms", "authors": [ "Jacob Hanimann", "Holger Moch", "Martin Zoche", "Abdullah Kahraman", "Jacob Hanimann", "Holger Moch", "Martin Zoche" ], "abstract": "Aligning protein isoform sequences is often performed in cancer diagnostics to homogenise mutation annotations from different diagnostic assays. However, most alignment tools are fitted for homologous sequences, leading often to alignments of non-identical exonic regions. Here, we present the interactive alignment webservice IsoAligner for exact mapping of exonic protein subsequences. The tool uses a customized Needleman-Wunsch algorithm including an open gap penalty combined with a gene-specific minimal exon length function and dynamically adjustable parameters. As an input, IsoAligner accepts either various gene/transcript/protein IDs from different databases (Ensembl, UniProt, RefSeq) or raw amino acid sequences. The output of IsoAligner consists of pairwise alignments and a table of mapped amino acid positions between the canonical or supplied isoform IDs and all alternative isoforms. IsoAligner’s human isoform library comprises of over 1.3 million IDs mapped on over 120,000 protein sequences. IsoAligner, is a fast and interactive alignment tool for retrieving amino acids positions between different protein isoforms. Its application will allow diagnostic and precision medicine labs to detect inconsistent variant annotations between different assays and databases. Availability: This tool is available as a Webservice on www.isoaligner.org. A REST API is available for programmatic access. The source code for both services can be found at https://github.com/mtp-usz/IsoAligner.", "keywords": [ "alignment", "protein isoform", "amino acid sequence", "protein ids", "exon-mapping", "amino acid position", "splice-variant" ], "content": "Introduction\n\nMapping isoform sequences to each other and identifying corresponding amino acids (AA) between isoforms is an important and prevalent task, especially in the interpretation of cancer mutations [Stephenson et al., 2019]. Most functional databases like COSMIC (RRID:SCR 002260), ClinVar (RRID:SCR 006169), or gnomAD (RRID:SCR 014964) use the longest protein isoform as a reference for their annotations. However in cancer diagnostics, shorter splice variants with different amino acid positions can be chosen, leading to confusions with respect to the existence of mutations in the aforementioned functional databases. Many inconsistent variant annotations exist [Tsai et al., 2021]. For example, the MET p.D1246N resistance mutation arising in lung cancers with MET exon 14 skipping events is commonly annotated as p.D1228N in diagnostic assays. While the first is annotated on the 1408 AA long NM 001127500.3, the latter is annotated on the 18 AA shorter transcript NM 000245.4. Simply looking for information on MET p.D1228N in functional databases might thus result in wrong conclusions. To find corresponding AAs in other databases, one general approach is to read off the corresponding positions from a pairwise global alignment like the Needleman-Wunsch algorithm (available for example at the EBI or SIB). However, the optimal solution to a global alignment can include the alignment of distinct exons giving the false impression that AA at these regions are corresponding to each other. To circumvent this problem, the Mirage [Nord et al., 2018] software performs a computationally expensive multiple sequence alignment of the corresponding sequences in the genome.\n\nHere, we introduce IsoAligner, the first web-service for effortless, fast, dynamic and interactive positional mapping of AA between isoform sequences. The tool applies a gene-specific minimal exon length function integrated into a Needleman-Wunsch algorithm to identify false-positive correspondences between amino acids of different isoforms. IsoAligner is simple, interactive, supports simultaneously gene/transcript/protein IDs from ENSEMBL (RRID:SCR 002344), UniProt (RRID:SCR 002380), RefSeq (RRID:SCR 003496), HGNC (RRID:SCR 002827) and UCSC (RRID:SCR 011624), and returns a ready-to-use mapping table between AA positions of protein isoforms. The source code for IsoAligner is available from GitHub and is archived with Zenodo (Hanimann & Kahraman, 2022).\n\n\nMethods\n\nThe challenge of aligning protein isoforms can be described as matching identical exons. The IsoAligner algorithm exploits this elementary characteristics of isoforms and applies custom parameters to the established Needleman-Wunsch algorithm followed by an evaluation of all subalignment lengths. Subalignments that do not meet the gene-specific minimal exon length, are discarded and marked as false-positive correspondences. The default global alignment parameters have been selected to support island-like solutions of the alignment with an heuristically predefined open gap penalty score. Gap extensions are not penalized (match: 1, mismatch: -2, open gap: -1, gap extend: 0). However, the user has the possibility to interactively change and adjust these parameters.\n\nThe IsoAligner software is written in Python v3.8 (RRID:SCR 008394). The alignment algorithm is based on the align.globalms function of the pairwise2 module from the Bio package (RRID:SCR 007173) and the website is built with streamlit. A REST API for programmatic access runs on the flask framework.\n\nThe Human Isoform Library forms the core of IsoAligner. The library is a comprehensive reference database comprising +1.3 million gene/transcript/protein IDs mapped on 120k protein sequences from multiple sequence reference databases namely Ensembl [Howe et al., 2021], UniProt [The Uniprot Consortium, 2021], RefSeq [O’Leary et al., 2016], HGNC [Tweedie et al., 2021] and UCSC [Damian Smedley et al., 2015]. The integration of the different databases was carried out by pairing IDs of protein sequences to each other by using the Biomart (RRID:SCR 002987) mapping tool and comparing raw amino acid sequences. The individual minimal exon length was required to be at least three AA and extracted from Ensembl’s GTF file (v104). For custom sequences provided by the user, we set the minimum exon length to 12 AA corresponding to the median length of all shortest exons in a gene. Using our adapted Needleman-Wunsch alignment approach we were able to map for the whole human isoform library with 106k alignments and a total of 40.5 million perfect AA matches (dataframe available online). However, we could also identify 862,136 false-positively aligned amino acids positions that could have resulted in a wrong amino acid position in an alternative isoform. Ultimately, our human isoform library provides a clean positional mapping table for corresponding AA for all alternative protein isoforms.\n\nSince the front-end of IsoAligner is built with streamlit, it is compatible with following browsers:\n\nGoogle Chrome (version 98 or newer)\n\nFirefox (version 97 or newer)\n\nMicrosoft Edge (version 98 or newer)\n\nSafari (version 14 or newer)\n\nWebsite. The input text field on www.isoaligner.org accepts various gene and protein IDs as well as custom sequence pairs (see Figure 1). Gene and protein IDs from different databases can be mixed and searched simultaneously. The workflow is as follows:\n\nQuick Start: Click on ’Show Example’ and then ’Search and Align’ to get a overview.\n\nEnter either one isoform ID per gene or two isoform IDs per gene or a list of genes names or two raw amino acid sequences. The input can be tab, comma or whitespace separated. Click ’Search and Align’ or ’Align’ to compute alignments.\n\nInformation to the chosen reference sequence and its alignments against all other isoforms can be displayed by using corresponding drop-down buttons.\n\nFurther down on the page, the computed mapping table is shown. On the left sidebar of the application, the function parameters of the Needleman-Wunsch algorithm and the minimal exon length function are displayed. Changing the values of the parameters, instantly updates the alignment visualisation and mapping table.\n\nThe mapping table can be filtered using the ’Filter table for exact value’ input field and pressing enter.\n\nThe entirety of the mapping table can additionally be complemented with associated isoform IDs and be downloaded as a csv or tsv file.\n\nFurther information on how to use the IsoAligner can be found at the ”Manual & About” section on the left sidebar of IsoAligner’s website.\n\nREST API. The REST API is built using Flask v1.1 and is accessible through the URL www.isoaligner.org/api. Currently, a get method for isoform IDs called ”map” is available for the retrieval of mapping tables between corresponding amino acid positions as well as the method ”align” to retrieve the alignment of two raw protein sequences.\n\nThe resource ”map” gives access to the human isoform library, computes alignments with specified parameters and retrieves whole mapping tables in json format. The only required parameter is id1, to provide the Isoform ID of interest. Additional parameters are:\n\nParameter: id1\n\nID of any type (Ensembl, Refseq, Uniprot, UCSC) to access the isoforms of a gene of the human isoform library. To define the reference protein sequence against which all other splice variants will be aligned, a specific isoform identifier should be used. Otherwise, the longest isoform is automatically chosen as the reference canonical sequence.\n\nRequest example: www.isoaligner.org/api/map?id1=EGFR-201\n\nResponse: Entirety of a mapping table in json format for EGFR with EGFR-201 defined as the reference sequence aligned against all other isoforms of the human isoform library.\n\nParameter: id2\n\nSpecific isoform ID (Ensembl, Refseq, Uniprot, UCSC) to use as the alternative splice variant to align with the reference sequence of id1.\n\nRequest example: www.isoaligner.org/api/map?id1=EGFR-201&id2=EGFR-207\n\nResponse: mapping table in json format for EGFR-201 aligned against EGFR-207.\n\nParameter: pos\n\nIn case of setting id1 and id2 in the request, a single corresponding AA positions on the alternative isoform sequence can be retrieved.\n\nRequest example: www.isoaligner.org/api/map?id1=EGFR-201&id2=EGFR-207&pos=1038\n\nResponse: 993\n\nParameter: min_ex_len\n\nThe alignment parameter for the minimal exon length (consecutive AAs) is gene-specific per default and can be manually defined as follows:\n\nRequest example: request:www.isoaligner.org/api/map?id1=EGFR-201&id2=EGFR-207&min ex len=23\n\nParameter: df_ids\n\nSequence database IDs to be included in the mapping table. Per default, the mapping table consists of the same type of IDs sent with the request. Available options are: [ensembl, refseq, uniprot, ucsc, hgnc].\n\nRequest example: request:www.isoaligner.org/api/map?id1=EGFR-201&id2=EGFR-207 &df ids=[ensembl,uniprot]\n\nParameter: match\n\nNeedleman-Wunsch alignment parameter to reward matches. This value must be ≥0.\n\nParameter: mismatch\n\nNeedleman-Wunsch alignment parameter to penalize mismatches. This value must be ≤0.\n\nParameter: open_gap\n\nNeedleman-Wunsch alignment parameter to penalize opening a gap. This value must be ≤0.\n\nParameter: gap_open\n\nNeedleman-Wunsch alignment parameter to penalize extending a gap. This value must be ≤0.\n\nWith the resource ”align”, one can align two raw amino acid sequences sent with the request and retrieve a mapping table in json format. The required parameters are seq1 and seq2. All alignment parameters: min ex len, match, mismatch, open gap, gap open are also applicable to this resource.\n\nParameters: seq1 and seq2\n\nReference and alternative raw amino acid sequences. Must be at least 7 AA’s long, for example:\n\nRequest: www.isoaligner.org/api/align?seq1=CRSSWTAAMELSAEYLREKLQRDLEAEHVE&seq2=YLREKLQRDLEAEHVEVEDTTLNRCSCSFRVLVVSAKFEGKPLLQRH\n\nResponse: mapping table in the json format.\n\n\nUse cases\n\nIsoAligner helps to transfer annotated mutational data from one transcript to another when working with different isoform database IDs. For example, the identification of the MET p.D1246N and p.D1228N resistance mutations in different transcripts as discussed in the introduction can be easily identified with IsoAligner. First, the user needs to paste one of the transcripts IDs, for example the RefSeq ID NM 000245.4 of the p.D1228N annotation into the ”Input” text field at the top of the website (see Figure 2).\n\nClicking the ”Search and Align” button, runs the IsoAligner algorithm and returns simple statistics on the transcript. In this case, there are 8 human isoform entries for the MET gene in the IsoAligner database. The Refseq ID given as the input (NM 000245.4) is automatically mapped to the ensemble transcript name (MET-202). Additional information such as the isoform sequence, various gene attributes and isoform IDs can be found by clicking the ”View details about this Isoform Entry” drop-down menu (see Figure 3).\n\nPairwise sequence alignments between all transcripts and the query transcripts can be found by clicking on the drop-down menu ”View Alignment Visualisations” (see Figure 4). The alignments update immediately, when ever any alignment parameters on the left-hand sidebar is adjusted.\n\nFurther down, the user can find the ”Mapped Amino Acid Positions” table that lists the corresponding amino acid positions between all MET isoforms in the IsoAligner database and the query isoform NM 000245.4. Typing the amino acid position of the resistance mutation 1228 into the ”Filter table for exact value” text field and pressing enter shows all mapped amino acid position to position 1228 (see Figure 5). The first listed position is the corresponding amino acid in the canonical transcript MET-201 with the RefSeq ID NM 001127500.3. The last column provides the information that the corresponding amino acid position of 1228 in the canonical transcript is 1246. Note, that a corresponding amino acid to a shorter isoform with a RefSeq ID NM 001324402.2 is also shown, mapping the position 1228 to 798, while the third hit corresponds to a map between position 1210 in the query transcript and 1228 in the canonical transcript.\n\nA ”Download Table” button is available to retrieve the entirety of the table in tsv or csv format. Consider clicking the checkbox ”Select all columns” to get additional database IDs for the transcripts. Alternatively, users might send an API request https://www.isoaligner.org/api/map?id1=NM_000245.4 to the REST API and retrieve a json file representation of the mapping table.\n\nThe data used in this use case can be found as Underlying data (Hanimann & Kahraman, 2022).\n\n\nConclusion\n\nIsoAligner is a fast and interactive protein isoform alignment webservice that uses a customised Needleman- Wunsch algorithm to specifically align protein alternatively spliced isoforms. The comprehensive library of IsoAligner comprises 1.3 million IDs and 120k protein sequences of 19k human genes which allows rapid positional mapping of amino acids across isoform IDs from Ensembl, RefSeq, UCSC and UniProt.\n\n\nData availability\n\nZenodo: IsoAligner: dynamic mapping of amino acid positions across protein isoforms. https://doi.org/10.5281/zenodo.6354488 (Hanimann & Kahraman, 2022).\n\nThis project contains the following underlying data:\n\nHuman Isoform Library Data (the datasets used to generate the Human Isoform Library)\n\nhuman isoform library v1.tsv.gz (the pre-computed mapped human isoform library)\n\nExample Manuscript (the input/output files from the example Use Case section).\n\n\nSoftware availability\n\nWebtool available at: https://www.isoaligner.org/\n\nREST API available at: https://www.isoaligner.org/api\n\nSource code available from: https://github.com/mtp-usz/IsoAligner\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.6354488 (Hanimann & Kahraman, 2022)\n\nLicense: CC0-1.0", "appendix": "Acknowledgements\n\nWe thank the members of the Clinical Computational Biology group at the University Hospital Zurich for their constant support and valuable inputs.\n\n\nReferences\n\nHanimann J, Kahraman A: IsoAligner: dynamic mapping of amino acid positions across protein isoforms (IsoAligner v1.2.0). Zenodo. 2022. http://www.doi.org/10.5281/zenodo.6354488\n\nHowe KL, Achuthan P, Allen J, et al.: Ensembl 2021. Nucleic Acids Res. 2021; 49(D1): D884–D891. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNord A, Carey K, Hornbeck P, et al.: Splice-Aware Multiple Sequence Alignment of Protein Isoforms. ACM BCB. 2018; 2018: 200–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Leary NA, Wright MW, Brister JR, et al.: Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016; 44(D1): D733–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmedley D, Haider S, Durinck S, et al.: The BioMart community portal: an innovative alternative to large, centralized data repositories. Nucleic Acids Res. 2015; 43(W1): W589–W598. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStephenson JD, Laskowski RA, Nightingale A, et al.: VarMap: a web tool for mapping genomic coordinates to protein sequence and structure and retrieving protein structural annotations. Bioinformatics. 2019; 35(22): 4854–4856. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Uniprot Consortium: UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021; 49(D1): D480–D489. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsai JM, Hata AN, Lennerz JK: MET D1228N and D1246N are the Same Resistance Mutation in MET Exon 14 Skipping. Oncologist. 2021; 26(12): e2297–e2301. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTweedie S, Braschi B, Gray K, et al.: Genenames.org: the HGNC and VGNC resources in 2021. Nucleic Acids Res. 2021; 49(D1): D939–D946. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "179554", "date": "05 Jul 2023", "name": "James D Stephenson", "expertise": [ "Reviewer Expertise Genomic variation analysis in protein coding regions." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 isoform alignment resource is a useful addition to the bioinformatics field. Whilst it is possible to extract equivalent positions across isoforms in other resources, the speed and ease, as well as the completeness and accessibility of IsoAligner make it a useful addition to the field.\nThe manuscript clearly explains the methods used and the breadth of resources used for the data which is a critical aspect of the resource. In addition to using all of the critical data sources for isoforms, IsoAligner also allows users to query using the various IDs used by these resources which makes retrieving data simple and intuitive. Multiple ways of searching make it easy for most users to use the tool with the data at hand rather than having to format or refactor it first. The availability of a bulk download is also a useful feature, as is the programmatic access via an API.\nThe code is deposited in GitHub with sufficient documentation which would allow replication of the software development by others and the methodology is clearly explained in the manuscript.\nThe example in the manuscript is useful to allow the interpretation of results generated using the tool. There is also sufficient help text in the user interface to help users understand the outputs. The fact that the example on the homepage is randomly generated rather than cherry picked is a good demonstration of the completeness of the tool and the confidence of the developers that the tool is robust. The performance of the user interface is also impressive, especially as it re-calculates alignments dynamically as the parameters are adjusted.\nThe conclusions stated are supported by the data and resource apart from one issue which requires addressing detailed below with regards to the number of proteins covered.\n\nMinor points to address:\nThe home page states that “The current human isoform library consists of ~19'000 protein coding genes”. However the bulk download stats show that there are 16432. In the paper conclusion it also states “IsoAligner comprises 1.3 million IDs and 120k protein sequences of 19k human genes” It would be useful to explain this difference in the paper and ensure that the statement on the home page is not misleading.\n\nAs a very minor additional observation, the gene number is written as “19’000” on the website but a comma is used in the manuscript, eg “862,136 false-positively aligned amino acids”. This does not necessarily need to be changed but should ideally be consistent.\n\nIt would be useful for the website and the manuscript to mention the maintenance plan and update cycle for IsoAligner. When was the data last updated and when will the data be refreshed? The paper mentions that it was updated at the time of initial publication but that was early 2022 I believe? From the Gitlab repository it looks like 'human isoform library' data has not been updated for two years.\n\nThere is a drop down menu for species but there only appears to be humans in the list. Does this list auto-populate based on the input or does it exist because further species are planned in the future? If it is the latter, might I suggest it is removed until other species are available.\n\nThe table download feature is useful, especially as the user can download only the columns selected. I don’t think that it is clear enough however so I would recommend just adding some brief text to the download table button such as “download table with currently selected columns”.\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": "185231", "date": "17 Aug 2023", "name": "Laurens van de Wiel", "expertise": [ "Reviewer Expertise Bioinformatics", "protein sequence and structure", "genomics", "comparative genomics", "evolutionary genetics", "software development" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHanimann et al. present IsoAligner as fast and dynamic web server which intuitively incorporates the Needleman-Wunsch algorithm to align all protein isoforms for a protein-coding gene of interest. I applaud the authors for the aesthetically pleasing web server the authors have made and compliment on how the extensive functionality is intuitively presented. The authors argue that one of the novelties of IsoAligner is that alignments are made from the perspective of translated exon regions instead of the entire isoform amino-acid sequence composition. They kindly acknowledge that the computationally more expensive Mirage alignment software also operates from an exon-specific perspective. IsoAligner as a software seems very useful for many bioinformatic teams in the field of (human) genetics for variant interpretations. Especially from a variant data-integration point of view I can see that IsoAligner could lead to more accurate condensations of datasets. I have the following remarks that I would like to see addressed:\nThe authors argue against using isoform-based alignments, as this often leads to alignments on non-identical exon regions. For this, the authors provide a single, but convincing, case on how this can lead to the wrong conclusions when referring to a protein-based variant without correctly adding the specific isoform. In terms of quantification, I can only find the number of falsely positive aligned amino acid positions that relate to this in the “Methods” section under “Human Isoform Library”. There is information lacking in this quantification:\nThe number of amino acids does not mean much outside of the context of genes and/or identical protein sequences between genes. How many of the 120k protein sequences are the exact same protein-product (suggesting to compare to UniProtKB) from their respective genes? From my own previous analysis in (DOI: 10.1002/humu.23798) which performed a similar analysis on only 2 sets: UniProtKB-SwissProt protein sequences with Ensembl GENCODE (hg19/v13) annotation sets: “there is an identical match of 79.4% of the Human Swiss-Prot protein sequences to one or more of 42,116 GENCODE transcripts. This means that 25.7% of the GENCODE transcriptions differ in messenger RNA (mRNA) but translate to the same Swiss-Prot protein sequence.”\n\nThe human genome and its protein products should not be taken out of context of genome build (e.g. GRCh37/hg19, GRCh38/hg38) and the corresponding genome annotation version (RefSeq, Ensembl/GENCODE, or combined as MANE). Adding this information would benefit the segmentation and transparency of the data in the manuscript and without it the web server would be less useful for the genomics community.\n\nIt would further aid the manuscript’s argument on exon vs isoform based alignment if the authors could expand by adding a table with a per-dataset comparison (annotation vs protein database) and a combined dataset comparison would help to compare if the exon-based alignment (e.g. there is no quantification on how many genes / protein sequences are extracted per dataset)\n\nWith these suggestions taken into account, some minor scientific oriented questions could be answered in the manuscript, such as: do falsely positively aligned amino acids differ between genome builds (hg19 vs hg38) or annotation source (e.g. RefSeq vs Ensemble)? Or the like.\n\nComparison with Mirage: IsoAligner is claimed to be faster than Mirage, and from my one-sided usage of IsoAligner and without previously having used Mirage. I can state that IsoAligner is indeed fast and responsive, but it would be beneficial to the argument of the manuscript to see some comparison with Mirage that shows the actual increase in speed and/or reduction of mathematical complexity and especially if this increase in speed sacrifices anything in terms of accuracy of output. Comparing a few challenging alignment cases would provide insights into differences, and a controlled environment speed test would further bolster this comparison.\n\nI have been able to cause a server-side crash lasting several hours twice by entering “TTN” (25 transcripts, varying in aa length from 48aa to 35,991aa) as a stress-test. I would like to see if the authors are able to indeed analyse TTN and/or find a work around to this and similar cases. To ascertain if the error was client-side, I tried accessing IsoAligner on multiple devices and browsers. However, I confirmed that neither other websites nor devices could access the web server. In all cases the web server displayed the error message “Oh no. Error running app. If this keeps happening, please contact support”. Although preferable to an unresponsive server, I have several suggestions for future improvements, which I've detailed under minor suggestions below.\n\nMultiple potential trackers/js are attached to the session (segment, googletagmanager), please add a privacy policy on what is tracked and for what purpose, see: https://ourworldindata.org/privacy-policy\n\nMinor comments and suggestions:\nIntroduction:\nConsider specifying or removing the term \"functional\" from \"functional databases\". The current phrasing is ambiguous, making it unclear if you're referring to operational databases or databases containing functional variations. If it's the latter, note that variations in gnomAD and ClinVar aren't inherently functional.\n\nThe variants in gnomAD and ClinVar are transcript-specific (I am unfamiliar with COSMIC), indicating every transcript based on user selection. The distinction between exon-based vs. isoform-based alignment might not be problematic unless authors provide examples for clarity.\n\nMethods:\nThe \"alignment approach\" section would be clearer with a step-by-step breakdown or even an illustrative figure. The figure showcasing two isoforms on the web server's \"about\" page could be integrated here, as it elucidates the challenge the authors address, even for non-experts.\n\nResults, such as \"... with 106k alignments and a total of 40.5 million perfect AA matches\", should be discussed in the \"Results\" section and not in \"Methods\".\n\nGeneral Observations\nFor clarity, the first mention of large numbers should be spelled out in full. Subsequent mentions can be abbreviated, but clarity is paramount. For approximations, consider using symbols like \"~\" or terms like \"about\", \"approximately\", or \"roughly\" to avoid ambiguity.\n\nWeb server improvements:\nAPI Output format: The API example at https://www.isoaligner.org/api/map?id1=EGFR-201 returns JSON strings with escaped characters (e.g., \\\"). This typically results from using double quotes for strings. While not a major concern, it necessitates additional processing for the output.\n\nAPI Error Handling: If commands that don't exist are submitted, ensure the system provides descriptive error messages. For instance, using dfids instead of df_ids didn't yield an informative error.\n\nThe page isoaligner.org/api instructs users to refer to the left sidebar on the main page for details on sending requests. It would be more user-friendly if the page directly detailed the necessary steps, especially if the main page became unavailable.\n\nIt may be beneficial to implement explicit URL routes for direct navigation to specific isoforms, e.g., “isoaligner.org/ENSEMBL//”. This would simplify error tracking, especially if a recorded link caused server downtime.\n\nGitHub:\nKudos to the authors for open-sourcing the code. It would further aid users if a detailed configuration guide is provided on their GitHub, allowing for deployment of IsoAligner in custom environments.\n\nUser-specific configuration files are present in the .idea and .streamlit directories, which is considered a bad-practice\n\nThe requirements.txt file merely lists \"Bio\" without specifying a version. This can be problematic as it defaults to downloading the latest version, potentially introducing instability in future iterations of the software.\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? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] } ]	1	https://f1000research.com/articles/11-382
https://f1000research.com/articles/11-381/v1	31 Mar 22	{ "type": "Data Note", "title": "Dataset for the adapted COVID stress scales (ACSS) including teaching anxiety and preparedness, and resilience of academic professionals in Mexico", "authors": [ "Gerardo R. Padilla-Rivas", "Juan Luis Delgado-Gallegos", "Daniel Arellanos-Soto", "Hector Franco-Villareal", "María de los Ángeles Cosío-León", "Gener Avilés-Rodríguez", "Erika Zuñiga-Violante", "Gerardo Salvador Romo-Cardenas", "Jose Francisco Islas", "Gerardo R. Padilla-Rivas", "Juan Luis Delgado-Gallegos", "Daniel Arellanos-Soto", "Hector Franco-Villareal", "María de los Ángeles Cosío-León", "Gener Avilés-Rodríguez", "Erika Zuñiga-Violante", "Gerardo Salvador Romo-Cardenas" ], "abstract": "Because of COVID-19, many social interactions have been continued through electronic means. This has been especially true for the educational experience, where professors and students have strived to move to the virtual classroom. The data sets presented here examined how academic professionals have dealt with the stress of the move, how they have strived to become more prepared and eventually more resilient to this change. Using an online platform, we obtained data at the end of the academic period during November and December 2020. The data analyzed three major areas: teaching anxiety and preparedness, resilience, and overall stress, by applying a modified version of the adapted COVID-Stress Scales. We statistically analyzed our data looking for important correlations, to help better understand the challenges educators have met though the pandemic, and how this can assist policy-makers in managing stress and looking for ways to enhance resilience in academic professionals.", "keywords": [ "Academic Professionals Stress", "Adapted COVID Stress Scales", "Academic Professionals Resilience" ], "content": "Introduction\n\nIn early 2020, the Mexican Ministry of Health issued a series of guidelines to prevent and reduce the risk of infection with COVID-19, in which there was a directive to suspend all in-person school activities, moving nearly two million academic professionals and over 30 million students from across México, from the traditional classroom setting to the virtual classroom, in order to reduce the spread of the disease.1 However, this placed the educational system under a significant amount of pressure and stress, needing to migrate to a digital teaching model, and adapt to diverse technological tools and develop computational skills. As expected, this move brought many challenges, particularly to academic professionals as they had to endure both the stress derived from the COVID-19 disease itself and the technological challenges brought by the development of the academic material, to continue immersing the students to obtain the desired academic experience.2,3 Furthermore, some teaching professionals required going to a physical location at least part-time, which implied the continuous use of protective personal equipment (PPE), and the combination of preparing material at a distance and in-person.4,5 Hence, this resulted in fear of COVID-19 infection, and emotional and physical fatigue. Stress overload can trigger the development of mild to severe psychiatric disorders such as depression, anxiety, burnout syndrome and sleeping disorders, putting the mental health of teaching professionals at risk.6,7 Fortunately, resilience is a dynamic adaptative process supporting a healthy psychological state developed by many academic professionals under these conditions.8\n\nDifferent mental health scales have been used to assess and evaluate diverse psychological conditions. In the present study, we evaluated three areas: teaching anxiety and preparedness, resilience and overall stress, by applying a modified version of the adapted COVID-Stress Scales (ACSS). The data presented in the database is important, as this is one of the first integrative evaluation on stress, anxiety and resilience in México during the COVID-19 pandemic and can be used to further study the impact of the COVID-19 pandemic in mental health.\n\n\nMethods\n\nOriginally, we used the ACSS to understate stress in healthcare professionals.5,9 Our current study continues the application of the ACSS, but now for academic professionals, as they faced challenges in bringing education to the virtual classroom. Briefly, to collect the data, we developed the questionnaire, as described in our earlier work, which was further distributed both by directed email and online using social media platforms.8\n\nResults from the questionnaire were (as described in our previous work) classified according to their accumulated sums.5,9 As stated earlier, several modifications were done, such as the inclusion of “teaching anxiety and preparedness”, and “resilience” sections. Contamination, Social Economical, and the fear of being an asymptomatic patient, all had additions to the number of questions. Results from all of our sections were classified as follows: section with two questions, responses including absent = 0-2, mild = 3-4, moderate = 5-6, severe = 7-8. Section with four questions: absent = 0-4, mild = 5-8, moderate = 9-12, severe = 13-16. Sections with six questions (original scale): absent = 0-6, mild = 7-12, moderate = 13-18, severe = 19-24. Sections with nine questions: absent = 0-8, mild = 9-17, moderate = 18-26, and severe = 27-36. Finally, the resilience section was categorized as: very low (0-4), low (5-8), normal (9-12), high (13-16), and very high (17-20). Tables 1 and 2 show the classification per section.\n\nThe intervals on Tables 1 and 2 were calculated diving the number of categories – 1 by the maximum result (Maximum points, Tables 1 and 2). For most of the sections, we used: absent, mild, moderate, and severe, or for resilience very low, low, normal, high, and very high.\n\nWe further calculated correlations between the results using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY, USA). Statistical tests included Pearson’s chi-squared (p < 0.05), calculation of degrees of freedom, verisimilitude and linear association. Our work showed that academic professionals through the COVID-19 pandemic became highly resilient, with as over 80% of all professionals scoring from high to very high in resilience; meanwhile for the ACSS, except for the “danger2 category (section 1) in which the majority of participants (> 40%) scored moderate, the rest of the “stress” categories participant majority was in the absent to mild range. Interestingly, most participants scored mild for teaching anxiety and preparedness and absent for the fear of being an asymptomatic patient. These results can be seen in Table 3 of our manuscript.8\n\nRAW.csv or RAW.xlsx does not contain sum of sections, total and total sections 1-6.\n\nThere are six files in the datasets, first the original version of the questionnaire in Spanish and a translated version in English (Questionnaire (English Version).docx, and Questionnaire (Spanish Version).docx), as well as the raw dataset (RAW.csv, and RAW.xlsx) obtained and a re-categorized/processed dataset (CEECS results.csv, and CEECS results.xlsx).\n\nThe RAW files (.csv or.xlsx) contain the original results from the questionnaire, including the newly developed sections of teaching anxiety and preparedness with four questions, and resilience (section R) with five questions, in addition to the modified ACSS questionnaire (sections 1-6). Notably, within the modified ACSS, additional questions were added to the contamination section (seven questions), social and economical section (eight questions), and fear of being an asymptomatic patient (two questions). All other sections comprised six questions.5 The teaching anxiety and preparedness, and the fear of being an asymptomatic patient sections, sections 1 – 4, and section R were evaluated as never (= 0), little (= 1), moderate (= 2), much (= 3), and extreme (= 4). Sections 5-6, were evaluated as never (= 0), rarely (= 1), sometimes (= 2), occasionally (= 3), and almost always (= 4).\n\nThe CEECS results files (.csv or.xlsx) are the processed files containing the data values, both individually and by sums. In addition, we collected the sum of values for each section in the “sum” sections. Sum sections were divided into individual sections: teaching anxiety and preparedness, sections 1 - 6, fear of being an asymptomatic patient, and section R; next was a “total” section which included the total values in the sum sections, and a sections 1-6 total which evaluated stress as in the COVID -Stress Scales with no added sections.10\n\nThe files contain identifier numbers (ID number) under order of participation, followed by start and completion times of the questionnaire, and a statement of willingness to take part in the questionnaire was recorded. Next, a sociodemographic section asked participants their gender, age (range), level of education, residence (by state), the number of household occupants, presence of comorbidities e.g., cardiac disease, pulmonary disease, diabetes, obesity, among others. Next, we asked about the academic level at which they taught (from Elementary to Graduate), the mode in which they give classes (in-person, online,), and finally the number of hours (range) they work with students. If in the modality question they answered in-person or mix, we asked the number of hours (range) they worked in a physical presence.\n\nAfter the social demographic section, both files contained teaching anxiety and preparedness, followed by sections 1 – 6 of the ACSS. Next, we asked if they had been diagnosed with COVID, followed by the fear of being an asymptomatic patient and section R of the questionnaire. The general structure of the dataset is shown in Table 3.\n\nThe study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Hospital La Misión, Monterrey NL. México. Protocol #PSY-CEECS-ESP-001.\n\n\nConsent\n\nParticipant consent was taken by electronic form. Before opting to partake in the study, the survey informed the participants about the nature of the study.\n\n\nData availability\n\nZenodo: Dataset of Teaching anxiety, stress and resilience of academic professionals in Mexico, using the adapted COVID-19 stress scales (ACSS), https://doi.org/10.5281/zenodo.635483811\n\nThe project contains the following underlying data.\n\n• CEECS results.xlsx\n\n• CEECS results.csv\n\n• RAW.xlsx\n\n• RAW.csv\n\n• Questionnaire (English Version).docx\n\n• Questionnaire (Spanish Version).docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe would like to thank the Departamento de Bioquímica y Medicina Molecular y Althian for the initial development of the survey. We also like to the Universidad Autónoma de Baja California and the Universidad Politécnica de Pachuca for their collaboration in the development of this manuscript.\n\n\nReferences\n\nMérida Martínez Y, Acuña Gamboa LA: Covid-19, Pobreza y Educación En Chiapas: Análisis a Los Programas Educativos Emergentes. Rev. Int. Educ. para la Justicia Soc. 2020; 9: 61–82. Publisher Full Text\n\nSánchez M, Martínez A, Torres R, et al.: Retos Educativos Durante La Pandemia de COVID-19: Una Encuesta a Profesores de La UNAM. Rev. Digit. Univ. 2020; 21: 1–24.\n\nBaptista Lucio P, Almazán Zimerman A, Alberto Loeza Altamirano C, et al.: Encuesta Nacional a Docentes Ante El Covid-19. Retos Para La Educación a Distancia National Survey to Teachers Facing Covid-19. Challenges for Distance Education. Rev. Latinoam. Estud. Educ. 2020; 50: 41–88.\n\nRajkumar RP: COVID-19 and Mental Health: A Review of the Existing Literature. Asian J. Psychiatr. 2020; 52: 102066. PubMed Abstract \| Publisher Full Text\n\nDelgado-Gallegos JL, Montemayor-Garza RJ, Padilla-Rivas GR, et al.: Prevalence of Stress in Healthcare Professionals during the Covid-19 Pandemic in Northeast Mexico: A Remote, Fast Survey Evaluation, Using an Adapted Covid-19 Stress Scales. Int. J. Environ. Res. Public Heal. 2020; 17. PubMed Abstract \| Publisher Full Text\n\nJandrić P, Hayes D, Truelove I, et al.: Teaching in the Age of Covid-19. Postdigital Sci. Educ. 2020; 2: 1069–1230. Publisher Full Text\n\nBesser A, Lotem S, Zeigler-Hill V: Psychological Stress and Vocal Symptoms Among University Professors in Israel: Implications of the Shift to Online Synchronous Teaching During the COVID-19 Pandemic. J. Voice. 2020; 36: 291.e9–291.e16. PubMed Abstract \| Publisher Full Text\n\nDelgado-Gallegos JL, Padilla-Rivas GR, Zuñiga-Violante E, et al.: Teaching Anxiety, Stress and Resilience During the COVID-19 Pandemic: Evaluating the Vulnerability of Academic Professionals in Mexico Through the Adapted COVID-19 Stress Scales. Front. Public Heal. 2021; 9: 669057. PubMed Abstract \| Publisher Full Text\n\nPadilla-Rivas GR, Delgado-Gallegos JL, Montemayor-Garza RDJ, et al.: Dataset of the Adapted COVID STRESS SCALES for Healthcare Professionals of the Northeast Region of Mexico. Data Br. 2021; 34. PubMed Abstract \| Publisher Full Text\n\nTaylor S, Landry CA, Paluszek MM, et al.: Development and Initial Validation of the COVID Stress Scales. J. Anxiety Disord. 2020; 72: 102232. PubMed Abstract \| Publisher Full Text\n\nIslas JF, Delgado-Gallegos JL, Padilla-Rivas G: Dataset of Teaching anxiety, stress and resilience of academic professionals in Mexico, using the adapted COVID-19 stress scales (ACSS) [Data set]. Zenodo. 2022. Publisher Full Text" }	[ { "id": "144066", "date": "18 Aug 2022", "name": "Stamatios Papadakis", "expertise": [ "Reviewer Expertise eLEARNING" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, the research process and the results could be explained more thoroughly to make the study more transparent and informative.\nThe introduction would benefit from a succinct description of what the paper is about and a stronger emphasis on the problem addressed by the research. The authors may briefly state the intended contribution of this research, given its theoretical background, and provide enough information to the reader about its significance.\nIt is suggested that the authors can consider the following papers relative to the study aims:\nKarakose, T., Ozdemir, T. Y., Papadakis, S., Yirci, R., Ozkayran, S. E., & Polat, H. (2022). Investigating the Relationships between COVID-19 Quality of Life, Loneliness, Happiness, and Internet Addiction among K-12 Teachers and School Administrators—A Structural Equation Modeling Approach1.\n\nMohammed, D. Y. (2022). The web-based behavior of online learning: An evaluation of different countries during the COVID-19 pandemic2.\n\nAguayo, J. M., Valdes, J., Cordoba, V. H., Nájera, M., Vázquez, F. R., Muñoz, E., & García Lirios, C. (2022). Digital activism in students of a university in central Mexico in the COVID-19 era3.\nThe paper needs to explain more clearly and in enough depth the research approach of the study.\nThe methods should be adequately described to show how the research was conducted to improve its transparency and trustworthiness.\nThus:\nThe rationale for creating the dataset(s) clearly is partly described. It must be explained why the protocols used are appropriate for the present study.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] }, { "id": "218958", "date": "30 Oct 2023", "name": "Anil Kakunje", "expertise": [ "Reviewer Expertise Psychiatry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere are my suggestions / comments to the article -\nThey have worked in an important and relevant area and specially during the pandemic period. They worked as a team to improve the mental health research in Mexico which is useful for people around the world.\n\nThe present paper seems to be extension of an earlier paper. The details are not clear on many things specially related to the methodology. It says we distributed by email and in social media. It was sent to how many people? how many responded ? How long did it take ? and so on..\n\nIn the pandemic period when was this study done exactly is not clear. The pandemic had a long course with varying peaks. Readers need to know the exact point at which the questionnaire was answered. People slowly started getting used to Covid-19, vaccination was widely used and the fear among the public slowly decreased.\n\nSomeone reading this paper only; finds it difficult to get a clear picture. without reading the earlier linked papers.\n\nWas a Data set added with ACSS scale in the earlier paper\n\nMore than 1/3 of the total 11 references are their own work. One big project is broken to multiple papers.\n\nAcknowledgement second line grammar can be corrected. The places of the Institutes also can be added.\n\nCan add a conclusion heading and give a take home message. The article has an abrupt ending with the data description.\n\nThe reference number 11 is the data availability link of authors own work in 2022.\n\nThe salient and important points of the data set could have been put in print in the paper to help the readers who do not want to go through the entire data set.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? No\n\nAre sufficient details of methods and materials provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-381
https://f1000research.com/articles/11-375/v1	31 Mar 22	{ "type": "Research Article", "title": "Understanding variation in patient care: A qualitative study of hospital (non-ST elevation myocardial infarction) practices", "authors": [ "Helen Cramer", "Jacki Hughes", "Maggie Evans", "Gene Feder", "Christi Deaton", "Katie Featherstone", "Adam Timmis", "Harry Hemingway", "Rachel Johnson", "Jacki Hughes", "Maggie Evans", "Gene Feder", "Christi Deaton", "Katie Featherstone", "Adam Timmis", "Harry Hemingway", "Rachel Johnson" ], "abstract": "Background\nVariation in care is often poorly understood but has a big impact on patients. Non-ST segment elevation myocardial infarction (NSTEMI, also known as non-ST elevation acute coronary syndrome or NSTE-ACS) is the most common form of heart attack. NSTEMI is frequently hard to diagnose, its management pathway poorly defined and there is considerable variation in clinical practice.\n\nMethods\nA qualitative study based on site visits, observation, and interviews with managers, clinicians and patients. The setting was 10 hospitals in England and Wales selected to represent variation in 30-day mortality. 199 hospital staff and 68 patients were observed; 142 staff and 53 patients were interviewed. Analysis was thematic and guided by the principles of grounded theory. We triangulated interviews, observational data and medical records and interpreted these findings with reference to national guidelines.\n\nResults\nWhile the majority of hospitals in our sample had specialist cardiac roles, variation in their remits, specifically their involvement in close monitoring, significantly affected patient management. Close monitoring was important in the identification and prioritisation of patients. Rapid responses with diagnostic and treatment procedures were facilitated by close monitoring but also heavily dependent on effective and flexible bed and catheter laboratory management.\n\nConclusions\nClose monitoring was a key area of variation. Guidelines for NSTEMI care specify what to do, but not how to do it. These findings are especially relevant for acute conditions with diagnostic and treatment uncertainty. Detailed examples of variation in care can inform quality improvement and potentially help improve patient outcomes.", "keywords": [ "Non-ST elevation myocardial infarction (NSTEMI)", "monitoring", "qualitative research", "acute coronary syndrome", "hospital mortality", "quality improvement", "uncertainty" ], "content": "Introduction\n\nUnwarranted variation in clinical care has been described by Atsma and colleagues (2020) as typically ‘large, omnipresent, persistent, and difficult to grasp’ (Atsma, Elwyn, & Westert, 2020). Variation in patient outcomes for myocardial infarction (MI) between hospitals is a consistent finding and remains after adjusting for case mix (Yeh et al., 2010). Factors identified by quantitative means may partly explain these variations: high numbers of MI patients (West et al., 2010), teaching hospital status (Patel et al., 2007), ward first admitted to (Moledina et al., 2021a), care by cardiologists (Birkhead, Weston, & Lowe, 2006; Moledina et al., 2021b) and adherence to guidelines (Chung et al., 2015; Engel, Damen, van de Wulp, de Bruijne & Wagner, 2017) are associated with better patient outcomes. However, residual variation in hospital outcomes for MI remains unexplained. Qualitative research examining variation in care and outcomes for patients with acute MI identified areas of care that distinguished high from low performing hospitals. These areas included organisational values and goals, senior management involvement, broad staff presence and expertise, communication and coordination among interdisciplinary teams, and problem solving and learning (Curry et al., 2011).\n\nResearch investigating variation in care for patients with MI has largely focused on ST-segment elevation myocardial infarction (STEMI), where there is a complete blockage of the coronary artery. This is relatively straightforward to diagnose with electrocardiography (ECG) and has a well-defined pathway of care including effective procedural treatment: percutaneous coronary intervention (PCI/angioplasty). Timely intervention for STEMIs has been a key focus globally and there have been clear improvements in time to treatment, supported by national initiatives in the UK (Laskey et al., 2010). Variation in clinical practice for STEMI patients has therefore been reduced. Non-ST segment elevation myocardial infarction (NSTEMI, also known as non-ST segment elevation acute coronary syndrome or NSTE-ACS), where there is only a partial coronary artery blockage, is more common and has a worse prognosis than STEMI if left untreated. Diagnosing NSTEMIs is often complex and involves integrating findings from the patient’s full clinical history, physical examination, ECG, and repeated blood tests to check for a rise in troponin levels (a protein that is released into the bloodstream during a heart attack). The management pathway is also less straightforward for NSTEMI patients compared to STEMIs, partly due to the lengthy process of establishing a diagnosis (Darling et al., 2013; Deaton et al., 2016; Gambhir, 2018; Koganti & Rakhit, 2015; Shepple et al., 2016; Wu et al., 2018). The variation in care for NSTEMI patients across different hospitals is large and may be linked to poor outcomes for patients.\n\nClinical guidance can inform criteria for assessing quality of care. Guidance covering NSTEMI care includes the UK’s National Institute for Health and Care Excellence guidance (NICE, 2010, 2016; NICE 2010, 2013) and the European Society of Cardiology (ESC, Roffi et al., 2016). Guidance recommends an ECG as soon as possible when patients present with chest pain and continued ECG monitoring even if the initial ECG is normal. If NSTEMI is suspected, continued monitoring is recommended and for a management plan to start as soon as an NSTEMI is suspected. An angiogram, where detectable dye is injected into the arteries, can visually aid diagnosis and NSTEMI patients at most risk of having another cardiac event or of clinically deteriorating should be offered an angiogram within a short time frame (e.g. 96 hours). Treatment of NSTEMI patients may either be with medication or the ‘scaffolding’ of collapsed arteries with PCI/angioplasty. Again, this is time sensitive and more likely to be effective if carried out within a short time frame. Although easily overlooked in the guidance, early and continued monitoring is the core activity from which other actions in NSTEMI care stem.\n\nIn this study (called ‘Variation In Cardiac Care’ or ‘VICC’), the research team examined differences in the management of NSTEMIs in 10 UK hospitals. In a previous publication related to VICC (Deaton et al., 2016) we focused on the varied roles and responsibilities of specialist cardiac and advanced practice nurses involved in the care of patients with NSTEMI. In Cramer et al. (2018), based on the VICC dataset, we developed a theoretical argument about the ‘boundary work’ (Abbott, 1988; Gieryn, 1983) of multidisciplinary teams and how this was central to the everyday organising and coordinating work of hospital staff. There were benefits for NSTEMI patients getting noticed as ‘boundary objects’ (Star & Griesemer, 1989) in the same way as STEMI patients. In this paper and using the same VICC dataset we focus on another key variation we found between hospitals: close monitoring. Focusing only on NSTEMI patients, we describe close monitoring differences in more detail and examine why this variation mattered. Detailed examples of variation in care can inform quality improvement and potentially help improve patient outcomes.\n\n\nMethods\n\nQualitative design, including observations of NSTEMI care, interviews with clinical staff and patients, as well as scrutiny of medical records.\n\nWe used the Acute Coronary Syndrome (ACS) registry for England and Wales (Myocardial Ischaemia National Audit Project, MINAP, Herret, Smeeth, Walker & Weston, 2010) to identify a sample of 30 hospitals in the top or bottom third of 30-day case-mix adjusted mortality for ACS. We purposively sampled eight hospitals: four from the top tertile and four from the bottom; they varied in coronary interventional facilities, teaching status, volume of cardiac admissions and geography. We piloted the field work methods in two hospitals before commencing on the main sample of eight hospitals. This pilot work gave us a greater insight into the complexity of NSTEMI care and, as a result, led us to focus exclusively on NSTEMI care in the main study. In total there were ten participating hospitals with variation in their organisation of care for NSTEMI patients (see Table 1). As a condition of the research the hospitals were not aware of their selection based on performance status for 30-day mortality. The research team was also blinded to hospital performance status until the analysis of qualitative data was completed.\n\n† Low = 1-249, medium = 250-499, high= 499+\n\n‡ Hospital variation in 30-day mortality with a final diagnosis of either ST-segment elevation myocardial infarction (STEMI) or Non- ST segment elevation myocardial infarction (NSTEMI) and adjusting for case-mix. Data are for all 236 hospitals in England and Wales with 413642 first time admissions from 1st January 2003 to 6th August 2009. These data were used for the initial sampling framework. Data rounded to the nearest whole number.\n\n§ Primary percutaneous coronary intervention (PCI/also known as angioplasty)\n\n\|\| Coronary Artery Bypass Graft\n\n¶ Privately owned catheter laboratories\n\n# Acute coronary syndrome\n\nBefore any data collection commenced the study was approved by the South West 5 Rec, UK National Health Service ethics committee (10/H0107/75, approval gained January 2011). For each hospital we obtained initial permission from the clinical lead for cardiology before approaching staff and teams in specific wards and departments. One hospital we approached declined to take part. As this was a study involving ward observations and staff shadowing of a clinical condition needing care in multiple hospital departments, it was accepted by our ethics committee that it would be impossible to gain written consent for all participants encountered. In agreement with our ethics committee we sought informed verbal assent from all participants being observed, followed by written consent of patients, staff and carers when the researcher observed their care more closely or requested an interview.\n\nData collection was conducted in five different ways: (i) observation of care for a purposive sample of NSTEMI patients from admission through to discharge (n=68); (ii) observation of staff at each site (n=199); (iii) interviews with clinicians and managers (n=143); (iv) interviews with patients including the same patients 30 days after discharge (n=53); and (v) patients’ medical records (See Table 2). Fieldwork was carried out over two weeks at each hospital (June 2011 - August 2012), focusing on the processes of admission, diagnosis, treatment and discharge. The researcher, JH, had previous experience in conducting ethnographic interviews and observations in hospital settings, including research for her doctoral thesis. Staff identified and introduced likely ACS patients to the researcher early in the patient’s hospital stay and the researcher then regularly visited them and asked staff about their care. The study was described to participants as trying to identify the different approaches to NSTEMI care with the aim of sharing the best practices more widely. Assurances of strict confidentiality of any critical feedback was given to all participants and, for patients, that their care would not be affected by anything they said. The researcher was White British and did not have a clinical background but was passionate about improving patient care and the potential of qualitative research to achieve this. To minimise social desirability bias, staff behaviour and accounts of care were compared with observations and accounts of care from other staff and from patients. Patient participants were purposefully selected to represent diversity of age and gender (see Table 3), with carers sometimes being included in the interviews. Staff participants were selected to represent member identified categories and variation of roles within each hospital (see Table 4). We also paid special attention to specialist cardiac nurse roles as part of the theoretical sampling strategy. Interviews with staff and patients varied with some lasting a few minutes and others lasting over one hour. Verbal data were audio-recorded and transcribed; observational data were written as field notes. The transcripts and fieldnotes were not returned to participants or hospitals for comment or correction.\n\n† limited number of ACS patients and/or restricted research access.\n\nThis table also appears in Cramer et al. (2018). 'Who does this patient belong to?' boundary work and the re/making of (NSTEMI) heart attack patients. Sociology of health & illness, 40(8), 1404–1429. https://doi.org/10.1111/1467-9566.12778. Table reprinted with permission from Wiley as part of the Copyright Transfer Agreement.\n\nData analysis aimed to identify variation in care, the possible reasons for observed differences and if and why it mattered. The interview topic guides and observations were informed by clinical guidelines available at the time of study (NICE, 2010, 2016; NICE 2010, 2013), existing literature on the quality of NSTEMI care as well as experiences at the two initial sites. We inductively identified categories from within the data, coded all of the data within these categories and used constant comparison to check items of coded data within each category for similarities and differences (Glaser & Strauss, 1967). The analytical approach followed many of the broad principles of grounded theory: simultaneous data collection and analysis; analytical codes developed from data not using preconceived logically deduced hypotheses; theoretical sampling; memo making as a bridge between coding and writing; and conducting literature reviews after developing an independent analysis. Our triangulation of data included both clinician and patient perspectives. Strong themes emerged on core areas which we felt gave us adequate information power (Malterud, Siersma, & Guassora, 2016) rather than data saturation as in such a complex clinical field, it is hard to claim full data saturation. The categories and code structure were developed by JH, HC and ME in collaboration with team members (KF, GF, CD and RJ). Half of the team had a clinical background (GF, RJ, CD, AT, HH) with specialisms in nursing (CD), cardiology (AT, GF, RJ) and general practice (GF, RJ) and the other half were non-clinicians with a variety of social science backgrounds (JH, HC, KF, ME).\n\n\nResults\n\nWe found variation in the care of NSTEMI patients in several (related) domains. The focus of this paper is on the domain of monitoring practices. We explore the variation in monitoring practices between hospitals by way of four key challenges for NSTEMI care: monitoring a wide group of patients; the prioritisation of high and medium risk patients; coordinating beds and catheter laboratory slots, and timely interventional responses. Patients and staff participants are: [P1, P2]; staff [S1, S2]. The different sources of data were: interview [I]; observation [OB]; audio-recorded [AR]; and field notes [FN].\n\nMonitoring a wide group of patients as they enter the hospital system and ongoing outreach work with patients and staff in multiple locations across the hospitals was part of the NSTEMI challenge. Identifying, and then closely monitoring NSTEMI and potential NSTEMI patients, was fundamental to care.\n\nPatients arriving in the Accident and Emergency department (A&E) are initially seen by generalist staff. NSTEMI diagnosis took time to emerge and, in many cases, required cardiac specialists to combine symptom interpretation and test results. As diagnosis was often complex and protracted, close monitoring in suspected cases was also very important. Having robust organisational arrangements whereby generalist staff (in places like A&E) could quickly, regularly and easily call on specialist cardiac staff for patients with symptoms like chest pain was essential for NSTEMI diagnosis but varied significantly across our hospital sample. Only half of the hospitals in the sample employed cardiac specialist nurses whose role focused on close monitoring to facilitate early identification of patients with NSTEMI (or possible NSTEMI). Other hospitals, for example, employed cardiac nurses whose main job was data reporting, or did not employ cardiac specialist nurses at all. In Hospital 3, the specialist nurse described how he undertook a huge variety of everyday monitoring related tasks and informal practices that might be needed in the support of identification in A&E:\n\nI try and catch people down in A&E and before they go to the wards … [] I go through the diagnosis and look to see if there’s anybody with chest pain … [emergency staff] they’ll catch me and say … [] I’ve got this patient on [ward] I’d like you to have a look for me. [] mopping up any loose ends as well because often they’re seen by junior doctors … [] And if tests are needed then I will expedite those tests. …. It’s all about being proactive. … [] I can get things moving (S12, I:AR).\n\nRaised troponin levels helped to identify possible NSTEMI patients. However, troponin may also be raised for other reasons such as kidney disease, making diagnosis difficult. One patient, who had kidney disease (renal failure), described his experience:\n\nThey put me on an observation ward … [] they didn’t actually know with the tests and the blood tests what was wrong with me because I’ve got renal failure as well. … [] it can give a false reading on my bloods. Erm they did one for the heart, they did my ECG’s … [] when they whittled everything down it come down to a heart attack … [] I’m having an angiogram to find out how if there’s a blockage, where the blockage is (P55, Hospital 8, I:AR)\n\nIdentifying possible NSTEMI patients who may have been missed at the time of admission or who had a cardiac event while in hospital required particular approaches to monitoring also involving specialist expertise. Most, but not all, hospitals in the sample used daily lists of troponin measurements as a method to try and identify NSTEMI and possible NSTEMI patients located around the hospital. Cardiac nurses in Hospital 8 described checking troponin lists daily as a laborious but necessary fail-safe method:\n\nIt is frustrating I can visit 10 different wards in a day and none of them turn out for me … [] but I may find that one of those patients has come in with a cardiac presentation or had a cardiac event whilst they’ve been in and I may find that nobody else has noticed that they had a raised troponin and ECG changes (S147, cardiac nurse, I:AR).\n\nThe danger of not having specialist cardiac staff closely accessible, available and checking over patients was that NSTEMIs could get easily overlooked. One senior cardiac nurse described in some detail how she persisted with her suspicions, monitoring and investigations on behalf of an NSTEMI patient in A&E, highlighting the complexity of diagnosis and frequent need for specialist interpretation:\n\nHis initial troponin had come back at 0.93 but he had … chronic kidney disease [] … So the [general emergency] consultant that saw him … [] … He wasn’t convinced it as an [MI] … [] So what we decided to do was to repeat the troponin to see if it … stayed elevated … [] I felt that it was an MI … [] it felt a little bit too high. And I didn’t think we could really ignore the chest pain … [I] just didn’t think that he was very good at describing his symptoms … [] So anyway, we, we repeated the troponin and it had come down to 0.48, so there had been a rise he went under [cardiologist] and when he reviewed him …. [] he said we definitely have to treat it as an MI (S150, Hospital 8, senior cardiac nurse, I:AR).\n\nOngoing close monitoring of NSTEMI or potential NSTEMI patients was required to prioritise patients at medium or higher risk of another cardiac event or, to identify patients who were clinically deteriorating. Again, good access to specialist cardiac support was often critical. For instance, the lack of specialist input in A&E may have meant that a patient in Hospital 9 was not prioritised appropriately and only received an intervention at the upper time limit recommended for interventions (96 hours maximum):\n\nA 57-year-old woman was brought to A&E and reported constant pain. She had ECG changes, a strong family history of heart disease, a big rise in troponin from 129 to 2033 and an initial diagnosis of NSTEMI. She went to catheter laboratory for an angiogram and angioplasty after 91 hours (P59, Hospital 9, OB:FN and medical notes).\n\nWhere ongoing pain is reported, risk is increased.[16] The patient in this case was told the blockage in her artery was large. In an interview she explained that her doctor thought she should have received an intervention much earlier:\n\nI was in constant pain from when I first come in from when I had the operation done yesterday … [] the doctor downstairs was going mad, the surgeon, he said: ‘By rights you should have been brought straight down as an emergency’. But they’d just been giving me paracetamol for the pain (P59, Hospital 9, I:AR).\n\nIn Hospital 9 where this patient’s treatment was slow, cardiac ward staff reported an inadequate A&E triage system and poor access to specialist cardiac advice. With very little initial filtering of NSTEMI or possible NSTEMI patients, as patients entered the hospital a wide assortment of chest pain patients were sent to the cardiac wards with staff acknowledging that a high proportion of these were being inappropriately admitted, for example, with gastric rather than heart problems. In the following example, the cardiologist refers to system as being ‘crumpled’ due to poor filtering and prioritisation systems at the hospital entry point in A&E:\n\nAssessment of NSTEMIs is at the moment a little bit crumpled, … [] an initial triage, yeah and that might help a little bit more … [] but at the end of the day, A&E are not going to be in a position to necessarily help, they just send it up … [] the sheer number, the economy of scale has been lost within numbers and the pressure (S170, Hospital 9, I:AR).\n\nHospital 5 stood out as having the most effective systems for NSTEMI patient triage and prioritisation. Part of this system included having an overnight cardiac registrar checking A&E and other wards for possible NSTEMI patients. While specialist cardiac support in the other hospitals was often reduced significantly at nights and weekends, in Hospital 5 the 24/7 integrated cardiac specialist approach meant that the overnight registrar helped to identify and prioritise NSTEMI patients and had a robust morning handover. Where all the other hospitals had daily ward rounds (or sometimes less frequently in the case of Hospital 4), Hospital 5 had two multidisciplinary ward rounds per day. In the following quote, a nurse explained how the overnight registrar doctor (or ‘reg’) duty system worked. The quote highlights how closely cardiac specialists worked with generalist staff and describes a clear process for double checking decisions with senior colleagues:\n\nWe have a registrar that comes on at 8 o’clock at night until they’ve done patient triage in the morning … If it’s an NSTEMI, ECG no changes, patient stable, the reg will say they’re happy for them to go to the admissions unit or might say I’m not quite happy with this patient I want them to go to coronary care …. If they get anyone in that is [troponin] positive for NSTEMIs, [A&E staff] ring the night reg, and the night reg goes down and reviews the patient … [so] at night the cardiac patients are seen reviewed and medications appropriate started and plans made on the night shift so that group of patients aren’t waiting until 8 o’clock [am] for a consultant to see them before a plan starts … [If] you’ve got a NSTEMI who’s having dynamic ECG changes [marker of increased risk of poor outcome] he’d ring the on-call cardiologist and say do you want to put him in the lab tonight? So the on-call team would come in for that if they’re unstable (S97, senior cardiac nurse, Hospital 5, I:AR).\n\nCertain categories of NSTEMI patients were not considered for prioritisation. Two hospitals automatically excluded patients over the age of 85 from cardiac wards, unless there were exceptional reasons. This conflicts with ESC guidance that older people benefit equally from intensive management (ESC 5.8 web addenda, Roffi et al., 2016; Kaura et al., 2020). For example, in Hospital 5 we traced the care of one 87year-old woman with a diagnosis of NSTEMI who stayed on a care of the elderly ward. On this ward she did not see a cardiologist, nor have her planned echocardiogram and did not benefit from the close cardiac monitoring necessary for the medical management of her condition. She died shortly after leaving hospital (P34, OB:FN and medical notes).\n\nEffective monitoring of an NSTEMI patient included being able to respond quickly and appropriately to changing patient needs. Making an accurate diagnosis with angiogram and, where appropriate treating disease with angioplasty, were both sometimes needed. As well as differences in the availability of specialist cardiac staff to support timely and appropriate responses, there were also considerable differences between hospitals in their ability to access catheter laboratory facilities (where angiograms and angioplasty were carried out), and in their bed management systems.\n\nGuidelines recommends an intervention window of 96 hours.[14,15,16] However, the wide variation in the availability and number of diagnostic and treatment facilities limited hospitals’ ability to respond within that window. Some hospitals had no catheter laboratories (Hospital 2), some hospitals only had facilities and trained staff for angiogram diagnosis (Hospital 8) and some hospitals had capacity and staff for both angiograms and angioplasty (Hospitals 1,3,4,5,6,7,9,10 – see also Table 1). NSTEMI patients needing angiogram/angioplasty who were initially admitted to smaller district hospitals with limited or no interventional facilities, required transfer to a hospital which did have the facilities. For patients needing transfer, delays in treatment (or worse, no treatment) were regularly reported. In one smaller hospital, although they had catheter laboratories and offered both angiograms and angioplasty, half of the six patients observed went home without their planned angiograms (Hospital 4, OB:FN). In one hospital the cardiologist highlighted regular delays when transfers were needed between hospitals:\n\nIf the patient comes from another [district] hospital it is not rare that the patient receives his cath [procedure in a catheter laboratory] after 4 or 5 days (S56, cardiologist, Hospital 3, I:AR).\n\nOne patient described their frustration at having to wait in a smaller district hospital to be transferred to a larger hospital with better facilities:\n\nThere was a lot of people back and forth seeing to me at one time. And they said I’d had the heart attack … [] one of the arteries was closed … [] so they said then that they can do the angiogram tests up there [district hospital]. But if I needed any work done they can’t put stents in [also known as PCI or angioplasty] …. this is why they transferred me down here [larger hospital] … I came in yesterday, the ambulance was booked for 9.15 in the morning …. [] but they didn’t come until about 7.30. Night. It was a long, quite a long wait. (P49, Hospital 7, I:AR)\n\nBeing admitted to a larger (‘tertiary’) hospital with all the facilities within the same hospital, however, did not always mean that required interventional work was available for NSTEMI patients. In the larger hospitals there was competition from STEMI patients who were always prioritised over NSTEMI patients in accessing a slot in the catheter laboratory.\n\nClosely related to being able to secure a slot in a catheter laboratory, hospital bed management was essential to support patients with the appropriate level of cardiac care, especially after interventional treatment. Hospitals managed their beds in various ways and we identified several different methods that appeared to be effective. For example, in Hospital 7, bed meetings took place four times per day and it was compulsory for all staff to attend these meetings. In many hospitals however, ineffective bed management practices at times undermined patient care:\n\nThere are times when they say ‘we’ve got no beds’ … [] if you just speak to the bed manager you’re just one of several people wanting a bed (S170, Hospital 9, cardiologist, I:AR).\n\nIn Hospital 5 a bed management system had been devised that was led by nurses and was very flexible and placements were not dependent on the approval of doctors (which often slowed how soon a bed could be secured). Referred to in Hospital 5 as ‘patient flow’, one fieldwork diary entry captured this constant need to juggle bed spaces according to patient needs:\n\nBed management overseen by nurses, constantly re-negotiated. No Dr’s authority needed. Had case last night where man did not wake after catheter laboratory sedation so needed intensive cardiac care bed urgently. Bed plans change minute to minute (Hospital 5, OB:FN).\n\nHaving a whole system approach facilitated responding in a timely manner. By this we mean the NSTEMI patient journey had been well thought through and there was a cumulative effect of having robust bed and catheter laboratory systems, underpinned by regular ward rounds as well as cardiac specialist input. For example, a patient case in Hospital 5 illustrates a seamless process for screening and swiftly treating patients. This patient was quickly found a bed in a specialist ward, his ongoing pain was under close and repeated review by specialist cardiac staff and there was a rapid interventional response:\n\nOne 72-year-old patient came back to hospital with chest pain having been discharged earlier that day. He was seen in A&E by a cardiologist and immediately given a bed in a cardiac specialist ward. Later that day a consultant reviewed his case and, unsure whether the ongoing pain was due to earlier angioplasty or angina, discussed the case with another cardiologist and agreed to an angiogram. Next day at the 8am ward round his symptoms were reviewed by a consultant, specialist cardiac nurse and night cardiac registrar and he had an angiogram later that day (P32, OB:FN and medical notes).\n\nBy contrast, in Hospital 4, bed management challenges, lack of an integrated cardiac system, and the limited access for NSTEMI patients to the catheter laboratories hampered timely intervention. In Hospital 4 there was a particular problem around beds because they had dual condition wards where both cardiac and respiratory patients were placed. Having dual conditions in one ward meant that there was sometimes confusion over which patients needed monitoring for which conditions and ultimately the responsibility for individual patients. On occasion, cardiac doctors missed seeing their patients on ward rounds. For example, one 82-year-old NSTEMI patient stayed for nine days in Hospital 4 but went home without her planned angiogram and two important medications missing. The nursing staff said that she had been missed on a ward round and this patient confirmed that she had only seen the doctor once:\n\nPatient: I’ve been here some 8 days today and I’ve been having scans on my liver and me kidneys and blood tests and scans and x-rays … [] I am hoping today that they have all the answers whether it’s go home or have the angiogram … []\n\nResearcher: Have you seen a cardiologist yet, have you seen the heart doctor?\n\nPatient: Yes I saw him last week, I’ve seen him once … [] the big mojo (laughs) that’s the one we are waiting for the decision (P27, Hospital 4, I:AR).\n\n\nDiscussion\n\nClose monitoring, often including good access to cardiac specialists, was a key area of variation. Specialist monitoring of potential NSTEMI patients helped manage the uncertainty and complexity of diagnosis. Active monitoring to identify undiagnosed patients (e.g. checking lists of repeat troponin measurements) and to prioritise patients required both specialist cardiac roles for interpretation and appropriate follow up. UK national guidelines recommend using clinical judgement to decide how often monitoring should be done until a firm NSTEMI diagnosis is made (NICE 2010, 2016), but they neither address how this monitoring should be organised, nor do they promote the important role cardiac specialists can play. We found that NSTEMI patients could be disadvantaged by initially presenting at smaller district hospitals with more limited or no interventional facilities and that catheter laboratory and bed management either inhibited or facilitated timely intervention. Apparently taking 12 years to achieve, with its integrated cardiology system approach, Hospital 5 stood out as having many of the best examples of monitoring and response, although patients in older age groups did not necessarily benefit. Aside from the recent Covid-19 pandemic which has impacted on treatment times for all heart attack patients (Gluckman et al., 2020), delays for NSTEMI patients getting a diagnostic angiogram, for example, are not improving significantly compared to STEMI patients (Wu et al., 2018). Looking at variation in care across hospitals can provide insights into how to provide timely interventions for conditions such as NSTEMI (Atsma and colleagues, 2020).\n\nGood coordination across diverse hospital teams is associated with reductions in overall length of stay for heart attack patients (Madell, Villa, Haywood, & Le Comte, 2015). One key study by Curry et al. (2011) that examined care for STEMI and NSTEMI patients, identified the importance of strong communication and coordination across disciplines and departments and empowered nursing staff. While there are some similarities between the findings of Curry and colleagues’ study and this one, the specific challenges of NSTEMI identification and diagnosis were not recognised. The importance of cardiac specialists being involved early in the diagnosis and the prioritisation of NSTEMI patients has been recognised elsewhere (de Belder 2012; Comer, 2021; Koganti & Rakhit, 2015; Stukel et al., 2010) including specialist nurse roles (Cramer et al., 2018; Deaton et al., 2016; Tierney et al., 2013). Other studies highlight the importance of frontline staff flexibility and autonomy (Cramer et al., 2018; Taylor, Clay-Williams, Hogden, Briathwaite, & Groene, 2015) and the need to resolve the catheter laboratory and bed space competition NSTEMI patients face from those with STEMI (Bellenger, Eichhofer, Crone, & Curzen, 2004; Koganti & Rakhit, 2015). However, care must be taken so that the trend towards recommending ever increasing specialisation is done with proper attention to the crucial and complementary generalist roles and their ongoing training.\n\nA major strength of this study was being able to observe the delivery of care as well as interview staff and patients about it. Other strengths included blinding of our research team to hospital performance indicators and including some smaller district hospitals (with limited intervention facilities) in the study. The inclusion of these smaller hospitals is important because many NSTEMI patients are cared for in district hospitals for some or all of their acute care. A limitation was that we visited hospitals for a relatively short period so would have been unable to detect possible changes or temporal variation in care. While our analysis of care is linked to evidence-based guidelines and based on careful triangulation between observations of patient care and clinical work, interviews and patient notes, our findings are hypothesis-generating and would benefit from further work testing the relationship of NSTEMI care to patient outcomes. In the time since data collection for this study occurred, a more sensitive blood test to check for troponin levels has come into use. This allows NSTEMI in some patients to be ruled out more swiftly, potentially reducing the number of patients admitted to hospital for observation. Nonetheless the data and findings on variation remain highly relevant to the ongoing challenges of diagnosis and management of patients with NSTEMI.\n\n\nConclusions\n\nVariation in care offers us a window into practices that could be improved (Atsma and colleagues, 2020). Cardiovascular disease is still a major cause of premature death and morbidity in developed countries and the proportion of NSTEMIs is increasing relative to STEMIs (Sanchis-Gomar, Perez-Quilis, Leischik, & Lucia, 2016). Many of the best examples of monitoring and response were dependent on the availability of cardiac specialists. Their role, whilst being labour intensive and harder to link to outcomes, is often recognised as crucial in connecting and coordinating care. These specialists are, however, also vulnerable to funding cuts. The example of monitoring in NSTEMI care is relevant to other acute conditions with diagnostic and treatment uncertainty, particularly in patients with multiple morbidities. Findings from the application of qualitative and observational methods can potentially inform quality improvement (Leslie, Paradis, Gropper, Reeves, & Kitto, 2013).\n\n\nData availability\n\nThe underlying data are not publicly available due to their containing information that could compromise the privacy of research participants and due to data security restrictions.\n\nThe research was given ethical approval on the condition that participants confidentiality would be maintained. We assured our committee ‘the absolute anonymisation of all data will protect the privacy of all patients’. Given the nature of the work the only reasonable way to ensure this confidentiality for both patients and staff is to not make data publicly available and to exercise care and caution of data requests. For example, we consider the data related to individual patient care such as patient’s medical notes to be too identifiable and in interviews, critical reflections of management and hospital care were discussed. Due to the small number of hospitals involved in the study, staff could be easily identified by their roles as there was often only one matron or specialist nurse role per hospital. The MINAP data on hospital performance (30-day mortality after admission) is not openly published and we do not have permission to disclose this. In our application to the MINAP committee we stated the research team would ‘not publish the hospital’s identity in any way or publish any linked information about individual hospitals and their outcome group (e.g. good or poor)’. We were specifically asked by our NHS ethics committee not to divulge to the participants that the research was looking at poor performing hospitals as well as top performing ones. The anonymised data that support the findings of this study are, however, available on request to bona fide researchers and will be considered on a case-by-case basis. We will consider, for example, requests for anonymised data that supports particular claims made by the authors. Approvals for such requests will require the consideration of the study team’s ethics committee but may also require some reassurance from the requesting researcher’s ethics committee on their planned use of data. Requests to access the data should be directed to South West-Frenchay UK National Health Service ethics committee, previously known as South West 5 Rec (frenchay.rec@hra.nhs.uk).\n\nZenodo: Topic guide and observation guide for Understanding Variation in Patient Care. https://doi.org/10.5281/zenodo.6360572 (Cramer et al., 2022).\n\nThis project contains the templates for the interview questions and observation guides used in this research.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAbbott A: The system of professions: An essay on the division of expert labour. Chicago: University of Chicago Press; 1988.\n\nAtsma F, Elwyn G, Westert G: Understanding unwarranted variation in clinical practice: a focus on network effects, reflective medicine and learning health systems. Int. J. Qual. Health Care. 2020; 32(4): 271–274. 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Publisher Full Text\n\nTaylor N, Clay-Williams R, Hogden E, et al.: High performing hospitals: a qualitative systematic review of associated factors and practical strategies for improvement. BMC Health Serv. Res. 2015; 15: 244. PubMed Abstract \| Publisher Full Text\n\nTierney S, Cook G, Mamas M, et al.: Nurses' role in the acute management of patients with non-ST-segment elevation acute coronary syndromes: an integrative review. Eur. J. Cardiovasc. Nurs. 2013; 12(3): 293–301. PubMed Abstract \| Publisher Full Text\n\nWest RM, Cattle BA, Bouyssie M, et al.: Impact of hospital proportion and volume on primary percutaneous coronary intervention performance in England and Wales. Eur. Heart J. 2010; 32(6): 706–711. PubMed Abstract \| Publisher Full Text\n\nWu J, Gale CP, Hall M, et al.: Impact of initial hospital diagnosis on mortality for acute myocardial infarction: A national cohort study. Eur. Heart J. Acute Cardiovasc. Care. 2018; 7(2): 139–148. PubMed Abstract \| Publisher Full Text\n\nYeh RW, Sidney S, Chandra M, et al.: Population trends in the incidence and outcomes of acute myocardial infarction. N. Engl. J. Med. 2010; 362: 2155–2165. Publisher Full Text" }	[ { "id": "216282", "date": "30 Oct 2023", "name": "Fuat Polat", "expertise": [ "Reviewer Expertise 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\nThis study investigates the impact of variations in close monitoring practices on NSTEMI patient management, revealing its significance in a context characterized by diagnostic and treatment uncertainty.\nI am writing to recommend the indexing of the research study titled \"Variation in Close Monitoring Practices and its Impact on Non-ST Segment Elevation Myocardial Infarction (NSTEMI) Patient Management: A Qualitative Study in English and Welsh Hospitals\".\nThis study, conducted by a team of dedicated researchers, offers valuable insights into the variations in healthcare practices and their influence on patient management, with a specific focus on Non-ST Segment Elevation Myocardial Infarction (NSTEMI) patients. The research methodology, including site visits, interviews, and observations, was rigorously applied, and data analysis was conducted in line with grounded theory principles. The findings shed light on the importance of close monitoring and its impact on patient outcomes in a context marked by diagnostic and treatment uncertainties.\nThe study's detailed examination of variations in close monitoring practices, as well as its implications for patient care, makes it a significant contribution to the field of healthcare. The research aligns with the goals and scope of Journal and would be of great interest to the journal's readership.\nI strongly recommend this study for indexing and believe it will provide valuable insights to the medical and healthcare community.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-375
https://f1000research.com/articles/9-1458/v1	15 Dec 20	{ "type": "Case Report", "title": "Case Report: Good responsiveness of metastatic sarcomatoid urothelial carcinoma with chondrosarcomatous differentiation to immune checkpoint inhibitor after radical surgery and adjuvant chemotherapy", "authors": [ "Hyung Ho Lee", "Hye Ju Kang", "Weon Seo Park", "Wonyoung Choi", "Ho Kyung Seo", "Sung Han Kim", "Hyung Ho Lee", "Hye Ju Kang", "Weon Seo Park", "Wonyoung Choi", "Ho Kyung Seo" ], "abstract": "Background: Sarcomatoid urothelial carcinoma with chondrosarcomatous differentiation (SUCCD) in the ureter has a poor prognosis and is a rare histological variant of ureteral cancer. The majority of ureteral cancers are urothelial carcinomas. Clinical case: We present a case of well-controlled metastatic SUCCD treated with an immune checkpoint inhibitor after radical surgery and failed adjuvant chemotherapy. The patient was a 68-year-old male with previous cure history of cT1 staged esophageal squamous cell carcinoma referred to the urology department for a right hydronephroureterosis complicating an intraureteral enhancing mass. After ureteroscopic biopsy and intraureteral urine cytology, atypical pleomorphic cell nests and chondroid tissue consistent with sarcomatoid urothelial carcinoma were observed. The patient underwent a successful radical right nephroureterectomy with bladder cuffing. The final diagnosis was a pT3N0 sarcomatoid urothelial carcinoma (heterologous component: chondrosarcoma > 95%) located at the right distal ureter and right renal calyx with infiltration of the periureteric fat and renal parenchyma of the renal capsule. On the postoperative one-month follow-up computed tomography scan, multiple enlarged lymph nodes and metastatic lung nodules were detected. The initiated adjuvant three cycles of gemcitabine-carboplatin therapy was marked by disease progression; thus, second-line therapy with atezolizumab was used for treatment. After five cycles of atezolizumab, the tumors showed a partial response without any grade 3 complications. Conclusion: The recurrent metastatic SUCCD showed good response to the immune checkpoint inhibitor after unsuccessful therapy with radical surgery and first line chemotherapy despite the unfavorable outcome of the pathology.", "keywords": [ "urothelial carcinoma", "sarcoma", "chondrosarcoma", "immune checkpoint inhibitor", "atezolizumab" ], "content": "Introduction\n\nUreteral cancer is rare, with a prevalence of less than 10% of all urinary tract urothelial carcinomas. Most (>90%) urothelial carcinomas occurring in the ureter are transitional cell carcinomas, while the others (<10%) include sarcomatoid urothelial carcinoma, squamous cell carcinoma, adenocarcinoma, and small cell carcinoma1. Among the rare histology types, urothelial carcinoma with chondrosarcomatous differentiation is the most rare2. A recent review by Lu et al. showed a poor survival outcome in 25 sarcomatoid ureter cancer cases with chondrosarcomatous differentiation2. In this case study, we present a 68-year-old patient recently diagnosed with sarcomatoid urothelial carcinoma with chondrosarcomatous differentiation of the right ureter discovered during esophageal cancer treatment. We thereby report a case of good response to the immune checkpoint inhibitor with an overall review of the histopathological characteristics of sarcomatoid ureteral cancer with some genetic background.\n\n\nCase description\n\nA 68-year-old male patient was referred to the urology department for right hydronephrosis complicating an incidental distal ureter stricture, which was found during an abdominal computed tomography (CT) scan for distal esophageal cancer. The patient was recently diagnosed with a 1.2 cm round-shaped distal esophageal squamous cell carcinoma, clinically stage T1, and was receiving a two-week 6600 cGy proton therapy in 33 fractions. The patient had a past medical history of restrictive lung disease, alcoholic liver cirrhosis (Child A), and was a heavy ex-smoker.\n\nThe CT treatment-planning scan showed right hydronephroureterosis due to distal ureteral stricture and ureter kinking. A CT urography was further performed at the urology department showing an abnormal ureteral kinking lesion with a distal intraureteral enhancing mass, just above the ureterovesical junction of the bladder (Figure 1A). Ureteral cancer was suspected given the findings in the right distal ureter: irregular wall thickening, and hydronephrosis with multiple small stones. Cystoscopy and microscopic urine analysis using the Nuclear Matrix Protein 22 test showed negative findings, except for benign hyperplasia of the prostate and a moderate trabeculated bladder without any voiding symptoms. Further, ureteroscopic biopsy with intraureteral urine cytology under general anesthesia found atypical cells, atypical pleomorphic cell nests, and chondroid tissue, consistent with sarcomatoid urothelial carcinoma (Figure 1B).\n\n(A) Preoperative CT urography and (B) ureteroscopy findings.\n\nAfter the first two-week proton therapy, the patient underwent a successful right open radical nephroureterectomy from the 11th intercostal incision and pelvic Gibson incision for bladder cuffing without a positive resection margin and intratumoral positive lymph nodes. Macroscopically, the pelvocalyx was enlarged, and the cut surface revealed multiple whitish solid tumors measuring up to 2.1 × 1.7 × 1.3 cm in the pelvis and extending to the cortex. The tumor also involved the distal ureter (Figure 2A). Microscopically, the tumor had a biphasic appearance of a high-grade urothelial carcinoma and a sarcomatous component with chondrosarcomatous differentiation (Figure 2B). Immunohistochemical analysis indicated that the sarcomatous areas were positive for vimentin, and the areas of malignant urothelial cells were diffusely positive for pan-cytokeratin and p63 (Figure 2C). Based on the histological and immunohistochemical findings, a pT3N0 sarcomatoid urothelial carcinoma (heterologous component of chondrosarcoma >95% positive for vimentin, p63, and pan-cytokeratin) located at the right distal ureter and a separate small tumor in the right renal calyx were diagnosed. The tumor in the renal calyx infiltrated the periureteric fat and renal parenchyma of the renal capsule.\n\n(A) Gross specimen of a right distal ureter mass and of a satellite renal pelvis tumor, (B) microscopic findings, and (C) immunohistochemical findings. (A) The cut surface of the resected kidney shows multiple whitish solid homogenous masses in the pelvic region extending to the renal parenchyma. The tumor in the distal ureter shows an irregular nodularity on the surface, a thickened wall, and periureteric fat invasion. (B) The tumor shows an area of urothelial carcinoma forming an epithelial component and sarcomatous area with abundant chondroid production, and the two components are fused together. (C) Immunohistochemical staining shows strong cytokeratin positivity in the urothelial carcinoma component and vimentin positivity in the cartilaginous sarcoma component.\n\nThe patient was discharged within 10 days without any complication including azotemia and resumed the two-week proton therapy for esophageal cancer. On the postoperative one-month follow-up CT scan, an increased size and necrosis of aortocaval lymph nodes and multiple metastatic lung nodules were detected. Three cycles of adjuvant gemcitabine and carboplatin (gemcitabine 1000mg/m2 D1, 8, and carboplatin AUC 5 D1, every 3 weeks) chemotherapy was administered due to the decreased renal function because of an underlying chronic kidney disease. The follow-up imaging after 3 cycles (9 weeks) of the initial treatment regimen indicated disease progression; thus, a second-line systemic therapy was initiated using an immune checkpoint inhibitor, atezolizumab (1200mg, every 3 weeks). After three cycles of atezolizumab, the multiple enlarged lymph nodes and lung nodules were no longer enlarged, and after five cycles of atezolizumab the overall size of multiple metastatic lesions decreased (Figure 3), indicating a partial response to atezolizumab. The patient has undergone seven cycles of atezolizumab without any grade 3 adverse event, and will continue to be treated.\n\n\nDiscussion\n\nChondrosarcoma is one of the most common malignant bone tumors in adults3. Chondrosarcomas are further stratified into conventional, mesenchymal, dedifferentiated, and clear cell subtypes. The conventional chondrosarcoma represents about 85% of all chondrosarcomas and is notoriously difficult to treat with chemotherapy3. According to the 4th edition of the World Health Organization Classification of Tumors, the International Agency for Research on Cancer defined the sarcomatoid variants of urothelial carcinoma as being histologically indistinguishable from those of sarcomas with a prevalence of 0.6% of all bladder tumors4. The most common symptom of SUCCD is gross hematuria with male predominance (ratio 3:1). The aggressive pathologic feature results in mostly nodal and visceral metastases at the time of diagnosis, like in our case. Our patient had an underlying alcoholic liver cirrhosis, a chronic pulmonary disease, and a distal esophageal cancer being treated with radiation therapy. He was diagnosed with pT3NxM0 chondrosarcoma with multiple sites at the distal (pT2) and the proximal ureter (pT3). Our case was similar to a surgical case of an 81-year-old Japanese female with a multifocal, synchronous pT2 sarcomatoid ureteral cancer and pT3 renal pelvic urothelial carcinoma with multiple visceral metastases diagnosed on postoperative month 115. Our patient had no nodal enlargement or metastases at the time of preoperative workups; however, the lung metastases and multiple nodal enlargement observed on postoperative month one strongly support the speculation that micrometastases were already present at the time of diagnosis of the right hydronephroureterosis and surgery. It is also worth noting that our patient presented with multiple gross hematurias during the esophageal cancer workups, and the right atrophic kidney with cystic changes and hydronephrosis were suggestive of a long-existent tumor.\n\nThe prognosis of SUCCD is dismal (3–24 months survival time)2 because the majority of urothelial carcinoma cases with sarcomatoid differentiation are known to be high grade, with an increased risk of micrometastases at the time of diagnosis due to late diagnosis, and have challenges associated with diagnosing a rare multifocal ureteral cancer6–9. Therefore, surgical resection is the primary treatment choice especially for chemoradiation-resistant tumors despite diagnosis at advanced stages. However, several retrospective case series and clinical trials have proposed the use of either anti-angiogenic inhibitors or immune therapy in the current genetic era.\n\nImmunotherapy has had enormous success in treating multiple cancer subtypes. Success has been particularly seen with immune checkpoint inhibitors, which are now approved as standard therapy in melanoma, lung cancer, and genitourinary cancers. Immunotherapy agents are increasingly demonstrating success in many cancer subtypes, and there have been preclinical suggestions that they may do the same in chondrosarcoma. The PI3K-Akt-mTOR, SRC, and Hedgehog pathways are the potential oncogenic targets for chondrosarcoma with VEGF2 inhibitors10,11. Other identified targets with next-generation sequencing are recurrent alterations in TP53, ACVR2A, COL2A1, YEATS2, and IDH12. These play a role in chondrosarcoma; therefore, they could be potential targets for immunomodulatory agents. In addition, immune checkpoint inhibitors have been successful treatments for sarcomas. Atezolizumab, an immune checkpoint inhibitor, has been approved for second-line therapy in ureteral cancer in Korea and in our case we observed a good response. A previous 67-year-old male patient treated with four cycles of nivolumab resulted in a near-complete response in metastatic pulmonary nodules13. Immune checkpoint inhibitors have been suggested to provide tumor-specific immune responses against cancer-specific antigens such as NY-ESO-1 or LAGE-1 in sarcoma patients with dedifferentiated chondrosarcoma, as well as chondrosarcoma cell lines13–15. The expected favorable outcome with the use of a single agent PD1 inhibitor with/without immunomodulatory agents in chondrosarcoma needs to be further investigated.\n\nIn conclusion, this recurrent metastatic case of carcinoma is a rare variant of multifocal synchronous ureteral cancers which responded well to atezolizumab. Our findings will help in early diagnosis, treatment planning, and better management with immune checkpoint inhibitors in case the current chemotherapy fails; thus, ensuring improved prognoses. In future, a series of case reports and studies would highlight the clinical benefit of checkpoint inhibitors with/without immunomodulatory agents in this disease clearly.\n\n\nEthical considerations and consent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient and the caregiver of the patient.\n\nThis retrospective study was approved by the Institutional Review Board (IRB) of the National Cancer Center (IRB No. NCC 2020- 0313-0001). This case report was proceeded in accordance with the tenets of the ethical guidelines and regulations of the World Medical Association Declaration of Helsinki-Ethical Principles for Medical Research Involving Human Subjects.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nSoria F, Shariat SF, Lerner SP, et al.: Epidemiology, diagnosis, preoperative evaluation and prognostic assessment of upper-tract urothelial carcinoma (UTUC). World J Urol. 2017; 35(3): 379–87. PubMed Abstract \| Publisher Full Text\n\nLu W, Wang Y, Li Y, et al.: Sarcomatoid urothelial carcinoma with chondrosarcomatous differentiation of the ureter: A case report and review of the literature. Oncol Lett. 2017; 13(3): 1331–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWagner MJ, Livingston JA, Patel SR, et al.: Chemotherapy for Bone Sarcoma in Adults. J Oncol Pract. 2016; 12(3): 208–16. PubMed Abstract \| Publisher Full Text\n\nMontironi R, Lopez-Beltran A: The 2004 WHO classification of bladder tumors: a summary and commentary. Int J Surg Pathol. 2005; 13(2): 143–53. PubMed Abstract \| Publisher Full Text\n\nTakemura K, Motoi T, Tonooka A, et al.: Multifocal Synchronous Upper Urinary Tract Carcinosarcoma (Sarcomatoid Carcinoma) With Rhabdomyoblastic Differentiation. Int J Surg Pathol. 2019; 27(5): 547–52. PubMed Abstract \| Publisher Full Text\n\nNicolas MM, Nazarullah A, Guo CC: Sarcomatoid urothelial carcinoma with chondrosarcomatous differentiation of the ureter: a case report. Anal Quant Cytopathol Histpathol. 2014; 36(2): 111–6. PubMed Abstract\n\nJohnin K, Kadowaki T, Kushima M, et al.: Primary heterologous carcinosarcoma of the ureter with necrotic malignant polyps. Report of a case and review of the literature. Urol Int. 2003; 70(3): 232–5. PubMed Abstract \| Publisher Full Text\n\nFleming S: Carcinosarcoma (mixed mesodermal tumor) of the ureter. J Urol. 1987; 138(5): 1234–5. PubMed Abstract \| Publisher Full Text\n\nPerimenis P, Athanasopoulos A, Geragthy J, et al.: Carcinosarcoma of the ureter: a rare, pleomorphic, aggressive malignancy. Int Urol Nephrol. 2003; 35(4): 491–3. PubMed Abstract \| Publisher Full Text\n\nPolychronidou G, Karavasilis V, Pollack SM, et al.: Novel therapeutic approaches in chondrosarcoma. Future Oncol. 2017; 13(7): 637–48. PubMed Abstract \| Publisher Full Text\n\nJones RL, Katz D, Loggers ET, et al.: Clinical benefit of antiangiogenic therapy in advanced and metastatic chondrosarcoma. Med Oncol. 2017; 34(10): 167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTotoki Y, Yoshida A, Hosoda F, et al.: Unique mutation portraits and frequent COL2A1 gene alteration in chondrosarcoma. Genome Res. 2014; 24(9): 1411–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWagner MJ, Ricciotti RW, Mantilla J, et al.: Response to PD1 inhibition in conventional chondrosarcoma. J Immunother Cancer. 2018; 6(1): 94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiraki A, Ikeda K, Yoshino T, et al.: Tumor-specific cytotoxic T lymphocyte responses against chondrosarcoma with HLA haplotype loss restricted by the remaining HLA class I allele. Biochem Biophys Res Commun. 2001; 286(4): 786–91. PubMed Abstract \| Publisher Full Text\n\nPollack SM, Li Y, Blaisdell MJ, et al.: NYESO-1/LAGE-1s and PRAME are targets for antigen specific T cells in chondrosarcoma following treatment with 5-Aza-2-deoxycitabine. PLoS One. 2012; 7(2): e32165. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "81387", "date": "18 Mar 2021", "name": "Wan Song", "expertise": [ "Reviewer Expertise Urologic 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\nIn this study, they presented a case of sarcomatoid urothelial carcinoma with chondrosarcomatous differentiation in the ureter treated with an immune checkpoint inhibitor after radical surgery and failed adjuvant chemotherapy. They showed that immune checkpoint inhibitor might be a successful option in case the current chemotherapy failed. Congratulations on good and inspiring work. It is pleasure to review high quality cases.\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": "100758", "date": "29 Nov 2021", "name": "Katsuhiro Ito", "expertise": [ "Reviewer Expertise Urology", "cancer immunotherapy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reported the case of sarcomatoid urothelial carcinoma with chondrosarcomatous component and the favorable response to the immune checkpoint inhibitor. Although sarcomatoid urothelial carcinoma of the ureter accounts for <10% of ureteral cancer, I think chondrosarcomatous lesion is relatively common among sarcomatoid variants. Nonetheless, it is an important report to share the outcomes of the immune checkpoint inhibitor for this rare disease.\nSpecific comments:\nIn the introduction, the authors said that the chondrosarcomatous differentiation is the rarest subtype and cited the study from Lu et al., however, in the study from Lu et al. the majority of the sarcomatoid UC is accompanied by the chondrosarcomatous component. Please correct the sentence.\n\nLu et al. reported sarcomatoid UC of the ureter, and not all were sarcomatoid ureter cancer cases with chondrosarcomatous differentiation. Please correct it.\n\nCase presentation is OK, but I recommend the authors show not only LN but also lung CT scan before/after atezolizumab.\n\n\"Adjuvant gemcitabine and...\": Adjuvant chemotherapy generally means chemotherapy to avoid recurrence. As the patient had already experienced lung mets, the term \"adjuvant\" should be avoided.\n\n\"(heterologous component of chondrosarcoma >95% positive for vimentin, p63, and pan-cytokeratin)\": This sentence seems unclear. Was more than 95% of the area positive for both vimentin and pan CK?\n\nAre there any findings that the metastatic lesion includes a chondrosarcomatous component?\n\nThe discussion was not straightforward. Since the patient was diagnosed as sarcomatoid UC, not chondrosarcoma, the first paragraph of the discussion seems irrelevant.\n\nThe authors discussed the genetic alteration of chondrosarcoma. Is there any evidence that these genetic mutations are present in sarcomatoid UC?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "7994", "date": "31 Mar 2022", "name": "Sung Han KIM", "role": "Author Response", "response": "We thank the editors and the reviewer (Prof Katsuhiro Ito) for his thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached herewith. Responses to Reviewer’s Comments 1. In the introduction, the authors said that the chondrosarcomatous differentiation is the rarest subtype and cited the study from Lu et al., however, in the study from Lu et al. the majority of the sarcomatoid UC is accompanied by the chondrosarcomatous component. Please correct the sentence. Response: We corrected the sentence according to the reviewer’s remark as follows (page 2 lines 60-62): “Among the rare histology types, urothelial carcinoma with chondrosarcomatous differentiation is relatively common among sarcomatoid variants.” 2. Lu et al. reported sarcomatoid UC of the ureter, and not all were sarcomatoid ureter cancer cases with chondrosarcomatous differentiation. Please correct it. Response: We corrected the sentences as follows (page 2 lines 63-64): “A recent review by Lu et al. showed a poor survival outcome in sarcomatoid ureter cancer cases with chondrosarcomatous differentiation among their 25 sarcomatoid ureter cancer cases2.” 3. Case presentation is OK, but I recommend the authors show not only LN but also lung CT scan before/after atezolizumab. Response: Due to the limited images according to the guidelines, we selected those targeted LN lesions better to show the therapeutic responses to the immune checkpoint inhibitors in this case report. 4. \"Adjuvant gemcitabine and...\": Adjuvant chemotherapy generally means chemotherapy to avoid recurrence. As the patient had already experienced lung mets, the term \"adjuvant\" should be avoided. Response: Thank you for the remark. We deleted the “adjuvant” in the abstract section and in the case presentation section of the manuscript (page 2, lines 46-48 and page 3, lines 106-107). 5. \"(heterologous component of chondrosarcoma >95% positive for vimentin, p63, and pan-cytokeratin)\": This sentence seems unclear. Was more than 95% of the area positive for both vimentin and pan CK? Response: We changed the phrase as follows: \"heterologous component of chondrosarcoma positive for vimentin, p63, and pan-cytokeratin\"(page 3, lines 100-101). 6. Are there any findings that the metastatic lesion includes a chondrosarcomatous component? Response: Unfortunately, we did not do target biopsy on the metastatic lesions. 7. The discussion was not straightforward. Since the patient was diagnosed as sarcomatoid UC, not chondrosarcoma, the first paragraph of the discussion seems irrelevant. Response: We deleted the first paragraph of the condrosarcoma explanation section to better clarify the sacomatoid variant of urothelial carcinoma (page 4, lines 117) 8. The authors discussed the genetic alteration of chondrosarcoma. Is there any evidence that these genetic mutations are present in sarcomatoid UC? Response: There exist several papers elucidating the importance of genetic profiling in sarcomatoid urothelial carcinoma. One article from Cell Press (Cell Rep. 2019 May 7;27(6):1781-1793.e4. doi: 10.1016/j.celrep.2019.04.048.) is where Bogdan Czerniak and his research team introduced research about a comprehensive genomic analysis of 28 cases of SARC and 84 cases of conventional urothelial carcinoma (UC), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. They found out that sarcomtoid urothelial carcinoma shows a distinct mutational landscape, with enrichment of TP53, RB1, and PIK3CA mutations which might give important implications for the development of more effective therapies, such as immune check-point inhibitors. We added this reference and their research articles to show whether it is possible that genetic profiling might be an important implication for future effective therpies using immune therapy in sarcomatoid urothelial carcinoma. We added some of the sentences to show the genetic profiling research in sarcomatoid urothelial carcinoma in the manuscript (page 4, lines 150-154). We also added a new reference (#12) as follows in the reference section (page 6 lines 211-212)." } ] } ]	1	https://f1000research.com/articles/9-1458
https://f1000research.com/articles/11-372/v1	30 Mar 22	{ "type": "Research Article", "title": "The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study", "authors": [ "Thomas Erwin Christian Junus Huwae", "Mohamad Hidayat", "Hidayat Sujuti", "Retty Ratnawati", "Mohamad Hidayat", "Hidayat Sujuti", "Retty Ratnawati" ], "abstract": "Background: Tourniquet is a common instrument used in Orthopedics field to reduce blood loss, providing a better operating field. One of the deleterious effects of tourniquet is ischemic-reperfusion injury, which occurs after deflation of the tourniquet. One of the possible ways to mitigate ischemic-reperfusion injury is by administrating antioxidants to reduce oxidative stress. Natural ingredients like Aloe vera are well known for its antioxidant and anti-inflammatory activities. This experimental study was conducted by fracturing the tibia of male Wistar strain rats (Rattus norvegicus) and administering Aloe vera gel orally as antioxidant. Method: A total of 18 rats were used in this study, divided into 6 groups. The first group was the control group, that included rats with fractured tibia not receiving torniquet and aloe vera treatment. The second and third groups were rats with fracture, who received torniquet application for one hour and two hours respectively without aloe vera treatment. The rest were the treatment groups, the rats with factures were given different dosages of Aloe vera extract: 40 mg/kg bodyweight (BW), 60 mg/kg BW, and 80 mg/kg BW. Three hours after administration, the tourniquet was inflated for two hours, followed by its deflation. The tibia was harvested to examine levels of superoxide dismutase (SOD), Malondialdehyde (MDA), and Bone morphogenetic protein 7 (BMP-7), and callus diameter, and osteoblast numbers were evaluated. Result: Administration of Aloe vera extract reduced the oxidative stress parameters (SOD and MDA). Tourniquet application decreased the osteoblast cell count and callus diameter, and administration of Aloe vera extract increased both variables close to the control value (p<0.05). Conclusion: The result of this study suggests that Aloe vera extract could be used to ameliorate ischemic-reperfusion injury.", "keywords": [ "Aloe vera", "fracture healing", "ischemic-reperfusion injury", "tourniquet", "Superoxide dismutase", "Malondialdehyde", "Bone morphogenetic protein" ], "content": "Introduction\n\nTourniquet is frequently used in orthopedic procedure involving fractures of long bone to achieve a bloodless operation field, or it can also be used in elective procedure such as total knee arthroplasty (TKA).1,2 The application of tourniquet, however, poses several complications including local neurovascular complication, systemic complication, nerve injury, post-tourniquet syndrome, etc.3 The deleterious effects of the tourniquet is caused by the direct compression and by the ischemic-reperfusion injury occurring after deflation.4 A study in Norway found that the incidence of tourniquet-related complications in orthopedic surgery was about 0.032%, more frequently affecting lower extremities compared to upper extremities.5\n\nIschemic-reperfusion injury is also one of possible serious complication of tourniquet application.6 Ischemic reperfusion can affect the limb itself (muscle and joint), or it may affect distant organ.4 Tourniquet-related ischemia-reperfusion injury can increase the superoxide synthesis and suppress endogenous antioxidant activity. Moreover, application of tourniquet by only 2 hours showed elevated creatine phosphokinase and lactic acid in animal model, suggesting that muscle damage occurs.7 On the other hand, the damage to distant organ is thought to be caused by superoxide ions entering circulation through venous blood flow.\n\nThere are several strategies suggested to ameliorate the ischemic-reperfusion injury after tourniquet application: ischemic preconditioning and postconditioning, reperfusion interval, and administration of pharmacologic agents.8–10 Reperfusion interval is proven to reduce the inflammatory markers in the distant organ, in this case the lung.10 However, the drawback of this method is the prolonged operating time. Thus, the authors would like to explore a measure which could be applied preoperatively to prevent this.\n\nOne of possible methods to reduce oxidative stress is by administering exogenous antioxidant, and this may be done preoperatively. Several antioxidants have been used in previous experimental studies: methylene blue, tramadol, magnesium sulphate, platonin, and others.11–14 One of naturally occurring antioxidant often used in various settings is Aloe vera. Aloe vera gel has been used to counter ischemic-reperfusion injury in the lung, kidney, and liver.15,16 However, the use of this agent in bone tissue is yet to be studied. Thus, the author aimed to evaluate the antioxidative effect of Aloe vera gel to mitigate ischemic-reperfusion injury after tourniquet application.\n\n\nMethods\n\nThis experimental study was conducted between January–March 2019 at the Faculty of Medicine, Universitas Brawijaya, Indonesia. The acclimatization, maintenance, and intervention of the samples were done in the Animal Model Unit, Laboratory of Pharmacology. The measurement of levels of superoxide dismutase (SOD), Malondialdehyde (MDA) were done in the Physiology Laboratory. The evaluation of Bone morphogenetic protein 7 (BMP-7) level was conducted in the Central Biomedical Laboratory. The callus and osteoblast evaluation were conducted in the Pathology Laboratory. The Ethics Committee of Faculty of Medicine, Universitas Brawijaya approved all animal protocols, and all subsequent experiments with the registration number: No. 72/EC/KEPK-S3/03/2020. All animal protocols, and all subsequent experiments were also carried out according to the ARRIVE guidelines and regulations.\n\nThe animal models in this study were 18 male rats (Rattus norvegicus), divided into 6 groups with 3 rats in each group. Male rats were used because the use of male rats is already established in fracture experimental study.17 Furthermore, the authors used only male rats to reduce variability because the previous study found that female rat fracture model exhibits different mechanical properties.18\n\nThe inclusion criteria were healthy 3-month-old male Rattus norvegicus weighing 180–200 g. The exclusion criteria were infection, death, abnormal extremity and broken/damaged cast. The animals were acclimatized for a week in a controlled condition of 12-h light/dark cycle at the temperature of 23.6°C with gentle handling, daily cage cleaning and close monitoring; they were fed a standard chow diet with free water intake.\n\nThe rats were divided into six groups. These groups were sacrificed 14 days after fracturing the tibia by dislocating their cervical to minimize the animal suffering. The details of the groups and their interventions are as follows:\n\nThe right tibia of the rat was fractured using the closed bone cutting technique.19 The animals were fasted for three hours before the procedure. The animals were anesthetized using 40 mg/kg bodyweight (BW) Ketamine HCl. The fracture was initiated by holding the rat’s leg on the proximal and distal parts using two forceps until complete fracture is achieved marked by false movement. The fracture was immobilized with long leg cast using plaster of paris (POP). The rats received analgetic medication in the form of paracetamol 10 mg/kg if there were signs of pain such as lethargy, difficulty in eating, and shivering.\n\nThe tourniquet used was a 4.5 oz orthodontic rubber band with a diameter of 1/8 inch. The orthodontic rubber band was applied to one leg of the rat on the proximal thigh. The use of orthodontic rubber band in the ischemic–reperfusion injury in the murine model has been established in the previous study.20 Tourniquet is often used in ischemic–reperfusion rats’ model because it provides adequate occlusion to femoral artery and its numerous collateral branches. Orthodontic rubber band is superior because it causes less soft tissue damage.20\n\nThe Aloe vera gel was created with Soxhlet method, then diluted with dimethyl sulfoxide (DMSO). The lowest dose of 40 mg/kg BW was determined using a conversion table by Laurence and Bacharach.21 The second dose of 60 mg/kg BW was determined as the half of the lethal dose of Aloe vera in rats (120.65 mg/kg BW/day).22 The third dose of 80 mg/kg BW was determined according to the interval between the first and second dose. The Aloe vera gel was administered three hours before application of torniquet and was given using a probe.\n\nSOD is an enzyme acting as an endogenous antioxidant.7 In this study, the authors measured the SOD level using SOD ELISA kit. The SOD levels were measured using the serum and the tissue (bone).\n\nMDA is the byproduct of lipid peroxidation, a process occurring in oxidative stress.23 The authors measured MDA level in the serum and bone tissue using Thiobarbituric acid reactive substances (TBARS) assay.\n\nBMP-7 is a subfamily of Transforming Growth Factor-β (TGF-β) which has osteoinductive activity. The serum and bone tissue level of BMP-7 were measure using BMP-7 Quantikine ELISA kit.\n\nThe callus formed was measured manually from the harvested tibia using a standardized caliper.\n\nThe number of osteoblasts in the callus tissue was measured microscopically. The number of cells was measured per high power field (HPF). The microscope used was Olympus BX-51 Dot Slide with Olympus XC10 camera.\n\nTo determine the significance of the comparison of the mean of various parameters, a hypothesis test was conducted using the One-Way ANOVA with an error rate of 5% or a confidence interval of 95%. However, before using parametric statistics, the requirements for normality and homogeneity of the data were tested first. The homogeneity of the data can be considered homogeneous because the number of samples in each group were the same. If the data was not normally distributed, the Kruskal-Wallis test was performed.\n\nFrom the normality test, all data were found to be normally distributed in all groups, except for the plasma SOD variable and the number of osteoblasts. The osteoblast variable had an abnormal distribution in all groups. Variables that were normally distributed were tested using parametric statistics, namely ANOVA, while variables that were not normally distributed were tested using non-parametric statistics, namely the Kruskal-Wallis test with a p-value of <0.05 which was considered significant. Further, all pair-wise comparisons were tested using Tukey test. If the assumptions were not met, the comparison was conducted using Kruskal-Wallis test and post-hoc Dunn test. Data analysis was performed by SPSS version 25 software (RRID:SCR_002865), NY Armonk, USA. URL: http://www-01.ibm.com/software/uk/analytics/spss/.\n\n\nResults\n\nThere were 18 Wistar strain rats used in this study. The measurement results of each variable in group 2 to 6 are depicted in Table 2.\n\n† p-value from one-way ANOVA test.\n\n‡ p-value from Kruskal-Wallis test.\n\n* p-value <0.05 is considered as significant.\n\nWe observed the bone and plasma SOD level did not increase much after administration of varying dose of Aloe vera, especially in plasma SOD. Meanwhile a dose-dependent increase was observed in bone SOD measurement. Both plasma and bone MDA decreased after administration of increasing dose of Aloe vera gel.\n\nThere was no significant difference in MDA levels between the groups of rats that were given a tourniquet or not. However, the administration of Aloe vera extract was shown to reduce bone MDA levels, although there was no significant decrease in plasma MDA levels. Both plasma and bone BMP-7 were seen to increase with the administration of Aloe vera extract. However, a greater increase in bone BMP-7 was seen compared with plasma BMP-7.\n\nAfter application of tourniquet, the authors observed a decrease of almost 30% of callus diameter formed (group 1 compared to group 3). Administration of Aloe vera gel increased the callus diameter close to diameter of control group. Similar reduction was observed in osteoblast cell count. In group 3, the number of osteoblasts decreased by 64.18 % compared to group 1. After administration of 40 mg/kg BW Aloe vera gel, the number of osteoblasts increased to 75% of the number in the control group. Moreover, with administration of 80 mg/kg BW Aloe vera gel, there was a 11% increase of osteoblasts number in group 6 compared to group 1.\n\n\nDiscussion\n\nIn fracture itself, oxidative stress can occur due to ischemic-reperfusion mechanism. In the first three days of the fracture healing phase, an ischemic phase occurs (no oxidative stress occurs). Then, at the stage of callus formation, new capillaries and proinflammatory cells will increase the production of reactive oxidative stress (ROS) that cause oxidative stress. With the increase in ROS, oxidative stress will arise which can be measured by MDA levels. Ischemic-reperfusion conditions that occur in fracture healing can be exacerbated by the use of a tourniquet. ROS cause loss of bone mass by inhibiting osteoblast differentiation and enhancing osteoclastogenesis.24 The results of this study showed that there were significant differences in the variables of bone SOD, bone and plasma MDA, bone and plasma BMP-7, callus diameter and number of osteoblasts. This significant difference was also found between the groups without a tourniquet and those with a tourniquet, as well as between the groups with or without Aloe vera use. Correlation analysis showed an inverse relationship only in plasma and bone MDA variables, with significant values.\n\nThe application of a tourniquet or administration of Aloe vera did not make a significant difference to plasma or bone SOD levels. These results may indicate that the administration of antioxidants such as ethanolic extract of Aloe vera is more beneficial, if given early in the onset of ischemic-reperfusion injury, and the benefits of its administration may be reduced or even absent if the injury is long-standing. In groups 1 and 3, it can be seen that the levels of SOD, both bone and plasma, did not have a significant difference, this could mean that SOD did not play a role in ischemic-reperfusion injury conditions due to oxidative stress, although the use of tourniquets.\n\nBoth the level of plasma and bone tissue MDA increased significantly after application of tourniquet. There was also no large decrease observed with the administration of a larger dose of Aloe vera ethanolic extract. This may indicate that the effect of antioxidants such as ethanolic extract of Aloe vera is more influential in the acute phase and the decrease in bone MDA on day 14 is a physiological process. This signified that the application of tourniquet and the subsequent reperfusion during its deflation caused a marked oxidative stress in local tissue and systematically. The same result was observed in the another preliminary study by the authors.6\n\nTurgut et al. used MDA levels in bone preparations to show an increase in oxidative state on bone healing in rats. MDA levels in murine model increased significantly on days 7 and 14 after tibia fracture.25 Turgut et al. stated that the first three days of the fracture healing phase were similar to the ischemia phase, whereas the second and third weeks were similar to the reperfusion phase of the ischemic reperfusion mechanism.25 This explains an increase in MDA levels and a decrease in SOD levels in the group after 14 days.24\n\nBone BMP-7 showed a significant increase after administration of Aloe vera ethanolic extract at doses of 40 mg/kg BW, 60 mg/kg BW, and 80 mg/kg BW. This can indicate that the administration of Aloe vera ethanolic extract has more effect on local tissue in bone than systemic. Bone BMP-7 levels also increased with the increase in the dose of Aloe vera given. Therefore, it can be interpreted that the administration of Aloe vera ethanolic extract can induce the production of BMP-7 in bone. This is supported by previous studies discussing the physiological conditions of bone healing. Cho, et al stated that BMP-7 acts in the osteogenic phase of the bone healing process where this phase is the final phase of the bone healing process (around days 14–21), the result of this phase is the formation of a hard callus.26 A previous study also has demonstrated that Aloe vera increased both number of osteoblasts and BMP-7 production.27\n\nThe results of the osteoblast count were comparable to the observations made on the callus diameter. In the group of rats who were given a tourniquet for either 1 hour or 2 hours, there was a significant decrease in the number of osteoblasts when compared to group 1. ROS are known to decrease the ability of osteoblastic differentiation in experimental animal bone marrow stromal cells (BMSCs), through Wnt/β inhibition, which plays a role in stimulating osteoblast differentiation, thereby increasing osteoblast apoptosis. When lipid peroxidation occurs due to ROS, aldehyde molecules are formed, such as MDA. Increased lipid peroxidation also increases apoptosis and osteoclastogenesis. Therefore, a high MDA expression indicates an injury due to post-ischemic reperfusion that has the potential to interfere with the bone healing process.24 This explains the increase in MDA as well as a decrease in the number of osteoblasts in the tourniquet-treated group. The administration of Aloe vera ethanolic extract could increase the number of osteoblasts and in group 6 (80 mg/kg BW ethanolic Aloe vera extract) the number of osteoblasts exceeded the number of controls. This was in line with previous research by Carson et al. who found that combination of whey protein and polyphenol of green tea (antioxidant) could increase osteoblast proliferation.28\n\nThe use of a tourniquet causes the diameter of the callus formed after 2 weeks to be smaller. In addition, the administration of Aloe vera extract could increase the diameter of the callus until it was close to the control group. The increase in callus diameter in the group given the ethanolic extract of Aloe vera could be caused by its antioxidant property. The phenolic compound found in Aloe vera is a strong, reductant and hydrogen donor.29 Other studies have demonstrated the benefit of Aloe vera in ameliorating ischemic-reperfusion injury in other organs.30,31 The results of our study suggest that Aloe vera gel may be used preoperatively to reduce the deleterious effect of ischemic-reperfusion injury after tourniquet application in bone fracture cases. Carson et al. also found that phenolic compound is the main antioxidant found in Aloe vera; this may explain the effect of Aloe vera in callus formation and osteoblast number in this study.32 There was a decrease in callus diameter in groups 3 when compared to group 1. These results indicate that the duration of using a tourniquet for 2 hours, the safety time to use a tourniquet, decreases the callus formation.\n\nThe strength of this study is that the results showed an improvement in callus diameter on administration of ethanolic extract of Aloe vera to the long bones (tibia) of white rats (Rattus norvegicus) in whom a tourniquet was used. This result is a very significant breakthrough in the management of ischemic-reperfusion injury. However, this study still has several limitations. Firstly, the authors did not identify the exact active polyphenolic or ethanolic compound responsible for the antioxidant activity of Aloe vera extract. The authors also did not measure the concentration of active compound in Aloe vera extract quantitatively. Moreover, there was a variation in weight of animal model used in this study. There was a possible difference in metabolic rate of Aloe vera extract and its active compound due to difference in bodyweight. This study only measured the parameters on two time points: directly after treatment and after 14 days. Future study could do measurements in more time points to better elucidate the metabolic changes imposed by ischemic-reperfusion injury and antioxidant administration. Nevertheless, this study could be used as a reference for future studies on the use of antioxidant in management of ischemic-reperfusion injury due to tourniquet use.\n\n\nConclusion\n\nAloe vera extract could be used as an antioxidant to reduce oxidative stress due to ischemic-reperfusion injury after application of tourniquet in fracture cases. Moreover, Aloe vera extract could improve proliferation of osteoblasts, callus formation, and BMP-7 production.\n\n\nAuthor contributions\n\nThomas Erwin Christian Junus Huwae: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Writing – Original Draft Preparation, Writing – Review & Editing\n\nHidayat Sujuti: Supervision, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\nRetty Ratnawati: Supervision, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\nMohamad Hidayat: Supervision, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\n\nData availability\n\nZenodo: Underlying data for ‘The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study’, https://doi.org/10.5281/zenodo.6258676.33\n\nThis project contains the following underlying data:\n\n• Raw data - Aloe vera for Ischemic-Reperfusion Injury after Tourniquet Application. (Include data for this study)\n\nZenodo: ARRIVE Checklist for ‘The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study’, https://doi.org/10.5281/zenodo.6258676.33\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nI am grateful to all my colleagues and resident in Orthopaedic and Traumatology Department, Faculty of Medicine Universitas Brawijaya - RSUD Dr. Saiful Anwar whom I have had the pleasure to work during this and other related projects.\n\n\nReferences\n\nKumar K, Railton C, Tawfic Q: Tourniquet application during anesthesia: “What we need to know?”. J. Anaesthesiol. Clin. Pharmacol. 2016; 32(4): 424–430. PubMed Abstract \| Publisher Full Text\n\nAhmed I, Chawla A, Underwood M, et al.: Tourniquet use for knee replacement surgery. Cochrane Database Syst. Rev. 2020; 2020(12). Publisher Full Text\n\nSharma JP, Salhotra R: Tourniquets in orthopedic surgery. Indian J. Orthop. 2012; 46(4): 377–383. PubMed Abstract \| Publisher Full Text\n\nLeurcharusmee P, Sawaddiruk P, Punjasawadwong Y, et al.: The Possible Pathophysiological Outcomes and Mechanisms of Tourniquet-Induced Ischemia-Reperfusion Injury during Total Knee Arthroplasty. Oxidative Med. Cell. Longev. 2018; 2018: 1–15. PubMed Abstract \| Publisher Full Text\n\nOdinsson A, Finsen V: Tourniquet use and its complications in Norway. J. Bone Joint Surg. Br. 2006 Aug; 88-B(8): 1090–1092. PubMed Abstract \| Publisher Full Text\n\nHuwae TECJ, Ratnawati R, Sujuti H, et al.: International Journal of Surgery Open The effect of using torniquets on fracture healing disorders: A study in wistar strain rats (Rattus norvegicus). Int. J. Surg. Open. 2020; 23: 48–52. Publisher Full Text\n\nTran TP, Tu H, Pipinos II, et al.: Tourniquet-induced acute ischemia-reperfusion injury in mouse skeletal muscles: Involvement of superoxide. Eur. J. Pharmacol. 2011 Jan; 650(1): 328–334. PubMed Abstract \| Publisher Full Text\n\nPac-Soo CK, Mathew H, Ma D: Ischaemic conditioning strategies reduce ischaemia/reperfusion-induced organ injury. Br. J. Anaesth. 2015 Feb; 114(2): 204–216. PubMed Abstract \| Publisher Full Text\n\nHausenloy DJ, Yellon DM: Preconditioning and postconditioning: underlying mechanisms and clinical application. Atherosclerosis. 2009 Jun; 204(2): 334–341. PubMed Abstract \| Publisher Full Text\n\nHuwae TECJ, Santoso ARB, Kesuma W, et al.: Reperfusion Interval as a Prevention of Lung Injury Due to Limb Ischemia–Reperfusion After Application of Tourniquet in Murine Experimental Study. Indian J. Orthop. 2020; 54(5): 704–710. PubMed Abstract \| Publisher Full Text\n\nWang L, Chen B, Lin B, et al.: Methylene blue attenuates lung injury induced by hindlimb ischemia reperfusion in rats. Mediat. Inflamm. 2018; 2018: 1–8. PubMed Abstract \| Publisher Full Text\n\nTakhtfooladi MA, Jahanshahi A, Sotoudeh A, et al.: Effect of tramadol on lung injury induced by skeletal muscle ischemia-reperfusion: an experimental study. J. Bras. Pneumol. 2013 Jun; 39(4): 434–439. PubMed Abstract \| Publisher Full Text\n\nKao MC, Jan WC, Tsai PS, et al.: Magnesium sulfate mitigates lung injury induced by bilateral lower limb ischemia-reperfusion in rats. J. Surg. Res. 2011; 171(1): e97–e106. PubMed Abstract \| Publisher Full Text\n\nHsu KY, Tsai PS, Lee JJ, et al.: Platonin mitigates acute lung injury induced by bilateral lower limb ischemia-reperfusion in rats. J. Surg. Res. 2011; 167(2): e255–e262. PubMed Abstract \| Publisher Full Text\n\nSehitoglu MH, Karaboga I, Kiraz A, et al.: The hepatoprotective effect of Aloe vera on ischemia-reperfusion injury in rats. North Clin. Istanbul. 2018 Oct; 6(3): 203–209. Publisher Full Text\n\nSahin H, Yener AU, Karaboga I, et al.: Protective effect of gel form of gastric gavage applicated aloe vera on ischemia reperfusion injury in renal and lung tissue. Cell. Mol. Biol. (Noisy-le-Grand). 2017 Dec; 63(12): 34–39. PubMed Abstract \| Publisher Full Text\n\nProdinger PM, Bürklein D, Foehr P, et al.: Improving results in rat fracture models: Enhancing the efficacy of biomechanical testing by a modification of the experimental setup. BMC Musculoskelet. Disord. 2018; 19(1): 1–8.\n\nMoreno LD, Waldman SD, Grynpas MD: Sex differences in long bone fatigue using a rat model. J. Orthop. Res. 2006 Oct; 24(10): 1926–1932.\n\nIbrahim N, Mohamad S, Mohamed N, et al.: Experimental Fracture Protocols in Assessments of Potential Agents for Osteoporotic Fracture Healing Using Rodent Models. Curr. Drug Targets. 2014; 14(14): 1642–1650. Publisher Full Text\n\nCrawford RS, Hashmi FF, Jones JE, et al.: A novel model of acute murine hindlimb ischemia. Am. J. Physiol. - Hear. Circ. Physiol. 2007; 292(2): H830–H837. Publisher Full Text\n\nMartina SJ: Antiplatelet Effectivity between Aspirin with Honey on Cardiovascular Disease Based on. Bleeding Time Taken on Mice. 2019. (Cvd).\n\nGuo X, Mei N: Aloe vera: A review of toxicity and adverse clinical effects. J. Environ. Sci. Heal. - Part C Environ. Carcinog. Ecotoxicol. Rev. 2016; 34(2): 77–96. Publisher Full Text\n\nAyala A, Muñoz MF, Argüelles S: Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal. Oxidative Med. Cell. Longev. 2014; 2014: 1–31. PubMed Abstract \| Publisher Full Text\n\nPrasad G, Dhillon MS, Khullar M, et al.: Evaluation of oxidative stress after fractures. A preliminary study. Acta Orthop. Belg. 2003; 69(6): 546–551. PubMed Abstract\n\nTurgut A, Göktürk E, Köse N, et al.: Oxidant status increased during fracture healing in rats. Acta Orthop. Scand. 1999 [cited 2022 Feb 24]; 70(5): 487–90. PubMed Abstract \| Publisher Full Text\n\nCho T-J, Gerstenfeld LC, Einhorn TA: Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 2002 Mar; 17(3): 513–520. PubMed Abstract \| Publisher Full Text\n\nAl-Hijazi AY, AL-Mahammadawy AKAA, Altememe EI: Expression of BMP7 in Bone Tissue Treated with Aloe vera. Int. Res. J. Nat. Sci. 2015 Nov; 13(2): 39–48.\n\nCarson M, Keppler JK, Brackman G, et al.: Whey Protein Complexes with Green Tea Polyphenols: Antimicrobial, Osteoblast-Stimulatory, and Antioxidant Activities. Cells Tissues Organs. 2018; 206(1–2): 106–118. PubMed Abstract \| Publisher Full Text\n\nMarzanna H, Dziedzic K: Hęś2019_Article_AloeVeraLWebbNaturalSourcesOfA(1).pdf. 2019; 255–65.\n\nYuksel Y, Guven M, Kaymaz B, et al.: Effects of Aloe Vera on Spinal Cord Ischemia-Reperfusion Injury of Rats. J. Investig. Surg. 2016 Dec; 29(6): 389–398. PubMed Abstract \| Publisher Full Text\n\nNejatzadeh-Barandozi F: Antibacterial activities and antioxidant capacity of Aloe vera. Org. Med. Chem. Lett. 2013; 3(1): 5. PubMed Abstract \| Publisher Full Text\n\nLópez Z, Núñez-Jinez G, Avalos-Navarro G, et al.: Antioxidant and cytotoxicological effects of Aloe vera food supplements. J. Food Qual. 2017; 2017: 1–10. Publisher Full Text\n\nHuwae TECJ: Data for Aloe vera for Ischemic-Reperfusion Injury after Tourniquet Application [Data set]. Zenodo. 2022. Publisher Full Text" }	[ { "id": "129336", "date": "22 Apr 2022", "name": "Langga Sintong", "expertise": [ "Reviewer Expertise Orthopaedic and Traumatology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article about ischemic reperfusion injury in fractured bone healing and its prevention with Aloe vera extract. The article provides a good presentation about how Aloe vera extract can prevent ischemic reperfusion injury after tourniquet application which is commonly used in orthopaedic surgery. The specific process of how the Aloe vera can prevent those injuries is also well explained in the article. The Aloe vera administration technique is also simple and easy to implement in clinical settings. The article provides sufficient background information for readers to understand the problem. The methods and statistical analysis in this article are technically clear and sound. I hope this study can be a recommendation for future studies about preventive treatment for ischemic reperfusion injury.\nFor the improvement of the article, I would recommend adding the step-by-step process of how to make Aloe Vera extract. I recommend adding that information to make the research more reproducible. Then, I would recommend adding some photographs to make the article more descriptive for readers.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "186509", "date": "17 Aug 2023", "name": "Saroj Kumar Shrestha", "expertise": [ "Reviewer Expertise Molecular biology", "Cancer", "Bone Remodelilng", "particulate matter", "vascular smooth muscle cell", "chondrogenesis", "Periodontitis" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is exciting to read about the ischemic reperfusion injury in fractured bone healing and the practical measure of aloe vera gel. The methods applied, and results and outcomes are excellent; however, the authors cannot express it briefly.\nThere are many minor grammatical mistakes; please once go over for English (grammar) checking. I would recommend that the author add some images during the experiment like, fracture of the tibia, administration of aloe vera gel, callus diameter, and osteoblast images.\n\nLine: 72-73: Tourniquet-related ischemia-reperfusion injury can increase superoxide synthesis and suppress endogenous antioxidant activity. This is reference number 7.\n\nLine: 73-75: Application of tourniquet for only 2 hours showed elevated creatine phosphokinase and lactic acid in animal models, suggesting that muscle damage occurs. This should be referenced as Heppenstall et al. (19791).\n\nLine 75-76: is this the authors' hypothesis, or did the authors forget to give the reference?\n\nIs it necessary to provide the details of the time of the experiment and the name of different laboratories? I suggest that the authors remove lines 92 to 96. However, the authors can thank the various labs in the acknowledge section.\n“The Ethics Committee of Faculty of Medicine, Universitas Brawijaya approved all animal protocols and all subsequent experiments with the registration number: No. 72/EC/KEPK-S3/03/2020. All animal protocols and all subsequent experiments were also carried out according to the ARRIVE guidelines and regulations”. Remove these sentences from the original position and copy them into the Animal model section.\nLine 107-108: “The exclusion criteria were infection, death, abnormal extremity, and broken/damage cast.” The condition of the rats like this is before or after the experiment? If this condition of rats is before the experiment, it is better to remove this sentence from the manuscript because the authors already mentioned that he used 18 healthy three months old rats.\n\nRemove Table 1 and write the divided groups' names and doses of Aloe vera gel into the animal model section in detail.\nIt is better to take a healthy control group with no fractured tibia group, so the authors can compare it to the other fractured and aloe vera gel applied groups (not necessary if not possible now).\nLine 116: Shortly (main points) describe the closed bone cutting technique.\n\nDescribe detail about the aloe vera gel extraction process. How much percentage of ethanol is used for gel extraction?\n\nLine 139: the authors used 60 mg/kg BW as the second dose. The authors mention that 60 mg/kg is half of the lethal dose of Aloe vera in rats, meaning if the authors give 60 mg/kg Aloe vera to 10 rats, 5 should have to die. But the authors do not mention any death of the rats in this experiment. Why did the authors choose these 60 mg/kg and 80mg/kg doses of aloe vera, knowing that it is toxic to rats?\n\nLine 141: Please clearly mention the route of aloe vera gel administration in rats (not only probe).\n\nLine 143: Describe shortly on SOD measurement. Provide the catalog number of the SOD ELISA kit. How do you prepare the bone for ELISA? Describe shortly.\n\nLine 147: Describe the MDA level measurement from the TBARS assay very shortly.\n\nLine 151: Describe shortly BMP-7 quantification and provide the catalog number of the BMP-7 Quantikine ELISA kit.\n\nLine 155: Describe shortly on callus formation measurement process.\n\nLine 158: is it possible to capture the image of osteoblast? If the authors can, please attach the pictures in the manuscript, which increases the quality of the authors' experiment and the article’s quality.\n\nLine 181: describe the time of the experiment after the fracture of the tibia. I think this analysis was done after 14 days of a tibia fracture. The authors should have mentioned it in the result section. Further the authors can mention shortly the preparation of samples from blood and bones.\n\nThere is no description of abcdefgh in Table 2, or is it not necessary to mention it?\n\nLine 211-216: References needed, and reference 24 is not matched for that statement.\n\nLine 217: the authors mention no more difference between the SOD of bone and serum in line 184, but the authors mentioned a significant difference in bone SOD in line 217. Please re-check it.\nSimilarly, please check lines 198 and 218-219 about the level of MDA with or without a tourniquet.\nLine 254-259: is these sentences fit reference 24?\n\nLine 283: the authors measured the parameters on two-time points: directly after treatment and after 14 days. Where are these two data? I have seen only one-time point data in Table 2.\n\nThank you. Best wishes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-372
https://f1000research.com/articles/11-370/v1	30 Mar 22	{ "type": "Research Article", "title": "Field programmable gate array implementation of an intelligent soft calibration technique for linear variable differential transformers", "authors": [ "Santhosh Krishnan Venkata", "Binoy Krishna Roy", "Preeti Mohanty", "Binoy Krishna Roy", "Preeti Mohanty" ], "abstract": "Background: Displacement is often used as an indirect indicator for monitoring multiple parameters, i.e. force, velocity, acceleration, and weight, making it an important variable for the measurement and control of processes. Sensors such as Linear Variable Differential Transformers (LVDTs) play a primary role in the design of any displacement measuring instrument. Calibration of an instrument is carried out to produce accurate results from the measuring instrument. Methods: The objective of this study is to calibrate the output of LVDT by designing a signal conditioning circuit so as to extend the linearity range of the sensor to 100% of the full scale input range, and also allows the measurement technique to adapt to variations in the physical parameters of the LVDT, the supply frequency, and the temperature. An optimized neural network is trained to produce linear and adaptive output from the raw data obtained from LVDT. Optimization is achieved by choosing the best neural network algorithm, number of hidden layers and transfer function of neurons which produce the least mean square error. The optimized neural network algorithm is implemented on a Field Programmable Gate Array (FPGA) chip for testing and validation in real life. Results: Experimental results show that the proposed technique was able to extend the linearity of LVDT and make the output adaptive for variations in physical parameters of LVDT, supply frequency and temperature. Conclusions: Accurate measurement of displacement is essential in many process applications, and a good calibration technique is required to produce accurate measurement. The presented calibration technique using optimized neural network algorithms has produced reliable measurements as desired.", "keywords": [ "Artificial neural networks", "Nonlinear estimation", "LVDT", "FPGA", "Sensor modelling" ], "content": "Introduction\n\nDisplacement reflects the dynamicity of the product under consideration. At certain instants, the dynamicity provides a metric of the stability of an object. Monitoring of the displacement is an important process in understanding the health of a structure. Several studies have discussed the relevance and importance of displacement measurements. For instance, the flexible loading of steel beams was analysed by measuring their displacement under a unit load using sensors such as Linear Variable Differential Transformers (LVDTs) and imaging techniques.1 Experiments have been conducted to study the fatigue behaviour of the Quisi and Ferrandet Bridges (twin 170-m-long steel railway bridges) using deflection sensors like strain gauges and LVDTs.2 The monitoring of irregularities on rail tracks has been carried out by measuring their displacement using an LVDT placed on a scaled vehicle.3 Instrumentation systems have been designed to measure the strain exerted on a reinforced polymer confined concrete beam using an LVDT,4 and the robustness of built structures has been tested by measuring the strain using LVDTs and accelerometers.5 LVDT displacement sensor is used to analyse the elasticity of asphalt and to measure the displacement caused by the application of load, axial, lateral, and Von Mises strain to a composite fibre reinforced polymer in confined and unconfined concrete slabs.6 These strain data can also be used to analyse the compressive behaviour of concrete slabs.7 Photogrammetric methods have been used to measure the displacement of geosynthetics during tensile tests.8 In Ref. 9, the flexural behaviour of cement pavement is analysed using distributed fibre optics and LVDT displacement sensors, while10 discusses the stress–strain behaviour of cement floors treated with soil and exposed to different types of seawater. The bonding behaviour of a reinforced concrete mixture subjected to corrosion and partial repair using self-compacting concrete is reported in Ref. 11, where the bonding strength is measured with respect to deformation using LVDTs. In Ref. 12, a fibre optic sensor array is deployed to analyse the strain experienced by sandstone under loading. The progressive damage suffered by masonry under cyclic compression loads and exposure to acoustic emissions is discussed in Ref. 13, with digital imaging and LVDTs used to measure the displacement. The use of LVDTs for the measurement of radial strain due to static and dynamic loading in geomaterials is reported in Ref. 14. Thus, displacement sensors are used in many applications and LVDTs are one of the more common sensor types. A brief study on the operation and characteristics of LVDT is now presented.\n\nSensor instruments typically consist of signal conversion, signal conditioning, and display unit blocks, each of which is needed to achieve error-free measurements. In Ref. 15, the design of a signal conditioning circuit using an amplifier, filter, and other elementary components is reported. The output of an LVDT is conditioned using modulation and demodulation circuits to obtain a calibrated result. The LVDT output is suitable for use in compensating a fibre optic interferometric instrument.16 A lookup table can be used to produce a linear/calibrated output from an LVDT.17 To compensate the nonlinearity in the LVDT response, a least-mean-square system has been developed as a compensator for nonlinearity.18 Signal conditioning using a dual slope converter circuit can be applied to an LVDT for the measurement of displacement.19 The alternating current (AC) output of the LVDT is converted to a direct current (DC) signal using a Root Mean Square (RMS)-to-DC converter circuit; the design and implementation of such a circuit are reported in.20 The physical design of the winding in an LVDT can be altered to achieve a higher range of measurement and complemented with an amplifier design for signal conditioning.21 The displacement caused by service loads on a rail bridge has been measured by an LVDT and transmitted using wireless transmission to a base station through a combination of sensors and the Audrino platform.22 Various flex sensors and LVDTs have been used to monitor the ground movement under the application of a load, with the output transmitted using the Bluetooth transmission principle.23 A combination of differential amplifiers and RMS-to-DC converter circuits have been used for signal conditioning of the LVDT output so as to obtain precise positioning results.24 A high-gain amplifier has been designed to improve the measuring range of LVDTs to the nanometre level without affecting their sensitivity,25 and an adaptive optimization circuit has been developed using a neural network to produce a linear output from an LVDT.26 A sine wave oscillator with stable amplitude and frequency phase-matching circuit with pulse width modulating functionality has been reported,27 where the circuit is designed to produce a linear output from the LVDT. A neural network algorithm has also been used to design the inverse function for the sensor characteristics, allowing a linear output to be produced from a sensor cascade in Ref. 28. Neural network algorithms can be used to offset the nonlinearity of LVDTs,29 and ant colony optimization can produce a similar effect.30 Reference 31 reports the design of a signal conditioning circuit for variable reluctance solenoids that enables accurate position and velocity measurements.\n\nCompact LVDTs can be designed to measure the displacement in a reactor, with the entire experimentation based on solving analytical equations that have been validated through an experimental setup.32 Reference 33 reports the enhancement of linearity in an ironless inductive sensor using an improved coil design. A thermal compensation algorithm can also be used to achieve higher accuracy. Analogue filters and neural network algorithms can be used to improve the characteristics of LVDTs,34 and application-specific integrated circuits have been developed to implement the signal conditioning circuits for LVDT sensors, consisting of an amplifier and a lookup table for producing a calibrated output.35 A combination of analogue amplifiers and multipliers can be used to provide the transfer characteristics,36 which are the inverse of the LVDT characteristics, resulting in a highly sensitive and linear output from a cascade. Reference 37 analyses the performance of an oscillator-based signal conditioning circuit for calibrating the output of LVDTs. Algorithms based on support vector machines have been used to design an automatic ranging functionality for LVDTs,38 and an analytical model of LVDTs has been developed to analyse their performance.39 Through the many techniques that have been developed to overcome the difficulties created by the nonlinear response characteristics of LVDTs, it is clear that linearization over a certain range of input scale is an important concept. Further, the output of LVDTs depends on several parameters, such as the number of turns of the primary and secondary windings, the dimensions of the primary and secondary windings, the excitation frequency, and environmental characteristics like the temperature. Most reported calibration techniques consider the LVDT parameters to be fixed, and so the calibration process must be repeated every time the parameters change.\n\nMost previous studies only consider the linearization of the LVDT over a portion of the input range, and do not consider any adaptation to the various LVDT parameters. These limitations motivated us to search for a better calibration technique for LVDTs. This paper describes an intelligent adaptive calibration technique using an optimized Artificial Neural Network (ANN) for displacement measurements by LVDT. This optimized ANN is trained to obtain linearity over the whole input range and allows the output to adapt to changes in the physical parameters of the LVDT, the excitation frequency, and the temperature. The proposed intelligent calibration technique is implemented on a field programmable gate array (FPGA) and temperature measurements are performed in real time.\n\n\nLVDT-based measurement system\n\nA block diagram of a displacement measurement system based on an LVDT is shown in Figure 1. A brief description of each block is presented below.\n\nAn LVDT is a transducer used to measure linear displacement that operates on the principle of mutual inductance. Figure 2 shows an illustration of an LVDT. There is moveable soft iron core positioned between three coils (one primary and two secondary). The primary coil is excited by an AC source of frequency f. The two secondary coils run along the same axis as the primary coil, and are connected in a series opposition way so as to monitor the change in displacement in both directions. Figure 3 provides a sliced view of the coils, clarifying their arrangement. Due to the excitation in the primary coil and in the vicinity of the secondary coils, a mutual inductance develops and produces voltages of V1 and V2 across the coils, respectively. If the core is displaced in either direction, the voltages across the secondary coils will vary, while that across the primary coil will remain constant. The variation in the secondary voltage will be proportional to the displacement of the core.40–43\n\nThe LVDT can be described by the following equations. The voltages generated in secondary coils 1 and 2 are given by Equations (1) and (2), respectively.\n\nIp - primary current due to excitation Vp\n\nx1 - distance penetrated by the armature towards secondary coil 1\n\nx2 - distance penetrated by the armature towards secondary coil 2\n\nnp - number of turns in primary winding\n\nns - number of turns in secondary winding\n\nf - frequency of excitation of primary coil\n\nTaking La = 3b, the differential voltage v = v1 - v2 is given by\n\nThe primary current Ip is given by\n\nThe inductance of a coil changes with variations in temperature. This relation can be written as44\n\nThe output signal of the LVDT is converted to a DC signal which is proportional to the displacement to be measured. This is done using an ‘LTC1967 AC to DC converter’. The mathematical relation used by the LTC196745 is given by\n\n\nProblem statement\n\nIt is important to understand how displacement-measuring instruments respond to variations in the properties of the LVDT. Thus, we consider the influence of the physical parameters of the windings, such as the ratio of the outer and inner coil diameters (ro/ri), ratio of primary and secondary coil lengths (b/m), number of primary windings (np), and number of secondary windings (ns), as well as the excitation frequency (f) and the temperature (t). The LVDT coil diameter, length ratio, and primary/secondary winding numbers can be varied to obtain different types of LVDT. The excitation frequency and temperature also vary under different working conditions. From the equations presented in the previous section, it is apparent that the output voltage obtained from the LVDT with respect to variations in displacement depend on one or all of the parameters discussed. The performance of an LVDT was therefore analysed by setting ro/ri to 2, 4, and 6; setting b/m to 0.25, 0.5, and 0.75; setting np to 100, 200, and 300; setting ns to 100, 200, and 300; setting f to 2.5 kHz, 5 kHz, and 7.5 kHz, and setting t to 25°C, 50°C, and 75°C. Under each of these conditions, the output responses obtained by the LVDT for various displacements are plotted in Figures 4–9.\n\nThe input–output responses of the LVDT plotted in Figures 4–9 show that the LVDT produces nonlinear characteristics, despite theoretically being a linear transducer. It can also be observed that the outputs depend on the physical parameters of the sensor and environmental factors. From the available literature, it is clear that researchers have worked on linearizing the LVDT output over 10–80% of its workable range, indicating that there is scope for improvement.2–24 Additionally, it has been reported that the instrumentation must be recalibrated whenever the physical parameters have undergone changes.27–31 Thus, the solution presented in this paper attempts to overcome these limitations.\n\nThe objective of this work is to design and implement an intelligent calibration technique on the Spartan-3E FPGA so that displacement measurements using an LVDT produce a linear output over the full range of the input scale. Additionally, the proposed system can adapt to variations in the physical parameters of the LVDT, the excitation frequency, and the temperature using the concept of an optimized ANN.\n\n\nProposed solution\n\nTo achieve the objective of designing an intelligent calibration technique which would increase the working linear range of the LVDT and make the instrument adaptive to variations in its physical parameters, a neural network model is proposed. A block diagram representation of displacement measurements with the proposed intelligent calibration technique is shown in Figure 10. The proposed instrumentation system consists of a program using neural network algorithms46–50 which will be trained to produce a linear output and resist the influence of changes in the physical parameters. Training the neural network is a process of tuning the neural network model to produce output such that the LVDT produces linear output for the full range, input data for the training is the output of data conversion circuit for varying displacement, b/m ratio, ro/ri, ns, np, supply frequency and temperature (the full training data has been provided in Underlying data51). Further, an attempt is made to enhance the functioning of the neural network model by considering network optimization and a transfer function. Matlab R2000b (RRID: SCR_001622) version is used for training the neural network. The custom code created as part of this research is available in Extended data. The code is also compatible with the open source software Scilab.\n\nANN, Artificial Neural Network; LVDT, Linear Variable Differential Transformer; FPGA, Field Programmable Gate Array.\n\nThe neural network is trained using the LVDT output from data conversion circuit for variation in displacement and the ratio of the outer and inner coil diameters (ro/ri), ratio of primary and secondary coil lengths (b/m), number of primary windings (np), and number of secondary windings (ns), as well as the excitation frequency (f) and the temperature (t). These data act as inputs to the neural network, and the linear output is a target matrix which is independent of variations caused by changes in physical parameters and relates only to the input displacement.\n\nIn matrix notation, the final output is obtained as\n\nIn the proposed solution, the neural network model employs a multilayer perceptron to train the system. The neural network model consists of an input layer, hidden layer, and output layer. The input layer is used to receive the inputs for the network model. The hidden layer considers a network of neurons arranged in multiple layers. Various algorithms can be used to train the neural network model. In this study, we consider five standard, openly available training algorithms, named (for the purposes of this study) AL1–AL5. These are, respectively, stochastic gradient descent,46 Nesterov accelerated gradient,47 adaptive gradient descent,48 adaptive moment estimation,49 and resilient backpropagation.50 The number of hidden layers and the transfer functions of the neurons can also be varied to obtain the desired objective from the neural network.\n\nThe designed program using neural network algorithm after training is downloaded on the FPGA chipset to be used in a displacement measurement system consist of LVDT. For the FPGA programming using MATLAB, the steps included adding details of hardware used (Spartan-3E FPGA kit), followed by code generation, synthesis, verification and testing. The memory and processing time of the chipset are vital parameters which should be considered when applied for testing in real-time applications. To achieve a neural network model with the best utilization of memory and processing time, it is vital to ensure an optimized network. To optimize the model, the number of hidden layers and the transfer functions of the neurons are varied and different combinations are tested. Table 1 presents the mean square error (MSE) obtained with various algorithms and numbers of hidden layers (see datalogging.xlsx in Underlying data). AL5 gives the lowest MSE with six hidden layers in the neural network algorithm. A higher number of hidden layers reduces the computation speed and increases the memory requirements. Hence, AL5 with two hidden layers is considered a suitable trade-off between the error component and the computational requirements.\n\nFor the chosen neural network model consisting of resilient backpropagation with two hidden layers, a suitable neuron transfer function must be determined to achieve an optimized neural network model. The MSEs obtained with the Tanh, Sigmoid, Linear Tanh, Linear Sigmoid, Softmax, Bias, Linear, Axon, Tansig, and Logsig transfer functions are listed in Table 2. From the table, it is clear that a neural network model with AL5, two hidden layers, and the Axon transfer function is optimal. The complete set of parameters in the final neural network model is given in Table 3.\n\n\nResults and analysis\n\nThis section presents the results obtained by the intelligent displacement measuring instrumentation system consisting of LVDT as a sensor, followed by data conversion circuit, and a trained optimized neural network model, when subjected to measurement. 182 Tests were carried out to evaluate the performance of the intelligent calibration technique for linearity and adaptation. For testing purposes, the range of displacement was considered from 0–100 mm, the range of ro/ri was set to 2–6, the range of b/m was set to 0.25–0.75, the range of ns was set to 100–300, the range of np was set to 100–300, the range of f was set to 2.5–7.5 kHz, and the temperature range was set to 25–75°C. The displacement measurements (available in train data.xlsx in Underlying data) with the proposed soft calibration technique based on the optimized ANN for various input displacements and combinations of ro/ri, b/m, ns, np, f, and temperature are listed in Table 4. The root mean square of the percentage error is 0.0431%.\n\nThe performance of the proposed intelligent displacement measurement technique was tested in a real-life scenario in a laboratory setup using an FPGA board. The experimental setup for this purpose is shown in Figure 11. Three different cases (to demonstrate the condition of the senser under particular variable values) were considered and the results are presented in Table 5 (datalogging.xlsx in Underlying data). Figure 12 shows the input output plot obtained from the designed work, it is seen from the figure that the output of the proposed system is varying linearly with the input over the full input range and the output of LVDT is independent of variation in physical parameters of the LVDT.\n\n\nConclusions\n\nThe calibration of any instrumentation system is an important process for ensuring the system behaves as expected. The process of calibration is time-consuming and involves substantial costs. Hence, it is very important to have an efficient calibration process. In this study, an intelligent calibration technique using a neural network model was designed. The neural network model was trained to produce a linear output from the nonlinear signal received from the data conversion circuits of an LVDT. The neural network was then trained to produce an output which would be independent of variations in the physical parameters of the LVDT sensor, such as the ratio of the inner and outer coil diameters, the ratio of secondary and primary coil windings, and the number of windings in the primary/secondary coils, as well as the excitation frequency applied to the LVDT primary winding and the atmospheric temperature around the LVDT.\n\nThe calibration system was developed on an FPGA board to allow for the physical implementation of the measurement system. Thus, an optimized neural network model was an important concern. The neural network model was optimized by testing various training algorithms, numbers of hidden layers, and transfer functions. In the optimization process, the MSE was considered as the cost function. The resilient backpropagation scheme with two hidden layers and the Axon transfer function was found to produce the optimal MSE, when considering the trade-off between accuracy and computational resources. The test results under simulation conditions and real-life conditions demonstrate that the reported calibration technique produces a linear output, offsetting the nonlinearity that exists in conventional measurement systems. Additionally, the system produces very low measurement errors, even with variations in the physical LVDT parameters, with a root mean square error of 2.8%. The system can be further improved by optimizing the algorithm and considering other variables in the sensor system which influence the measurement output.\n\n\nData availability\n\nOpen Science Framework: LVDT. https://doi.org/10.17605/OSF.IO/94NPQ.51\n\nThis project contains the following underlying data:\n\n• train data.xlsx (voltage output of LVDT, used for training neural network)\n\n• datalogging.xlsx (data corresponding to LVDT measurement parameters)\n\nOpen Science Framework: LVDT. https://doi.org/10.17605/OSF.IO/94NPQ.51\n\nThis project contains the following extended data:\n\n• program.sce (code that can be used to replicate the reported work in MATLAB (R2020b). The code is also compatible with the open source software Scilab.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nÖzsariyildiz ŞS, Düğenci O, Sulev E: Determination of displacements on built-up steel beams during experiments by image processing. J. Constr. Steel Res. 2021; 179: 106560. Publisher Full Text\n\nBertolesi E, Buitrago M, Adam JM, et al.: Fatigue assessment of steel riveted railway bridges: Full-scale tests and analytical approach. J. Constr. Steel Res. 2021; 182: 106664. Publisher Full Text\n\nUrda P, Aceituno JF, Muñoz S, et al.: Measurement of railroad track irregularities using an automated recording vehicle. Measurement. 2021; 183: 109765. Publisher Full Text\n\nPour AF, Gholampour A, Ozbakkaloglu T: Influence of the measurement method on axial strains of FRP-confined concrete under compression. Compos. Struct. 2018; 188: 415–424. 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Engineering Vibration, Communication and Information Processing. Singapore: Springer; 2019; (pp. 549–560).\n\nYang YS, Bae KY: The Modelling and Design of a Linear Variable Differential Transformer. Int. J. Precis. Eng. Manuf. 2021; 1–10.\n\nNeubert HKP: Instrument Transducers: An Introduction to Their Performance and Design. 2nd ed.New Delhi: India, Oxford University Press; 2003.\n\nLiptak BG: Instrument Engineers Handbook-Process Measurement and Analysis. 4th ed.CRC Press; 2003.\n\nMurty DVS: Transducers and Instrumentation. PHI publication India; 2003.\n\nPaul PL: Regitien, “Inductive and Magnetic Sensors”. Sensor for Mechatronics. 2012; 125–159. Publisher Full Text\n\nGroszkowski J: The temperature coefficient of inductance. Proceedings of the Institute of Radio Engineers. 1937; 25(4): 448–464. Publisher Full Text\n\nWilliams J: Instrumentation circuitry using RMS-to-DC converters. Linear Technology Corporation, Application Note. 2007; 106: 8–9.\n\nLei Y, Hu T, Li G, et al.: Stochastic gradient descent for nonconvex learning without bounded gradient assumptions. IEEE transactions on neural networks and learning systems. 2019; 31(10): 4394–4400. PubMed Abstract \| Publisher Full Text\n\nGu P, Tian S, Chen Y: Iterative learning control based on Nesterov accelerated gradient method. IEEE Access. 2019; 7: 115836–115842. Publisher Full Text\n\nLydia A, Francis S: Adagrad—an optimizer for stochastic gradient descent. Int. J. Inf. Comput. Sci. 2019; 6(5): 566–568.\n\nKhan AH, Cao X, Li S, et al.: BAS-ADAM: an ADAM based approach to improve the performance of beetle antennae search optimizer. IEEE/CAA Journal of Automatica Sinica. 2020; 7(2): 461–471. Publisher Full Text\n\nErkaymaz O: Resilient back-propagation approach in small-world feed-forward neural network topology based on Newman–Watts algorithm. Neural Comput. Applic. 2020; 32(20): 16279–16289. Publisher Full Text\n\nVenkata KS, Roy BK, Mohanty P: LVDT. [Dataset].2022, March 24. Publisher Full Text" }	[ { "id": "292182", "date": "20 Jun 2024", "name": "Bassam J. Mohd", "expertise": [ "Reviewer Expertise FPGA", "NN", "Security", "DSP", "Hardware" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a smart calibration method for LVDT displacement sensors. An optimized neural network learns to linearize sensor output across its entire range and adapt to variations in temperature, frequency, and LVDT properties. This NN is implemented on an FPGA chip for real-time temperature measurements. The design produced reliable measurements.\nIn general, the paper is well-written and provides comprehensive introduction and description of the system. However, there are issues in the paper that I strongly recommend addressing.\nI. Important issues:\nWhy FPGA platform was used? Why not a simple controller or DSP processor?\nFPGA design should be explained more. How many neurons were implemented in the HW? What is the time required to complete one run of the NN? Power and energy numbers would be great.\nTable 3: the training set is really small; it is important to expand dataset for better results and avoid over fitting.\nDesign results should be compared with other published work. The comparison should be with NN-based designs (e.g. ref 29, ref 30, ref 35) and non-NN algorithms (e.g. ref 38 which uses SVM).\nII. Minor issues:\nThe introduction section can be split into two sections: introduction (introduce LVDT and its application) and literature review (present researches relates to LVDT).\nFigures 4-9: are those figures produced by the presented equations or sampled from the actual LVDT?\nA block diagram of NN would be useful, with number of neurons in the layers.\n\nTable 1: No_of_hidden_ayer=4 has different trend from other cases. Any explanation?\nTable 2: since NN is implemented in HW, ReLU activation function should have been considered. Also, I am not sure too many knows AXON function. Explain this function.\n\nTable 5 (with results from real-time system) has higher error rate compared with Table 3 (simulated results). You need to justify the differences.\nFigure 12: what are the three cases that are plotted? Why case2 seem to deviate more (compared with case 1 and case 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": [] }, { "id": "327149", "date": "23 Oct 2024", "name": "Ngoc-Thang Bui", "expertise": [ "Reviewer Expertise deep learning for medical applications", "FPGA for ultrasound 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\nAfter reviewing the manuscript \"Field programmable gate array implementation of an intelligent soft calibration technique for linear variable differential transformers\", the reviewer has some comments as follows.\n1. The manuscript presents the implementation of an ANN model on the Spartan 3E FPGA to perform calibration technique for linear variable differential transformers.\n2. The reviewer has some comments as follows:\n2.1. First, I noticed 2 problems: 1). The ANN model is implemented on the Spartan 3E, 2). The author uses MATLAB v2000 for this research. To my knowledge, ANN and deep learning models are often implemented on FPGA chips with ARM cores inside (i.e., Zynq). The authors use the Spartan 3E chip which is quite old and can be said to be unsuitable because this chip line is not supported by newer software (i.e., Vivado). The authors use MATLAB v2000 for a study in 2024. Is this really appropriate?\n2.2. In Fig. 11, the author presents the experimental model, However, it is not clear. 1). How does the FPGA read data from the sensor? What type of communication is used? How does the FPGA process data? Does the FPGA transmit data or display the calculation results?\n2.3. The authors need to describe in detail and fully the components implemented in the study including both hardware and software. How did the study use the FPGA? How to implement the ANN model on FPGA.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-370
https://f1000research.com/articles/11-369/v1	30 Mar 22	{ "type": "Research Article", "title": "Lateral Multimodal Learning in a Saudi EFL Context: Investigating the Perceptions of Teachers and M2E Female Learners", "authors": [ "Arif Ahmed Mohammed Hassan Al-Ahdal", "Fahd Hamad Alqasham", "Mohammed Ali Mohammed Qarabesh", "Fahd Hamad Alqasham", "Mohammed Ali Mohammed Qarabesh" ], "abstract": "Background: ELT scenario in Saudi Arabia has undergone a sea change since the pandemic. With an aim to maximize resource utilization and ensure wide learner base, college students (male and female) are taught simultaneously, the former in a face-to-face mode and the latter in an audio-only mode. The nomenclature given to this unique classroom design by the researchers is Lateral Multimodal Learning (LML), one which has its own advantages and disadvantages. This mode of learning puts a great deal of pressure on the teachers as they must attend to a huge number of students with different needs and levels of competence, whereas it ensures best utilization of infrastructural and human resources by the administrations. Being a newly developed educational model, it is important to assess the efficiency of this type of learning. Methods: This study evaluates the model from the point of view of students (99), using a questionnaire, and that of teachers (06), using semi-structured interviews. Results: The results show that Saudi female students present high perceptions of learning via LML (M=4.03); are satisfied with this type of learning (M= 3.81) and the aids applied in learning via LML (M= 4.02). Findings also show moderate perceptions on the difficulties they encountered while emerging in LML mode (M =3.39). Furthermore, the study shows correlation between the four domains, i.e., perceptions, satisfactions, challenges, and aid. The highest correlations were between perceptions and satisfactions (r=.719); perceptions and aids (r=.659), and satisfaction and aids (r=.656). The teachers’ interviews show their agreement on the efficacy of LML as being professionally fulfilling and one that they would like to continue with in the future too. Conclusions: The study concludes with recommendations, which would be of great benefit and help for all parties or stakeholders involved.", "keywords": [ "COVID-19 Epidemic", "Distant Education", "e-learning", "English as a Second Language (EFL)", "English Language Teaching (ELT)", "Remote Education", "Saudi University Students and Teachers" ], "content": "Editorial note\n\nEditorial Note (26th July 2023): The F1000 Editorial Team is currently substantiating that this research was presented at an IIARP conference and should be included in the IIARP Gateway. The Editorial Team is currently requesting further information from the authors regarding this. This Editorial Note will be updated as new information is provided.\n\n\nIntroduction\n\nLateral Multimodal Learning (LML) is a pedagogical concept that took shape in the unique educational circumstances presented by the novel coronavirus (COVID-19) pandemic to the Saudi teaching community. Given the nature of gender segregated classrooms in the country, teachers (whether male or female) are wont to teach multimodally in lateral classrooms. The design is that learner groups are still gendered but each learns with the same teacher, who teaches one group F2F and the other remotely in an audio-only mode. To fulfil the social and cultural restrictions, only male students can talk to and see the teacher; whereas female students can only talk to, but not seeing the teacher, nor can the teacher see them. The global society was severely impacted by COVID-19 pandemic, which brought the world to a standstill in 2020. Educators worldwide were forced to migrate to online learning in such unusual circumstances. In-person instruction posed an unacceptable danger of getting and transmitting the illness. This abrupt and unexpected transition brought unforeseeable ramifications to educators, as the globe continues to see its impacts. In this background, the current study is guided by the following research questions:\n\n1. What perceptions do Saudi EFL language instructors have about LML?\n\n2. What perceptions do Saudi EFL female students have about LML?\n\n3. Are Saudi EFL female students satisfied with LML Learning mode?\n\n4. Are there any correlations between EFL Saudi female students' perceptions, satisfaction, difficulties and aids toward LML mode?\n\n5. Do Saudi EFL female students get help from other aids while learning though LML?\n\nThese topics are addressed via an examination of the most recent research released in the fields of EFL and language instruction after the pandemic hit in 2020. The issue was investigated in available literature using search terms such as ‘English language’, ‘Saudi universities’, ‘COVID-19’, and ‘EFL’. The next parts examine Saudi Arabia’s reaction to the epidemic, how schools and colleges continued to educate students after closures, and prior research on e-learning in the Saudi context. Following that, the most significant problems faced by Saudi university EFL professors and students in online learning are discussed, as well as the positive consequences noticed as a result of the transition to online learning. Many studies were conducted regarding Covid-19 (Al-Ahdal & Alqasham, 2020; Hazaea et al., 2021; Avelar et al., 2019; Sefara et al., 2019; Vanslambrouck et al., 2019; Vattøy & Smith, 2019) around the globe and in Saudi. All the previously mentioned studies either focused on the challenges that instructors faced while teaching online (Al-Ahdal, & Alqasham, 2020; Hazaea et al., 2021) or students’ attitudes towards online learning, or even the psychological statues of the learners (Truong & Wang, 2019; Tsai, 2019). To the best knowledge of the researchers, no previous research nationally or internationally evaluates the LML mode. Hence, this study is an endeavor to conceptualize and determine the boundaries of this new mode of learning.\n\n\nLiterature review\n\nLML is a new mode of teaching, which stands for Lateral Multimodal Learning, i.e., a classroom situation in which a teacher teaches male and female students simultaneously in segregated classrooms, the former being face-to-face and the latter being remote. National and cultural sentiments are honored in this model by restricting the interaction of female learners to a strictly audio mode.\n\nSince the outbreak of the pandemic, the Kingdom of Saudi Arabia (KSA) has been vocal in its humanitarian message. That is, regardless of color, gender, religion, or nationality, human life is more valued than everything else. As a result, throughout the first few days of March 2020, an emergency plan saw the closure of everything that threatened to jeopardize people's health and well-being, including schools, universities, and public and private organizations (Avelar et al., 2019).\n\nHowever, the country’s high ambitions and aspirations were not harmed by these physical closures. On the contrary, it exacerbated them. As a result of the closures, all Saudi sectors, including education, embraced digital technology to assist sustain services while limiting the disease (Aylett et al., 2021). Within a short period of time, the whole nation started transitioning to remote learning settings, whether via television broadcasts or communication through different online platforms: Telegram, Zoom, Teams, WebEx, and Blackboard. However, during the start of the epidemic, the situation was far from optimal. Many students and instructors lacked the digital literacy and internet capacity required to fully utilize these facilities. Despite this, they rose to the occasion and eventually adapted to the new educational standard.\n\nThe Saudi Ministry of Education established Madrasati (meaning ‘my school’), a national Digital Teaching Platform (DTP), in the 2020-2021 academic school year. The objective was to establish a centralized platform for providing online teaching to nearly six million students enrolled in Saudi public institutions from kindergarten to twelfth grade (Canals & Al-Rawashdeh, 2019). With the start of the new school year, instructors started to regain lost learning. Middle and high-school students took online lessons during designated morning hours, while primary school students had afternoon sessions. These efforts guaranteed that each level received an appropriate amount of learning time and helped alleviate internet traffic congestion caused by a large number of concurrent users. The afternoon hours allotted for elementary school kids enabled parents to help their young children in attending their online lessons and to assist them with their learning (Canals & Al-Rawashdeh, 2019).\n\nOn the other hand, since most Saudi institutions had previously integrated digital communication and learning technologies though on a different scale, they were inherently better equipped to migrate to an online learning environment. For example, university students are provided with an official email address upon enrollment. As a result, contact between students and their institutions was not disrupted. Additionally, most Saudi institutions used Blackboard®, a Learning Management System (LMS) that supports both synchronous and asynchronous forms of instruction. However, before the pandemic, this software was not widely utilized and functioned just as a complement, and its e-learning customers are still discovering its benefits.\n\nNumerous phrases are often used when addressing the use of technology to offer educational materials, including e-learning, online learning, electronic learning, digital learning, and technology-enhanced learning. All these terms refer to ‘a collection of technology-mediated instructional approaches that may be utilized to promote student learning and include features of evaluation, tutoring, and teaching’ (Chan, 2021). In other words, digital and online technologies are the main and sole means of providing educational information and teaching. This may be synchronous (i.e., teaching happens in real time with both students and teachers present online) or asynchronous (i.e., the content and instruction are available for the students to access any time via recordings of the lessons or independent online activities).\n\nA vast amount of prior research has examined the efficacy of integrating technology into EFL instruction at KSA colleges. However, these studies used technology tools in conjunction with face-to-face teaching, a process known as blended learning. Blended learning is described as ‘any formal education program in which a student learns at least in part via online learning’. That is, just a portion of the instructions are sent online; the remainder are delivered in person. This strategy has been demonstrated to be effective in learning English in the Kingdom of Saudi Arabia. Additionally, Correa-Baena et al. (2018) discovered that supplementing in-person teaching with online activities (i.e., viewing lesson-related videos and engaging in discussion forums) enhanced university EFL learners’ listening and speaking abilities. Other research examining university EFL students’ evaluations of the blended learning model’s efficacy in acquiring English reveal favorable sentiments regarding this strategy (Fu et al., 2019; Hackl & Ermolina, 2019; Hux et al., 2021; Hwang et al., 2019).\n\nNonetheless, these studies look at technology as a complement to face-to-face training, not as a substitute, as was the situation during the epidemic. The blended learning paradigm is distinguished from the e-learning model by the fact that the former includes some in-person teaching, whilst the latter is totally online, rendering the outcomes incomparable and warranting separate discussion.\n\nObstacles and hindrances in English language teaching (ELT) that existed before the COVID-19 epidemic were worsened by the abrupt change to online schooling and the resulting mandatory adaptations. One of the issues most often addressed in ELT is the motivation of language learners to learn (Lau & Gardner, 2019). Language instructors in Saudi Arabia have often noted a lack of students’ motivation (Macalister & Nation, 2019), which is most likely due to students’ poor competency (Marcoux et al., 2021), but may also be due to other causes (Mira & Fatimah, 2020). According to previous research, online learning environments are more engaging and favorable to raising students’ motivation (Nakayama, 2018). However, distant learning is ‘situationally sensitive’ (Newton & Nation, 2020), impacted by external variables such as time restrictions, grades, and the learning environment of students. The epidemic exacerbates these problems. Indeed, a recent study of over 1,000 academic English teachers in 99 countries found that one of the respondents’ primary worries during the epidemic was student motivation (Prakash & Murthy, 2019).\n\nAccording to a study by Rayganand Moradkhani (2020), 68 percent of respondents stated that students are less motivated to learn online during the COVID-19 epidemic. This demotivation is not always attributable to the online learning environment. According to other sources, the major reasons of student demotivation during the pandemic include being socially isolated, having a slow internet connection, dealing with distractions at home, and being unable to meet class goals (Schmid et al., 2020). Since practically all KSA education is now delivered online, students are required to attend class from their homes, where they may feel isolated from their classmates or distracted by siblings and other family members who are also studying or working remotely. Only those who need practical training or laboratory work continue to engage in face-to-face instruction.\n\nA further factor for students’ demotivation might be mental health concerns, considering the strong correlation between motivation and anxiety (Sefara et al., 2019). Numerous studies found that many students had anxiety, sadness, and post-traumatic stress disorder as a result of the epidemic, particularly during its first stages (Truong & Wang, 2019; Tsai, 2019). Learning during the COVID-19 pandemic may be very difficult for kids owing to the rapid changes taking place around them and their fears about the sickness (Vanslambrouck et al., 2019; Vattøy & Smith, 2019). Consequently, students may experience psychological anguish, as a result of their anxiety of being unable to continue their academic advancement (Villegas et al., 2020). While students and instructors have continued the educational process, they are doing so under less-than-optimal settings and face multiple obstacles, all of which may influence motivation.\n\nAnother challenge raised by the 2020 pandemic is digital preparedness. Digital readiness may be described as ‘the degree to which individuals succeed or struggle while attempting to navigate their surroundings, solve issues, and make choices’ via the use of technology (Wang, 2019). The transition to remote learning surprised the globe and left many students and educators unprepared for the new duties they were required to undertake. Wilson et al. (2020) conducted a Blackboard readiness assessment with 25 students enrolled in Taif University’s English language department. The results implied that students are digitally unprepared and lack technical abilities. Additionally, the author discussed how she trained her pupils to download and annotate PDF files, as well as demonstrating the importance of assisting students through tough moments. Indeed, in many situations, English language teachers have been forced to assume the job of a technical support expert, instructing students on how to download, upload, and distribute their work, among other things. Those with lesser English proficiency levels, particularly when working with language learners, may have extra challenges using digital technology, given that majority of technology use is offered in English (Yasuda et al., 2021).\n\nAdditionally, students may only access instructional information and online courses if they have access to adequate equipment and a dependable internet connection (on both the students’ and instructors’ end). Thus, internet connectivity and bandwidth have a substantial impact on learners’ online experiences, since their learning experiences are contingent on the dependability of their internet connection (Monteiro et al., 2020). Fandiño et al., (2019) who conductedqualitative interviews with 12 EFL university students at King Saud University found that students had technical difficulties when taking lessons on Blackboard, ranging from incompatible equipment to audio interruptions to being locked out by the site. These difficulties become more prevalent at specific periods of the day as a result of higher internet traffic using the platform concurrently. Similarly, Vattøy (2020) studied the effects of online assessment on 20 EFL students at Onaiza institutions and reported that 64.3 percent of respondents experienced ‘frequent disconnections’ that harmed their learning and online assessments. Interruptions caused by a sluggish or insufficient internet connection degrade the quality of the online learning experience. They may also have a detrimental effect on learning, since they may result in student dissatisfaction and demotivation (Jiang, 2018).\n\nAdditionally, there is a problem with inadequate technical help. Due to the high volume of students and professors requiring assistance at any one moment, students do not get assistance when they are in need (Jansen et al., 2021). Additionally, students with impairments encounter accessibility challenges and may not get the necessary help when they visit their educational institutions in person. For instance, persons with visual impairments may have difficulty seeing the digital whiteboard, reading instructor-posted material, or engaging in classroom conversation. They need access to voice-to-text software, which, at the time of writing, does not support all digital platforms and file types. Similarly, students who are deaf or hard of hearing are not always able to access closed captioning or subtitles for oral or video courses. These accessibility concerns for students with disabilities exclude them and prohibit them from fully using online education (Butler & Le, 2018).\n\nIt is worth noting that one of the most amazing accomplishments of the Saudi Ministry of Education in terms of general education was the creation of 23 educational television channels dubbed iEn for individuals without access to the internet. These channels feature translations in Saudi sign language for deaf pupils. Additionally, they have established three channels for students enrolled in special education. These instructional adjustments assist in mitigating some of the accessibility difficulties associated with distance education for students with disabilities. Nonetheless, such services are lacking at the university level.\n\nOn the other hand, EFL learners face substantial challenges due to a lack of visual input during online learning. Due to cultural limits and a desire to protect users’ privacy, students and instructors are not needed to switch on cameras in the virtual classroom, particularly at the tertiary level. However, ELT theory and research support the relevance of non-verbal information and facial movements in the growth of language learners. Visual cues are vital for conveying critical information that communicates the message’s overall meaning (Mackay, 2019). Thus, in an online language class, the listener’s lack of visual feedback from the speaker is a disadvantage for EFL students. As a result, several studies on online learning during the epidemic revealed that in the Saudi EFL setting, face-to-face communication with peers and instructors is preferred (Bernstein &Woosnam, 2019). Indeed, participants in Mackay’s (2019) qualitative focus groups said that they preferred in-person sessions since they ‘lacked eye contact with the lecturer’.\n\nAssessment is another issue when ELT is conducted remotely for a variety of reasons (Al-Ahdal& Alqasham, 2020; Hazaea et al., 2021). To begin with, many language teachers have the challenge of ensuring that the work submitted by students is their own. Despite the widespread availability of anti-cheating software and plagiarism detection technologies, not all teachers are well educated to use them, and students circumvent them using a variety of academically dishonest tactics.\n\nVattøy (2020) observed that one of the challenges in online English instruction is the ‘ease with which the exam material may be penetrated’. They discuss numerous strategies for preventing cheating, including utilizing a plagiarism checker to verify students’ written responses, rewriting the substance of objective type questions, showing single questions in random order, and shortening the test’s allocated time. Nonetheless, there is no assurance that the student is completing the evaluation on his or her own without being physically present or using surveillance software (which would breach students’ privacy). Additionally, it complicates the process of evaluating pupils’ academic development. Mackay (2019), for example, discovered that students utilized a second device during tests to do internet searches or simply copy and paste results from other sources. Remote evaluations do not adequately portray students’ growth as a result of these evaluation difficulties.\n\nAdditionally, online learning and teaching are more time-consuming than in-person sessions, adding another layer of difficulty. Jansen et al. (2021) discovered that just 10 percent of English language instructors spent most of their teaching time online prior to the epidemic. However, 55 percent of instructors polled now spend 100 percent of their instructional time online. Besides teaching online, language teachers prepare content, produce materials, send and respond to emails, post tasks for students, and grade students’ work online, to mention a few of their new duties. Consequently, language instructors spend 10 to 12 hours every day in front of a computer screen (Canals & Al-Rawashdeh, 2019). Similarly, in Jansen et al. (2021), in a poll of Saudi EFL university students, 85.7 percent said that preparing for online lectures takes them longer than preparing for in-person sessions. It is also troubling since, as Vattøy (2020) indicates, increased time spent online, particularly on mobile devices, is strongly associated with academic procrastination and social media addiction. Additionally, prolonged screen time and online interactions leave many users fatigued and depleted, a condition dubbed Zoom Fatigue (Avelar et al., 2019).\n\nAdditionally, there are concerns with learning systems. Apart from Blackboard LMS, students and teachers are wont to use various programs and technologies, although these have raised concerns about privacy and security. Among these is WhatsApp, a smartphone application that enables users to share text messages, photographs, videos, voice notes, and even make free phone calls to family and friends. According to Correa-Baena et al. (2018), WhatsApp is one of the most popular applications among university EFL students, who see the service as a tool for improving their reading, writing, and grammatical skills. Al-Ahdal and Hussein (2020) studied the use of WhatsApp as a learning tool for developing Saudi EFL learners’ wiring skills. Similarly, Ali and Bin-Hady, (2019) found that WhatsApp reduced Saudi EFL learners’ stress and helps them to overcome the anxiety associated with language learning. Al-Ahdal and Alqasham (2020) explored EFL Saudi instructors’ use of WhatsApp in classroom. However, several privacy issues have surrounded the application due to the application’s usage and sharing of users’ data.\n\nZoom, a videoconferencing program, had the same issues. Prior to the COVID-19 epidemic, nearly no one used Zoom for teaching, but as schools became virtual, almost everyone did. Zoom’s teacher-friendly features include live audio and video conferencing, a digital whiteboard, screen sharing, and the ability to upload teaching resources. It was also beneficial to university students. Bernstein and Woosnam (2019) discovered a favorable association between Saudi EFL university students’ Zoom usage, their assessed usefulness and acceptability of the technology. Nonetheless, security flaws were uncovered during a series of Zoombombings, in which an uninvited participant enters a Zoom session with the potential for overt disruption. Another issue was Deepfakes, which is defined as ‘the imposition of another person's face onto another person's body in video format using Artificial Intelligence algorithms’. Therefore, the majority of Saudi institutions have advised their teachers against using Zoom for teaching and online conferencing and have asked them to switch to a more secure option, such as Blackboard LMS, even if it lacks some of the useful features offered by Zoom.\n\nDespite the disadvantages of online English training, some beneficial features of this have emerged. One of the most major benefits of online education is the flexibility it provides. Remote learning enables students and instructors to attend courses from any location and at any time (if learning asynchronously). Additionally, it enables students and teachers to choose from a variety of devices and applications. The study of Avelar et al. (2019) with Saudi university students indicated that they are more likely to utilize their mobile phones in e-learning settings, which makes accessing their classes and learning materials simpler. Additionally, the authors said that virtual learning environments allow learners to study the course whenever and wherever they are. Chan (2021), for example, noted that EFL students attended classes while at work, in the automobile, sat with family, or from the comfort of their bed, even though this is not always desired. Additionally, this flexibility saved students time spent travelling to and from their academic campuses (Bernstein & Woosnam, 2019). Surprisingly, even teachers trapped across borders—as a result of quarantine regulations and air travel restrictions—could teach from nations separated by many time zones (Aylett et al., 2021). Thus, online education during the pandemic alleviated physical restrictions of distance and time, opening the door for a new approach to learning that is not constrained by these shortcomings.\n\nAnother advantage noted because of the shift to online English training is that students who were previously too timid to speak out in front of a big class may now be more inclined to do so in front of a screen from the comfort of their homes. For example, Villegas et al. (2020) studied 311 EFL university students from five major Saudi institutions and discovered that attending lessons through Blackboard (rather than in-person) assisted learners in overcoming their shyness in class discussions. Similarly, it has been said that online language learning increases introverted students' confidence in class and promotes peer interaction. The (partial) anonymity provided by e-learning creates a secure environment that helps pupils overcome their fears while speaking a foreign language.\n\nThe transition to online training has also hastened the development of new abilities in both language students and educators, since many have been forced to navigate unfamiliar territory. English language instructors are using new abilities for the first time since the epidemic, including teaching online, making presentations online, providing students with online practice, utilizing electronic copies of coursebooks, and conducting online evaluations (Wright, 2021). Similarly, the virtual learning environment aided in the development of EFL students’ language skills, particularly their listening and speaking abilities (Canals & Al-Rawashdeh, 2019), as well as their internet searching abilities (Bernstein & Woosnam, 2019). Despite the challenges individuals had in adjusting to online settings, the new experiences stimulated the learning of unique talents and sharpened existing competence.\n\nSimilarly, the shift to digital learning has resulted in accidental learning (i.e., learning without intending to study) of English via casual exposure to the language. Because English is the most frequently used language online, accounting for 25.9 percent of all internet content, EFL learners are exposed to more English outside of their language sessions than they would in a face-to-face classroom. Additionally, claims that Saudi EFL students’ usage of the internet facilitates informal language learning by providing access to material that appeals to their interests, such as music, movies, YouTube videos, and videogames. Thus, internet education may be advantageous for students’ informal English learning.\n\nAdditionally, the need for unique abilities for online teaching and learning has resulted in a widespread demand for professional development opportunities. With the growth of COVID-19 cases and the tightening of quarantine rules, online training and instructional material have exploded in popularity. As a result, everyone now has access to a plethora of information through free internet webinars, conferences, courses, and workshops (Jansen et al., 2021). It has never been easier to attend educational and training activities in several locations without being restricted to a single location. Saudi universities all took part in these activities, granting admission to both university affiliates and non-affiliates. Additionally, some professors, educators, libraries, and even publishers made their resources freely accessible to guarantee that, even if the rest of the world grinds to a halt, education does not.\n\n\nMethods\n\nThe study used a mixed methods research design to collect data from the participants. On the quantitative part, a questionnaire was used, and semi-structured interviews with specific themes were used to collect teachers’ data. The former research paradigm allows for the researchers to demonstrate their survey to a large number of participants. So, the population will usually be represented thoroughly.\n\nTwo five-point Likert scale questionnaireswere designed by the researchers themselves. The first questionnaire was designed to check Saudi female students’ perceptions on the participation in LML. The students’ questionnaire consists of four domains: (1) students’ perceptions on LML, (2) students’ satisfaction for being indulged in such learning, (3) the difficulties that Saudi female students encountered and (4) the aids they received from others. The first domain includes 17 items, the second comprised six items, while the third has four items and the last domain includes five items. The questionnaire was sent to three ELT professors to referee its content validity. All their comments were considered. The teachers’ questionnaire includes only 11 items and grouped in one domain that is to examine their perceptions toward LML mode.\n\nThe participants of this part of the study comprised Saudi EFL female students studying for their bachelor’sdegree in English language and Translation at Qassim University, College of Language and Translation via the LML mode. They enrolled in different levels from one into four. Their median age is 23 years. The researchers shared the link of the digital questionnaire with all the female students. 101 responses were received and two of them were deleted because they were incomplete. All the ethical considerations were ascertained. Students were assured that they did not need to divulge their names or other personal information.\n\nIt is important to briefly mention how the authors performed the research ethics during this research. Beginning from the top to the bottom, the researchers got a consent letter (16-Eng-2021) from the committee of ethical issues in the college of Language and Translation at Qassim University. The consent letter was submitted to the head of the English department to follow-up the procedures including scheduling a time for the researchers to meet with the student and teachers to explain them the aim of the study. Likewise, all participants including 99 female students and 6 EFL teachers, were assured that their names are not required to be mentioned on the survey and were requested to select as precise alternatives as possible to assure the truthfulness of the study. Finally, the researchers promised them to share with them the finding of the study as soon as the study being completed. Students orally accepted to participate in the study and also agreed that their data would be published anonymous publicly.\n\nThe qualitative data were collected from the six EFL teachers that form the total faculty of EFL at the university where the study was conducted. The researchers isolated themes that have been shown by prior research to be predominant issues in EFL classrooms from the teachers’ point of view. Out of these, 11 themes relevant to the Saudi EFL context were picked to form the questions of the semi-structured interviews. The last question, numbered 12, was an open ended one which required teachers to list the positive and negative points of the LML practices in Saudi Arabia. Data from the 11 interview questions were quantified to establish leading trends.\n\n\nData analysis and results\n\nAfter collecting the students’ responses to the questionnaire over the period of a week, the data were analyzed using SPSS version 23. The data were coded in which the alternative strongly agree was coded into 5 and strongly disagree into 1. Both descriptive and inferential analyses were adopted.\n\nTable 1 shows the Cronbach Alpha reliability test for the domains the study focused on. According to the table, all the domains got a strong to very strong reliability values except the instructors’ questionnaire which is considered as acceptable at (P= 0.69). The other domains, i.e., students graded from good reliability as in the second and the third domain (P= .79) for both to very high as in the first domain where the reliability was shown as (P = .94).\n\nTo answer the first research question (What perceptions do EFL instructors have about LML?),we turn to Table 2 which presents the Saudi EFL instructors’ views on LML. Six Saudi instructors provided their perceptions on their teaching experience using LML. According to the Table, instructors provided a moderate perception with a mean score and standard deviations of 3.59 and 0.44 respectively. The items show variations in levels of perceptions. A high perception in item 3 (4.17) reflected that instructors perceived the aids that LML provided them during their teaching experience. Yet, instructors showed low perceptions (2.33, 0. 82) as in item 11, which represented their dissatisfaction with the teaching experience while pursuing such kind of learning.\n\nTo answer research question 2 (What perceptions do Saudi EFL female students have about LML?), we turn to Table 3 which reflects Saudi EFL female students’ perceptions of LML. As the Table shows, 99 respondents answered to this category. They show a high mean score of (4.03,0.61) with a relative importance equal to 80.63 percent. Students’ perceptions were gathered viz-a-viz their responses to 17 items as Table 3 shows. Through all the items from 1 into 17, students showed high perceptions between 3.82 to 4.39, in which the highest perception was recorded in item 14, where students showed their enthusiasm towards such type of learning that they can learn without being put under stress. Gradually, students responded fall to the lowest point in (high level) where they scored (3.82, 0.87) as they showed their perceptions on their practice of English language in LML as they usually do in face-to-face mode.\n\nTo answer the third research question (Are the Saudi EFL female students’ satisfied with their experience on LML?),we turn to Table 4 which presents Saudi EFL students’ perceptions on their satisfaction while learning through LML. As the Table exhibits, students perceived the LML learning mode satisfactorily, they scored a total mean score of 3.88 with a standard deviation of 0.67, which is considered as high according to the statistical measures. Their satisfaction regarding their performance to the course requirements (item 18) and their present progress (item 19) scored high with mean scores 4.01 and 4.02 respectively. Despite the high level of satisfaction as calculated in the total mean scores, items 21 and 23 scored moderately, where their mean scores and standard deviations were (3.56, 1.00 & 3.58, 0.99) respectively. They showed moderate perceptions regarding the progress they achieved in the language skills and contents attainted within the course they studied.\n\nTo answer the fourth research question (Do Saudi EFL female students face difficulties while learning through LML?), we see that Table 5 presents Saudi EFL female students' perceptions on the difficulties they faced while appearing in LML. Students showed that they face difficulties in learning though LML, such difficulties rated as moderate though. 68 of them (M = 3.39) agreed that they face difficulties on LML learning mode as a result of missing the facial expression of the instructors or instructors’ dominance in lecturing or the difficulties stem from the robotic or mechanical feeling they got while learning when the instructors are not seen or interacted with. To answer the last research question, Do Saudi EFL female students get help from other while learning though LML. Table 6 represents Saudi EFL Saudi female students’ perceptions on the source of aids they use integrated with LML learning. According to Table 6, students showed high perceptions on the aid they got with a total mean score of (4.02) and a standard deviation of (0.78). The aids gradually rated from the family as high as (M = 4.31, Std. = 0.74) to the aids they got from the college administration which scored as (M = 3.83, Std. = 0.99).\n\nThis study hypothesizes that there are significant correlations between the four domains at a point of 0.05.\n\nTable 7 presents the correlation between the four domains, which are, students’ perceptions, satisfactions, difficulties, and aids. Pearson correlations coefficient was used to show the relationships amongst the previously mentioned domains. The findings show that there are correlations between students’ perception and satisfaction (r = .719) regarding the studying through LML mode, students’ perceptions and aids (r = .659.) Still there is a correlation between students’ perceptions and difficulties that face, such correlation is not available, not strong though, (r = .351). Table 7 also presents the correlation between the second domains, i.e., students’ satisfactions with the difficulties and the aids they got while learning through LML mode, (r = .582, r = .656) respectively. The third domain, i.e., students’ difficulties were also correlated with the aid they got, yet that correlation is not significant (r = .261).\n\nThe qualitative data collected from the teachers through the semi-structured telephonic interviews established clear trends in their perceptions of LML. The results are quantified in Table 2.\n\nTeachers’ responses show that the Saudi academia is ready to follow the current method of LML, which is a happy situation for all stakeholders including administration, funding agencies, teachers and learners who have become well adapted to the mode in use in the last two years. It is notable that teachers perceive this as ranking high on the professional satisfaction scale, with as much as 93.33% relative importance being given to LML on the benefits accrued to teaching experience. Professional growth (83.33%), LML for future teaching plans (76.67%), and teachers’ degree of involvement while using LML (73.33%) are encouraging outcomes. On the flip side, difficulties in the teaching experience (56.67%), obstacles in delivering the LML course pack (60.0%), and the mechanical nature of the experience (66.67%) are the areas that need to be checked in the future.\n\nFinally, responding to the open-ended question that elicited responses on the positive and negative aspects of the practice, teachers listed some challenges such as learning how to fulfill the demands of the new mode, making the classes interesting to ensure learner engagement, and using annotations as support materials for the main teaching. Two of the teachers interviewed also pointed out that the nature of communication in M2E is abstract which is sometimes a challenge for both teachers and learners, though they agreed that this could be a fallout of the general psychological ramifications of the pandemic-imposed restrictions, and not necessarily attributable to the M2E mode of learning. Apart from this, one teacher felt that knowledge transfer was incomplete in M2E as the entire gamut of paralinguistic communication was shelved in this method with learners being totally invisible to the teacher. On the other hand, the benefits were more robust, including the usefulness of the method in developing learners’ oral communication, presentation skills, and listening skills.\n\n\nDiscussion\n\nThis study found that Saudi EFL students perceived LML in moderate level. This finding can be interpreted to indicate the success of the support that the Kingdom of Saudi Arabia supplied to the virtual learning initiative, though this type of learning does not yet reach the level of conventional learning due to many reasons, say the long of time needed to prepare virtual learning (Jansen et al., 2021), or the non-readiness of the organization to cope up with the transfer (Hazaea et al., 2021). The transition to online education has brought to light numerous additional challenges impeding students’ advancement. One of the issues identified is a lack of digital ready skills among students which is exacerbated by a lack of technological assistance. Additionally, students spend considerable effort resolving such challenges and traversing online territory.\n\nThe findings of this study further showed that Saudi EFL female students have high positive perceptions on studying through LML. This finding opens our expectations to think about the future of virtual learning in the Saudi context. This finding is in line with Villegas et al. (2020) who found that Saudi students positively view virtual learning as such kind of learning break down their shyness which usually associated with conventional classrooms.\n\nRemote learning enables students to connect to their virtual classes from any location. However, distant learning’s adaptability is a double-edged sword. On the one hand, it reduces physical constraints such as location and time, enabling access to learning and development opportunities around the globe and reducing travel times to and from university campuses. On the other side, it provides for more distraction from educational activities. The home setting is fraught with disturbances, especially when additional family members work and study from home. Additionally, the lack of separation between the home and school settings makes it more difficult for students to differentiate between leisure and academic time.\n\nFurthermore, EFL Saudi female students expressed high positive satisfactions towards LML learning mode. This finding can be interpreted socio-culturally. Female students living in a conservative society and guided by the Islamic creeds finding their target in such kinds of learning and without being put in any embarrassing situations arising from co-education or even with facing instructors.\n\nThis study also explored the difficulties that female students encounter in indulging in LML. The students reported that face difficulties in a moderate level. These difficulties may stem from the missing of instructors’ facial expression which may help in understanding the content. LML learning is different from virtual learning that no-one-sees-the-others. Other may see this learning as a robotic learning where no real interaction occurred. Students need visual input of spoken language, primarily for language development. Not using video elements throughout the class may jeopardize the students’ development of speaking and listening abilities. Alternatively, students’ vocabulary and research abilities may develop as a result of incidental learning that happens as a result of browsing the internet and all its English information. Nonetheless, testing the development of these language abilities online has shown to be a challenge that needs instructors to take additional care to ensure the validity of the assessment.\n\nStudents reported highly positive attitudes that they got help from others which help them in pursing this learning most, the highest help was supplied by their family, in addition to the college administration. It is the secret behind the high positive perceptions that students reported earlier. The study found that there is good correlation between students’ perceptions and satisfaction in this learning more (r = .72) and perceptions and the aids they got (r = .66).\n\nTeachers, though only a small number could be interviewed given the limited faculty size in the university, appeared upbeat about the LML mode of teaching-learning and expressed readiness to continue with the mode in the long run.\n\n\nSignificance of the Study\n\nThe significance of this study stems from two axes. Firstly, the reach and unavoidability of the online educational mode in EFL in the coming future with new waves of the pandemic being forecasted. Secondly, the goals of Saudi Vision 2030 clearly state the desire of the administration to produce a generation of educated young people who can exploit the vast potential of the global job market. In this background, and of the so far subdued socio-economic status of Saudi women, this study is vastly significant.\n\n\nConclusion\n\nThis study reported the perceptions of Saudi EFL instructors and students toward a new mode of learning called LML. They study reported medium preference of instructors to such kind of leanings. They also showed some points which needs to be worked out to efface this learning mode, like how to deliver the content of the course effectively and attaining students’ motivations. Furthermore, the study explored four domains in Saudi EFL female students, i.e., perceptions, satisfaction, difficulties and aids. The study reported good correlations between perceptions and satisfaction and aids. It also reported good correlations between satisfaction and aids. In conclusion, educators and stakeholders must examine the aforementioned challenges while assessing the effectiveness of e-learning in EFL and assessing its use and applicability after the epidemic. Future study in this area should examine the disparity in learner motivation between those who are more likely to engage in online peer conversations and those who are less motivated to participate when courses are conducted online. Additionally, larger-scale research is required to determine the academic EFL learning results at the university level in Saudi Arabia.\n\nThis research may be expanded to include the perspectives, opinions, and impressions of educational institution stakeholders, such as ELT department chairs, curriculum designers, and publishers, as well as the students themselves. The research may always be replicated in another EFL setting using identical parameters. Nevertheless, the current study has significant shortcomings that should be addressed in future research. Qualitative components such as group discussions and interviews might be added to the quantitative data to bolster them. The efficiency of online learning through Blackboard was determined by a poll of students’ views, not actual learning results. This may have been accomplished more effectively by assessing learning outcomes and comparing them to those of a control group that did not use Blackboard for instruction. Additionally, the research was limited to a single institution; if data were gathered from several Saudi universities, broader conclusions may have been drawn.\n\n\nRecommendations\n\nWhereas the current study concluded that female EFL students in Saudi Arabia perceive online language learning positively, this finding contradicts many other previous studies like Macalister and Nation (2019), Prakash and Murthy (2019), and Raygan and Moradkhani (2020). Such studies reported that virtual leaning demotivates learners. This contradiction is important and should be diligently reinvestigated in future studies. It can be justified to the context of the participants and their ethical and religious heritage. Furthermore, the studies reported herein have been conducted at the climax of the pandemic, now after the cease of the curve, the situation becomes better and both students and instructors accustomed and be familiar with virtual learning. Therefore, it is also recommended that before generalizing the results of this study, deeper investigations into other online learning environments and conditions in EFL should also be investigated. Similarly, the number of teachers enrolled in the study was small at six, though it was a condition beyond the researchers’ control, future studies may be undertaken with a larger teacher base.\n\n\nLimitations\n\nThe gender specificity of this study was a limitation but one which could not be overcome due to the sociocultural milieu of Saudi Arabia. Another limitation was the wholly quantitative data and that too, to the exclusion of the EFL teachers’ point of view.\n\n\nData availability\n\nFace-to-face and mouth-to-ear modes - https://doi.org/10.6084/m9.figshare.18667568.v1.\n\nQuestionnaire for Instructors.csv - https://doi.org/10.6084/m9.figshare.18667571.v1.\n\nStudents' Questionnaire.csv - https://doi.org/10.6084/m9.figshare.18667574.v1.\n\nQuestionnaire for Instructors - https://doi.org/10.6084/m9.figshare.18667577.v1.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAl-Ahdal AAMH, Alqasham FH: Saudi EFL learning and assessment in times of covid-19: Crisis and beyond. Asian EFL Journal. 2020a; 27(4.3): 356–383.\n\nAl-Ahdal AAMH, Alqasham FH: WhatsApp in language classroom: Gauging Saudi EFL teachers' roles and experiences. Opcion. 2020b; 36: 1667–1680.\n\nAl-Ahdal AAMH, Hussein NMA: WhatsApp as a writing tool in EFL classroom: An study across two universities in Saudi Arabia. Asian EFL Journal. 2020; 27(3.1): 374–392.\n\nAli JKM, Bin-Hady WRA: A study of EFL students' attitudes, motivation and anxiety towards WhatsApp as a language learning tool. Arab World English Journal (AWEJ) Special Issue on CALL. 2019; (5): 289–298. Publisher Full Text\n\nAvelar ABA, da Silva-Oliveira KD , da Silva Pereira R : Education for advancing the implementation of the Sustainable Development Goals: A systematic approach. The International Journal of Management Education. 2019; 17(3): 100322. Publisher Full Text\n\nAylett MP, Clark L, Cowan BR, et al.: Building and designing expressive speech synthesis. The Handbook on socially interactive agents: 20 years of research on embodied conversational agents, intelligent virtual agents, and social robotics Volume: Methods, behavior, cognition. 2021; (pp. 173–212). 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PubMed Abstract \| Publisher Full Text\n\nHazaea AN, Bin-Hady WRA, Toujani MM: Emergency remote English language teaching in the Arab league countries: Challenges and remedies. Computer-Assisted Language Learning Electronic Journal. 2021; 22(4): 201–222.\n\nHux K, Wallace SE, Brown JA, et al.: Perceptions of people with aphasia about supporting reading with text-to-speech technology: A convergent mixed methods study. Journal of Communication Disorders. 2021; 91: 106098. PubMed Abstract \| Publisher Full Text\n\nHwang GJ, Hsu TC, Hsieh YH: Impacts of different smartphone caption/subtitle mechanisms on English listening performance and perceptions of students with different learning styles. International Journal of Human–Computer Interaction. 2019; 35(4-5): 333–344. Publisher Full Text\n\nJansen T, Vögelin C, Machts N, et al.: Judgment accuracy in experienced versus student teachers: Assessing essays in English as a foreign language. Teaching and Teacher Education. 2021; 97: 103216. Publisher Full Text\n\nJiang L: Digital multimodal composing and investment change in learners' writing in English as a foreign language. Journal of Second Language Writing. 2018; 40: 60–72. Publisher Full Text\n\nLau K, Gardner D: Disciplinary variations in learning styles and preferences: Implications for the provision of academic English. System. 2019; 80: 257–268. Publisher Full Text\n\nMacalister J, Nation IP: Language curriculum design. Routledge; 2019. Publisher Full Text\n\nMarcoux K, Cooke M, Tucker BV, et al.: The Lombard intelligibility benefit of native and non-native speech for native and non-native listeners. Speech Communication. 2021; 136: 53–62. Publisher Full Text\n\nMira AS, Fatimah S: Students' paraphrased texts and their perceptions of paraphrasing in academic writing. Lingua Didaktika: Jurnal Bahasa danPembelajaran Bahasa. 2020; 14(1): 55–69. Publisher Full Text\n\nMonteiro J, Alam J, Falk TH: Generalized end-to-end detection of spoofing attacks to automatic speaker recognizers. Computer Speech & Language. 2020; 63: 101096. Publisher Full Text\n\nNakayama S, Tjandra A, Sakti S, et al.: Speech chain for semi-supervised learning of Japanese-English code-switching Asr and Tts. 2018 IEEE Spoken Language Technology Workshop (SLT). IEEE; 2018, December; (pp. 182–189). Publisher Full Text\n\nNewton JM, Nation ISP: Teaching ESL/EFL listening and speaking. Routledge; 2020. Publisher Full Text\n\nPrakash JJ, Murthy HA: Analysis of inter-pausal units in Indian languages and its application to text-to-speech synthesis. IEEE/ACM Transactions on Audio, Speech, and Language Processing. 2019; 27(10): 1616–1628. Publisher Full Text\n\nRaygan A, Moradkhani S: Factors influencing technology integration in an EFL context: investigating EFL teachers’ attitudes, TPACK level, and educational climate. Computer Assisted Language Learning. 2020; 1–22. Publisher Full Text\n\nSchmid M, Brianza E, Petko D: Developing a short assessment instrument for technological pedagogical content knowledge (TPACK. xs) and comparing the factor structure of an integrative and a transformative model. Computers & Education. 2020; 157: 103967. Publisher Full Text\n\nSefara TJ, Mokgonyane TB, Manamela MJ, et al.: HMM-based speech synthesis system incorporated with language identification for low-resourced languages. 2019 International Conference on Advances in Big Data, Computing and Data Communication Systems (icABCD). IEEE; 2019; (pp. 1–6). Publisher Full Text\n\nTruong TNN, Wang C: Understanding Vietnamese college students’ self-efficacy beliefs in learning English as a foreign language. System. 2019; 84: 123–132. Publisher Full Text\n\nTsai PH: Beyond self-directed computer-assisted pronunciation learning: A qualitative investigation of a collaborative approach. Computer Assisted Language Learning. 2019; 32(7): 713–744. Publisher Full Text\n\nVanslambrouck S, Zhu C, Pynoo B, et al.: A latent profile analysis of adult students’ online self-regulation in blended learning environments. Computers in Human Behavior. 2019; 99: 126–136. Publisher Full Text\n\nVattøy KD: Teachers’ beliefs about feedback practice as related to student self-regulation, self-efficacy, and language skills in teaching English as a foreign language. Studies in Educational Evaluation. 2020; 64: 100828. Publisher Full Text\n\nVattøy KD, Smith K: Students' perceptions of teachers' feedback practice in teaching English as a foreign language. Teaching and Teacher Education. 2019; 85: 260–268. Publisher Full Text\n\nVillegas DFM, Varona WH, Sanchez AG: Student teachers’ identity construction: A socially-constructed narrative in a second language teacher education program. Teaching and Teacher Education. 2020; 91: 103055. Publisher Full Text\n\nWang CJ: Facilitating the emotional intelligence development of students: Use of technological pedagogical content knowledge (TPACK). Journal of Hospitality, Leisure, Sport & Tourism Education. 2019; 25: 100198. Publisher Full Text\n\nWilson ML, Ritzhaupt AD, Cheng L: The impact of teacher education courses for technology integration on pre-service teacher knowledge: A meta-analysis study. Computers & Education. 2020; 156: 103941. Publisher Full Text\n\nYasuda Y, Wang X, Yamagishi J: Investigation of learning abilities on linguistic features in sequence-to-sequence text-to-speech synthesis. Computer Speech & Language. 2021; 67: 101183. Publisher Full Text" }	[ { "id": "129325", "date": "07 Apr 2022", "name": "Prof. Abdul-Hafeed Ali Fakih", "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 manuscript is well-written, and ideas are outstandingly presented and greatly organised. It discusses an under-researched area of Applied Linguistics: Lateral Multimodal Learning. The topic identified is relevant, significant, and the authors' style of writing is engaging and impressive. The analysis is very fit and critical. It displays the authors' thorough understanding and carefully presented argument. The manuscript, in a nutshell, presents, compares/contrasts perspectives, and draws original and meaningful conclusions with future implications. The authors have also managed to satisfactorily answer the research questions in a very interesting and lucid manner. That apart, the manuscript is also free from grammar, punctuation, spelling errors, and typos.\nThere are, however, two issues that need to be rectified, as pointed out below:\nThe expression Mouth-to-ear (M2F) should be given in full in the title. I find the coinage really appealing. Kudos!\n\nIn lines 3 and 4 of \"Academic institutions in KSA\", the in-text citation (Canals & Al-Rawashdeh, 2019) should be replaced with (Alqahtani, 2022), considering the publication date and appropriacy of the reference. Thus, in the reading list, too, the following reference (a, below) should be used in place of b.\na. Alqahtani, M. H. (2022). Post pandemic era: English language teachers' perspectives on using the Madrasati e-learning platform in Saudi Arabian secondary and intermediate schools. World Journal of English Language, 12(2), 102-116. 1\nb. Canals L, Al-Rawashdeh A: Teacher training and teachers’ attitudes towards educational technology in the deployment of online English language courses in Jordan. Computer Assisted Language Learning. 2019;32(7):639-66\n\nIs the work 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": "8063", "date": "07 Apr 2022", "name": "Arif Ahmed Mohammed Hassan Al-Ahdal", "role": "Author Response", "response": "Thank you so very much for the encouraging words and insightful comments, Prof. That, indeed, makes great sense. Thanks heaps once again!" } ] }, { "id": "138925", "date": "30 May 2022", "name": "Wafa Hazaymeh", "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\nA well-written article with well-presented and arranged topics. It focuses on an up-to-date field of Applied Linguistics. The topic selected is current and vital, and the writers' writing style is engaging and exceptional. The analysis is precise and critical. It reflects the writers' deep grasp and well-presented argument. In overall, the essay gives correct discussion and derives new and significant discoveries with future implications. The writers have also addressed the research questions correctly and in a very interesting and transparent manner. Furthermore, there are no grammatical, punctuation, spelling, or typographical errors in the document. However, please be careful with the following points:\n\nCertain references should be changed to include the DOI of each reference of the following:\nAl-Ahdal AAMH, Alqasham FH: Saudi EFL learning and assessment in times of covid-19: Crisis and beyond. Asian EFL Journal. 2020a; 27(4.3): 356–383. Al-Ahdal AAMH, Alqasham FH: WhatsApp in language classroom: Gauging Saudi EFL teachers' roles and experiences. Opcion. 2020b; 36: 1667–1680. Al-Ahdal AAMH, Hussein NMA: WhatsApp as a writing tool in EFL classroom: An study across two universities in Saudi Arabia. Asian EFL Journal. 2020; 27(3.1): 374–392. Hazaea AN, Bin-Hady WRA, Toujani MM: Emergency remote English language teaching in the Arab league countries: Challenges and remedies. Computer-Assisted Language Learning Electronic Journal. 2021; 22(4): 201–222.\n\nMoreover, some references are not highlighted in the research as the rest of the references such as:\nAl-Ahdal& Alqasham, 2020 Mackay 2019 Wright, 2021\n\nIn addition, there should be a space between:\n\nbachelor’s degree (What perceptions do EFL instructors have about LML?), we. Please use the researchers instead of we in the article\n\nI certify that I have read this work and have the requisite level of expertise to ensure that it fulfills an adequate research standard.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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/11-369
https://f1000research.com/articles/11-364/v1	29 Mar 22	{ "type": "Systematic Review", "title": "Bibliometric analysis and network visualization mapping of global research in Q fever vaccine", "authors": [ "Tauseef Ahmad", "Haroon Haroon", "Eric David Bicaldo Ornos", "Husam Malibary", "Akbar Hussain", "Mukhtiar Baig", "Eman Y. Santali", "Jeehan H. Alestad", "Muzaheed Muzaheed", "Ali A. Rabaan", "Harapan Harapan", "Haroon Haroon", "Eric David Bicaldo Ornos", "Husam Malibary", "Akbar Hussain", "Mukhtiar Baig", "Eman Y. Santali", "Jeehan H. Alestad", "Muzaheed Muzaheed", "Ali A. Rabaan" ], "abstract": "Background: Query fever (Q fever), caused by Coxiella burnetii, is a highly infectious zoonotic infection to humans and livestock. Despite extensive efforts to develop effective vaccines against this disease, only one vaccine is licensed and available. The aim of this study was to investigate the global research trends, keystone bibliometric parameters, and network visualization mapping in Q fever vaccine from 1941 to 2021.\n\nMethods: A retrospective bibliometric followed by a visualized study was conducted. The searches were conducted in the Science Citation Index Expanded (SCI-E) Edition of Web of Science Core Collection (WoSCC). The following keywords were used: \"Q fever\" OR \"Query fever\" OR \"Coxiella burnetii\" OR \"Coxiella-burnetii\" OR \"C. burnetii\" (Topic) AND \"Vaccin\" OR \"Immuniz\" OR \"Immunis\" (Topic) without any limitation. The data were plotted for co-authorship countries, co-occurrence keywords plus, and bibliographic coupling sources network visualization mapping. The VOSviewer version 1.6.17 was used for network visualization.\n\nResults: The bibliographical search resulted in a total of 478 publications which were included in this study. The publications were mainly published in English (n=436), while the major document types were articles (n=391). The most productive year was 2014 (n=33), while the most cited year was 2020 (n=1026). The extensively studied research areas were immunology and veterinary science, and the most used keywords plus were Q-fever and Coxiella-burnetii. Kazar J (n=17) was the leading author, while the famous journal was Acta Virologica (n=23). The most active institution was the Slovak Academy of Sciences (n=32), and the leading country was the US (n=129).\n\nConclusion: A rapid increase has been observed in Q fever vaccine publications and citations in the past 20 years. This study might be of great interest to provide standard bibliographic information and keystones parameters in Q fever vaccine research.", "keywords": [ "Q fever", "Q fever vaccine", "Bibliometric analysis", "Web of Science", "Visualization mapping" ], "content": "Introduction\n\nQuery fever (Q fever) is a highly infectious zoonotic infection to humans and livestock caused by the etiological agent Coxiella burnetii, an intracellular gram-negative bacterium widespread throughout the world.1,2 Domestic ruminants are believed to be the primary source of Q fever in humans.3,4 In humans, the infection is primarily transmitted via inhalation of aerosols from contaminated soil and animal excrement, most notably parturient fluids.4–9 Consumption, particularly raw milk, is also likely a route of C. burnetii transmission. Although C. burnetii has been isolated from arthropods, primarily ticks, it is unlikely that arthropod-borne transmission of Q fever is significant in humans.5,10 However, C. burnetii can infect other animal species, including pets and birds and cause human cases of Q fever.3,6,8,11 The majority of animal species infected with C. burnetii exhibit no symptoms.4,6 However, in goats and sheep, Q fever's most common clinical manifestations are abortion and stillbirth. Q fever has been linked to sporadic abortion, infertility, and metritis in cattle.6,12 Abortion epidemics in livestock have been reported in endemic regions, resulting in severe economic consequences.1,13,14 Abortion can result in the excretion of up to 1 billion C. burnetii per gram of placenta.15\n\nIn humans, the disease presents as an acute flu-like illness with a debilitating headache and cyclic fever as its hallmark symptoms.1,5 The typical signs and symptoms of symptomatic infection include headache, pyrexia, and respiratory tract infection, including atypical pneumonia; hepatitis is also a possibility.16 Chronic infection is well-known, most commonly manifesting as Q fever endocarditis.5,16–18 Correlations between C. burnetii infections and the onset of atherosclerosis, chronic fatigue syndrome, and other cerebrovascular events also have been suggested.1,5,18,19 Despite extensive efforts to develop an effective vaccine against human Q fever, only Q-Vax® is commercially available, and its licensed use is limited to Australia.20–22 Thus, the current study was conducted to explore the global research outputs, research areas, and frontiers, and to establish the visualization mapping of research in the Q fever vaccine.\n\n\nMethods\n\nA bibliometric review followed by a visualized study was conducted (See underlying data).23 The review is reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping review (PRISMA-ScR) guidelines.23 The Science Citation Index Expanded (SCI-E) Edition of Web of Science Core Collection (WoSCC) was used to search for relevant publication on the Q fever vaccine as of October 16, 2021. The Web of Science (WoS) is the world's oldest database belonging to the commercial provider.24,25 Currently, the WoS is hosted by Clarivate Analytics.26 The WoSCC database is a selective citation index of scholarly and scientific publishing covering books, data compilations, proceedings, and journals.25 The WoSCC is a commonly used database for bibliometric studies.27–33 Previously published bibliometric studies conducted in medical and health sciences and other areas used SCI-E Edition of the WoSCC.34–37 Therefore, in the current study, SCI-E Edition was utilized.\n\nThe following keywords were used: “Q fever” OR “Query fever” OR “Coxiella burnetii” OR “Coxiella-burnetii” OR “C. burnetii” (Topic) AND “Vaccine” OR “Immunize” OR “Immunis” (Topic). The searches were performed without any limitation in the topic field. The Topic field searches title, abstract, author keywords, and keywords plus.\n\nWe extracted many attributes such as author name, year of publication, journal, document type, institution, and country. The data were downloaded both in Comma-separated value and Tab delimited files. The collected information was entered into a Microsoft Excel spreadsheet, and the values were presented in frequencies and percentages.\n\nThe data were exported in Tab delimited file into VOSviewer software version 1.17.1 for macOS for network visualization mapping. VOSviewer is a widely available tool for network visualization, overlay visualization, and density visualization mapping.38 The retrieved data were plotted for co-authorship countries, co-occurrence keywords plus, and bibliographic coupling sources network visualization mapping. After plotting the data, clusters were formed, and each color designates a different cluster. The minimum cluster size for co-authorship countries was fixed at 10. The countries with zero total link strength (TLS) were excluded from the plotting. The thicker line between the countries represents the stronger collaboration, while the larger node or label represents the higher the weight vice versa.39 The co-occurrence of a keyword plus was selected at 5. The keywords plus terms are generated from the titles of articles/documents cited by the author of the article being indexed. The articles/documents whose references are not linked to source items will not have keywords plus.40 The data were further plotted for bibliographic coupling sources based on the cited references. The minimum number of items of a source was selected at 5. Alongside network visualization, the density visualization mapping was generated. In density visualization, same as network and overlay visualization, the items are represented by their label. In density visualization, each point has a color that represents the density of items at that point. In the neighborhood of a point in density visualization, the larger the number of items, and the weights of the neighboring items, the closer the color of the point is to yellow color.39\n\n\nResults\n\nThe initial search retrieved a total of 478 documents, and all the documents were included in the final bibliometric analysis and visualization mapping. The documents were published in nine languages: English (n=436), German (n=20), French (n=11), Czech (n=3), Dutch (n=3), Russian (n=2), Italian (n=1), Polish (n=1), and Slovak (n=1).\n\nThe included publications were cited 12,434 times (26.01 average citations per item) and 9,378 times without self-citations (19.62 average citations per item). The overall H-index value was 51 in the published documents. The most productive year of publications was 2014 (n=33, 884 citations), while the most cited year was 2020 (n=20, 1,026 citations), as shown in Figure 1. The documents published in 2021 (n=20) were cited 817 times.\n\nThe majority of the documents were published as original/research articles (n=391), followed by review articles (n=51), and proceeding papers (n=24) (Figure 2A). The most studied research areas were immunology, veterinary sciences, and infectious diseases (Figure 2B). The most prolific author was Kazar J (n=17) (Figure 2C) and most of the article published in Acta Virologica (n=23) (Figure 2D).\n\nIn total, 60 records did not contain institution names, while 34 records did not contain country names. The institution with the most publications (n=32) was the Slovak Academy of Sciences (Figure 3A). The top three leading countries in Q fever vaccine research were the USA (n=129), Australia (n=62), and the Netherlands (n=54), as shown in Figure 3B.\n\nOf the total countries, 18 countries with TLS zero were excluded from the mapping. For the rest of the countries, the TLS ranged from 1 to 36. USA was the leading country with the highest TLS with other countries (n=36), followed by Netherlands (n=28), Switzerland (n=27), France (n=21), and England (n=21). A total of two clusters were formed; cluster 1 consists of 23 countries, while cluster 2 had 16 countries (Figure 4).\n\nOf the total keywords plus, only 105 met the criteria. At the same time, the cluster size was fixed at 10. A total of four clusters were formed; cluster 1 consists of 45 keywords plus, followed by cluster 2 (n=23), cluster 3 (n=20), and cluster 4 (n=17) (Figure 5). The widely used keywords plus based on occurrence and TLS was Q-fever (n=110, TLS=402), followed by Coxiella-burnetii (n=77, TLS=266), infection (n=67, TLS=338), vaccination (n=64, TLS=287), and an outbreak (n=57, TLS=275).\n\nOf the total publication sources, only 23 met the criteria and were plotted. Two clusters were formed; cluster 1 consisted of 12 items, while cluster 2 had 11 items. Based on TLS value, Vaccine, Infection and Immunity, and PLOS One were the leading sources of publication (Figure 6).\n\n\nDiscussion\n\nThis study has analyzed Q fever vaccine research trends and characteristics from 1941 to 2021. The key topics and research type in Q fever vaccine, main contributors and generators of knowledge in this field, and collaborations among researchers were presented. This study has shown an increase in publications and citations on Q fever vaccine research, especially for the last 20 years. This mirrors the trend of increasing vaccine-related publications throughout the years.41 However, publications on Q fever vaccinations are lagging other infectious diseases such as Ebola and HIV.42,43 This might be due to the status of Q fever as a neglected and understudied disease.44\n\nThe top research areas on Q fever vaccination include immunology, infectious disease, and microbiology. Furthermore, keyword analysis revealed that research areas include epidemiological surveys and pathophysiologic and genetic studies. This signifies the importance of fully understanding the disease entity to generate knowledge for vaccine production.22 Also included in the top research areas are veterinary sciences, public environmental, occupational health, and agriculture. This relates to the importance of Q fever not only for human health but also for the industry, particularly agriculture and animal husbandry.45\n\nThe USA is the leading country in terms of the number of publications in Q fever vaccination. Furthermore, 8 out of the top 10 leading institutions in the field are from the USA. This is consistent with the high scientific productivity of the USA across different fields.46 The USA is currently the country with the highest research expenditure and highest gross domestic product, which might explain its high productivity on research.47,48 Moreover, we have shown that USA authors had the highest TLS with other countries, signifying its rich collaborations. Cooperation and collaboration have been shown to correlate well with increased research productivity.49\n\nNotably, some countries with a high disease burden, including Morocco, Zimbabwe, and Nigeria, are not the countries with the highest research production.44 This indicates an imbalance can be seen between research production and the burden of disease. Therefore, there is a need to increase research activities among these countries to provide more evidence on Q fever.\n\nMost of the top 10 journals in the Q fever vaccine field are international journals. Furthermore, 6 out of the top 10 journals have an impact factor greater than 3.0. Journal selection is influenced by different factors including journal ranking system, reliability of reviewing, university and national policies, usefulness of reviewers’ feedback, and research funding bodies.50\n\nThis is the first bibliometric study to summarize the global research output and trends in the Q fever vaccine indexed in WoSCC. The key bibliometric indices and visualization mapping were generated. This study used a single database WoSCC which may bias the results. Therefore, further studies are recommended to utilize multiple databases to evaluate the publication frequency and citation analysis.\n\n\nConclusion\n\nThis study used bibliometric analysis to provide an insight on the Q fever vaccine research, worldwide. In the past two decades, a rapid increase has been observed in Q fever vaccine publications and citations. USA was the leading country, and immunology was the most studied research in this field. The current study will provide standard bibliographic information and keystone parameters in Q fever vaccine research.\n\n\nData availability\n\nFigshare: Bibliometric analysis and network visualization mapping of global research in Q fever vaccine.\n\nDOI: https://doi.org/10.6084/m9.figshare.18316595.23\n\nThis project contains the following underlying data:\n\nQ-fever data.text: (Data of the publications downloaded from the Web of Science that were used in this bibliometric analysis study)\n\nFigshare: PRISMA-Scoping review checklist for “Bibliometric analysis and network visualization mapping of global research in Q fever vaccine”\n\nDOI: https://doi.org/10.6084/m9.figshare.18316595.23\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe acknowledge Southeast University, Nanjing, China, for providing free access to the WoSCC database. 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PubMed Abstract \| Publisher Full Text\n\nAkintunde TY, Musa TH, Musa HH, et al.: Mapping the global research output on Ebola vaccine from research indexed in web of science and scopus: a comprehensive bibliometric analysis. Hum. Vaccin. Immunother. 2021; 17: 4246–4258. PubMed Abstract \| Publisher Full Text\n\nNye J, D'Souza MP, Hu D, et al.: Research productivity and collaboration of the NIH-funded HIV vaccine trials network: A bibliometric analysis. Heliyon. 2021; 7(1): e06005. PubMed Abstract \| Publisher Full Text\n\nHonarmand H: Q Fever: an old but still a poorly understood disease. Interdiscip. Perspect. Infect. Dis. 2012; 2012: 1–8. PubMed Abstract \| Publisher Full Text\n\nMori M, Roest H-J: Farming, Q fever and public health: agricultural practices and beyond. Archives of Public Health. 2018; 76(1): 2–9. PubMed Abstract \| Publisher Full Text\n\nWhite K: Publications output: US trends and international comparisons. National Science Foundation; 2019.\n\nMariotto S, Mantovani A: Scientific productivity in neurology: impact of the socio-economic status. Neurol. Sci. 2021; 42(4): 1563–1566. PubMed Abstract \| Publisher Full Text\n\nUSR: UNESCO science report: Towards 2030.2015. Reference Source\n\nAbramo G, D’Angelo AC, Murgia G: The relationship among research productivity, research collaboration, and their determinants. J. Informet. 2017; 11(4): 1016–1030. Publisher Full Text\n\nRowley J, Sbaffi L, Sugden M, et al.: Factors influencing researchers’ journal selection decisions. J. Inf. Sci. 2020: 016555152095859. Publisher Full Text" }	[ { "id": "129250", "date": "26 Apr 2022", "name": "Carrie Mae Long", "expertise": [ "Reviewer Expertise Coxiella burnetii", "Q fever", "vaccinology", "immunology", "bacterial pathogenesis", "bacteriology", "host-pathogen modeling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe submitted systematic review manuscript aims to investigate historic and modern Q fever vaccine-related publications. The authors conducted analysis of general research trends, global distribution, and other features related to these publications. The authors concluded that there has been a notable increase in Q fever vaccine-related publications in the past twenty years and that the presented study may provide utility as a bibliographic resource for researchers. The manuscript addresses an important and timely topic. The submitted work was easy to read and the data was presented in a concise matter. While the concept of the review is with merit, I have several concerns regarding the data inclusion criteria and the main figures.\nMajor comments:\nThe study design and database search were well-described and are suitable for replication. I have several concerns regarding the inclusion criterion for \"Q fever vaccine research\":\nThe authors refer to the research of interest as \"Q fever vaccine research\" yet the criterion for this moniker does not appear to be well-defined. Given the wide breadth of studies that would be identified by the described search keywords, were publications identified based on type of manuscript (e.g. review, primary study) and level of relevance to C. burnetii/ Q fever vaccine research (e.g. ancillary association or study primary focus). I feel that these criteria need to be formally defined for the resultant data to be useful.\n\nSimilarly, it might be prudent to refer to the manuscripts in question as \"Q fever vaccine-related publications\".\n\nHistorically, C. burnetii has been referred to by a number of names, including Rickettsia burnetii, Rickettsia burneti, and Coxiella burneti. I am concerned that works published prior to formal naming of the bacteria and disease may be excluded on the current basis of the search.\n\nFigure 5B appears to be identical to Figure 4B.\n\nI was unable to access the Q-fever data.text file. Access to this file is very important given that one of the main conclusions addresses the utility of this data.\n\nMinor Comments:\nThe figure legends are very terse and could potentially be improved with the addition of relevant information. For example, in Figure 3 it is unclear how authorship and study country are defined (e.g. first author, last author, any author affiliation?). This could be outlined in the figure legend for clarity.\n\nThe authors state that \"6 out of the top 10 journals have an impact factor greater than 3.0\". How was impact factor determined? Some journals have chosen to refrain from self-reporting. 1 So I assume that impact factor was determined based on a third party source?\n\nI suggest replacing words such as \"attractive\" and \"famous\" in reference to journals with less subjective alternatives.\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": [] }, { "id": "336171", "date": "05 Nov 2024", "name": "Festus Mulakoli", "expertise": [ "Reviewer Expertise 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\nSummary of the article This bibliometric analysis of the Q fever vaccine offers valuable insights into the trends and patterns in vaccine-related publications. By systematically examining the publication data, this analysis sheds light on the evolving research landscape surrounding Q fever vaccines, including the frequency of publications over time, key contributors in the field, and emerging areas of focus. Additionally, this analysis serves as a vital resource for government agencies, researchers, and other stakeholders, enabling them to closely monitor these trends. These data can inform decision-making processes related to public health policies, funding allocations, and vaccination strategies on a global scale. By understanding the trajectory of research on Q fever vaccines, stakeholders can address public health needs and respond to emerging challenges in infectious disease prevention. Comments to authors Abstract\nInclude a summary of visualization and recommendations\nBackground\nThe authors must provide a comprehensive overview of the global burden of Q fever, specifically focusing on its prevalence across different regions. This analysis should also delve into the various determinants of the disease, including environmental, socioeconomic, and biological factors that influence its spread and impact in diverse populations. By highlighting these elements, we will enhance our understanding of the significance of Q fever on a global scale and identify potential areas for intervention and further research.\nMethod section\nWhy review publications since 1941?\nResults section Several improvements are recommended to enhance the clarity and understanding of figures 1-6, I recommend making several improvements.\nFirst, it is beneficial to distinctly separate the concepts of network and density in Figures 4, 5, and 6. This separation helps viewers differentiate between the two elements being presented more easily. Additionally, incorporating a variety of colors into graphics can significantly improve visual distinction and comprehension. Finally, increasing the resolution of the images ensures that all details are sharp and clear, facilitating a better interpretation of the data.\nDiscussion section\nThe discussion section should be thorough and well-rounded, incorporating citations from a variety of relevant studies to provide context and support for the findings. It is essential to critically assess the existing literature and identify gaps, inconsistencies, and areas of agreement, thereby situating the current research within this broader framework. In addition, the authors should articulate how their findings contribute to a larger body of knowledge, emphasizing their significance and potential implications for future research and practice. By doing so, the authors not only reinforce the relevance of their work but also invite further dialogue and investigation within the academic community.\nConclusion section To enhance the clarity of the conclusion,\nI suggest that the authors emphasize the key findings derived from this study and highlight their implications for the effective management of Q fever.\n\nAdditionally, we should see how these findings can inform better preventive strategies and health policies aimed at reducing the incidence of Q fever in the affected populations. By clearly articulating these points, this study provides valuable insights that contribute to ongoing efforts in managing this disease.\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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-364
https://f1000research.com/articles/11-362/v1	29 Mar 22	{ "type": "Research Article", "title": "Improving memorability using Emojis in a shoulder surfing resistant authentication method", "authors": [ "Mohamed Mahrous Mahrous Amer", "Yvonne Hwei-Syn Kam", "Aiman Hussein Elkhedrawi", "Mohamed Mahrous Mahrous Amer", "Aiman Hussein Elkhedrawi" ], "abstract": "Background: Emojis are icons that are familiar and fun to add pizzazz and colour to communication. They have also been used in authentication where the emojis form memorable pictogram story-like passwords. Emojis, which are graphical, are in general vulnerable to shoulder surfing attacks (SSAs). This paper studies whether graphics such as emojis offer better memorability than numerics when implemented in a shoulder-surfing resistant authentication method. Thus, the proposed method aims to meet both needs of being shoulder-surfing resistant as well as being memorable. Methods: In this paper, a SSA resistant method (DragPIN) is used as a reference system on which to implement emojis in place of numerics. Additionally, a new feature, cue questions was implemented for added security. In the proposed method, users composed emoji-based stories using personalised cue questions that served as memory aids. Moreover, these self-chosen cue questions were less comprehensible to shoulder-surfing observers. There were two variants of the DragPIN method, manual and automatic-sliding. To compare the differences, both the reference configuration and modified versions based on the proposed method were implemented. Thirty people participated in user testing. A pre- and post-survey appraised user experience. User testing and survey on both methods and their variants for performance, memorability, and usability were performed. Results: All implementations successfully resisted shoulder surfing. The time taken for login in the manual variant using the proposed methodology was shorter than using the reference method. After four to six weeks, login performance taking into account intermediate failures was better for the proposed method (86.7-91.7%) than the reference method (76.7-78.3%). Hypothesis testing also showed significance in the results. This could point to higher memorability in the proposed method. Conclusion: The study provides testing of emoji-based compared to PIN-based implementation in authentication. Emoji-based stories may form memorable passwords while personalised cue questions may aid memorability.", "keywords": [ "Graphical Authentication System", "PIN", "Password", "Emoji", "Shoulder Surfing" ], "content": "Introduction\n\nIn general, graphical passwords are more memorable than text passwords because of the picture superiority effect.1–3 Graphical authentication has been a widely researched topic. At the time of writing this paper, 1,090 articles were retrieved by Google Scholar with the search terms “shoulder-surfing” and “graphical authentication”.\n\nThere was an uptrend of publications from 2012 to 2017 which plateaued until 20204 (Figure 1), with mean citations of 15.18 per paper. In dimensions.ai4 the search phrase involving combinations of “emoji”, “picture”, “password” and “authentication” retrieved 587 publications.\n\nTable 1 shows a comparison of previous works. Emojis have been used in authentication5 but are in general more vulnerable to shoulder surfing attacks (SSAs). DragPIN6 and the methods in7,8 are resistant to SSA. The automatic sliding variant implemented by DragPIN has the advantage of the display not being static, so the displayed state may not necessarily correspond to the password, which makes it shoulder surfing resistant. However, methods7,8 are vulnerable to intersection attacks after multiple recorded observations. DragPIN is resistant to SSAs but uses numbers, which are less memorable than pictures. EmojiAuth5 is not SSA resistant but uses emojis, which are more memorable.\n\nBoth methods have strengths and disadvantages. Therefore, a modified DragPIN that uses emojis instead of digits addresses both systems’ drawbacks as well as maintaining their respective advantages.\n\n\nMethods\n\nA DragPIN prototype was constructed for testing. A signup screen, as shown in Figure 2, allows a user to create a login and register a 4-digit pin. Users could sign in by choosing either the manual or automatic tabs (Figure 3). Conceptually, the implementation (shown in Figure 3) is similar to the original DragPIN.6\n\nFigure 3 shows the DragPIN interface implementation. A prototype for EmojiAuth was also made: the signup page and login screen with an emoji keyboard are shown in Figures 4 and 5, respectively. Unlike the implementation in the original DragPIN, which had only a choice of 20 emojis, the prototype allowed users to make use of a wider set of emojis.\n\nOperation\n\nIn this section, we will describe how the software works. We implemented EmojiSlide (the proposed method) and DragPIN (the reference method) as a web application. All the dependencies required to run the source code are managed by Pipenv version 2020.11.15. The software is provided in the repository as mentioned under the Software availability section. Installation instructions are included in the README.md file archived in release v0.1-beta in the repository. Memory (RAM) 512 MB and 1× CPU cores are the minimum system requirements. Django was the framework used to build this web application. Figure 6 describes the flow of the web application that was used to evaluate the differences between DragPIN and the proposed method (EmojiSlide).\n\nAt the start of the program, the user will be prompted to select the authentication method desired, either EmojiSlide or DragPin. Thereafter, users will have two options, which are login or sign up. If the user navigates to the signup page in DragPIN or Emoji-Auth, an empty form is generated and passed to the frontend. The form data received by the backend via POST HTTP request is validated and the user profile is saved in the database, following which users may use their credentials to log in via the earlier chosen method.\n\nThe proposed method uses emojis instead of numerics in the reference method, DragPIN. The user registers two 4-emoji passwords. For each 4-emoji password, the system generates six other random emojis, for a total of ten emojis. The set of these ten emojis is the challenge set. The challenge set forms the table (column) indexes used in authentication (shown in Figure 10). The challenge set is fixed per user. This ensures that a user's password cannot be deduced from observing the emojis displayed upon subsequent reloading of the challenge webpage.\n\nTo increase memorability and security, cue questions were introduced (Figure 7), which were not present in DragPIN. Users wrote a cue question for each emoji password which also served as the password prompt. Resistance to SSA is increased by having randomly chosen cue questions. Each user must register two cue questions and two passwords, each of which consists of four emojis.\n\nThe proposed method was designed as a web application called EmojiSlide.9\n\nThe username entry page is shown in Figure 8.\n\nFigure 9 shows that a security measure to prevent Cross Site Request Forgery (CSRF) has been implemented. A CSRF Token is a private, unique, and unpredictable value generated by a server-side application to protect CSRF-vulnerable resources. When the later request is made, the server-side application checks that it has the expected token and rejects it if it is absent or incorrect.\n\nAfter entering the username, the authentication screen is shown. During authentication (Figure 10), a user chooses either the manual or automatic sliding scheme. The procedure is similar to DragPIN, except that the digits have been replaced with emojis. Figure 10 shows the manual scheme. As an example, the user's emoji password is , , , . The login process is started by the user mentally choosing an alphabet from the available alphabets. Let the chosen alphabet be ‘D’. One of the D’s in each row is aligned with the password emojis in the correct sequence. The icons look slightly different in Figure 10 due to emojis being customized on different platforms.\n\nFigure 11 shows the automatic sliding variant. The same emoji password example is used. The space bar was used to capture the moment the sliding marker ‘B’ aligned with the password emoji. The “enter” key commenced the sliding of the next row. In this instance, the user pressed the spacebar during alignment and pressed the “enter” key after the marker had slid beyond the password emoji. As a result, the letter ‘B’ was no longer aligned with the password emoji. This misalignment resists SSA.\n\nEthical considerations\n\nEthics approval was obtained from the ethics committee of the Multimedia University for the research (approval number EA04420201). The demographics chosen was university students and adults (>18 years old). These would likely be using authentication daily in their lives and have experience with different methods of authentication. An invitation message was sent to potential participants who were acquaintances of the authors. The participants were mostly students in MMU, with a few working adults. The invitation included a website link to a presurvey. Consent was implicit as participants would answer this survey and submit their email if they chose to participate in further testing. No monetary reward was given for participation.\n\nBetween 30 to 100 participants is considered a medium-sized sample (Bošnjak & Brumen, 2020). In a review of authentication methods (Binbeshr et al, 2021), most of the user studies (51 out of 55 articles) had between 10 to 50 participants, with 30 being the most common. Thus, in the experiments conducted, the chosen number of participants was 30 or more.\n\nSeveral questions were chosen in the presurvey to gain insight into the users’ willingness to use emojis as password characters. The survey consisted of six questions (Box 1).\n\n1. On a scale of 0 to 5, 5 being strong and 0 being weak, how strong do you think your password is?\n\n2. How would you rate your ability to recall this password?\n\n3. Would you consider using emojis as your password?\n\n4. Use 6 to 10 emojis only to tell a story about yourself. mine would be: “”\n\n5. How would you rate your ability to recall this emoji story?\n\n6. Would you still consider emojis as a password?\n\nUser testing of EmojiSlide\n\nUser testing was done in two phases. Phase 1 tested for login accuracy and time taken, as well as SSA resistance. Phase 2 tested for memorability by measuring login accuracy. Participants in phase 2 were the same as those in phase 1 to achieve reliable memorability statistics.\n\nPhase 1\n\nIn phase 1, participants with ages ranging from 18 to 40 were invited to a Google meeting, which was recorded for further evaluation of the scheme’s capability to resist SSA. EmojiSlide’s motivations were briefly described. Then a test user was created. The participant (user) then learned how to login, using each of the variants in both the proposed and reference methods (EmojiSlide Manual, EmojiSlide Auto-sliding, DragPIN Manual, and DragPIN Auto-sliding). After familiarisation, users then registered and attempted to authenticate in each variant. Participants were given three attempts to login. The time taken for a successful authentication attempt was recorded. A usability survey on the proposed method was given after completion. Shoulder surfing was performed on video recordings of user logins. Four \"shoulder surfers” went through the familiarisation procedure as described before attempting SSA.\n\nA survey was provided to the participants (the questions can be found in Box 2). Questions 3 to 6 used a Likert scale. The first three questions were for gathering demographic information. The remaining questions were used to ascertain users’ experience with the proposed method.\n\n\n\n1. What is your age group?\n\n2. What is your occupation?\n\n3. How computer savvy are you?\n\n4. How would you rate your overall experience?\n\n5. How hard was it to recall your emoji password compared to a textual password?\n\n6. Would you trust this system to prevent a shoulder surfer?\n\nSystem usability survey\n\nAt the end of the phase 1 experiment, the participants were given a System Usability Survey (SUS) which is a Likert scale (shown in Box 3). Each question’s response was converted to points and the result was graded according to Ref. 10.\n\n\n\n1. I think that I would like to use this system frequently.\n\n2. I found the system unnecessarily complex.\n\n3. I thought the system was easy to use.\n\n4. I think that I would need the support of a technical person to be able to use this system.\n\n5. I found the various functions in this system were well integrated.\n\n6. I thought there was too much inconsistency in this system.\n\n7. I would imagine that most people would learn to use this system very quickly.\n\n8. I found the system very cumbersome to use.\n\n9. I felt very confident using the system.\n\n10. I needed to learn a lot of things before I could get going with this system.\n\nPhase 2\n\nIn phase 2, held 4-6 weeks later, the same users from phase 1 were invited to re-login to test for password memorability.\n\nSignificance testing\n\nHypothesis testing was performed to compare the differences between EmojiSlide(E) with DragPIN (DP) in both manual (m) and auto (a) variants. The software used was Microsoft Excel version 2011. The factors for comparison are the time taken for login, t and the mean number of intermediate failures, f. The null hypotheses are that there are no differences. The method’s name and variant form the subscript in Table 4, e.g. the time taken for Emojislide manual is tEm. For statistical analysis of results, we applied paired t-tests. A p value of p < 0.05 was considered statistically significant.\n\n\nResults and discussion\n\nThe datasets for the user results are available as Underlying data.11–13\n\nA total of 50 participants took part in the presurvey. The questions were not compulsory to answer thus not all questions had 50 responses. In the presurvey, participants were asked to create an emoji story about themselves using six to ten emojis. For question no. 3, ‘Would you consider using emojis as your password?’ (n = 50) about 72% answered Yes or “I am not sure”, and one person (2%) gave a comment about the possibility of emoji passwords being guessed, while 26% answered No (Figure 12). To ascertain their answer with practical experience, those who did not answer “No” went on to create their emoji stories in question 5. After creating emojis, (n = 37) answered the repeated question of ‘Would you still consider using emojis as your password?’ (question 6). Only 1 person answered No, indicating that there was a willingness to try using emoji passwords.\n\nThe respondents who created their emoji stories (n = 37) also rated their ability to recall the emoji story they created on a scale of 0 = weak and 5 = strong (Figure 13). Option ‘5’ had the highest number of responses, indicating that most respondents felt confident of their ability to remember their emoji password.\n\nA total of 30 participants took part in user testing. Figure 14 shows the age groups: most participants were aged 20-30 years old (76.7%). Table 2 shows the demographics of the participants.\n\nTable 3 shows the average time taken to login for successful attempts. Users logged in slightly faster using EmojiSlide (proposed method) compared to DragPIN. Results also showed that login to auto-sliding variants took longer than the manual variants.\n\nPost experiment, users were requested to state whether they would trust the system to resist SSA. Figure 15 shows that 76.7% answered yes, 23.3% were unsure and none answered no, showing that the system was judged capable by most participants.\n\nNone of the shoulder surfers were able to get any full PIN or emoji password. They commented that slowing or reversing the recorded videos availed little, especially for the automatic variants. They were only able to obtain two emojis, from three users, which was due to those users pointing their mouse cursor at their desired emoji. All participants logged in successfully within three attempts (100% login accuracy). Most of the mistakes occurred during phase 1, for the DragPIN auto variant where three participants used three login attempts to login.\n\nFigure 16 shows the average successful login rates when the number of intermediate failures before succeeding is taken into account. If a successful login takes one attempt (0 failures), the success rate = 100%, if it takes two attempts (1 failure), the success rate = ½ or 50% and if three attempts (2 failures), success rate = 1/3 or 33.33%. This is calculated per user. The average success rate is shown in Figure 16.\n\nAfter 4-6 weeks, the login accuracy for both the auto sliding and manual variants ranged between 76.7-78.3% for the reference method and 86.7-91.7% for the proposed method.\n\nReferring to Table 4, the null hypothesis for (1) is that there are no differences in the mean login time between the manual EmojiSlide (E) & DragPIN (DP). The t-test gives t(29) = 2.13, p = 0.04, which shows that the mean login time differs. The mean login time is shorter for the EmojiSlide. However, for (2), the time differences between the autosliding versions of E & DP were not significant.\n\nThe null hypotheses for (3) and (4) are that there are no differences in the number of intermediate failures (during Phase 1) in the manual and auto EmojiSlide and DragPIN versions respectively, while the alternative hypotheses are that the EmojiSlide versions have fewer failures. The one tailed t-test for manual variants (3) gave t(29) = 1.99, p = 0.028. The auto versions (4) had t(29) = 2.25, p = 0.016. In Phase 2, hypothesis set (3) gave t(29) = 2.11, p = 0.02 but in hypothesis set (4), the null hypothesis was not rejected. Thus, in Phase 1, EmojiSlide (manual and auto) had a lower number of failures compared to DragPIN, and this trend continued in Phase 2 for the manual variant.\n\nThis suggests higher memorability in the proposed method. The login accuracy was higher even though the users had two sets of emoji passwords to remember versus one PIN.\n\nThe SUS showed that the average score per user was 88.5% (Excellent). The score distribution is shown in Figure 17.\n\nAs the emoji-based implementation was based on one method, whether the memorability gains will extend to other authentication methods is yet undiscovered. Also, the sample comprised mostly young people thus the effect on older adults was not tested. Larger scale testing with a higher number and variety of participants can provide more insight. One of the system’s future upgrades is the use of the most recent version of emojis.\n\n\nConclusion\n\nIn this paper, a graphical authentication method was proposed where emojis were used in place of numerics and cue questions were added. Results indicate the proposed method and reference method resisted SSA where no passwords were compromised. Passwords remained memorable after 4-6 weeks where the proposed method had a login accuracy of 86.7-91.7% compared to 76.7-78.3% for the reference method. The results indicate that the use of emoji-based stories may have higher memorability than numbers. Personalized cue questions may also aid memorability.\n\n\nData availability\n\nFigshare: Using Emojis in a Shoulder-surfing Resistant Authentication Method, Pre-survey.csv. (Pre-survey results.). https://doi.org/10.6084/m9.figshare.14872062.v1.11\n\nFigshare: Using Emojis in a Shoulder-surfing Resistant Authentication Method, Phase1&2.csv. (User testing results). https://doi.org/10.6084/m9.figshare.17163470.v1.12\n\nFigshare: Using Emojis in a Shoulder-surfing Resistant Authentication Method, SUS.csv (System Usability Survey results.). https://doi.org/10.6084/m9.figshare.14872059.v1.13\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSource code for EmojiSlide available from: https://github.com/mahrous-amer/FYP/tree/v0.1-beta\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.55743879\n\nLicence: MIT\n\n\nGrant information\n\nThis work was supported by the IRFund grant [grant number MMUI/210071], Multimedia University, Malaysia.", "appendix": "Acknowledgments\n\nAn earlier abridged version of this work was presented at the iCatse International Conference on IT Convergence and Security 2021. 14\n\n\nReferences\n\nPaivio A, Csapo K: Picture superiority in free recall: Imagery or dual coding?. Cogn. Psychol. 1973 Sep.; 5(2): 176–206. Publisher Full Text\n\nBiddle R, Chiasson S, Van Oorschot PC: Graphical passwords: Learning from the first twelve years. ACM Comput. Surv. September 2013; 44(4). ISSN 0360-0300. Publisher Full Text\n\nSnodgrass JG, Asiaghi A: The pictorial superiority effect in recognition memory. Bull. Psychon. Soc. 1977 Jul.; 10(1): 1–4. Publisher Full Text\n\nEmoji authentication In publications—Dimensions: n.d. Retrieved September 1, 2021. Reference Source\n\nGolla M, Detering D, Drmut M: Emojiauth: quantifying the security of emoji-based authentication.2017. Publisher Full Text\n\nSrinivasan R: DragPIN: A secured PIN entry scheme to avert attacks. Int. Arab J. Inf. Technol. 2018.\n\nSalman M, Li Y, Wang J: A graphical pin entry system with shoulder surfing resistance. 2019 IEEE 4th International Conference on Signal and Image Processing (ICSIP). 2019. Publisher Full Text\n\nKasat OK, Bhadade U: Revolving flywheel pin entry method to prevent shoulder surfing attacks. 2018 3rd International Conference for Convergence in Technology (I2CT). 2018. Publisher Full Text\n\nAmer M: mahrous-amer/FYP: EmojiSlide-Prototype (v0.1-beta). Zenodo. 2021. Publisher Full Text\n\nBangor A, Kortum P, Miller J: Determining what individual SUS scores mean: Adding an adjective rating scale. J. Usability Stud. 2009; 4(3): 114–123.\n\nAmer M: Pre-survey.csvUsing Emojis in a Shoulder-surfing Resistant Authentication Method. figshare. Dataset. 2021. Publisher Full Text\n\nAmer M: Phase1&2.csvUsing Emojis in a Shoulder-surfing Resistant Authentication Method. figshare. Dataset. 2021. Publisher Full Text\n\nAmer M: SUS.csvUsing Emojis in a Shoulder-surfing Resistant Authentication Method. figshare. Dataset. 2021. Publisher Full Text\n\nAmer MM, Kam YH, Goh VT: A Study on Using Emojis in a Shoulder Surfing Resistant Authentication Method. Lecture Notes in Electrical Engineering 2021." }	[ { "id": "129237", "date": "13 Apr 2022", "name": "Gerard Bastiaan Remijn", "expertise": [ "Reviewer Expertise perceptual psychology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 implemented and tested the use of Emojis in a shoulder-surfing resistant authentication method (“DragPIN”). Users were asked to construct a PIN-like password consisting of a series of emojis, and they could use personalized cue questions to aid memorability of the series. Shoulder surfing resistance was tested under a manual input condition and an automatic-sliding method, while login time and memorability were tested against the original (numerical input) method. Results showed that both the new implementations indeed were shoulder-surfing resistant and that manual login for both methods was faster than automated login. Login performance after 4-6 weeks was better for the emoji method, which the authors attribute to better memorability.\nOverall, the usability of the new method has been sufficiently described and tested with a fair amount of users (n=30). However, the manuscript overall lacks appropriate descriptions of key terminology and the motivation behind the study is not clearly explained. In order to improve replicability, clearer descriptions of the methods/procedures are necessary as well. Added to this, there are numerous small figures that could be combined, and the captions overall are rather uninformative. These main issues, along with minor suggestions/questions, are described in detail below.\nMain:\nThe Introduction does not provide any specific reasoning as to why this topic is important and as to why the reader should read the article, other than that “there is an uptrend of publications”, which is not clear from the data in Figure 1 in the first place – rather, the number of publications in this area seems pretty stable over the last decade. Furthermore, an essential concept such as shoulder surfing is not explained. More background about the importance of visual passwords, shoulder-surfing, memorability, etc. would be informative to the general reader.\n\nThe lack of clarification continues in the Literature Review, e.g., the “automatic sliding variant” or “intersection attacks” are mentioned as if the reader should know about this already. A proper explanation and research background on these issues would improve the manuscript considerably. Related to this, based on the literature, the authors chose “DragPIN” as the reference method and listed 4 references of related works (Table 1). A quick literature scan (Google Scholar) on graphical password and emojis, however, yielded at least 20 seemingly relevant references. Again, a more elaborate description of the research background on the use of emojis as graphical passwords would provide a more solid ground for the current study. The article has just 14 references (however, see minor point 9 below), and 5 are self-referenced to the dataset.\n\nThe manuscript contains 17 figures and 4 tables. Many of the figures (e.g., Figs 3 and 4, and Figs 4 and 5) can be combined or seem unnecessary (e.g., Figure 8 just shows a sign-in bar). Moreover, the captions are not informative at all. Ideally, one should be able to understand a paper by just checking the figures and reading the captions, without the main text. For example, the caption of Figure 6 is “Flow diagram”, that of Figure 7 is “Cue question registration”. Why not provide full descriptions that include names of methods, etc., as a service to the reader.\n\nMinor:\nAbstract, Line 15, “Moreover … observers”. The sentence suggest that this was tested. If so, it should be moved under “Results”.\n\nThere are some unclear/inconsistent terms in the abstract, which make it difficult to grasp in one read: - Line 18-21 “modified versions” L18, unclear here. - Results. “All implementations...shoulder surfing”. Maybe a line on how this was tested? - Conclusion, “..PIN-based” means “numeric”?\n\nIntroduction, line 6: explain “dimensions.ai”? If this is a reference or URL, please provide.\n\nLiterature review, P3, L5: The statement that “DragPIN ... uses numbers, which are less memorable than pictures” is debatable, since highly personalized numerical PINs (e.g., date/year of birth) are often chosen specifically because they are easy to remember.\n\nLiterature review, P3, L7: “Both methods” meaning exactly which methods? Help the reader with clear descriptions.\n\nFigure 1. Other than suggested in the Introduction, I do not see a clear uptrend of publications in the last decade or so. Also, please explain whether these numbers include papers related to shoulder surfing or not. Furthermore, Y-axis: what does 9K mean in relation to 75, 50, 25?\n\nFigure 5. Why and how were these emojis selected?\n\nPage 5, para 4, “Resistance to SSA is increased by having randomly chosen cue questions”. Unclear. For the sake of replicability, were these dummy questions or selected from the user database? Were they presented on the screen along with the \"correct\" cue question for each user?\n\nUser test study, P7: both references not numbered and not in the reference list.\n\nP8, Phase 1, line 7: “Four shoulder surfers...attempting SSA”. For the sake of replicability, did all 4 perform SSA on each single user? Were they standing behind a user, at what given distance? Please provide details.\n\nP8, System usability survey. Likert scale questions – what was the scale range and what were the end points?\n\nP9, Figure 12: Y-axis (Frequency) shows the number of participants, while all other figures show percentages. X-axis: “Bin” is uninformative. I assume “0” means weak and “5” means strong, but please provide exact information on the axis label. What is “more” on the x-axis?\n\nP11, Figure 16. X-axis. Unnecessary “0”s behind period of each value (100.0%, etc.)\n\nP11. Login time and login attempts were recorded, but what was the average number of emojis used in the passwords? Users could use in between 6-10 emojis and a higher number likely results in a longer login time and more login mistakes. Please provide information.\n\nP11, Significance testing. I don’t see any specific justification for using a one-tailed test for hypotheses 3. and 4. DragPIN (reference 6) apparently has been tested and used before, so there is no reason to think by default that it can only render more failures than EmojiSlide. Two-tailed seems appropriate.\n\nP11, second sentence from bottom: “This suggests higher memorability in the proposed method.” Login failures indeed could be due to poor memorability of a password, but is it possible that they also could have been due to differences in system navigation (e.g., button press misses, or swiping mistakes) between both methods?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "8137", "date": "28 Apr 2022", "name": "Yvonne Kam", "role": "Author Response", "response": "Thank you to the reviewer for the detailed, insightful comments. Regarding the major points raised by reviewer #1, We will provide more reasons and motivations for this work and explain the concept of shoulder surfing. We will clarify the terms and add more references. The number of figures and content will be reviewed and the captions made more clear. We will also address the minor comments. We will revise the manuscript after receiving the 2nd reviewer report. We will endeavor to clarify concepts and give more details to facilitate the reader's understanding in the coming revision." } ] }, { "id": "159005", "date": "02 Feb 2023", "name": "Nur Haryani Zakaria", "expertise": [ "Reviewer Expertise Information security" ], "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\nSummary: The research work proposed an enhancement of DragPIN scheme using Emojis instead of numerical characters and claimed to be shoulder surfing resistant. The proposed scheme rely on personalized cue questions to aid memorability. The experiment conducted to evaluate the proposed scheme against referenced scheme (i.e.: DragPIN) using 2 variants (manual and auto-sliding). Findings indicate that the proposed scheme has better performance and to certain extend improved users’ memorability.\nIs the work clearly and accurately presented and does it cite the current literature? The manuscript did not provide an adequate review of the literature on what was the main issue that motivate the study to be carried out. According to my understanding, the work is proposing an enhancement to existing DragPIN scheme by leveraging the usage of Emojis (as being used in EmojiAuth). Limitation of the existing schemes should be highlighted clearly and what are targeted enhancement that the study intended to achieve.\nIs the study design appropriate and is the work technically sound? The design of the study seems inappropriate and was not written neatly. The flow of sections did not provide good understanding in terms of the procedures that were taken throughout the experiment. For example, it was not clear in terms of the differences between manual and auto-sliding version. Why is it necessary to have these two variants? It was also not clear how the “shoulder surfers” was selected? Are they part of the 30 participants recruited? Measurements used for the parameters were not mentioned clearly. For example, how do you consider a successful or failure of shoulder surfing act? How do you measure the success of login rate versus failure rate?\nAre sufficient details of methods and analysis provided to allow replication by others? I think the Methodology section need to be revised. It should remove the discussion proposed method and the CSRF security to other section. These two are not part of the method. Protocol of the experiment was not listed and elaborated which makes it difficult to follow and what more to replicate.\nIf applicable, is the statistical analysis and its interpretation appropriate? The hypotheses of the study were not listed properly which makes it difficult to comprehend the findings. Majority of the statistical analysis was done descriptively which only reports on frequency (percentages basis). T-test was used to measure the hypotheses testing but then again, since hypotheses statements is missing, it would be difficult to comprehend the findings.\nAre all the source data underlying the results available to ensure full reproducibility? The source of data was shared but then again it is challenging to follow the write-up of the manuscript since the sections need to be revised accordingly to fix the coherence aspect.\nAre the conclusions drawn adequately supported by the results? It is difficult to agree with the conclusion drawn when authors did not clearly mention how the parameters were measured. It was mentioned earlier in my comment above about how the protocols of the experiment was conducted? How about the measures taken for the success and failures of login accuracy.\nOther comments In general, the manuscript needs to be rewritten to improve the quality of the write-up. At its current stage, it is difficult to follow the narrative of the manuscript that can assist readers’ understanding of the study being carried out. I would encourage the author(s) to resubmit again the manuscript after considering the comments and suggestions given.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/11-362
https://f1000research.com/articles/11-361/v1	29 Mar 22	{ "type": "Research Article", "title": "Ponseti method under general anesthesia is an effective method of treatment for neglected congenital talipes equino varus: a cohort study.", "authors": [ "Panji Sananta", "Respati Suryanto Dradjat", "Tofan Margaret Dwi Saputra", "Muhammad Alwy Sugiarto", "Respati Suryanto Dradjat", "Tofan Margaret Dwi Saputra", "Muhammad Alwy Sugiarto" ], "abstract": "Background:Neglected congenital talipes equino varus (CTEV) deformity is common in poorer developing countries. If ignored, children with CTEV result in callosity, potential bone and skin infection, and a significant mobility limitation resulting from stiffness. There are many management options that can be used to manage neglected CTEV. Until now, the Ponseti casting technique is a gold standard for treating CTEV without surgery. Ponseti methods effectively correct CTEV deformity in all ages. However, patients treated with the Ponseti process will suffer pain during correction in daily practice. Therefore, it is necessary to give anesthesia to reduce pain and relax soft tissues to achieve a satisfactory outcome when correction is carried out. Methods:This study design is a retrospective. Our study consisted of 32 patients, divided into two groups. Group A is the group that was treated with the Ponseti using general anesthesia (GA), and group B is the group without using GA. The children were anesthetized using isoflurane inhalation with 1-2 mcg/kg. After that, we performed serial casting every week and evaluated the outcome and number change cast between using GA and without GA. Lastly, we used a paired t-test statistical analysis to determine the relationship between before and after therapy. Results:In group A, the mean Pirani score significantly reduced from 5.81 ± 0.403 to 0.625 ± 0.40. In contrast with roup B, where the mean Pirani score slightly decreased from 5.81 ± 0.403 to 4.437 ± 1.093. After the last serial cast, in group A, only four cast replacements were needed to achieve a good outcome, whereas, in group B, the results remained unsatisfactory after 10 cast changes. Conclusions: Ponseti method under GA is an effective treatment and reduced the number of cast changes for neglected CTEV.", "keywords": [ "Clubfoot", "Congenital Talipes Equino Varus", "Neglected", "Walking Age", "Children", "General Anesthesia." ], "content": "Introduction\n\nThe congenital talipes equinovarus (CTEV) is one of the most frequent and complex congenital deformities.1,2 The incidence of CTEV is estimated to be 1 to 2 per 1,000 live births.3,4 CTEV has four components: ankle equinus, hindfoot varus, forefoot adductus, and midfoot cavus.5 When ignored in children, CTEV can result in callosity, potential bone and skin infection and a significant mobility limitation resulting from stiffness.6,7 In older patients with CTEV, the soft tissue becomes fixed and more difficult to manage.8 This condition is usually found in neglected CTEV deformity.\n\nNeglected CTEV is a common issue in poorer developing countries.6 The terminology neglected CTEV is somewhat unclear. According to some previous studies, neglected CTEV is not treated until the age that most treatments are expected to be successful deformity correction, in which optimally start straight from birth.7,9 Subsequently, neglected CTEV may be defined as any CTEV which has not received any treatment before the age of 2 years.8,10 In developing countries, late presentation causes neglected conditions commonly due to a lack of awareness, treatment availability, or referral delays, and many parents choose to postpone treatment to seek traditional treatments(i.e. massage management).11\n\nThere are many options that can be used to manage neglected CTEV deformities, such as the Ponseti casting technique, Achilles tenotomy, Achilles tendon lengthening, plantar fasciotomy, etc.9 Over the last two decades, the Ponseti casting technique has become the gold standard for treating CTEV without surgery.5,12 The Ponseti method of serial casting has increased in popularity due to its effectiveness in correcting all components of CTEV in over 90% of cases.1,5,6,13 Despite this, another study reported that Ponseti also had weaknesses, although in small numbers such as leg discrepancies or reccurence.13,14\n\nPonseti methods effectively correct CTEV deformity in all ages. However, in daily practice, patients treated with the Ponseti method will suffer pain during correction.8,15,16 Furthermore, the neglected condition in an older child created difficulties correction due to stiffness and problem gait pattern.17 Therefore, it is necessary to give anesthesia to reduce pain and relax soft tissues to achieve a satisfactory outcome when correction is carried out.\n\nTo our knowledge, only a few studies discuss the outcome using the ponseti correction method with general anesthesia (GA), especially in a neglected condition. Therefore, the purpose of this study was to evaluate the outcome of Ponseti casting for neglected CTEV with GA if compared without using GA. The authors hypothesize that the result will be better with GA, especially in neglected patients. This article has followed STROBE checklist and guidelines.\n\n\nMethods\n\nThis study was approved by The Ethical Committee of the Medical Research Faculty of Universitas Brawijaya with number 400/036/K.3/302/2022. Informed consent was obtained verbaly from patient’s guardians for collection of the data for this study. Written infformed consent was obtained from the patient’s guardians for the publication of the data collected.\n\nThis study design is a cohort retrospective study using medical records as secondary data. The population in this study is patients who underwent Ponseti methods at Saiful Anwar Hospital, Malang, from January 2017 until December 2019. From medical records, we collected data on the age, sex, pirani score before and after ponseti method, and total number of cast change.\n\nThe total patient with neglected CTEV admitted to our hospital between January 2017 and December 2019 were 35 patient, but 3 patient were excluded by the exclusion criteria. two patient were treated with traditional treatment, and the data was incomplate in one patient. The final total sample of the study was 32 patients which consisted of 16 patients who had been treated with the Ponseti method using GA and 16 patients treated with Ponseti without GA (those whos gaurdians opted against using it). Therefore, we divided the sample into two groups, group A and Group B. In group A, isoflurane inhalation with doses of 1-2 mcg/kg were used as GA drugs. All patients either unilateral or bilateral using ponseti methods for CTEV repair with the supine position.\n\nInclusion criteria were patients over two years old with congenital talipes equinovarus and consent to participate. Exclusion criteria included neurological problems, spine or hip disorder, and children treated with other methods before performing Ponseti Method such as traditional treatment, incomplete medical records data.\n\nThe severity of deformity result was assessed using the Pirani scoring system at the beginning and end of treatment. Figures 1 and 2 provide examples of before and after treatment. two independent general practicion who did not contribute on this study collecting data to prevent bias; however, Ponseti correction was performed by a single orthopaedist in a Tertiary Hospital. Based on the Pirani score, the foot was given a score between 0 and 6. The cases of 32 children with CTEV had been treated and observed for one year. The serial casting was performed every week for manipulation, and the number of casting changes for the treatment was also evaluated. The cast was made using plaster of paris, and strengthened with fiberglass as an outer layer to prevent it from breaking in older children.3 The minimum number of casts used was four and the maximum number of cast changes was 12.17\n\nWhen the last cast was removed, the outcome follow up was carried out for one month. The parents were instructed to follow a home-based exercise program. The exercise program included 50 repetitions of dorsiflexion and foot abduction exercises, which were to be repeated five times daily for the first two years and then three times daily.18 We divided the outcome scores (recorded at the time treatment was completed) between 0 and 1 were excellent, between 1.5 and 2.5 for good score and scores > 3 for poor score after treatment.18\n\nThe statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) Version 23.0 (RRID:SCR_002865). The demographic data and other characteristics were measured in terms of numbers and percentages. A paired t-test was used to determine differences in Pirani scores between treatment groups. We also performed a chi-square test to evaluate the difference hypothesis between the two groups. The significance value < 0.05 was considered statistically significant with sensitivty 95% confidence intervel.\n\n\nResults\n\nIn this study, the total number of patients included is 32. There were 18 females (56.3%) and 14 male (43.8%). The mean age of all patients in this study is 8.937 ± 5.22, with the range age between 2 to 24 years old. The most common age group was 6-10 years with 11 Patients (34.4%) (Table 1). The distribution of group A (group with GA) is 6 male children and 10 female children with an average age of 9.5 ± 5.83. Then, in group B (group without GA), 8 patients are male, and 8 patients are female. In groupB, the mean age is 8.37 ± 4.66. An example of a patient before treatment is shown in Figure 1.\n\nPirani score with GA\n\nThis study categorized the distribution age of children with CTEV under GA as group A. There are 4 children in the age group of children 2-5 years that consists of one child (25%) with Pirani 5, and 3 children (75%) with Pirani score 4. Then, there are 5 children in the age group 6-10 years that all (100%) have a Pirani score of 5. And then, there are five children in the age group 11-15 years that consists of is two children (40%) with a Pirani score of 2 and three children (60%) with a Pirani score of 3. Lastly, for age over 15 years old, there are two patients in this group, 16 years and 24 years old, these patients had the same Pirani score of 6. In group A, the mean Pirani score before the Ponseti cast was 5.81 ± 0.403. Subsequently, after the Ponseti treatment with GA, the Pirani score reduced with mean was 0.625 ± 0.403 (Table 2).\n\na Paired t test result group A.\n\nb Paired t test result group A.\n\nWe evaluated the number of cast replacements for the patients the range was restricted from 4 to 12 times. In this group Ponseti mean cast change 4.125 ± 1.50 to achieve a satisfactory result shown in Figure 2 (a Pirani score of between 0 and 1).\n\nPirani score without GA\n\nPirani score without GA was categorized as group B. Similar to group A, we divide the distribution into four categories. In group B, there are five children in the age range 2-5 years old that consists of four children (75%) with Pirani score 6 and one child (25%) with Pirani 5. Then, there were five children in the age group 6-10 years that consisted of three children (50%) with a Pirani score of 6, two children (33.3%) with a Pirani score of 5, and one child (16.7%) with Pirani 4.For the age group 11-15 years, there were four children (100%) that had a Pirani score of 6. Lastly, for over 15 years old, there was only one patient (100%) who had a Pirani score of 6. Unlike the results above, the outcome after the Ponseti method did not achieve satisfactory results. In group B, we obtained the Pirani score before the Ponseti cast mean of 5.81 ± 0.403. Hereafter, Pirani score after the Ponseti treatment without GA was only slightly reduced, with a mean was 4.437 ± 1.093.\n\nIn this group, the ponseti mean cast change was 10.25 ± 3.53, even after 12 serial casting the condition of foot of group B patient can not achieved good outcome. (Table 2) Eventually, after Ponseti cast management using GA (group A), the Pirani score significantly (p < 0.000) achieved an excellent outcome (Pirani score 0-1) in all patients (100 %) after the last serial cast was performed (group A). In contrast, when the Ponseti method is used without GA (group B), 15 patients (93.8%) did not achieve a reduction in their condition and remain in a poor outcome status (Pirani score > 3). Fortunately, at least one patient in group B (6,3%) had improved to be in good status after the final follow-up (p < 0.000).\n\n\nDiscussion\n\nSeveral other studies reported satisfactory clinical outcomes using the Ponseti method, which requires a series of procedures and serial sets of casting.13,15,19 The treatment needs to be started as soon as possible and should be followed under close supervision. At an early age, the outcome of the Ponseti casting technique yielded satisfactory anatomical and functional results with simple, effective, minimally invasive, inexpensive, and shor duration to achieve correction.5,20 This is the opposite in the case of the neglected condition, where the soft tissue becomes rigid when treatment is started.8 Moreover, according to the author's experience, children over four years old often seem scared of undergoing treatments such as serial casting, which increases the initial stiffness. Therefore, in older children the outcome may not be optimal.\n\nIn this study, excellent clinical outcome was achieved using Ponseti methods under GA (group A) on 16 patients (100%), although this was only confirmed in severe cases. This finding is consistent with Hallaj-Moghaddam (2015), who stated that the Ponseti method of manipulation and casting is beneficial in severe clubfoot.21 His study was also conducted using general anastesia, for a satisfactory outcome.21 From the current study in group A, satisfactory outcomes were reached with an average of four cast replacements; whereas in group B, after an average of ten cast changes, the outcomes remained unsatisfactory.\n\nSometimes, Ponseti’s classic manipulative technique does not correct some feet, this is especially common in neglected cases in older patients. These feet are clinically characterized by an extremely stiff equinus, severe plantar flexion of all metatarsals, and the perception of a shortened foot even after several casting serial performances.4 In this study, we found that 15 patients (93.8%) who did not receive anesthesia (group B) did not receive a satisfactory correction. The authors assume this outcome is due to the patient experiencing pain, also there will be an increase in soft tissue tension. So that, the manipulation and correction of the deformity will not be optimal and affect the outcome of Ponseti management.\n\nIn a study by Pavone et al., the average of pirani score result was 5.56, therefore, his result suggest for percutaneus tenotomy. After casting and tenotomy was perfomed, 98.78% patients had normal passive range of movement.22 Unfortunately, in this study, we were limited to discussing the outcomes of conservative clubfoot management with the Ponseti method only without comparing the outcomes using ponseti with tenotomy. Hereafter, because of unsuccessful treatment, in this study, 16 participants in group B underwent tenotomy to repair their clubfoot condition.\n\nBefore Ponseti casting started, patients underwent foot manipulations held for a few minutes before casting to help the soft tissue structures relax prior to applying an above-knee cast.23 However, in some cases, where a patient has neglected clubfoot, the soft tissue is rigid and difficult to correct. If this is forced, it will cause severe pain. Several anesthetic techniques have been utilized from previous issues, including local anesthesia, GA, and spinal anesthetic to reduce rigidity.24–26\n\nSome previous studies used general anastesia for management of CTEV in adults as used by Ponseti during the first years of treatment. Parada (2009) stated that GA could be administered safely to children who underwent clubfoot management.26 In another study conducted by Haje (2020), he succesfully treated adult patients with neglected CTEV using the Ponseti method under GA.10\n\nIn this decade, various studies have shown that GA for children is totally safe.26,27 In this study, an inhalation anesthesia agent that is reported as having a lower risk of postoperative apnea in children was used.27 This finding is in line with the research that we have done, in which hospitalization was necessary for no more than one day in all patients who received GA without any complications to be reported.\n\nThis study has several limitations, such as the small sample in our study and the short follow-up period. Therefore, a further study long-term follow-up and a significantly larger sample size are recommended. On the others hand, the strength of this study can be reference for another study who discuss Ponseti method in neglected cases, and then this can help clinician about management that can be choosen for neglected CTEV cases.\n\n\nConclusion\n\nOur study concludes that Ponseti method under GA is an effective treatment and reduces the number of cast changes for neglected CTEV.\n\n\nData availability\n\nZenodo: Ponseti method under general anesthesia is an effective method of treatment for neglected congenital talipes equino varus. https://doi.org/10.5281/zenodo.593902828\n\nThis project contains the following underlying data:\n\n- Raw Data.sav (Raw cast change, Pirani data and age data)\n\n- Raw Data.xlsx (Raw cast change, Pirani data and age data)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nMalhotra R, Mohapatra A, Arora G, et al.: Ponseti technique for the management of congenital talipes equinovarus in a rural set-up in india: Experience of 356 patients. Children. 2018; 5(4): 1–13. PubMed Abstract \| Publisher Full Text\n\nDobbs MB, Rudzki JR, Purcell DB, et al.: Factors Predictive of Outcome after Use of the Ponseti Method for the Treatment of Idiopathic Clubfeet. J Bone Jt Surg - Ser A. 2004; 86(1): 22–27. PubMed Abstract \| Publisher Full Text\n\nLynn Staheli M: Clubfoot: Ponseti Management [3rd Edition] - help_cfponseti.pdf.2009; 32. Reference Source\n\nMaranho DAC, Volpon JB: Congenital clubfoot. Acta Ortopédica Bras. 2011; 19(3): 163–169. Publisher Full Text\n\nGanesan B, Luximon A, Al-Jumaily A, et al.: Ponseti method in the management of clubfoot under 2 years of age: A systematic review. PLoS One. 2017; 12(6): e0178299–e0178218. PubMed Abstract \| Publisher Full Text\n\nPenny JN: The neglected clubfoot. Tech Orthop. 2005; 20(2): 153–166. Publisher Full Text\n\nEidelman M, Kotlarsky P, Herzenberg JE: Treatment of relapsed, residual and neglected clubfoot: Adjunctive surgery. J Child Orthop. 2019; 13(3): 293–303. PubMed Abstract \| Publisher Full Text\n\nAlves C, Batlle AE, Rodriguez MV: Neglected clubfoot treated by serial casting: a narrative review on how possibility takes over disability. Ann Transl Med. 2021; 9(13): 1103–1103. PubMed Abstract \| Publisher Full Text\n\nVan Bosse HJP: Treatment of the Neglected and Relapsed Clubfoot. Clin Podiatr Med Surg. 2013; 30(4): 513–530. PubMed Abstract \| Publisher Full Text\n\nHaje DP: Neglected Idiopathic Clubfoot Successfully Treated by the Ponseti Method: A Case Report of an Adult Patient who Started Treatment at 26 Years of Age. J Orthop Case Rep. 2020; 10(4): 74–77. PubMed Abstract \| Publisher Full Text\n\nRasit A, Azani H, Zabidah P, et al.: Clubfoot: The Treatment Outcome Using Quantitative Assessment of Deformity. Malaysian Orthop J. 2012; 6(4): 2–5. PubMed Abstract \| Publisher Full Text\n\nLindahl O: Congenital clubfoot: Fundamentals oftreatment IGNACIO V. PONSETI. Sven läkartidningen. 1963; 60: 441–459.\n\nAl-Mohrej OA, Alshaalan FN, Alhussainan TS: Is the modified Ponseti method effective in treating atypical and complex clubfoot? A systematic review. Int. Orthop. 2021; 45(10): 2589–2597. PubMed Abstract \| Publisher Full Text\n\nJayasomeswar DN, Kumar DDM, Babu DBK: The outcome of Ponseti technique for idiopathic clubfoot. Int J Orthop Sci. 2019; 5(2): 774–777. Publisher Full Text\n\nIppolito E, Farsetti P, Caterini R, et al.: Long-term comparative results in patients with congenital clubfoot treated with two different protocols. J Bone Joint Surg Am. 2003; 85(7): 1286–1294. PubMed Abstract \| Publisher Full Text\n\nHayes CB, Murr KA, Muchow RD, et al.: Pain and overcorrection in clubfeet treated by Ponseti method. J Pediatr Orthop Part B. 2018; 27(1): 52–55. PubMed Abstract \| Publisher Full Text\n\nKhan SA, Kumar A: Ponseti’s manipulation in neglected clubfoot in children more than 7 years of age: A prospective evaluation of 25 feet with long-term follow-up. J Pediatr Orthop Part B. 2010; 19(5): 385–389. PubMed Abstract \| Publisher Full Text\n\nSheta RA, El-Sayed M: Is the Denis Browne Splint a Myth? A Long-Term Prospective Cohort Study in Clubfoot Management using Denis Browne Splint Versus Daily Exercise Protocol. J Foot Ankle Surg. 2020; 59(2): 314–322. PubMed Abstract \| Publisher Full Text\n\nColburn M, Williams M: Evaluation of the treatment of idiopathic clubfoot by using the Ponseti method. J Foot Ankle Surg. 2003; 42(5): 259–267. Publisher Full Text\n\nSaif Ullah M, Ferdous KMN, Shahjahan M, et al.: Management of Congenital Talipes Equino Varus (CTEV) by Ponseti Casting Technique in Neonates: Our Experience. J Neonatal Surg. 2013; 2(2): 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHallaj-Moghaddam M, Moradi A, Ebrahimzadeh MH, et al.: Ponseti Casting for Severe Club Foot Deformity: Are Clinical Outcomes Promising?. Adv Orthop. 2015; 2015: 1–5. PubMed Abstract \| Publisher Full Text\n\nPavone V, Testa G, Costarella L, et al.: Congenital idiopathic talipes equinovarus: an evaluation in infants treated by the Ponseti method. Eur Rev Med Pharmacol Sci. 2013; 17(19): 2675–2679. PubMed Abstract\n\nPeterson N, Prior C: Correction of the Neglected Clubfoot in the Adolescent and Adult Patient. Foot Ankle Clin. 2020; 25(2): 205–220. PubMed Abstract \| Publisher Full Text\n\nRadler C: The Ponseti method for the treatment of congenital club foot: Review of the current literature and treatment recommendations. Int Orthop. 2013; 37(9): 1747–1753. PubMed Abstract \| Publisher Full Text\n\nWu JP: Pediatric Anesthesia Concerns and Management for Orthopedic Procedures. Pediatr Clin N Am. 2020; 67(1): 71–84. PubMed Abstract \| Publisher Full Text\n\nParada CSA, Baird GO, Auffant RA, et al.: Safety of percutaneous tendoachilles tenotomy performed under general anesthesia on infants with idiopathic clubfoot. J Pediatr Orthop. 2009; 29(8): 916–919. PubMed Abstract \| Publisher Full Text\n\nAlsuhebani M, Martin DP, Relland LM, et al.: Spinal anesthesia instead of general anesthesia for infants undergoing tendon Achilles lengthening. Local Reg Anesth. 2018; Volume 11: 25–29. PubMed Abstract \| Publisher Full Text\n\nSananta P, Dradjat RS, Saputra TMD, et al.: Ponseti method under general anesthesia is an effective method of treatment for neglected congenital talipes equino varus.Publisher Full Text" }	[ { "id": "139314", "date": "13 Jun 2022", "name": "Kelly Gray", "expertise": [ "Reviewer Expertise Research expertise: congenital talipes equinovarus" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this study - it describes the use of oral GA to support the use of Ponseti casting in those with neglected clubfoot.\n\nThis is a challenging piece to review, because the use of general anaesthetics and potential side effects must be weighed against the benefits of the outcomes. With the information provided it is difficult to undertake a sound ethical analysis. Furthermore, there are study design considerations which are required to strengthen this work.\n\nFirstly, the use of the Pirani score is challenging. While the Pirani score is widely used in the very young population, it is unable to provide the full picture of correction in an older population. The use of dorsiflexion and abduction, the Dimeglio Scale and/or QOL measured with a scale such as the Clubfoot DSI, can provide a clearer picture of the correction/function of the foot of the older child. While I can appreciate the difference in the Pirani score outcomes between the two groups, I am unable to determine whether the foot is functionally significantly better to justify the use of GA.\n\nSecondly, a revision of the study design would increase confidence in results. Specifically:\nIndividuals would benefit from being matched in each group. This may be for severity, age, unilateral/bilateral and gender to allow comparability between groups. Group B had individuals with a Pirani score of 6 in 3/4 combined age groups, while the GA group had this in the >15 yr age group only. In the 11-15 year age group, Gp B had all participants with a Pirani of 4 while the GA group have individuals with a Pirani of 2 and 3.\n\nThe combining of unilateral and bilateral cases as individual feet can violate the assumption of independence. In bilateral cases, right and left feet may be affected by the same forces from the one individual - for example meaning that if one is less prone to correction, the other foot is likely to be the same. This violation can be overcome in a number of ways - statistical advice can support here.\n\nThe follow-up time of one month is unlikely to capture the potential for recurrence. While it may be assumed that a foot with better foot alignment is less likely to recur (or at least may take a longer period of time to recur) - this is challenging to definitively conclude within the follow-up period utilised.\n\nFinally, the ethical argument of utilising GA for each cast change may be more justified in this case than others. Firstly, the use of regular oral GA needs to be weighed against the use of a single potentially more invasive GA for tenotomy. The authors noted that Group B required tenotomies - in centres this may be justified as a lesser risk than regular GA - however, in this setting this may be more justified and should be explored. Secondly, the argument to complete casting in a more timely manner may be justified in some settings. For example, Harnett et al. in 2011 justified the use of an accelerated Ponseti technique to reduce casting time in those from rural and remote settings for those who could not afford to be away from home or work for extended periods. The justification of completing casting faster to outweigh the risk of GA here requires further exploring.\n\nThank you again for the opportunity to review this paper. Further consideration of the outcome measures used, the study design and ethical justification for the use of GA in this setting would greatly improve this 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? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "139316", "date": "27 Jun 2022", "name": "Manon Pigeolet", "expertise": [ "Reviewer Expertise Orthopedic surgery" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for offering me the opportunity to provide feedback on this work. I appreciate the research question a lot, and I think that the practical and technical difficulties that can arise during Ponseti casting in older children is a topic that has not received the attention it deserves in the literature. Casting an older child can be challenging, and whether outcomes are better when casting is done under GA is a valid question to be raised. It is also important to take the potential risks and side effects of GA into consideration.\nHowever, I do have several points of concern that should be addressed before this study can be accepted for publication:\nThe use of Pirani scoring in older children is known to be less valuable. Also, in this context, solely measuring a decrease in Pirani feels like an artificial end-point. There are potentially more useful measurements that can be used to assess the value of using GA for casting: number of cast complications like pressure sores, pain experiences by children, number of casts needed to achieve a functional plantigrade foot or an operable foot in older children, number of casts needed to achieve a plantigrade foot (this data seems to have been collected but not fully addressed in the paper).\n\nTable 1 and the results section are confusing as to how many patients and how many feet were included in the final analysis. The table shows that 16 children included in each group. While at the same time the table included 8 unilateral feet and 4 bilateral feet accounting for 12 children with 16 included feet for group A. So it is unclear to me whether there are 32 children in this study or 32 feet belonging to less than 32 children given that there are certain bilateral cases involved.\n\nHaving an age range of 2-24 years and only 32 feet/children involved constitutes a highly diverse group and it is unclear to me what the value is of drawing any conclusions on this set of cases. It is known that Ponseti treatment becomes less effective as a sole form of treatment in adolescents and young adults. This fact has been partially addressed by breaking down the groups into sub-groups by age. However, this approach creates such small sub-groups that the statistical relevance becomes negligible.\n\nIt is unclear as to why certain children were given GA and others weren't. Was there a specific reason why parents refused? A more detailed description of the decision making process of whether to do casting under GA or not would make the paper stronger.\nIn order for this paper to be acceptable for indexing I recommend the following to the authors:\nI would kindly like to advise the authors to look into other assessment tools than the Pirani score as an outcome measure. Tools like the \"Bangla clubfoot tool\" for example lends itself well for use in an LMIC setting and with a population of older children. This tool assesses whether the foot is functional in daily life, and focuses less on anatomic landmarks and factors.\n\nThe authors should consider expanding the cohort to a larger number before resubmitting the paper. The authors should also consider limiting the age range of children included\n\nIn order to avoid bias from parental preference (or other influencing factors) regarding the usage of GA, a prospective randomized study should be considered by the authors\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "139315", "date": "27 Jun 2022", "name": "Karthick Rangasamy", "expertise": [ "Reviewer Expertise Paediatric Orthopaedics", "club foot" ], "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\nFirst of all, I thank the F1000Research team for asking me to review this article on Neglected CTEV correction under GA by using the Ponseti method.\nAlthough the authors presented their work in a reasonable way, there are many concerns in this paper:\nThe age group of both the groups had a wide range from 2 to > 15 years, with the oldest being 24 years. The severity of the deformity also varies as the age advances and the child walks with a deformed foot. Although the Ponseti method of casting is the most common method of treatment to start the deformity correction even in old age and neglected cases too, I am not able to believe that the correction was achieved in 100% of cases in the GA group by casting alone without the need for any plantar fasciotomy, Achilles tenotomy or tendon lengthening, or minimal posteromedial soft tissue release surgeries in any of the cases in that group.\n\nAssessment by Pirani scoring is good for kids less than one year of age. I may suggest Dimeglio scoring must be used for assessing the deformity before, during, and after the correction on older kids. The final outcome by means of functional assessment by using ICFSG score or Laaveg and Ponseti method may be more satisfying.\n\nAfter the last cast removal, apart from exercises, is any foot orthosis (FAO) used to maintain correction?\n\nThe authors didn't mention any complications or reoccurrences obtained in their cohort. Maybe the follow-up is less, to assess the correct reoccurrence rates we need a long-term follow-up.\n\nThe risks involved in giving weekly general anesthesia to these children are debatable and the effect of these drugs on the brain development of these children needs to be studied.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-361
https://f1000research.com/articles/11-196/v1	16 Feb 22	{ "type": "Case Report", "title": "Case Report: Bocavirus Infection Radiologically Resembling a Congestive Heart Failure in a Patient with Metastatic Castration-Resistant Prostate Cancer Case-Report", "authors": [ "Javier David Benitez Fuentes", "Alicia de Luna Aguilar", "Paloma Flores Navarro", "Alfonso Lopez de Sa Lorenzo", "Carmen Toledano Rojas", "Berta Laguna Fonseca", "Richa Shah", "Clara Frick", "Alejandro Francisco Jimenez Ortega", "Natalia Vidal Cassinello", "Alicia de Luna Aguilar", "Paloma Flores Navarro", "Alfonso Lopez de Sa Lorenzo", "Carmen Toledano Rojas", "Berta Laguna Fonseca", "Richa Shah", "Clara Frick", "Alejandro Francisco Jimenez Ortega", "Natalia Vidal Cassinello" ], "abstract": "Background: Human bocavirus (HBoV) is a viral pathogen from the genus Bocaparvovirus (family Parvoviridae, subfamily Parvovirinae) discovered in 2005. Most of available literature is about HBoV in children and adults with hematological malignancies and in otherwise healthy children with respiratory infections. Information regarding infection in the adult population with solid tumors is scarce. Case Report: We report the case of a 51-year-old male with metastatic castration resistant prostate cancer undergoing chemotherapy treatment who presented with fever, dyspnea, dry cough, and pleuritic pain. Imaging techniques showed signs of congestive heart failure. Symptoms, laboratory tests and echocardiography revealed a more probable infectious etiology. Antibiotic therapy was started. A polymerase chain reaction (PCR) test of nasopharyngeal exudate for respiratory viruses was positive for HBoV. The rest of the microbiological tests were negative. Bronchoalveolar lavage (BAL) was performed. Bacterial culture of BAL was negative while respiratory virus PCR confirmed positivity for HBoV. Antibiotic therapy was discontinued. The patient gradually recovered. Conclusions: Emerging infectious diseases are a notorious threat for immunocompromised populations such as solid tumor patients. This case is unique because to our knowledge this is the first case report article of HBoV in a solid tumor patient and because imaging techniques exhibited signs of congestive heart failure that did not correlate with the rest of the tests. It shows that unusual pathogens should be considered when managing serious clinical complications with uncommon presentations in cancer patients. Notable diagnostic efforts should be made to reach a diagnosis in these cases.", "keywords": [ "Case Report", "Bocavirus", "Respiratory Tract Infection", "Prostate Cancer", "Immunocompromised Host", "Emerging Communicable diseases" ], "content": "Introduction\n\nHuman bocavirus (HBoV) is a single-stranded DNA viral pathogen belonging to the genus Bocaparvovirus (family Parvoviridae, subfamily Parvovirinae) which comprise four genotypes (HBoV1-4).1 It was discovered in 2005 by Tobias Allander and coworkers at the Karolinska University Hospital, Stockholm, Sweden in nasopharyngeal aspirates from children with respiratory tract infection (RTI).2 It can be found in respiratory secretions in high quantities during the acute phase and it can persist at low viral loads for months.3 Besides respiratory samples, HBoV has been detected in faeces, urine, saliva, blood, tonsils, and cerebrospinal fluid.3 Since its discovery this pathogen has gained recognition as a virus with a wide global distribution. It has an estimated global prevalence of about 6%, depending on the region being studied its prevalence ranges from 1 to 56% of respiratory samples and from 1.3 to 63% of stool samples.3\n\nIt is most likely transmitted by air and commonly associated with coinfection with other viruses making it difficult to assert if the main pathogen causing the symptoms is HBoV.4 There is evidence that HBoV1 is associated with respiratory disease especially in children.5 HBoV1 has also been associated with long periods of persistence in the mucosa of the respiratory tract which might play a role in its frequency of co-infections with other well recognized respiratory pathogens. This concurrent detection of other viral respiratory pathogens is high, some studies show a concurrent detection rate of other viral respiratory pathogens in more than 50% of respiratory specimens.4,5\n\nMost of the articles published focus on HBoV infection in the pediatric population and case reports are usually about immunocompromised pediatric patients, especially pediatric hematopoietic cell transplant recipients and hematologic malignancy pediatric patients.5–8 Some studies have shown that HBoV is an uncommon pathogen in adult patients with severe pneumonia.9 In many cases described, in the adult population, in literature HBoV is associated with increased mortality.9 HBoV is closely associated with an immunocompromised state and severe comorbidities such as structural lung disease and hematologic malignancy.9 Most original articles, case reports and case series published in the immunocompromised adult population show hematopoietic cell transplant recipients and hematologic malignancies as comorbidities with few exceptions. There are few articles regarding the adult population with solid tumors. We found only one study describing the prevalence of HBoV in the adult population with solid tumors from Li et al10 done in Wuhan (China) and published in 2011. This study revealed a prevalence percentage of HBoV infection in adult solid tumor patients of 39.74%.10 In another study from Lee et al11 done in Korea and published in 2019, a total of 185 adult subjects that were diagnosed with HBoV infection between January 2010 and December 2017 and were enrolled into the study. From these 185, 28 had solid tumors.11 Their clinical characteristics and risk factors for pneumonia were retrospectively evaluated.\n\nIn this case report we examine the case of a 59-year-old patient with a history of metastatic castration-resistant prostate cancer suffering from HBoV infection. To our knowledge this is the first case report article of a solid tumor patient infected with HBoV.\n\n\nCase report\n\nWe report the case of a 59-year-old Caucasian male barbershop owner, former smoker, diagnosed with achalasia in January 2019 treated with Heller myotomy and Toupet fundoplication on the 6th of May 2021. In November 2019 he complained of bone pain at different anatomic locations and was diagnosed with metastatic prostate cancer and bone only disease.\n\nThe patient was started on androgen deprivation therapy, immunotherapy with ipilimumab and nivolumab as well as chemotherapy with docetaxel in December 2019 as part of a clinical trial (NCT03879122). He initially received two cycles of intravenous ipilimumab 3 milligrams/kilogram (mg/kg) with intravenous nivolumab 3 mg/kg once a day on day one every three weeks for 6 weeks. Ipilimumab was discontinued due to grade three diarrhea, which was treated with a course of high dose oral steroids (equivalent to 2 mg/kg of prednisone) for two weeks until complete recovery. He then received four cycles of intravenous docetaxel 75 milligrams/square meter of body surface area (mg/m2) with intravenous nivolumab (3 milligrams/kilogram) once a day on day one every three weeks for twelve weeks, followed by maintenance with intravenous nivolumab at the same dose and schedule as part of the study protocol. The treatment continued unchanged until November 2020 when it was stopped due to bone and serologic progression. After November 2020 the patient was started on abiraterone (1000 mg once a day) plus 5 milligrams of prednisone twice a day and received radiotherapy over the bone metastases in progression. In May 2021 treatment was changed due to serologic and bone progression to intravenous cabazitaxel (20 mg/m2) once a day every three weeks plus prednisone 5mg twice a day every day. Dexamethasone 4 mg once a day everyday was added at the beginning of June 2021 due to bone pain.\n\nOn the 26th of June 2021, the patient arrived at the emergency department complaining of fever at home, dyspnea, dry cough, and left side pleuritic chest pain for the last seven days. The sequence of events is detailed in the timeline (Figure 1). Oral levofloxacin (500 mg) every 24 hours was started one week before the arrival of the patient to the emergency department with no improvement. His blood pressure was 90/50 mmHg (hypotension defined by 90/60 mmHg or below), temperature was 36.8°C (normal temperature level is between 36.1°C and 37.2°C), heart rate was 95 bpm (normal heart rate for adults 60 to 100 beats per minute), respiratory rate was 24 bpm (normal respiratory rate for adults 12 to 16 breaths per minute), peripheral oxygen saturation was 88% (normal level equal or more than 95%) with no supplemental oxygen.\n\nPhysical exam was pertinent with generalized decreased breath sounds with no signs of peripheral edema.\n\nLaboratory tests with complete blood count, coagulation panel, comprehensive metabolic panel, liver function tests, renal function tests and acute phase reactants revealed hemoglobin level of 8 grams/deciliter (normal level for males, 13.5 to 17.5 grams/decilitre), C-Reactive Protein (CRP) of 47.9 milligrams/liter (normal level less than 10 milligrams/liter), procalcitonin of 0.14 nanograms/milliliter (normal level less than 0.1 nanograms/milliliter), sodium of 127 milliequivalents/liter (between 135 and 145 milliequivalents/liter), lactate of 0.8 millimoles/liter (Normal lactate range is less than 2.3 millimoles/liter), the rest of the complete blood count, coagulation panel, comprehensive metabolic panel, liver function tests and renal function tests were normal (Figure 2). Arterial blood gases showed a partial pressure of oxygen of 57 mmHg and a partial pressure of carbon dioxide of 27 mmHg.\n\nN-terminal pro-brain natriuretic peptide (NT-proBNP), C-Reactive Protein (CRP).\n\nCardiac enzymes and N-terminal pro-brain natriuretic peptide (NT-proBNP) were negative. An electrocardiogram showed no remarkable alterations. A chest radiograph (Figure 3) exhibited an enlarged cardiac silhouette. A computed tomography angiogram of the chest was performed to confirm or rule out the possibility of a pulmonary embolism. It showed no signs of pulmonary embolism but confirmed an enlarged cardiac silhouette and exhibited a mild bilateral pleural effusion. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR), influenza PCR and Streptococcus pneumoniae and Legionella pneumophila urinary antigen tests all came back negative. Finally, blood, urine and sputum samples were collected for culture.\n\nBased on the respiratory and heart rate, hypotension, CRP elevation and fever, a serious infection could not be ruled out. Having a negative NT-proBNP did not point to a congestive failure despite imaging techniques.12,13 Wide spectrum antibiotics with intravenous meropenem (2 grams/8 hours), oral linezolid (600 milligrams/12 hours) and intravenous trimethoprim/sulfamethoxazole (320/1600 milligrams/6 hours) as well as intravenous fluid therapy, and symptomatic treatment were started following hospital protocols. Oral dexamethasone previously prescribed for bone pain was increased to 4 mg twice a day as part of the treatment of acute respiratory insufficiency. The patient was transferred to the medical oncology inpatient ward.\n\nDuring his stay an echocardiogram was performed revealing a normal size left ventricle, with no segmentary alterations and a left ventricular ejection fraction within normal values.\n\nBlood, urine, and sputum cultures were negative for bacterial and fungal pathogens. The medical team then requested a respiratory viruses PCR in nasopharyngeal exudate that displayed positivity for HBoV. The other viruses tested for (Influenza A, Influenza B, Influenza C, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, Enterovirus B, Rhinovirus, Coronavirus 229, Adenovirus, Metapneumovirus A, Metapneumovirus B, Respiratory syncytial virus A, Respiratory syncytial virus B) were negative. Based on the common concurrent infection rates with other pathogens bronchoscopy was performed and bronchoalveolar lavage (BAL) was done to collect samples. Culture and a respiratory virus PCR were performed in BAL. The culture was negative for bacteria and fungi. The PCR was again, positive for HBoV. Antibiotic therapy was discontinued based on these results and progressive clinical and analytical improvement was shown with gradual decrease in acute phase reactants.\n\nThe patient gradually recovered from the dyspnea, chest pain and dry cough having no more episodes of fever during hospitalization. A second chest radiograph (Figure 4) before discharge shows a normal size cardiac silhouette while maintaining the mild bilateral pleural effusion. After eight days from hospital admission the patient was discharged home. He was re-evaluated two weeks after discharge in medical oncology outpatient clinic reporting no further complications with normal laboratory tests with complete blood count, comprehensive metabolic panel, and acute phase reactants.\n\n\nPatient perspective\n\nI usually never complain but this time the pain and lack of breath were unbearable I could not keep waiting for these symptoms to go away. The first days I spent at the hospital I had lots of diagnostics tests done before the medical team could reach a diagnosis but at least they were done fast. During the time at the hospital started to feel better slowly but in the end, I could completely recover.\n\n\nDiscussion\n\nWe report an infection with an emerging pathogen in a solid tumor patient. The case is rare as it presented contradictory diagnostic test results with an uncommon radiological image.\n\nEmerging viral pathogens represent a growing threat for all people but especially for immunocompromised populations. Ultimately, we have seen an extremely prevalent example of this problem with the SARS-CoV-2. The coronavirus disease 2019 (COVID-19) pandemic takes a bigger mortality and morbidity toll when affecting cancer patients.14\n\nHBoV was first described in 2005,2 however we do not have much information about its prevalence in patients suffering from solid neoplasms. With the one exception being the study from Li et al10 that shows an almost 40% (62/156) HBoV prevalence among solid tumor patients compared to 3.51% (33/941) in children with respiratory tract infections. However, this study only reflects the population from Wuhan, and it does not have any clinical information regarding the patients enrolled. Meaning this data is likely to be different globally.\n\nIn a study by Lee et al11 185 patients infected with HBoV were enrolled. From the 185 patients, 76 (41.08%) were immunocompromised. From the immunocompromised patients 28 (36.84%) suffered solid malignancies treated with chemotherapy within 6 months of HBoV diagnosis, 11 (14.47%) had hematologic malignancies, 19 (25%) had solid organ transplantations, and 18 (23.68%) received hematopoietic cell transplantation. Of the 185, 110 had pneumonia. CT findings were analyzed in 34 of the 185 patients, from which 16 were immunocompromised. There was no significant difference in CT patterns between immunocompetent and immunocompromised patients and the most frequent findings in both groups were bilateral consolidation and/or ground glass opacities. Pleural effusion was observed in 50.0% patients.\n\nIn our case report the patient presented with laboratory tests that pointed to an acute infectious process with a potential respiratory origin, however imaging techniques showed conflicting data revealing an enlargement of the cardiac silhouette indicating a possible congestive heart failure. Vital signs, physical examination, a normal echocardiogram, clinical judgment, and the clinical picture all together tipped the balance towards an infectious diagnosis.\n\nAfter the negative results in the sputum, urine and blood culture, a PCR in nasopharyngeal exudate was performed, presenting a positive result for HBoV. Thus, revealing the probable cause of the patient's current pathologic process. Based on the fact that it is very common to have coinfections with other viruses HBoV had to be confirmed in BAL which revealed no other pathogen.5 This finding was concordant with the bilateral pleural effusion based on available published articles11 but not with the cardiac silhouette enlargement. After clinical improvement was noted a new chest radiography was performed that did not display an enlarged cardiac silhouette.\n\nTo our knowledge this is the first case report article of a solid tumor patient infected with HBoV. The discordant imaging and laboratory tests show the need for a clinical judgement that considers all aspects of the clinical picture and reinforces the necessity of further diagnostic efforts when diagnostic test results are conflicting. This is especially true in immunocompromised populations at risk of serious complications in case of a delayed diagnosis and treatment.\n\n\nData availability\n\nAll data associated with this article are available in the paper and no additional source data is required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details, clinical images, and views on the treatment was obtained from the patient.", "appendix": "References\n\nLüsebrink J, Wittleben F, Schildgen V, et al.: Human bocavirus - insights into a newly identified respiratory virus. Viruses. 2009 Jun; 1(1): 3–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllander T, Tammi MT, Eriksson M, et al.: Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc. Natl. Acad. Sci. U. S. A. 2005 Sep 6; 102(36): 12891–12896. Epub 2005 Aug 23. Erratum in: Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15712. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPolo D, Lema A, Gándara E, et al.: Prevalence of human bocavirus infections in Europe. A systematic review and meta-analysis. Transbound. Emerg. Dis. 2021 Jul 12. Epub ahead of print. PubMed Abstract \| Publisher Full Text\n\nJiang W, Yin F, Zhou W, et al.: Clinical significance of different virus load of human bocavirus in patients with lower respiratory tract infection. Sci. Rep. 2016 Feb; 6(6): 20246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChristensen A, Nordbø SA, Krokstad S, et al.: Human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection. J. Clin. Virol. 2010 Nov; 49(3): 158–162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan SB, Shin JA, Kim SK, et al.: Respiratory Viral Infections in Children and Adolescents with Hematological Malignancies. Mediterr J. Hematol. Infect. Dis. 2019 Jan 1; 11(1): e2019006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPochon C, Voigt S: Respiratory Virus Infections in Hematopoietic Cell Transplant Recipients. Front. Microbiol. 2019 Jan; 9(9): 3294. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchenk T, Strahm B, Kontny U, et al.: Disseminated bocavirus infection after stem cell transplant. Emerg. Infect. Dis. 2007 Sep; 13(9): 1425–1427. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi SH, Huh JW, Hong SB, et al.: Severe Human Bocavirus-Associated Pneumonia in Adults at a Referral Hospital, Seoul, South Korea. Emerg. Infect. Dis. 2021 Jan; 27(1): 226–228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Dong Y, Jiang J, et al.: High prevelance of human parvovirus infection in patients with malignant tumors. Oncol. Lett. 2012 Mar; 3(3): 635–640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee HN, Koo HJ, Kim SH, et al.: Human Bocavirus Infection in Adults: Clinical Features and Radiological Findings. Korean J. Radiol. 2019 Jul; 20(7): 1226–1235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSinger M, Deutschman CS, Seymour CW, et al.: The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016 Feb 23; 315(8): 801–810. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPonikowski P, Voors AA, Anker SD, et al.: 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur. J. Heart Fail. 2016 Aug; 18(8): 891–975. PubMed Abstract \| Publisher Full Text\n\nYarza R, Bover M, Paredes D, et al.: SARS-CoV-2 infection in cancer patients undergoing active treatment: analysis of clinical features and predictive factors for severe respiratory failure and death. Eur. J. Cancer. 2020 Aug; 135: 242–250. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "125565", "date": "09 Mar 2022", "name": "Macarena Torrego-Ellacuría", "expertise": [ "Reviewer Expertise Pharmacy", "clinical 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\nInteresting, detailed, and justified article. Some minor suggestions to improve:\nIntroduction, third paragraph, line 1: change \"articles published\" for \" published articles\" (structure adjective + noun).\n\nIntroduction, third paragraph, penultimate line. Replace \"From these 185\" with \"Of these 185, 28 (15,13%) had solid tumours\".\n\nUse the International System of Units (SI) symbols for the expression of all dose or concentrations related to biological samples, always leaving a space between the figure and the symbol used (pages 3,4,5, and figure 2). For the pharmacological treatment regimen (e.g. 2 grams every 8 hours or 600 milligrams every 8 hours) the current expression can be retained, without abbreviations.\n\nFigure 1: include figure legend with abbreviations used in the figure.\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": "7982", "date": "29 Mar 2022", "name": "Javier David Benitez Fuentes", "role": "Author Response", "response": "From your suggestions, I have followed points 1, 2, 4. Point 3 was done this way based on the comments from the editors of the journal so I will keep them that way. Thank you" } ] }, { "id": "123948", "date": "17 Mar 2022", "name": "Marcos Roberto Tovani-Palone", "expertise": [ "Reviewer Expertise Pathology", "General medicine", "Dentistry", "Global and Public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article that describes and discusses the case of a 59-year-old patient with a history of metastatic castration-resistant prostate cancer suffering from HBoV infection.\nThe article is well structured, with a complete description of the case and relevant discussion. Furthermore, the references used are up to date and appropriate and the figures are very illustrative.\nHowever, revisions are required to refine the article. Authors should reread the article in an attempt to review it for grammatical mistakes and writing style.\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": "7985", "date": "29 Mar 2022", "name": "Javier David Benitez Fuentes", "role": "Author Response", "response": "Article has been reread by various author correcting minor mistakes and improving writing style. We have also added one image from the computed tomography angiogram of the chest to increase the visual aspect of the case." } ] } ]	1	https://f1000research.com/articles/11-196
https://f1000research.com/articles/10-851/v1	25 Aug 21	{ "type": "Brief Report", "title": "An outbreak of Salmonella Enteritidis food poisoning following consumption of chicken shawarma: A brief epidemiological investigation", "authors": [ "Surendran Deepanjali", "Mandal Jharna", "Bammigatti Chanaveerappa", "Dhandapani Sarumathi", "Pallam Gopichand", "Kaliyappan Anupriya", "Mandal Jharna", "Bammigatti Chanaveerappa", "Dhandapani Sarumathi", "Pallam Gopichand", "Kaliyappan Anupriya" ], "abstract": "Background: Shawarma, a popular meat-based fast food could be a source of foodborne outbreak due to non-typhoidal Salmonella . A clustering of acute gastrointestinal (GI) illness following intake of chicken shawarma occurred primarily among the staff and students of a tertiary care hospital in southern India. Methods: A case-control study was conducted among 348 undergraduate medical students (33 cases, 315 controls). Data was collected using direct interviews and a simple online questionnaire. Epidemiological associations of GI illness were evaluated at three levels of exposure namely - eating food from any restaurant, eating food from the implicated food outlet, eating chicken shawarma from the implicated outlet. Results: Of 33 cases, 26 had consumed food from a particular food outlet, 4 from other outlets, and 3 did not report eating out. Consumption of food from the suspected food outlet was significantly associated with GI illness (odds ratio 121.8 [95% CI 28.4 to 522.7]; P<0.001); all the 26 cases who had eaten from the particular outlet had eaten chicken shawarma. In comparison, only one of the 315 controls had eaten this dish. Of the 27 persons (cases as well as controls) who had consumed chicken shawarma from the outlet, 26 fell ill. Culture of stool samples from 10 affected individuals and implicated food item yielded Salmonella Enteritidis. Conclusions: Thus, it can be concluded that meat-based shawarma is a potential source of NTS infection.", "keywords": [ "Salmonella Enteritidis", "foodborne disease outbreaks", "gastroenteritis" ], "content": "Introduction\n\nShawarma is a meat-based dish of Middle Eastern origin.1 It has become a popular food item across many countries including India. Nontyphoidal Salmonellae (NTS) are known to contaminate meat and poultry products resulting in foodborne disease outbreaks.2 There have been a few recent reports of foodborne disease outbreaks related to NTS contamination of chicken shawarma.3–5 A few countries have issued guidelines for safe preparation and serving of shawarma,6,7 but such guidelines do not exist in many developing countries. Further, foodborne disease outbreaks are often under-reported, and the necessary epidemiological investigations are not always carried out.8,9 Here, we report an epidemiological investigation of a foodborne disease outbreak caused by consumption of chicken shawarma, which mainly affected the students and staff of a teaching hospital.\n\n\nMethods\n\nThis brief outbreak investigation was carried out during the months of July and August 2019 at the Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India. Since the data was collected as part of an outbreak investigation which was a mandatory public health exercise, an exemption from review was granted by the Institute’s Ethics Committee. The participating subjects were aware that their data was being collected as part of a foodborne disease outbreak investigation which was a mandatory public health exercise. However, since the decision to publish the findings was taken many months after the outbreak investigation, an informed consent for publication was not explicitly taken.\n\nThe index case was a postgraduate resident who presented to the emergency department at 4 AM on July 22nd, 2019 with abdominal pain and multiple episodes of diarrhea which started about 7.5 hours after consuming biryani (a mixed rice dish) and chicken shawarma (a Middle Eastern dish made of thinly sliced cuts of meat marinated and cooked after stacking in a vertical skewer) from a food joint near the hospital. The index case developed high grade fever and multiple episodes of vomiting after hospital admission. Subsequently over the next 2 days, 19 more cases were admitted with similar illness, of whom 16 were either students or staff of the hospital. Three of the admitted cases reported about 6 other cases with similar illness treated at other health facilities, thus making the total number hospitalized cases to 26. All 26 cases reported consuming chicken shawarma from the same outlet.\n\nPatients who were admitted in Department of Medicine with the history of acute onset fever and gastrointestinal symptoms such as diarrhea and vomiting after consumption of chicken shawarma from the suspected food outlet on the dates July 22nd -July 24th were categorized as cases. Clinical history was collected through direct interview from individuals who were still hospitalized when data collection started and through telephonic conversation from individuals who could not be directly interviewed. The clinical history of those who were treated in pediatrics department of our institution for similar gastrointestinal complaints and also a few individuals treated elsewhere were obtained from patients who were admitted in Department of Medicine and who happened to share the chicken shawarma meal.\n\nStool samples of 14 hospitalized individuals could be submitted for the microscopic examination. Primary culture of stool samples as well as a specimen taken from shawarma which was obtained from the restaurant on the same day were carried out. Cultures were done in MacConkey, XLD, DCA, TCBS with selenite F enrichment and alkaline peptone water. Identification of the bacterial colonies were done using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS version 3.2, VITEK MS, Biomerieux). Stool polymerase chain reaction (PCR) was done using Eppendorf AG HAMBURG 22331 for identifying diarrheagenic E. coli and Campylobacter spp.\n\nWe conducted a case-control study for confirming the source of contaminated food. Since undergraduate medical students belonging to the third to ninth semesters constituted a major proportion of affected individuals, we considered them representative of the population at risk. We collected information from them by direct interview (202 individuals) which was carried out by meeting the third to ninth semester students when they assembled for scheduled theory classes. Those who could not be directly interviewed (146 individuals) were requested to fill up a web-based (Google forms) questionnaire which was circulated through the respective classes’ social media groups. The data was collected over the time period from July 25th to August 2nd, 2019. We asked three questions: ‘Where did you have your dinner on July 21?’ (the day prior to presentation of index case); ‘What did you eat?’; and ‘Did you have any gastrointestinal (GI) symptoms in the form of abdominal pain, vomiting or diarrhea during the index dates from July 22 to 24?’.\n\nWe defined cases as those who presented with GI symptoms on dates July 22nd -24th irrespective of need for hospital admission. Controls were those who reported no GI symptoms. We compared the odds illness at 3 levels of exposure among cases and controls at 3 levels. At each level the odds ratio was calculated as ratio of odds of illness in the exposed group to the odds of illness in the non-exposed group. Level 1 was eating at any place other than their hostel or home on July 21st; Level 2 was consumption of any food item from the suspected food outlet on July 21st among those who ate outside; and Level 3 was consumption of chicken shawarma among those who had dined at the suspected outlet. To assess the causality of the observed epidemiological association, we applied the Bradford Hill’s criteria adopted for foodborne disease outbreaks.10\n\n\nResults\n\nOf the 26 individuals who sought medical attention in our hospital and elsewhere, 17 were male and 9 were female. Their median age was 22 (18–25) years. The median (IQR) incubation period of symptom onset was 9.5 (8–12) hours.\n\nApart from the index case and his co-diner who had taken biriyani along with chicken shawarma from the implicated restaurant, the other 24 people had consumed only chicken shawarma. All cases had greenish loose watery diarrhea. Of 26 cases, 23(88.5%) had high grade fever and vomiting and 25(96.1%) had abdominal pain. Of the 20 cases admitted at our center, 3 required intensive care unit admission because of severe dehydration. All admitted patients recovered completely and were discharged home.\n\nMicroscopic examination of the stool samples was done for 14 affected individuals. In 13 individuals it revealed pus cells without any ova or cysts. In 10 patients, the stool culture revealed black colonies, which were identified as subsp. enterica serovar Enteritidis. Salmonella Enteritidis was also isolated from the shawarma sample. Stool PCR was negative in all 14 cases.\n\nThe case-control study involving undergraduate students identified 7 more cases of GI illness (not requiring hospitalization), thus taking the total number of cases to 33. Among the 33 cases, 26 had consumed food from the particular food outlet, 4 had consumed food from other outlets, and 3 did not report eating out (Table 1). Consumption of food from the implicated outlet was significantly associated with GI illness (odds ratio 121.8 [95% CI 28.4 to 522.7]; P < 0.001); 26 of 27 persons who had consumed chicken shawarma from that outlet developed GI illness. Applying the Bradford Hill’s criteria, the observed association was deemed to be causally linked; only the criterion of biological gradient was not fulfilled (Table 2).\n\n\nDiscussion\n\nWe found that the outbreak of gastroenteritis caused by Salmonella Enteritidis was epidemiologically linked to the consumption of contaminated chicken shawarma from a particular food outlet. Gastroenteritis outbreaks caused by NTS have been previously reported from India and other countries.11–13 Poultry meat contamination by NTS is also reported.14 Importantly, a study from Jordan found high rates of contamination of chicken meat used in shawarma by Salmonella spp.15 Previously, an NTS (Salmonella Thompson) outbreak caused by consumption of chicken shawarma was reported from Canada.3 Microbial contamination of shawarma can occur during the storage, cooking and serving of the meat. Generally, NTS does not survive high temperatures when the cooking process is adequate. However, the important step of secondary cooking of cut slices of meat might be overlooked when the food outlet becomes busy. It is important that food safety authorities enforce guidelines for safe preparation and sale of shawarmas and similar products.\n\nTwo important steps helped in quick containment of the outbreak in our setting: early identification of the contaminated food source and timely intimation of food safety authorities for prohibitory action. Also, since the contaminated food sample was procured while it was still on sale, we could isolate NTS from the source. Moreover, we demonstrated the epidemiological link by performing a case-control study.\n\nOne possible limitation of our investigation was that we could not obtain specimens for microbiological testing from the food handlers and the water used for cooking could also not be tested. Notwithstanding, our report helps to highlight shawarma as a potential source of food poisoning.\n\n\nConclusion\n\nIn conclusion, chicken shawarma is a potential source of food poisoning due to NTS. Epidemiological investigation of foodborne outbreaks could yield important information.\n\n\nData availability\n\nFigshare: Foodborne disease outbreak version 2. https://doi.org/10.6084/m9.figshare.15022065.v2.16\n\nThis project contains the following underlying data:\n\nData file 1. Deepanjali salmonella data (1).xlsx (Foodborne disease outbreak version 2)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nConsent\n\nThe data represented in the manuscript was collected as part of an outbreak investigation. An exemption from review was granted by Institute Ethics Committee.", "appendix": "Acknowledgments\n\nWe thank Prof. Rakesh Aggarwal, Director, JIPMER for encouraging to take up the outbreak investigation and giving critical inputs on the manuscript. We acknowledge the undergraduate medical students for their participation.\n\n\nReferences\n\nWikipedia contributors, “Shawarma,” Wikipedia, The Free Encyclopedia. (Accessed July 16, 2021).Reference Source\n\nHohmann EL: Nontyphoidal salmonellosis. Clin Infect Dis. 2001; 32: 263–269. PubMed Abstract \| Publisher Full Text\n\nGaulin C, Fiset M, Duchesne C, et al.: Salmonella Thompson outbreak associated with consumption of chicken shawarma and the usefulness of genome sequencing in the investigation. Can Commun Dis Rep. 2017; 43: 186–192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaigul AA: Salmonella poisoning and chicken shawarmas, western Riyadh, june 1997. Saudi Epidemiol. Bull. 1997; 4: 18–19.\n\nBremnar J: Mass Food Poisoning as Child Dies, 800 Hospitalized After Eating at Shawarma Restaurant [Internet]. Michigan poison and Drug Information Center. 2020 [cited 16 July 2021]. Reference Source\n\nGuidelines for Safe Preparation of Shawarmas or Similar Meat Products. Eastern Ontario Health Unit. 2021 [cited 16 July 2021]. Reference Source\n\nFood Protection Services: Donairs, Shawarmas and Similar Products [internet].2012 [cited 16 July 2021]. Reference Source\n\nWorld Health Organization: Foodborne disease outbreaks: guidelines for investigation and control. World Health Organization; 2008. (Accessed July 16, 2021).Reference Source\n\nArendt S, Rajagopal L, Strohbehn C, et al.: Reporting of foodborne illness by U.S. consumers and healthcare professionals. Int J Environ Res Public Health. 2013; 10: 3684–3714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLauchlin J, Little C, Nichols G, et al.: Gillespie I (2007) Epidemiology. In: McLauchlin J, Little C eds. Hobb’s Food Poisoning and Food Hygiene. 7th ed.UK: Hodder Arnold; 2007. p. 114–143.\n\nHuusko S, Pihlajasaari A, Salmenlinna S, et al.: Outbreak of Salmonella enteritidis phage type 1B associated with frozen pre-cooked chicken cubes, Finland 2012. Epidemiol Infect. 2017; 145: 2727–2734. PubMed Abstract \| Publisher Full Text\n\nHobbs JL, Warshawsky B, Maki A, et al.: Nuggets of Wisdom: Salmonella Enteritidis Outbreaks and the Case for New Rules on Uncooked Frozen Processed Chicken. J Food Prot. 2017; 80: 703–709. PubMed Abstract \| Publisher Full Text\n\nDikid T, Hpalya SS, Thakur JS, et al.: Salmonella food poisoning outbreak in Kharar town of Punjab. Indian J Public Health. 2009; 53: 265. PubMed Abstract\n\nSaravanan S, Purushothaman V, Murthy TR, et al.: Molecular Epidemiology of Nontyphoidal Salmonella in Poultry and Poultry Products in India: Implications for Human Health. Indian J Microbiol. 2015; 55: 319–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNimri L, Abu Al-Dahab F, Batchoun R: Foodborne bacterial pathogens recovered from contaminated shawarma meat in northern Jordan. J Infect Dev Ctries. 2014; 8: 1407–1414. PubMed Abstract \| Publisher Full Text\n\nDeepanjali S, Jharna M, Bammigatti C, et al.: Foodborne disease outbreak version 2. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "96979", "date": "04 Nov 2021", "name": "Cibin Veronica", "expertise": [ "Reviewer Expertise Collocation and management of data on zoonoses and zoonotic agents with focus on salmonella", "epidemiology od zoonotic agents", "food safety", "isolation/characterization of zoonotic agents" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper describes a food borne outbreak occurred in India focusing the attention on the epidemiological investigation that was carried out during the two months after the episode.\nThe paper is valuable since it contributes to provide data on potential sources of food borne diseases; it was in fact possible to identify the source of infection, the zoonotic agent and the setting. Moreover the authors provide a good selection of the current literature. The language is simple and immediate, but the way the contents are organized is chaotic and this makes the text not fluent or fully understandable. For example, in the paragraph “methods”, some results are described, while in the section “results”, in the paragraph “Stool examination”, the microscopic examination of samples is reported and this refers to a method.\nIt seems the authors did a very good “in the field” job but the way they describe it is not clear enough. The main issue is that it is not clear whether the outbreak investigation was performed as a learning tool (an exercise?) or if it was needed to identify the source of infection and thus to implement the sanitary measures to avoid additional cases.\nIn the section “stool examination” it seems that a sample of shawarma was analysed (but this matrix is not congruent with the “title”) as well and resulted to be contaminated with Salmonella Enteritidis but it is not clear when this analysis was performed. This result, with the information obtained from the hospitalized cases, was sufficient to confirm the source of infection; further investigations were probably not needed.\nAdditionally, the flow of the diagnostic approach (section “confirmation of cases for NTS infection”) is not clear. What was done as first analysis? With which purpose? Is there a sort of protocol to be followed in order to exclude step-by-step the potential hazards? As far as the serotyping, it is not clear which was the method performed.\nBelow some other suggestions:\nAbstract: “NTE” is used for the first time, please explain this abbreviation.\n\nTable 2 ”Coherence”: it has to be clarified the link between the question asked and the applicability of the criterion.\n\nCase control study: we suggest here to use the original reference when you cite the Bradford hill guidelines.\n\nConclusion: it is quite poor and generic; authors could add information on possible strategies in order to avoid similar outbreaks in the future. For example, the consideration “it is important that food…similar products.” of the discussion could be moved here.\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? 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": [ { "c_id": "7511", "date": "06 Dec 2021", "name": "Surendran Deepanjali", "role": "Author Response", "response": "We thank the reviewers for their kind appraisal of our manuscript and their constructive comments. Please find below a point-by-point response to the comments. We hope that reviewers find the changes satisfactory. We will be happy to respond to any further comments or suggestions by the reviewers. Comment: The paper describes a food borne outbreak occurred in India focusing the attention on the epidemiological investigation that was carried out during the two months after the episode. The paper is valuable since it contributes to provide data on potential sources of food borne diseases; it was in fact possible to identify the source of infection, the zoonotic agent and the setting. Moreover, the authors provide a good selection of the current literature. Response: We thank the reviewers for their encouraging words. Comment: The language is simple and immediate, but the way the contents are organized is chaotic and this makes the text not fluent or fully understandable. For example, in the paragraph “methods”, some results are described, while in the section “results”, in the paragraph “Stool examination”, the microscopic examination of samples is reported and this refers to a method. Response: We have now moved all the methodological aspects to the Methods section. The findings through clinical, microbiological and the epidemiological study have been moved completely to the Results section. We have also renamed the section “confirmation of cases of NTS infection” as “Microbiological investigations” for better clarity. Comment: It seems the authors did a very good “in the field” job but the way they describe it is not clear enough. The main issue is that it is not clear whether the outbreak investigation was performed as a learning tool (an exercise?) or if it was needed to identify the source of infection and thus to implement the sanitary measures to avoid additional cases. Response: To avoid this ambiguity we have now added further clarification in the Settings section - “The data presented in the study was collected as part of an outbreak investigation which was a public health exercise to identify the source of infection and taking steps to prevent further infections.” Comment: In the section “stool examination” it seems that a sample of shawarma was analysed (but this matrix is not congruent with the “title”) as well and resulted to be contaminated with Salmonella Enteritidis but it is not clear when this analysis was performed. This result, with the information obtained from the hospitalized cases, was sufficient to confirm the source of infection; further investigations were probably not needed. Response: The section ‘stool examination” is renamed as “Isolation of NTS”. The analysis of shawarma sample was done on the same day when the outbreak was identified and this is now made clear in the section “Microbiological investigations”. Comment: Additionally, the flow of the diagnostic approach (section “confirmation of cases for NTS infection”) is not clear. What was done as first analysis? With which purpose? Is there a sort of protocol to be followed in order to exclude step-by-step the potential hazards? As far as the serotyping, it is not clear which was the method performed. The first step was the identification of a possible foodborne disease outbreak which was suspected when many cases with gastrointestinal illness was admitted after consumption of the same food item from a single outlet. An investigation to confirm the contaminated source was done as part of the public health exercise. The subsequent case control study was undertaken to ascertain that the clinically observed association is indeed true. Although no pre-specified protocol was followed, the case-control study was envisaged by the Head of our institution to make sure that the observed association of the illness with shawarma consumption was a scientifically valid one. The method used for serotyping is now made clear in the section “Microbiological investigations”. Comment: Abstract: “NTE” is used for the first time, please explain this abbreviation. Response: We thank the reviewers for pointing this out. The suggested change has been made. Comment: Table 2 ”Coherence”: it has to be clarified the link between the question asked and the applicability of the criterion. Response: We have revised the explanation provided for this criterion in Table2. Comment: Case control study: we suggest here to use the original reference when you cite the Bradford hill guidelines. Response: The original reference has been added now. Comment: Conclusion: it is quite poor and generic; authors could add information on possible strategies in order to avoid similar outbreaks in the future. For example, the consideration “it is important that food…similar products.” of the discussion could be moved here. Response: As suggested by the reviewers, we have moved the point about need for food safety guidelines to the Conclusions section." } ] } ]	1	https://f1000research.com/articles/10-851
https://f1000research.com/articles/10-327/v1	27 Apr 21	{ "type": "Study Protocol", "title": "A public health intervention package for increasing tuberculosis notifications from private practitioners in Bandung, Indonesia (INSTEP2): A cluster-randomised controlled trial protocol", "authors": [ "Panji Fortuna Hadisoemarto", "Bony Wiem Lestari", "Katrina Sharples", "Nur Afifah", "Lidya Chaidir", "Chuan-Chin Huang", "Susan McAllister", "Reinout van Crevel", "Megan Murray", "Bachti Alisjahbana", "Philip C Hill", "Panji Fortuna Hadisoemarto", "Katrina Sharples", "Nur Afifah", "Lidya Chaidir", "Chuan-Chin Huang", "Susan McAllister", "Reinout van Crevel", "Megan Murray", "Bachti Alisjahbana", "Philip C Hill" ], "abstract": "Background. A significant proportion of tuberculosis (TB) patients globally make their initial visit for medical care to either an informal provider or a private practitioner, and many are not formally notified. Involvement of private practitioners (PPs) in a public–private mix for TB (TB-PPM) provides an opportunity for improving TB control. However, context-specific interventions beyond public–private agreements and mandatory notification are needed. In this study we will evaluate whether a tailored intervention package can increase TB notifications from PPs in Indonesia. Methods. This is a cluster-randomized trial of a multi-component public health intervention. 36 community health centre (CHC) areas will be selected as study locations and randomly allocated to intervention and control arms (1:1). PPs in the intervention areas will be identified using a mapping exercise and recruited into the study if they are eligible and consent. They will receive a tailored intervention package including in-person education about TB management along with bimonthly electronic refreshers, context-specific selection of referral pathways, and access to a TB-reporting app developed in collaboration with the National TB programme. The primary hypothesis is that the intervention package will increase the TB notification rate. The primary outcome will be measured by collecting notification data from the CHCs in intervention and control arms at the end of a 1-year observation period and comparing with the 1-year pre-intervention. The primary analysis will be intention-to-treat at the cluster level, using a generalised mixed model with repeated measures of TB notifications for 1 year pre- and 1 year post-intervention. Discussion. The results from this study will provide evidence on whether a tailored intervention package is effective in increasing the number of TB notifications, and whether the PPs refer presumptive TB cases correctly. The study results will guide policy in the development of TB-PPM in Indonesia and similar settings.", "keywords": [ "tuberculosis", "private practitioner", "notification", "protocol" ], "content": "Introduction\n\nTuberculosis (TB) is the leading infectious disease cause of global mortality. In 2018, an estimated 10 million people had TB and, including those living with HIV, 1.4 million died from the illness. However, approximately 3 million new TB cases are estimated to not be notified to health authorities, 2 million of which reside in the 20 high-burden countries that contribute 70% of the total global TB incidence.1 Case notification plays an important role in disease surveillance by identifying cases as potential sources of new infections and their contacts, measuring the disease burden, and prompting the implementation of timely curative treatment. Achieving the End TB target of TB elimination by the year 2030 relies on the completeness of case notifications.\n\nIn Asian countries with high TB incidence, between 70–85% of TB patients first seek care in the private sector.2 Among the different types of providers in the private sector, medically trained general practitioners or specialists who work either in solo practice or private clinics (private practitioners (PPs) play an important role because of their proximity to the community. An analysis of TB patient pathways in five low-middle income countries (LMICs) demonstrated that, on average, 26% of TB patients first sought care at primary care clinics, of whom only 13% of patients were tested with appropriate TB diagnostics, and only 7% were notified to the National TB Programme (NTP).3 PPs’ knowledge about correct TB management is highly variable and provision of substandard TB treatment is not uncommon.4 Hence, involving PPs in a TB public–private mix (TB-PPM) strategy has been recognized as an opportunity to improve TB control. It is challenging for NTPs to address these issues due to their scale, relatively unregulated PP practice, and limited resources.5\n\nThere were an estimated 845,000 incident cases of TB in Indonesia in 2018, the third highest after India and China.1 Approximately 20% of these are seen and treated in private, primary care health facilities.6 However, according to Indonesia’s TB inventory study, 41% of the incident TB cases were not notified to the NTP. Among health facilities, PPs had the lowest reporting rate (4%) followed by public/private hospitals (38%), both of which were lower than publicly funded Community Health Centres (CHC) (Pusat Kesehatan Masyarakat/Puskesmas) (85%).7\n\nAlthough TB-PPM has been recognized as an important component in Indonesia’s TB control programme, engagement of the private sector by donor-funded district-based TB-PPM pilot projects has been mostly limited to hospitals and specialists.2 As a part of advancing TB-PPM, the government made TB notification mandatory in 2016, but evidence of impact and compliance is lacking.8 In general, the benefits of PPM initiatives have yet to be fully realized across the country.9\n\nInterventions aimed at increasing PPs engagement need to be based on a sound understanding of contextual factors relevant to PPs and how PPs relate to the public sector with respect to the diagnosis, treatment and reporting of TB cases.10 We have previously conducted a study to describe and understand healthcare pathways of patients seeking treatment for TB and the quality of TB case management by PPs in the city of Bandung.11 In 30 CHC catchment areas, we identified 1200 PPs, of whom 245 had diagnosed at least one TB case in the previous 3 months. Among the 870 TB cases diagnosed by these PPs, fewer than 20% were notified. In an earlier feasibility study,12 we showed that 40% of participating PPs had inadequate knowledge of TB symptoms and/or signs. A digital application designed to assist with referral and notification was well received, utilized and showed potential to increase TB notifications, although not all PPs had phones that supported it. In this cluster-randomised trial, we will evaluate a further-refined tailored intervention package to increase notifications of TB by PPs in Bandung, Indonesia.\n\nWe will evaluate, first, whether a tailored intervention package increases notifications of TB from PPs in Bandung, Indonesia by calculating the difference in the change in number of TB notifications after a 1-year intervention between intervention and control arms, and second, to measure the proportion of referrals from PPs to CHCs in the study arms that are actually diagnosed with TB.\n\n\nMethods\n\nThis study has been registered on clinicaltrials.gov, December 2019, NCT04187313. It is a cluster-randomised controlled trial of a multi-component public health intervention to increase notifications of TB from PPs in Bandung, in Indonesia. Clusters will be defined as CHC catchment areas and the intervention will be administered directly to PPs in areas randomised to the intervention arm. No intervention will be given to PPs in the control arm. The CHCs in both intervention and control arms will be informed about the study and asked, through the local Health Office, to make their notification data available. Notifications will be obtained directly from local NTP registry data, with accompanying information gathered about the address of the patient and referring doctor. Notified TB cases are, by definition, TB cases who have started TB treatment. Data on address of the patient will identify the extent of the “contamination” between arms.\n\nBandung, a city of 2.5 million inhabitants, is served by 73 CHCs, each covering a defined geographic administrative area. Of these, 30 were randomly selected in proportion to population size for a previously conducted study.11 The 30 selected CHCs will be randomly allocated into intervention and control arms with 15 CHCs for each arm (Figure 1). The study population consists of all the people who may visit a PP in any of the selected CHC study areas during the year before and the year after the intervention. The outcome event is TB notification reported to the CHC by PPs in the selected study area.\n\nThirty Community Health Centre (CHC) areas were randomly selected and assigned to the invention arm (dark gray shade) and control arm (light gray shade) for INSTEP2 study.\n\nAll PPs in the intervention arm who reported having diagnosed at least one TB case in the past 3 months or who are practicing at the same private clinics where at least one PP reported having diagnosed at least one TB case in the past 3 months will be eligible for the intervention. PPs will be identified through a community-based mapping exercise conducted in our previous study and updated for the current study. Eligible PPs will intend to work in the current location for the duration of the study as their primary place of private practice. They will be ineligible if they are unable to use an electronic device for referral, anticipate more than 3 months of non-practice during the study period, or they are not qualified to practice according to the Indonesian Medical Association.\n\nWe will personally approach, at their place of work, each of the identified PPs in the intervention areas. They will be fully informed about the study, invited to participate, and provide written informed consent. A schedule will be generated for their education and their follow-up (once every two months) during the intervention period. We expect around 80% of approached PPs will be willing to participate in the study. PPs participating in the intervention will be offered a non-financial incentive of a certificate of proficiency issued by the Indonesian Medical Association that provides them with credits that can be used as proof of continuing medical education for renewing their license to practice.\n\nThe study intervention will comprise five components:\n\nIntervention 1: Education. We will develop educational material on TB management specifically aimed for PPs. It will cover both the NTP guidelines (published in 2016) and the most current recommendations. The educational material will cover not only clinical management of TB patients but also public health and regulatory aspects of TB, from TB identification, to provisional diagnosis and referral, how to use the mobile phone app, and strategies to improve communication and increase adherence. From the feasibility study,12 we found that not all PPs have time to attend a 1-day training session; therefore, the education will be delivered as two 1-hour in-person sessions each separated 1-week apart (Table 1). We will do a follow-up visit, one month after the completed education, for every PP. In addition, important aspects of daily TB management will be made available as desk references.\n\nIntervention 2: Patient management pathways. Data from a previous study11 will inform standardized TB management pathways for each PP, which take into account the context around each PP’s practice, identifying the most efficient and feasible diagnostic approach and notifying TB patients.13 In brief, we will offer three possible pathways for diagnosing TB, all ensuring that bacteriological confirmation will be attempted for all patients. The first pathway starts with a chest radiograph, while the second starts with sputum smear microscopy, and the last, reserved for patients with suspected drug-resistant TB, starts with a sputum Xpert test (Figure 2). Based on the NTP guidelines, Xpert use is currently limited for patients with presumptive drug-resistant TB.14 PPs will be asked for their preferred pathway, although they may choose any pathway for any particular patient.\n\nSputum Xpert examination will be reserved for patients suspected for having drug-resistant TB, as per the local National Tuberculosis Programme guideline.\n\nIntervention 3: Electronic system. This system is a refinement of the previously developed electronic referral system using a mobile phone app compatible with both android and apple operating systems (Figure 3).12 The system will enable essential data to be uploaded, consistent with the NTP forms, which includes patient identity, diagnostic examinations, TB type, and referral plan. Participating PPs will inform the patients that the clinic is participating in a study about a TB notification system, and that patient information will be relayed to the Ministry of Health as per the regulatory requirement. Inputted information will be sent to a secure centralised server and will be monitored by authorized individuals in a web-based application. The server will generate an automated text message every time participating physicians add a record to the database. System development and piloting will include consultations with the NTP. Study investigators will manually report patient information to the NTP via CHC, as the electronic system is not currently linked to the NTP. If necessary, the study staff can contact the PPs or CHC officers to complete patient follow-up (Figure 4).\n\nParticipating PPs are free to withdraw from the intervention at any time upon request. On the other hand, an investigator may discontinue or withdraw a PP from the intervention if the PP is unable to receive the study intervention within one month or if the PP meets an exclusion criterion that precludes further study participation. The reason for PP discontinuation or withdrawal from the study will be recorded.\n\nA follow-up will be made one month following the second educational visit. Due to the COVID-19 pandemic, the follow-up can be either an in-person visit or online meeting. This will facilitate additional discussion and to ensure that the PPs do not have any difficulties in understanding the recommended diagnostic pathways and using the reporting app. At the one-month visit, adherence to the intervention will be assessed according to pre-determined indicators, and a standard form filled with the results. At the end of the visit, any issues will be rectified through re-education. The intervention period will then be complete, while electronic refreshers will continue through fortnightly electronic messages. Once the PPs have received the intervention, real-time monitoring of referral practice of patients for diagnosis and notification will be undertaken through a secure web-link to the app.\n\n\nOutcomes\n\nThe primary endpoint is notifications of TB and will be measured in the 12 months before and the 12 months after the intervention is fully implemented. The change in the number of notifications will be compared between intervention clusters (n = 15) and control clusters (n = 15).\n\n\nParticipant timeline\n\nThe follow-up for participating PPs will be conducted as explained in Figure 5.\n\n\nSample size\n\nThe primary analysis will compare the change in total number of notifications of patients between intervention and control areas. Less than 10% of PPs work privately in more than one CHC area. Those who also practice within the public system should not affect notifications from that public system, as it has a high notification rate (>85%) already. While the majority of notified TB patients are diagnosed and treated in their own CHC area, a number are diagnosed outside of their CHC area (up to 35%; data from our previous study). Those from a different CHC area will have an approximately one in five chance of being from another intervention area, a one in five chance of being from a control area, and a three in five chance of being from a non-study CHC area (n = 43 non-study CHC areas within Bandung). Therefore “contamination” of the intervention into control areas is estimated to be <10% (35% × 1/5 = 7%).\n\nAssuming 1) PPs in each arm diagnose at least 500 TB patients in 12 months; 2) 65% of PP TB diagnoses are patients from their CHC area and 35% are from outside their area; and 3) a 15% baseline notification rate changing to 50% post-intervention in the intervention arm: PP notifications in the control arm areas will change from 75 to 87 notified cases while the change will be from 75 to 201 cases in the intervention arm. Total notifications (adding 475 per arm from non-PP) will change from 550 to 562 and 550 to 676, respectively. Taking the above into account, we will have approximately 90% power to detect a rate ratio of 1.2 (676/562) at the p = 0.025 level (one-sided).\n\n\nRecruitment\n\nIn the intervention arm, all eligible PPs who are identified through an updated list will be invited to participate in the study. For those whose contact numbers are obtained, research assistants will send up to three text/electronic messages to schedule a phone call to offer participation in the study; any PP who cannot be contacted by phone or does not respond to all three messages will be approached at their clinic. PPs whose contact numbers are not available will be directly visited by a research assistant. On the scheduled phone call/visit, a research assistant will explain about the study and offer participation after ascertaining that the PP meets all of the eligibility criteria. Upon agreement to participate, the research assistant will schedule a visit to obtain written informed consent and deliver the first education session.\n\n\nAssignment of interventions\n\nWe will randomly allocate 15 CHC areas as intervention areas, repeating the randomisation 100 times. From this we will select the 10 allocations that meet the criteria of 1) including having the least number of adjacent CHC areas between intervention and control arms, to minimize contamination between the arms, and 2) balancing by CHC numbers of TB cases diagnosed per annum. Then we will randomly select the final allocation from these ten.\n\nInvestigators, participating PPs and CHC staff will not be blinded to study arm as this is not practical in this type of trial. Staff conducting diagnostic investigations (laboratory personnel and radiologists) will not be part of the study team and will be blinded to study arm. Notification data will be abstracted from the local NTP registry so the data will not be influenced by study staff. Treatment allocation will be masked during data analysis. Trial randomisation codes will not be broken until the study is closed and analysis is commenced. Since there are no anticipated serious adverse events, no criteria for breaking the codes have been set.\n\n\nData collection\n\nFrom PPs in the intervention arm, prior to the intervention, we will collect information on their name, gender, age, and self-reported number of TB cases diagnosed in the last 3 months. This will enable comparison of basic characteristics between those who participate in the intervention and those who do not. PPs who refer patients for confirmation of diagnosis and notification will enter the following data about the patient into the app: name, gender, age, basis of presumptive/definitive diagnosis, other practice addresses, and the referral plan for the patient. The following data will be abstracted from routine records of TB cases at the CHCs: referring facility/practitioner, date, gender, patient identification, patient address (to the level of CHC area), sputum status, and basis of TB diagnosis.\n\nData abstraction from routine notification data will be done by trained enumerators. The 12-month period before intervention commences will be defined clearly and data abstraction will take place as soon as possible at the end of this period. The 12-month follow up period will be defined clearly, starting immediately after the last PP to receive the intervention has had their one-month follow up visit. Data abstraction will be done as soon as possible after this 12-month period is completed.\n\n\nData management\n\nInputted information from PPs will be sent to a secure centralised server and will be monitored through log-in by authorized trial staff in a secure web-based application. The coordinating investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness of the data reported. Hardcopies of study visit worksheets will be provided for use as source document worksheets for recording data for each PP enrolled in the study. Data recorded in the electronic case report form derived from source documents will be consistent with the data recorded on the source documents.\n\nClinical and laboratory data, will be entered into a password-protected REDCap electronic database15 and checked automatically for data that appear inconsistent, incomplete, or inaccurate. Clinical data will be entered directly from the source documents. Study documents will be retained for a minimum of 10 years after completion of the trial. No records will be destroyed without the written consent of the sponsor, if applicable. It is the responsibility of the sponsor to inform the investigator when these documents no longer need to be retained.\n\n\nStatistical analysis\n\nThe primary analysis will be intention-to-treat, where all patients notified by intervention and control PPs to a CHC will be included. Analysis will be carried out at the cluster level. 95% confidence intervals will be calculated and a significance level of 0.025 (one-sided) will be used. Study arms will be compared on baseline characteristics, including demographics of the PPs and the diagnosed TB cases, using descriptive statistics. We will estimate the rate ratio comparing TB notifications in the intervention and control groups. A generalised mixed model will be used with repeated measures of TB notification (pre- and post-intervention) and treatment×time interaction, a log link, Poisson errors and a random effect for PKM to account for over-dispersion.\n\nFor the secondary objectives, the proportions of bacteriologically confirmed TB cases among the reported TB cases by PPs will be compared in intervention and control arms using a generalised estimation equation. The model will be fitted to repeated measures of the proportions of true positive TB notifications for each CHC (pre- and post-intervention) with a treatment by time interaction, a log link, and Poisson errors, with robust standard errors to allow for both the binary data and over-dispersion. Additionally, the primary trial analysis will be repeated excluding patients who were notified by a PP from a different CHC to their area of residence. Safety and interim analyses are not planned for the study.\n\n\nSafety\n\nSafety oversight by a Data and Safety Monitoring Board (DSMB) will not be required for this public health intervention trial. However, an internal Data Monitoring Committee (DMC) will be established to oversee the study, focused on data quality. A quality management plan will be developed to monitor a site’s quality management. Quality control (QC) procedures will be implemented beginning with the data entry system and data QC checks that will be run on the database will be automatically generated on a weekly basis and any quality issues identified will be reviewed by the DMC and a plan put in place for resolution.\n\nA series of standard operating procedures (SOPs) will be written to guide the research team and ensure the trial is conducted and data are generated, documented, and reported in compliance with the protocol.\n\n\nEthics and dissemination\n\nProtocol and the consent forms have been reviewed and approved by the University of Otago human ethics committee (#H19-052), translated into Bahasa Indonesia and reviewed and approved by Universitas Padjadjaran ethics committee (#1089/UN6.KEP/EC/2019).\n\nAny major modifications to the protocol including changes of study objectives, study design, sample sizes, study procedures, or significant administrative aspects will be submitted for approval by the University of Otago and Universitas Padjadjaran’s ethics committees.\n\nConsent forms describing the study are given to the head of each CHC institution. For PPs undergoing intervention, consent forms describing in detail the study intervention, study procedures, and risks are given to the PP. The investigator will explain the research study to the participant PP and answer any questions that may arise. A verbal explanation will be provided in terms suited to the PP’s comprehension of the purposes, procedures, and potential risks of the study and of their rights as research PPs. PPs will have the opportunity to discuss the study with their family or surrogates, carefully review the written consent form and ask questions prior to signing. PPs will be informed that participation is voluntary and that they may withdraw from the study at any time, without prejudice. The consent process will be conducted the form signed prior to any intervention and a copy of the document given to PPs for their records. The rights and welfare of the PPs will be protected by emphasizing to them that there will be no negative repercussions if they decline to participate in this study, including no reporting of discontinuation to government authorities.\n\nParticipating PPs’ confidentiality and privacy are extended to cover any information relating to them. Therefore, the study protocol, documentation, data, and all other information generated will be held in strict confidence. No information concerning the study or the data will be released to any unauthorized third party without prior written approval of the sponsor. All research activities will be conducted in a setting that is as private as possible.\n\nPP research data, which are for purposes of statistical analysis and scientific reporting, will be stored. This will not include the PP’s contact or identifying information. Rather, individual PPs and their research data will be identified by a unique study identification number. The study data entry and study management systems will be secured and password protected. At the end of the study, all study databases will be de-identified and archived.\n\nAny actual conflict of interest of persons who have a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be required to have such conflicts managed in a way that is appropriate to their participation in the design and conduct of this trial. The study leadership has established policies and procedures for all study group members to disclose all conflicts of interest and will establish a mechanism for the management of all reported dualities of interest.\n\nPaper-based study data will be stored in a locked cabinet in the Tuberculosis Working Group office in Bandung. All electronic and web-based data will be secured by appropriate security protocols, password protected and only accessible to study investigators and designated research assistants.\n\nData collected for this study will be analysed and stored. After the study is completed, the de-identified, archived data may be transmitted to and stored, for use by other researchers including those outside of the study. Permission to transmit data will be included in the informed consent.\n\nThis study is not subject to any particular publication and data-sharing policies and regulations. However, we aim to publish study results in scientific conferences and a peer-reviewed journal, as well as presenting the results to relevant stakeholders in TB control in Indonesia.\n\n\nDiscussion\n\nTo our knowledge, the use of a randomized controlled trial to evaluate the effect of intervention to increase PPs involvement in TB control has been reported just once in the past ten years.16 Yellapa et al. implemented a package of interventions to PPs in the city of Tumkur, South India, and reported an almost a two-fold increase in case referral from intervention-arm PPs.16 Our study has a larger planned sample size and more focus on increasing notifications which was not sufficiently impacted in the aforementioned study. Additionally, using cluster-randomization and taking into account clustering in the analysis is more appropriate to measure the effect of programmatic intervention where implementation is being done at the cluster level. Hence, our study will contribute to providing robust evidence about the effect of intervening PPs on increasing TB patient notification and referral, in particular in the context of an urban setting in a lower middle-income country.\n\nThe study intervention is given as a package; therefore, it will be difficult, if not impossible, to separate out any individual intervention’s effect on TB notification and referral. However, pre- and post-test data may be used to measure the impact of in-person education on PPs’ knowledge and, insofar as knowledge correlates with practice, evidence of an increase in knowledge may suggest that education can potentially improve practice. However, it is understood that knowledge does not directly translate into practice and a “know–do” gap has been observed.17\n\nFor practical reasons, it will not be possible to blind the study investigators and research assistants to treatment allocation. To reduce potential bias due to differential rigor in data collection and analysis, the allocation will be concealed from enumerators responsible for collecting the study outcome data as well as in the data set used in analyses.\n\nThe study area is relatively small and homogeneous, we also limit participation to general practitioners and a limited number of specialties (internists and pulmonologists) who are the most likely PPs to refer TB cases.16 This study nonetheless will provide evidence of effectiveness for the package of interventions aimed at PPs and will be generalizable to similar settings in Indonesia, which, in itself, is important in the context of TB control.\n\nAs mentioned, a small amount of contamination between intervention and control arms is likely. PPs can practice in more than one place that may be located in both of the study arms. Further, PPs may also practice in the public sector, including at CHC. Lastly, it will not be possible to conceal the study intervention from TB programme officers in the control arm because these officers are in regular communication with one another. We assume that the degree of contamination will be small.\n\nCluster-randomised trials can be subject to selection bias if participants are recruited after randomisation, as is being done in this study.18 However, selection bias can be minimized because inclusion and exclusion criteria have been set a priori and participation will be offered to every PP meeting those criteria identified from mapping conducted before the start of the study. Lack of adherence of PPs in completing interventions was found to be a common problem in similar studies.19 Although participation is voluntary and hence PPs do not have to provide reasoning for withdrawing from the study, we will record reasons if they are provided and this information can be used to inform strategies to increase adherence.\n\nIncreasing PPs involvement in TB-PPM requires a PP-centred approach. This study will provide valuable evidence on whether a PP-centred intervention package is effective in increasing the number of TB notifications and referrals. The study results will be important to guide policy in TB-PPM in Indonesia and other similar settings.\n\n\nData availability\n\nNo data are associated with this article\n\nOpen Science Framework: Extended data for ‘Protocol for increasing notifications of tuberculosis from private practitioners (INSTEP2): A randomised controlled trial’, https://doi.org/10.17605/OSF.IO/KAGNB.20\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 2013 checklist for ‘Protocol for increasing notifications of tuberculosis from private practitioners (INSTEP2): A randomised controlled trial’, https://doi.org/10.17605/OSF.IO/KAGNB.20\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nPFH: Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Writing – Original Draft Preparation, Writing – Review & Editing. BWL: Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Writing – Original Draft Preparation, Writing – Review & Editing. KS: Methodology, Formal Analysis, Supervision, Writing – Review & Editing. NA: Data Curation, Investigation, Project Administration, Writing – Review & Editing. LC: Data Curation, Writing – Review & Editing. CCH: Methodology, Formal Analysis, Writing – Review & Editing. SM: Supervision, Writing – Review & Editing. RvC: Methodology, Writing – Review & Editing. MM: Funding Acquisition, Methodology, Resources, Supervision, Writing – Review & Editing. BA: Conceptualization, Data Curation, Funding Acquisition, Investigation, Methodology, Resources, Supervision, Writing – Review & Editing. PH: Conceptualization, Funding Acquisition, Methodology, Resources, Supervision, Writing-Original Draft Preparation, Writing – Review & Editing.", "appendix": "References\n\nWorld Health Organization: Global Tuberculosis Report 2019. Geneva: World Health Organization; 2019.\n\nWorld Health Organization: Engaging private health care providers in TB care and prevention: a landscape analysis. Geneva: 2018.\n\nHanson C, Osberg M, Brown J, et al.: Finding the Missing Patients With Tuberculosis: Lessons Learned From Patient-Pathway Analyses in 5 Countries. J Infect Dis. 2017; 216(suppl_7): S686–S95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBell CA, Duncan G, Saini B: Knowledge, attitudes and practices of private sector providers of tuberculosis care: a scoping review. Int J Tuberc Lung Dis. 2011; 8. PubMed Abstract \| Publisher Full Text\n\nUplekar M: Involving private health care providers in delivery of TB care: global strategy. Tuberculosis. 2003; 83(1): 156–64. PubMed Abstract \| Publisher Full Text\n\nSurya A, Setyaningsih B, Suryani Nasution H, et al.: Quality Tuberculosis Care in Indonesia: Using Patient Pathway Analysis to Optimize Public–Private Collaboration. J Infect Dis. 2017; 216(suppl_7): S724–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Health Indonesia: Tuberculosis Inventory Study in Indonesia 2016-2017. Jakarta:2018.\n\nUplekar M, Atre S, Wells WA, et al.: Mandatory tuberculosis case notification in high tuberculosis-incidence countries: policy and practice. Eur Respir J. 2016; 48(6): 1571–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReviono R, Setianingsih W, Damayanti KE, et al.: The dynamic of tuberculosis case finding in the era of the public–private mix strategy for tuberculosis control in Central Java, Indonesia. Glob Health Action. 2017; 10(1): 1353777. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPethani A, Zafar M, Khan AA, et al.: Engaging General Practitioners in Public–Private Mix Tuberculosis DOTS Program in an Urban Area in Pakistan: Need for Context-Specific Approach. Asia Pac J Public Health. 2015; 27(2): NP984–NP92. PubMed Abstract \| Publisher Full Text\n\nLestari BW, McAllister S, Hadisoemarto PF, et al.: Patient pathways and delays to diagnosis and treatment of tuberculosis in an urban setting in Indonesia. Lancet Regional Health - Western Pacific. 2020; 5: 100059. Publisher Full Text\n\nLestari BW, Arisanti N, Siregar AYM, et al.: Feasibility study of strengthening the public–private partnership for tuberculosis case detection in Bandung City, Indonesia. BMC Res Notes. 2017; 10(1): 404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArtawan Eka Putra IW, Utami NW, Suarjana IK, et al.: Factors associated to referral of tuberculosis suspects by private practitioners to community health centres in Bali Province, Indonesia. BMC Health Serv Res. 2013; 13: 445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Health Republic of Indonesia: National Guideline for TB control. Jakarta; 2016.\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): 377–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYellappa V, Battaglioli T, Gurum SK, et al.: Involving private practitioners in the Indian tuberculosis programme: a randomised trial. Trop Med Int Health. 2018; 23(5): 570–9. PubMed Abstract \| Publisher Full Text\n\nDas J, Kwan A, Daniels B, et al.: Use of standardised patients to assess quality of tuberculosis care: a pilot, cross-sectional study. Lancet Infect Dis. 2015; 15(11): 1305–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrierley G, Brabyn S, Torgerson D, et al.: Bias in recruitment to cluster randomized trials: a review of recent publications. J Eval Clin Pract. 2012; 18(4): 878–86. PubMed Abstract \| Publisher Full Text\n\nEldridge SM, Ashby D, Feder GS, et al.: Lessons for cluster randomized trials in the twenty-first century: a systematic review of trials in primary care. Clin. Trials. 2004; 1(1): 80–90. PubMed Abstract \| Publisher Full Text\n\nLestari BW: INSTEP2 Study.2021, February 28. Publisher Full Text" }	[ { "id": "90373", "date": "12 Aug 2021", "name": "Saurav Basu", "expertise": [ "Reviewer Expertise Public health", "Epidemiology and Community Medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study protocol describes the implementation of a public health intervention package through a cluster RCT to improve TB notification rate and awareness of PPs who are involved in the management of patients with TB.\n\nThe intervention comprises of three components including educational, patient management pathways, and an electronic system.\n\nThe protocol needs to provide more detail on the development of the intervention package, especially in context of the educative module (what is the baseline knowledge of TB in the PPs?). Reasons for non-notification also need to be explained and how this intervention package will promote notification (reasons other than lack of knowledge can also exist).\nYou can include secondary outcomes such as delay in treatment initiation (time from presentation to PP and initiation of ATT).\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "7978", "date": "25 Mar 2022", "name": "Bony Wiem Lestari", "role": "Author Response", "response": "We thank the reviewer for their valuable comments. Please find below our specific responses. Comment-1: The protocol needs to provide more detail on the development of the intervention package, especially in context of the educative module (what is the baseline knowledge of TB in the PPs?). Reasons for non-notification also need to be explained and how this intervention package will promote notification (reasons other than lack of knowledge can also exist). Response: We have revised the manuscript to include a more detailed information about the development of the intervention package, especially the education component. Comment-2: You can include secondary outcomes such as delay in treatment initiation (time from presentation to PP and initiation of ATT). Response: Indeed, health care facilities/providers are required to report the time of diagnosis, the time of treatment initiation, and the means by which TB is diagnosed. The potential effect of the intervention to these outcomes may be measurable, although we will not be able to measure the delay to treatment initiation in the control arm because we are not collecting data from PPs in this arm. We have added this in the discussion section." } ] }, { "id": "95006", "date": "27 Oct 2021", "name": "Charity Omenka", "expertise": [ "Reviewer Expertise Access to TB 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\nThis protocol describes a cluster-randomised control trial involving deployment of a multi-component intervention package. The methods, including the setting and eligibility criteria are clearly defined.\nHowever, the authors need to clarify the 5 components of the intervention as only 3 interventions are defined.\nIt would also be useful if the authors compared the primary outcome - change in notifications - between the 3 diagnostic methods mentioned - chest radiograph, sputum microscopy and Xpert testing.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "7979", "date": "25 Mar 2022", "name": "Bony Wiem Lestari", "role": "Author Response", "response": "Thank you for your valuable comments. Please find below our specific responses: Comment-1: However, the authors need to clarify the 5 components of the intervention as only 3 interventions are defined. Response: We apologise for the inconsistency. The five components of intervention are: 1) electronic referral and notification system, 2) standardized education materials, 3) an individualised plan for each PP with respect to their approach to the diagnosis and management of TB suspects, 4) 1 month follow-up visit from the study team, and 5) 2-monthly electronic reminder/refresher. However, components 4 and 5 are included as parts of the standardized education component. We have revised the statement in our manuscript to state that the intervention consists of three components. Comment-2: It would also be useful if the authors compared the primary outcome - change in notifications - between the 3 diagnostic methods mentioned - chest radiograph, sputum microscopy and Xpert testing. Response: Thank you for your suggestions. Indeed, health care facilities/providers are required to report the time of diagnosis, the time of treatment initiation, and the means by which TB is diagnosed (e.g., via sputum microscopy vs clinically). The potential effect of the intervention to these outcomes may be measurable, although we will not be able to measure the delay to treatment initiation in the control arm because we are not collecting data from PPs in this arm. We have added this in the discussion section." } ] } ]	1	https://f1000research.com/articles/10-327

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