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https://f1000research.com/articles/10-783/v1	10 Aug 21	{ "type": "Case Report", "title": "Case Report: A case of tuberculosis lymphadenitis mimicking a gastric tumor", "authors": [ "Dhafer Hadded", "Alia Zouaghi", "Meryam Mesbahi", "Fatma Khanchel", "Nawel Bellil", "Yazid Benzarti", "Zeineb Benzarti", "Anis Ben Maamer", "Dhafer Hadded", "Alia Zouaghi", "Fatma Khanchel", "Nawel Bellil", "Yazid Benzarti", "Zeineb Benzarti", "Anis Ben Maamer" ], "abstract": "Solitary tuberculosis of the upper gastrointestinal tract is a rare pathology that usually mimics the clinical and radiological features of malignant tumors. A gastric subepithelial tumor is usually detected during diagnostic endoscopy. Stomach tuberculosis, in particular, can appear as a subepithelial tumor of the stomach wall. Several cases of gastric tuberculosis imitating subepithelial gastric tumors have been reported recently. We describe the case of a patient with tuberculous lymphadenitis that mimics a submucosal gastric tumor. A 52-year-old female was admitted to our surgical department for epigastric pain and weight loss. Endoscopy was inconclusive; it revealed either a submucosal compression or an anterior submucosal lesion with erosive anterior gastropathy and a fistulous orifice located in the bulb. The patient was diagnosed with a gastric tumor and an endoscopic ultrasound demonstrated a rounded hypoechogenic antral lesion that was not vascularized and was distant from the gastric wall, whose five layers appeared of a normal aspect. The patient underwent an exploratory laparotomy. A biopsy was sent intraoperatively for frozen section examination, and concluded that the diagnosis was tuberculous intraperitoneal lymphadenitis. The patient received anti-tuberculosis treatment. This case demonstrates that gastric tuberculosis remains a challenging diagnosis.", "keywords": [ "Gastric tuberculosis", "abdominal tuberculosis", "Subepithelial tumor", "Tuberculous Lymphadenitis", "case report" ], "content": "Introduction\n\nAbdominal tuberculosis (TBC) can affect many organs in the peritoneal cavity such as the gastrointestinal tract, peritoneum, lymph nodes, spleen, and liver. It can affect one organ or many in combination.1 Gastrointestinal TBC’s presentation is varied, depending on the site that is involved.2 Its diagnosis is especially difficult. This particularity is explained by differential diagnoses that can mimic the various manifestations of gastrointestinal TBC, including infectious and noninfectious causes.3 TBC of the stomach is the rarest form and is generally misdiagnosed because it may mimic a gastric tumor.4\n\nHere, we report a case of mesenteric tuberculous lymphadenitis that had involved the gastric wall and mimicked a gastric submucosal tumor with no evidence of tuberculosis elsewhere.\n\n\nCase report\n\nA 52-year-old woman, with a history of hypertension, was hospitalized in our department of surgery following three months of epigastric pain and discomfort with weight loss. She had neither fever nor respiratory symptoms. Physical examination revealed mild tenderness in the upper abdomen associated with an palpable and painful epigastric mass measuring 4 cm. There was no cervical lymphadenopathy or hepatosplenomegaly, and laboratory data were normal. There were no abnormalities on the chest X-ray.\n\nThe upper endoscopy was inconclusive and showed either submucosal compression or an anterior submucosal lesion with erosive anterior gastropathy and a fistulous orifice located in the bulb (Figures 1 & 2). A biopsy was performed and concluded there was no malignancy and no evidence of tuberculosis. The endoscopic ultrasound revealed a rounded lesion approximately 3026 mm located in the antrum. The lesion was hypoechogenic and discretely heterogeneous, not vascularized, and distant from the gastric wall whose five layers appeared of a normal aspect (Figure 3). By positioning the probe next to the bulbar fistulous orifice, it was found that there was a second lesion with a hypoechogenic center (Figure 4) with a hypoechoic fistulous path.\n\nEsophagogastroduodenoscopy shows an extrinsic compression of the gastric wall and an anterior submucosal lesion, with a fistulous orifice located in the bulb (images were edited in Microsoft PowerPoint 2016 to remove patient’s scan data).\n\nAn abdominal computed tomography (CT) scan was performed and showed an exophytic, heterogenous gastric formation with an axial necrotic center measuring 4424 mm. After injection of contrast agent evoking peritoneal carcinosis nodules, the formation was found to be 24 mm and associated with multiple tissue nodules of enhanced infra centimeter. There were hepatic hilum and coeliomesenteric lymph nodes, one of which had a necrotic center measuring 9 mm in diameter corresponding to the one described on the endoscopic ultrasound (Figures 5 & 6).\n\nThe patient underwent an exploratory laparotomy with a prediagnosis of suspected gastric cancer. The surgical findings indicated a bulky mass adjacent to the antrum with posterior development invading the transverse mesocolon (Figures 7A & 7B), associated with multiple adenopathies of the mesentery, the transverse mesocolon, and the greater omentum, and organized inflows. There was a second mass of 3 cm located in the small omentum in contact with the left gastric artery, probably corresponding to a voluminous adenopathy (Figure 7C). A biopsy was taken and sent for frozen section examination, which found numerous epithelioid and giganto-cellular granuloma and central caseous necrosis, confirming the diagnosis of tuberculous intraperitoneal lymphadenitis (Figure 8). Indeed, gastrotomy was not performed because of the benign nature of the pathology. The patient was administered anti-tuberculosis treatment and was closely monitored. A CT scan taken six months after surgery revealed a total regression of all the gastric lesions and nectrotic lymph nodes previously described, and the disease was fully controlled.\n\nPatient’s lymph node showing numerous necrotizing and non necrotizing epithelioid and giganto-cellular granuloma. Giant cells of Langhan’s type are multinucleated (white arrow). In the center of a granuloma, necrosis is eosinophilc and granular; this is the microscopic appearance of caseous necrosis (black arrow) (HE×10)\n\n\nDiscussion\n\nAbdominal TBC is always misdiagnosed because of its various clinical manifestations. It is known as a great mimicker, especially when it affects abdominal organs without pulmonary infection, and malignant tumors are the most incriminating as a preoperative diagnoses.2,5 Gastric TBC is an extremely rare form whether it is a primary or secondary infection.1 Debi et al.6 explain the reasons for its rarity such as the bactericidal properties of gastric acid, the scarcity of lymphatic tissue in the gastric wall and the thick gastric mucosa in an intact stomach.\n\nMoreover, mesenteric tuberculose lymphadenitis is an extremely rare cause of intestinal manifestations involving the gastric wall, such as in our case.1 According to the literature, only a few cases have been reported showing tuberculosis lymphadenopathy mimicking submucosal gastric tumors.\n\nPrimary gastric TBC, that does not involve any other organ, is generally located on the antrum or prepyloric region involving the duodenum. This location is explained by the presence of lymphoid follicles.1,4 There are six types of gastric tuberculosis in pathological forms: tubercular ulcers; miliary tubercles; hypertrophic tuberculosis; tuberculous pyloric stenosis; solitary tuberculoma; and tubercular lymphadenitis.2\n\nClinically, patients generally present with nonspecific upper abdominal pain such as epigastric pain, associated with weight loss, anorexia, and weakness.4 The majority of patients with gastric tuberculosis are often diagnosed after surgery because of the lack of specific symptoms.1\n\nAn endoscopy is helpful to diagnose this pathology, especially by biopsy results. Endoscopies can show ulcers, masses, or extrinsic compression.7 However, in our case, gastric cancer was suspected and the biopsy did not help to confirm the diagnosis. The poor yield of the biopsy is explained by the submucosal lesion that may not reveal granulomas and is difficult to obtain tissues from.7,8 Endoscopic ultrasonography is also very helpful, especially in the case of submucosal lesions or related lymph node enlargement,7 as it can differentiate between an extrinsic compression and a subepithelial gastric tumor by identifying the relationship between the lesion and the gastric wall.9 Morphologically, no specific imaging findings can help diagnose tuberculosis rather than malignancy, because there are no pathognomonic characteristics that show radiological modalities.5\n\nUsing combined radiographic and endoscopic imaging can facilitate an early diagnosis without unnecessary surgical resection. However, it is always difficult to have a final diagnosis by endoscopic biopsy, so it becomes necessary to perform a surgical biopsy using frozen section examination.8\n\n\nConclusion\n\nAbdominal lymphadenitis tuberculosis presents a diagnostic challenge and a dilemma for clinicians. It may mimic a long list of differential diagnoses, such as in our case of tuberculosis lymphadenitis eroding the gastric wall. In these cases, endoscopy biopsy is the best modality to identify the pathology. Nevertheless, it could sometimes not be made preoperatively and may require surgery for diagnosis by intraoperative frozen biopsy.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nThe patient has provided both verbal and written informed consent for the publication of their clinical details and images. It was made sure that his identity will be kept a secret at all levels. A copy of a written request is available for review if requested.\n\n\nAuthor contributions\n\nAll authors were involved in the researching, writing, and editing of the manuscript.", "appendix": "References\n\nKim DY, et al.: Tuberculous mesenteric lymphadenitis involving the gastric wall: case report. Gastrointest. Endosc. nov. 2005; 62(5): 799–802. PubMed Abstract \| Publisher Full Text\n\nDebi U, et al.: Gastrointestinal Tuberculosis: An overview. Arch. Clin. Med. Case Rep. 2020; 4(5). Publisher Full Text\n\nLowbridge C, et al.: How can gastro-intestinal tuberculosis diagnosis be improved? A prospective cohort study. BMC Infect. Dis. déc. 2020; 20(1): 255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaudhary P, Khan AQ, Lal R, et al.: Gastric tuberculosis. Indian J. Tuberc. juill. 2019; 66(3): 411–417. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbu-Zidan FM, Sheek-Hussein M: Diagnosis of abdominal tuberculosis: lessons learned over 30 years: pectoral assay. World J. Emerg. Surg. déc. 2019; 14(1): 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDebi U: Abdominal tuberculosis of the gastrointestinal tract: Revisited. World J. Gastroenterol . 2014; 20(40): 14831. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShah J, Maity P, Kumar P, et al.: Gastroduodenal tuberculosis: a case series and a management focused systematic review. Expert Rev. Gastroenterol. Hepatol. sept. 2020; 1–10. PubMed Abstract \| Publisher Full Text\n\nEray IC, et al.: Primary gastric tuberculosis mimicking gastric cancer. Turk. J. Surg. sept. 2014. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim SB, Kim TN, Kim KH: Tuberculous Lymphadenitis Mimicking Gastric Subepithelial Tumor Diagnosed Using Endoscopic Ultrasound-guided Fine-needle Aspiration. Korean J. Helicobacter Up. Gastrointest. Res. 2018; 18(1): 65. Publisher Full Text" }	[ { "id": "102300", "date": "04 Jan 2022", "name": "Nonthalee Pausawasdi", "expertise": [ "Reviewer Expertise Gastroenterology and endoscopy." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors reported the rare case of mesenteric tuberculosis lymphadenitis, which mimicked gastric tumor. The authors should be commended on their work. There are a few areas where additional information would enhance the manuscript's clarity.\nQuestions/comments\nBased on the text, Figure 1 demonstrates an endoscopic image of the gastric wall, and Figure 2 depicts an image of the duodenal bulb. However, figure 1 & 2 composite does not correspond to the text (Fig 1 is the fistula opening, and Fig 2 is the bulging gastric wall).\n\nThe red arrow in Figure 1 should be made more noticeable. Please consider changing the color or bold the arrow.\n\nThe site of tissue biopsies is critical for pathological diagnosis. Please clarify where the mucosal biopsies were performed (bulging gastric wall vs. the fistulous orifice in the duodenal bulb). If the biopsies were performed from the gastric wall, which technique was applied (conventional mucosal biopsies vs. tunnel biopsies)?\n\nDoes the presence of necrotic mesenteric lymphadenopathy on CT scan suggest tuberculosis lymphadenitis?\n\nWhy was EUS-guided tissue acquisition not performed? EUS-FNA/B might have helped make the diagnosis in this case before performing surgery.\n\nThe diagnosis of abdominal tuberculous lymphadenitis can be challenging despite combined radiologic and endoscopic findings. However, endoscopic guided tissue acquisition for culture for tuberculosis can be helpful (Ref: Chang et al. 20201). This issue should be addressed in the discussion, and additional information related to bacteriological methods (e.g., AFB smear, culture TB, or PCR for TB) may be added if available.\n\nThe treatment regimen for mesenteric tuberculosis lymphadenitis is crucial. It would be helpful for the readers if the authors report the treatment regimen and duration.\n\nIt would be nice if the authors could demonstrate follow-up endoscopic images of the fistulous opening or CT scan after treatment completion.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "297280", "date": "11 Jul 2024", "name": "Farhan Shahzad", "expertise": [ "Reviewer Expertise gastroenterology", "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\nReview for the Case Report: A case of tuberculosis lymphadenitis mimicking a gastric tumor Following changes are suggested:\nChanges “3026 mm” to “30×26mm”, “2228 mm” to “22×28 mm” and “44*24 mm” to “44×24mm”. “After injection of contrast agent evoking peritoneal carcinosis nodules, the formation was found to be 24 mm and associated with multiple tissue nodules of enhanced infra centimeter”, rephrase this sentence and make it easier to understand. Why you didn't go for QuantiFERON gold Tb test as it is the standard guidelines nowadays to diagnose TB? The role of MRI examination has been neglected in your discussion. Because in liver tuberculosis, it can be useful. As the diagnosis was not clear, you should have gone for diagnostic laparoscopy with biopsy rather than laparotomy?\n\nI have research paper on isolated abdominal tuberculosis, please cite that paper. Ref-[1]\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-783
https://f1000research.com/articles/10-780/v1	09 Aug 21	{ "type": "Method Article", "title": "Contraceptive Use Dropout-adjusted Unmet Need for Family Planning", "authors": [ "Budi Utomo", "Hariyanti Hariyanti", "Sabarinah Prasetyo", "Robert Magnani", "Sukma Rahayu", "Budi Utomo", "Hariyanti Hariyanti", "Sabarinah Prasetyo", "Robert Magnani" ], "abstract": "Background: In the last two decades, unmet need for family planning in Indonesia remains stagnant, and contraceptive discontinuation has increased. These two indicators describe the risk of unwanted pregnancy in a population. Therefore, this study aims to develop an accurate calculation of the unmet need for family planning in Indonesia. Method: The study uses 2017 IDHS data to compare unmet need at survey-time and five years preceding the survey, measured by contraceptive calendar data that measured history of contraceptive use within five years preceding the survey. Unmet need at five years preceding the survey is measured by calculating the proportion of months not using contraceptive to the duration of months exposed to pregnancy in a period of 69 months. The study population is married women in Indonesia, with a sample size of 35,681.\n\nResults: Unmet need with contraceptive calendar calculation is higher than unmet need at survey-time. A difference of 3% concerns an additional of nearly 1.6 million unwanted pregnancies. This study proves that the high number of contraceptive discontinuations is directly proportional to higher unmet need with contraceptive calendar calculation. Conclusion: In Indonesia, with a relatively high contraceptive discontinuation rate, the calculation of unmet need using the calendar method is more precise than at survey-time method. The study results suggest the use of unmet need calendar for countries with high contraceptive discontinuation rate and provision of primary health care that is responsive to a potential unwanted pregnancy.", "keywords": [ "Contraceptive calendar data", "Family planning", "Contraception", "Contraceptive discontinuation", "Unmet need." ], "content": "Introduction\n\nUnmet need for family planning refers to those who are fecund and sexually active, but are not using any method of contraception, and report a desire to delay or limit childbearing. Women with unmet need are exposed to risk of unwanted pregnancy, unsafe abortions, and maternal death.1 Globally, there are 80 million unwanted pregnancies and 40 million abortions every year due to unmet need.2 In developing countries, around 222 million women experience unmet needs, and in Indonesia, around 1.000 births are unwanted or wanted later.3,4\n\nReferring to the history of family planning in Indonesia, in the 1970s, the family planning program was a national program aimed at population control. The indicators used were Contraceptive Prevalence Rate (CPR) and Contraceptive Years Protection (CYP), which were used to assess the decline in fertility. In 1994, the International Conference for Population & Development (ICPD) was held to introduce the concept of reproductive health and in 2012, the 2020 Family Planning was held to emphasized rights-based reproductive health.5 The perspective of the family planning program then has been oriented towards reproductive health and measured by the unmet need indicator to assess the risk of unwanted pregnancy which has an impact on maternal mortality.6 Furthermore, to achieve universal access to sexual and reproductive health and rights as a part of universal health coverage, the World Health Organization (WHO) and the United Nations Fund for Population Activities commiting to strive for zero unmet need for family planning information and services, and universal availability of quality, accessible, affordable and safe modern contraceptives.7\n\nThe achievement of family planning programs in Indonesia based on the 1991 to 2017 DHS shows that the unmet needs are in stagnant trend in last two decades while contraceptive discontinuation has increased. Unmet needs and contraceptive discontinuation capture the same phenomenon: women who do not use contraception are at risk of unwanted pregnancies. However, the data for these two indicators show contradictions.8–13\n\nThe progress of family planning program achievement becomes the basis for reviewing unmet need measurements in Indonesia. The standard of unmet need measurement is calculated based on the status of contraceptive use at survey time. This study develops an unmet need measurement based instead on contraceptive use within the five years preceding the survey which is termed the unmet need with calendar data.14 It takes into account the duration of contraceptive use, pregnancy and marriage, so conceptually the method would be more precise in estimating the risk of unwanted pregnancy in Indonesia.\n\n\nMethods\n\nThe study using a cross-sectional research design by analyzing the 2017 the publicly available database of Indonesia Demographic Health Survey that conducted by National Population and Family Planning Board, Statistics Indonesia, Ministry of Health and The DHS Program or ICF. The IDHS 2017 data are considered adequate in quality for statistical analysis as the survey report has been officially published and the survey data have been used in published studies. The studied population were currently married women with total of 35,681 women.\n\nThis study used reproductive and contraceptive calendar data which records several dimension on the history of reproductive and contraceptive events, such as: 1) Birth history; 2) Pregnancy; 3) Birth termination; 4) Current contracpetive use; 5) Episodes of contraceptive use during calendar period. The study focused on duration not protected by contraceptive use, described as measured in women of reproductive age who did not use contraception in the five years preceding the survey, which in IDHS 2017 was 69 months. These measurements were made after determining the study population, which then retrospectively reviewed the history of contraceptive use at 69 months prior to the survey. The data was analyzed by SPSS version 23.0 statistical packages (IBM SPSS Statistics, RRID:SCR_019096).\n\nThe detailed calculations of duration not protected by contraceptives are shown in Figure 1.\n\nFigure 1 explains that survey-time unmet need is measured based on contraceptive use status at the time of survey, while unmet need with calendar data is measured based on contraceptive use within the 69 months preceding the survey. The figure shows that unmet need at survey-time is limited by the marital status, which is the inclusion criteria in this study. The duration of not protected by contraceptive was carried out indirectly from calculating the duration of marriage, duration of contraceptive use, and pregnancy duration. This is to avoid the period before marriage within five years preceding the survey taken into account in the measurement.\n\nDuration of contraceptive use is calculated by adding up the person months of contraceptive use, either modern or traditional, over a period of 69 months for each individual. Pregnancy events, live births, stillbirths, abortions and duration of marriage were calculated as the duration of pregnancy in the same way as duration of contraceptive use. The formula to calculate the duration of not protected by contraceptive use is obtained as follows:\n\nBased on the formula, the calculation of unmet need with contraceptive calendar data is as shown below:\n\nDifferences between calculation of unmet need at survey time and with contraceptive calendar data are explained on Table 1.\n\n\nResult\n\nTable 2 shows that the unmet need at survey time among married women was 10.5%, lower than unmet need with calendar data (13.1%). This difference of about 3% concerns almost 1.6 million additional women at risk of unwanted pregnancy. The magnitue of the difference depends on the contraceptive prevalence rate, method mix, and discontinuation rate in the coutry at the time of the survey.\n\nIt should be noted that measurements of unmet need in the field are dynamic. Reproductive intentions as part of the calculation of unmet need are known to be unstable over the long-term, even in the span of few weeks. Fertility intentions were also unstable among a reference group of family planning users.15 This study did not assess the status of contraceptive use before pregnancy and treated the duration of pregnancy as not to have unmet need. Treating women who are pregnant or postpartum amenorrheic as not in need of family planning decreases the level of unmet need. Intendedness of pregnancies are likely to be biased as pregnant women don’t generally view themselves as having unmet need, regardless whether their pregnancy was accidental.\n\nAnother finding (Figure 2) shows that average duration of not protected by contraceptive use is 30.6% meaning that unwanted pregnancy might occur in one-third of the marriage period. Only one-third of respondents used contraception in the full observation period (69 months), and 13.6% of respondents have never used contraception at all.\n\n\nDiscussion\n\nUnmet need for family planning in the IDHS report was measured using point prevalence whereas in this study, it was measured using period prevalence. This aims to focus on the duration of not protected by contraceptives and at risk of unwanted pregnancy. Previous studies suggest that the measurement for standard unmet need does not reflect the percentage of women at risk of unintended pregnancy who are not using contraception, and a complementary measure of unmet need will be helpful to identify the target population in need of contraceptive services. Another research modified unmet need measurement by removing the categories of women included in standard unmet need calculation by limiting the population study to age 44 years, removing permanent (female and male sterilization) family planning users, infecund women, currently pregnant women who wanted the pregnancy, and non-pregnant women who want a child within two years.16,17\n\nThis study show that the unmet need estimated at survey-time is lower than unmet need with calendar data which illustrates the risk of unwanted pregnancy more accurately in a population, but calendar data does have the potential for under-reporting and recall bias. On the other hand, the under-reported potential might show that the unmet need for family planning in the population is much higher than the results of this study.14\n\nThe measurement of unmet need with calendar data is more appropriate than standard methods in countries where contraceptive discontinuation rates are still high. This is indicated by the higher value of unmet need proportion with calendar data compared to the standard method. The study prove that contraceptive discontinuation rates are directly proportional to unmet needs using the calendar method. The results found that nearly 1.6 million additional married women in Indonesia are at risk of experiencing unwanted pregnancy.\n\nThe calculation of the duration of not protected by contraceptives was measured in women who did not use contraception in a period of 69 months based on the status of unmet need at the time of the survey, while the status of no need at the time of the survey was used to calculate the duration of not using contraception for women who wanted to become pregnant within 69 months. The duration of no need is not included as the numerator for calculating the unmet need with the calendar method. Calculation of duration with no need status or wanting to get pregnant in the study was calculated as 35% (duration not protected by contraception because you want to get pregnant divided by the total duration not protected by contraception). This result is not much different from the 2017 IDHS report which explains the contraceptive discontinuation with ‘wanted to get pregnant’ reason is 30%, while 70% do not want to get pregnant. Thus, the assumption of contraceptive use status during the survey, which is then used as the basis for calculating the duration of unprotected contraception, is acceptable.\n\nPrevious research stated that health reproductive age couples who had frequent unprotected sexual intercourse have a 20% chance of getting pregnant in one cycle.18 The pregnancy rates increase over time, namely 57% of women will become pregnant in the first three months and then rates increase 15% in the next three months. Another study showed that 30% of women became pregnant in first month, 50% pregnancies occuring within two months, and 80% of pregnancies occuring within six months, and for couples who do not use contraceptive method within 12 months, 85% are at risk of pregnancy.19,20 This study showed that 35% of women experienced discontinuation and were at risk of unwanted pregnancy. A previous study described cumulative pregnancy rate for one year after contraceptive discontinuation as 72-85% for IUDs, 75-81% for pills, 72-76% for implants, and 65-67% for injectables.21\n\nBased on the above findings, it is necessary for the government of Indonesia to equip primary health care services in a way which is responsive to unwanted pregnancies. The 2019 Health Facilitiy Research show that Indonesia only has 30.4% Puskesmas with Basic Obstetric and Neonatal Emergency Service (PONED) with Banten (59.7%) for the highest and West Papua (9.6%) for the lowest.22 Policy adjustments are needed in order to anticipate the low demand satisfied in Indonesia.\n\n\nConclusion\n\nUnmet need of family planning is a crucial measure to assess the achievement of family planning programmes. The results show that for Indonesia context with a relatively high contraceptive discontinuation rate, the measurement of unmet need for family planning need adjusment, where using contraceptive calendar data for five years preceding the survey is more appropriate than at survey time. The results also showed that the average percentage of duration not using contraception was one-third of marriage period and at risk of unwanted pregnancies. Policy makers such as National Family Planning Coordination Board (BKKBN) and Ministry of Health need to adjust the calculation of demand satisfied considering the high contraceptive discontinuation in Indonesia. They also need to equip primary health care services with PONED in order to be responsive for unwanted pregnancy.\n\n\nDeclarations\n\nThis article is part of a dissertation result that has received approval from the Ethics Comission for Research and Community Health Service, Faculty of Public Health, Universitas Indonesia, Letter Number 670/UN2.F10/PPM.00.02/2019, on November 13th, 2019.\n\nThe underlying data for this study is owned y the DHS Program (https://dhsprogram.com/methodology/survey/survey-display-522.cfm). The electronic data is available from the DHS Program under its terms of use (https://dhsprogram.com/Data/terms-of-use.cfm) . Before downloading the data, users must register as a DHS user for reasons laid out on the DHS Program website (https://www.dhsprogram.com/data/Registration-Rationale.cfm) and dataset access is only granted for legitimate research purposes.\n\nAuthor 1 (BU) – Conceived of the presented idea, developed the theory and supervised the findings of this work.\n\nAuthor 2 (H) – Carried out the analysis and wrote the manuscript.\n\nAuthor 3 (SBP) – Verified the analytical methods and supervised the findings of this study.\n\nAuthor 4 (RM) – Verified the analytical methods and supervised the findings of this study.\n\nAuthor 5 (SR) – Together with Author 2, wrote the manuscript with input from all authors.", "appendix": "Acknowledgment\n\nI would like to thank the Poltekkes Ministry of Health Jakarta I for providing assistance and support in completing this research and FPH UI as a place to study and gain knowledge so that this research was completed properly on time.\n\n\nReferences\n\nBradley SEK, Croft TN, Fishel JD: Revising Unmet Need for Family Planning: DHS Analytical Studies No. 25.2012; 25(January): 63.\n\nSedgh G, Singh S, Hussaina R: Intended and Unintended Pregnancies Worldwide in 2012 and Recent Trends. Stud Fam Plann. 2014; 45(3): 301–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUNFPA: Choices not Chance- UNFPA Family Planning Strategy 2012-2020. Unfpa. 2012.\n\nBKKBN, BPS, Kemenkes, ICF: Survei Demografi dan Kesehatan Indonesia 2017.2018.\n\nWilopo SA: Hasil Konferensi Kependudukan di Kairo: Implikasinya pada Program Kesehatan Reproduski di Indonesia. Populasi. 1994; 5(2): 1–29. Publisher Full Text\n\nBKKBN, BAPPENAS, Kemenkes, UNFPA, CANADA: Program Keluarga Berencana Berbasis Hak untuk Percepatan Akses terhadap Pelayanan Keluarga Berencana dan Kesehatan Reproduksi yang Terintegrasi dalam Mencapai Tujuan Pembangunan Indonesia.2012.\n\nBKKBN: Tantangan Keluarga Berencana Dan Kesehatan Reproduksi Menjadi Pembahasan Utama Pada Konferensi Internasional. SIARAN PERS No. RILIS/103/B4/BKKBN/IX/2019. 2019.\n\nCentral Bureau of Statistic, National Family Planning Coordinating Board M of H: Indonesia 1991 DHS Summary Report (English).pdf. 1991.\n\nBPS CB of S. 1994 SR.pdf.1994.\n\nBPS CB of S. DHS 1997.pdf.1997.\n\nBadan Pusat Statistik BK dan KB: Laporan Survei Demografi dan Kesehatan Indonesia 2007.2008;\n\nBadan Pusat Statistik, Badan Koordinasi Keluarga Berencanan Nasional, Departemen Kesehatan, Macro International: Survei Demografi dan Kesehatan Indonesia 2012. Sdki. 2013: 16.\n\nBKKBN, Badan Pusat Statistik, Kementerian Kesehatan, IFC International: Survei Demografi dan Kesehatan Indonesia 2017. Sdki. 2017: 16.\n\nUSAID: DHS Contraceptive Calendar Tutorial.2018. 4–48 p.\n\nStaveteig S: Understanding unmet need in Ghana: Results from a follow-up study to the 2014 Ghana Demographic and Health Survey. DHS Qual Res Stud No 20 [Internet]. 2016; (January). Reference Source\n\nBradley SE, Casterline JB: Understanding Unmet Need: History, Theory, and Measurement. Stud Fam Plann. 2014; 45(2): 123–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoreau C, Shankar M, Helleringer S, et al.: Measuring unmet need for contraception as a point prevalence. BMJ Glob Heal. 2019; 4(4): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMakar RS, Toth TL: The Evaluation of infertility. Hum Reprod Update. 2002; 1(6): 586–606. PubMed Abstract \| Publisher Full Text\n\nJoffe M: Time to pregnancy: A measure of reproductive function in either sex. Occup Environ Med. 1997; 54(5): 289–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHatcher RA, Trussell J, Nelson AL, et al.: Contraceptive Technology. reproducti. ob/gyns, reproductive medicine MDs and primary care physicians, editor.2007; 874 p.\n\nGayatri M: Menepis Mitos Kemandulan Akibat Kontrasepsi: Analisis Kesintasan Data Kalender Kontrasepsi Dan Kehamilan SDKI 2007, 2012 Dan 2017.Universitas Indonesia; 2019.\n\nKemenkes RI: Riset Fasilitas Kesehatan Rifaskes 2019.2020." }	[ { "id": "98880", "date": "17 Nov 2021", "name": "Ilene S Speizer", "expertise": [ "Reviewer Expertise Demography", "fertility", "family planning", "unintended pregnancy 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 study presents a novel measure of unmet need using 5-year calendar data. The authors demonstrate that the standard survey-time unmet need underestimates overall unmet need in Indonesia. While I find this to be an intriguing study, I did have a number of major and minor comments.\nMajor comments:\nThe authors state that unmet need between 1991 and 2017 is stagnant while discontinuation is on the rise. The values for unmet need and discontinuation over time are not presented nor is contraceptive use and method mix provided. This left me wondering if discontinuation is on the rise because of a change in method mix (i.e., transition from IUD to implants where side effects may be more common and causing discontinuation). Also, given that in 2017 unmet need is 10.5% (shown in Table 2), it is possible that it will not decline much more given that it is a research (and not individual) defined measure and women may choose not to use.\n\nFrom my understanding of the approach to calculate the novel measure, any period when a woman is in union and not pregnant and not using has an unmet need. The authors argue that fertility intentions are fluid and thus not reliable and thus not part of the calculation; however, intentions are used for the “duration with no need measure” it seems. This seems inconsistent. Also, what time point is used for the fertility intentions in that measure – is it at the time of the current survey and then applied retrospectively? Further, in Table 2 you indicate that women do not want children (or want to delay) in the unmet need with calendar data measure presented on the right – so where does the information on fertility desires come from (and what time period)?\n\nThe rationale for this measure is not completely clear. Unmet need in its current form is already hypothetical and meant to be used at the aggregate and not individual level. As the authors rightly point out, there are flaws with unmet need including that it treats pregnant women with an unintended pregnancy as having an unmet need and does not reflect women’s desire or intentions to use a method. But their hypothetical measure has the same problems, and others, so why develop a measure that requires a lot of data to capture (including calendar data that is not always obtained and has known flaws) and does not fix the identified problems.\nMinor comments:\nYou say that your sample is currently married women (and you give a specific number of women). My guess is that this is the number of women at the time of the survey but since the analysis is by episodes and some women may have been married earlier (and not married at the time of the survey), I wasn’t sure what the number of women was representing.\n\nThere are a lot of statements about “unwanted pregnancy” prevention and that the new measure demonstrates more women at risk of unintended pregnancy. However, this assumes that all women who are non-users want to delay or avoid childbearing and that they intend to use contraception. Many women may choose not to use contraception and then might not declare a subsequent pregnancy as unintended.\n\nDetails are provided on obstetric and neonatal care services but not on access to family planning services and provision of a full range of methods. Perhaps this oversight is because 100% of facilities offer family planning which would be great. But it is not clear why recommendations are made about obstetric and neonatal care based on this analysis.\n\nCYP is couple years of protection (and not contraceptive years of protection).\n\nThe last sentence of the first paragraph of the results “The magnitude of the differences…” This is a key point, but no information is given on these key issues (see major point above).\n\nThe first paragraph of the discussion says that the new method is aiming to determine the duration of not protected by contraception and at risk of unwanted pregnancy, but this assumes that fertility desires are measured. It is not clear where fertility desires are coming from here.\n\nThe paragraph on the calculation of the duration not protected in the discussion lost me. It says it is based on the status of unmet need at the time of the survey and also mentions focusing on women who wanted to become pregnant within 69 months. I read the paragraph multiple times and am still not sure what it is demonstrating and how fertility intentions are being used (from the time of the survey and applied retrospectively?).\n\nThe grammar throughout this paper needs to be checked.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "98878", "date": "17 Nov 2021", "name": "Siswanto Agus Wilopo", "expertise": [ "Reviewer Expertise Reproductive health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author stated that: “the achievement of family planning programs in Indonesia based on the 1991 to 2017 DHS shows that the unmet needs are in stagnant trend in last two decades while contraceptive discontinuation has increased.” This statement is the reason to develop a new method of unmeet-need calculation. The existing unmet need is based on the prevalence (%) calculation while their method is based on a rate of person month calculation (rate from cohort observation). Certainly, we can have difference results due to these calculations. The unmet need calculation includes traditional method which result of low unmet need for Indonesia. Will the result show a similar unmet need when calculation is limited to modern of contraceptives method? Moreover, their model did not explain impact of the birth termination due to abortion. This information will influence the duration of not using contraception which ultimately to an unmet need. Authors need to explain why two methods are not giving same results.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-780
https://f1000research.com/articles/10-779/v1	09 Aug 21	{ "type": "Case Report", "title": "Case Report: Case report: Mixed infection of Plasmodium vivax and Plasmodium falciparum in a tertiary hospital", "authors": [ "Abeer M. Al-Subaie" ], "abstract": "Mixed infections with two or more species of Plasmodium are frequently reported due to vector factors, parasite factors (formation of hypnozoites) and host factors (residing in endemic areas, travel to endemic areas, inadequately treated previous infection, lack of compliance to therapy). Here we report a case of a 33-year-old Saudi female who had a significant travel history, and a peripheral blood smear (PBS) revealed mixed infection with P. falciparum and P. vivax. The case was successfully treated with a combination therapy of artemisinin and primaquine with follow up testing at three, seven, 14, and 28 days. Mixed malaria infections are especially reported in travelers to endemic areas. Hence, adequate diagnosis and appropriate treatment of the cases contributes majorly to preventing relapse and controlling the disease. Travel consultations should be given to all travelers before their trips to endemic countries.", "keywords": [ "Malaria", "Falciparum malaria", "Plasmodium vivax", "Plasmodium falciparum", "case report" ], "content": "Introduction\n\nMalaria is a vector-borne disease caused by protozoan parasites belonging to genus Plasmodium, and is considered to be one of the most important parasitic infections throughout the world, resulting in high mortality rates1. There were previously four known species responsible for human malaria: P. falciparum, P. vivax, P. ovale, P. malariae. Recently, it was discovered that there is also a fifth species, known as P. knowlesi2. P. falciparum and P. vivax have the widest distribution, while P. malariae has a very low prevalence3.\n\nThe Kingdom of Saudi Arabia (KSA) has reported 61 and 38 indigenous malaria cases in the years 2018 and 2019 respectively (World Health Organization (WHO), World Malaria Report 2020). The indigenous cases in KSA have decreased drastically over the years (272 cases in 2016 to 38 in 2019) due to the implementation of the National Malaria Control Program (NMCP), the establishment of mobile units with specific focus on undocumented migrants from neighboring countries where diagnosis, treatment and medical care are provided free of charge to all imported cases4 (WHO, 2020).\n\nThe majority of malaria infection cases in KSA are due to P. falciparum (97%) followed by P. vivax (2%). P. falciparum causes malignant tertian malaria occasionally associated with severe complications in infected individuals. Another concern with P. falciparum infection is recrudescence, resulting in treatment failure and emergence of antimalarial drug resistance to chloroquine (WHO, 2020).\n\n\nCase report\n\nA 33-year-old Saudi female, Eastern region resident was admitted to the ‘Infectious Diseases’ ward of our teaching hospital King Fahad Hospital of the University, Imam AbdulRahman bin Faisal University with ‘fever of other and unknown origin’. The patient presented with a seven-day fever with chills and complained of decreased appetite and generalized body pain associated with dark urine, pale stool and diarrhea, with history of fever with chills and generalized body pains for one week. On taking detailed history, the patient stated that she had recently returned from a 20-day trip to the Ivory Coast. The patient didn’t have any form of comorbidities and denied any history of animal contact, immunocompromised status, or intake of any kind of prescribed or illicit drugs or raw milk ingestion during this period. Family history of the patient revealed absence of hereditary blood diseases and was of insignificant relation to the patient’s clinical presentation. The patient did not seek any medical facility prior her admission to our hospital and has only self-administrated paracetamol tablets in response to the fever and body aches.\n\nOn recording the vital signs (measurement of body temperature, weight, blood pressure and pulse rate), the patient was febrile with an oral temperature of 39.9°C, tachycardic with a pulse rate of 118 beats/minute, blood pressure of 135/85 mmHg, and respiratory rate of 20 breaths/minute.\n\nUpon physical examination, the patient appeared ill and had pale conjunctivae. Lymphadenopathy was noted in the left axillary (1×1 cm and 0.5 × 1.5cm) and left inguinal region (0.5 × 2cm). Skin appeared normal and there were no petechiae. The results of hematological testing revealed the patient had normocytic normochromic anemia, pancytopenia (Table 1), and liver function test (LFT) were borderline with significantly high levels of lactate dehydrogenase indicating an active haemolysis (Table 2). The peripheral blood smear (PBS) depicted P. falciparum gametocytes (crescent) and P. vivax gametocytes (of ameboid shape with Schüffner's dots). PBS was then repeated on three, seven, 14, and 28 days. C-reactive protein (CRP) plasma level and erythrocyte sedimentation rate (ESR), which are both inflammatory blood markers that indicate an ongoing infection, were higher than the normal levels (Table 3). Serological testing included measuring the antibody levels (both their Immunoglobulin (Ig) IgG and IgM forms) for Epstein–Barr virus (EBV), and Cytomegalovirus (CMV). The patient’s results were positive for both EBV IgG and CMV IgG (Table 4). It is worth mentioning that there were no challenges encountered either in clinically examining the patient or getting access to the laboratory diagnostic tests.\n\nThe patient was treated as follows: oral doxycycline 100 mg once a day, artesunate 200mg orally once a day for three days, and sulfadoxine and pyrimethamine 1500mg + 75mg single dose orally on day one. On the third day, after excluding glucose-6-phophate dehydrogenase (G6PD) deficiency by measuring the G6PD enzyme level, oral primaquine 30 mg/day for three days was added to the treatment regimen. After seven days of the treatment, the patient's symptoms had resolved; the PBS was repeated and revealed the absence of morphological forms of both parasites along with normal CBC and LFT results. Treatment and care provided to the patient was free of any financial charges. During the treatment course the patient did not develop any adverse effects due to the administrated medications. Figure 1 summarizes the timeline of the case management course.\n\nCBC Complete blood count; LFT Liver function test; LDH Lactate Dehydrogenase; ESR Erythrocyte sedimentation rate; CRP C-reactive protein; PBS Peripheral blood smear.\n\n\nDiscussion\n\nSeveral studies conducted worldwide and in KSA report that malaria mixed infections are not uncommon5–8. The majority of malaria studies in KSA were studies conducted in the Southwest area and included cases of mixed P.falciparum and P.vivax infections9–11. However, in the Eastern province of KSA where this case is reported, there have been few cases of single or mixed infection malaria reported due to effective control measures which resulted in the termination of malaria transmission11.\n\nMixed infections in malaria can be attributed to one or more of four reasons: several species of Anopheles can carry more than one species of the parasite; P.vivax can remain as hypnozoites in the liver, which can be cause a relapse; recrudescence in P.falciparum infection due to drug resistant forms; and incomplete treatment of a previous infection12.\n\nMicroscopic examination of PBS for the morphological forms of the malarial parasite remains the gold standard for laboratory confirmation of the infection11. However, making a diagnosis of malaria mixed infection based on a PBS microscopic diagnosis is challenging. This difficulty can be due to: low levels of parasitemia; altered morphology of the parasite due to self-treatment; or the similarity in Plasmodium species, e.g. P. knowlesi is often misdiagnosed as P. malariae or P. falciparum12.\n\nSeveral tests can be used in conjunction with the PBS such as rapid diagnostic tests (RDT), antibody detection using indirect fluorescent antibody tests (IFA), and polymerase chain reaction (PCR) to identify the infection. However, all these methods have their own limitations. The RDT cannot detect low level parasitemia while IFA is time consuming, subjective, requires fluorescence microscopy and a trained observer13. However, molecular tests such as PCR can assist in definitive species determination and also can detect low level parasitemia (about one parasite/μL of blood) (Centers for Disease Control and Prevention)13. It was not possible to perform a PCR test in the time this patient was diagnosed in our hospital due to the unavailability of the test in our laboratory, which was a limitation in the diagnostic approach.\n\nThe main strategy in treatment of uncomplicated malaria consists of oral therapy with a combination of two agents, the artemisinin and a partner drug that eliminates the remaining parasites, (in the case of chloroquine resistance) or chloroquine monotherapy (in the case of chloroquine sensitivity) (WHO, 2020). Treatment for chloroquine-resistant P. falciparum should be administered in the setting of known exposure to a chloroquine-resistant region, unknown prevalence of chloroquine resistance, or uncertain exposure history. As a result, artemisinin-based combination therapies (ACT) have become the first line of treatment for P. falciparum and mixed malaria infections. The ACT regimens have few side effects and are cidal to all the asexual stages of the parasite in the blood, causing rapid clearance of the infection14.\n\nIn this reported case, the patient received an ACT regimen comprising artesunate, sulfadoxine and pyrimethamine. Doxycycline was also chosen as it has high patient compliance and can be given once daily, and the seven-day course of these tablets finishes after the ACT regimen. Primaquine is given in malarial mixed infections along with ACT as it is the only drug recommended to eliminate the intrahepatic forms (hypnozoites) of P. vivax and gametocytes of P. falciparum. However, the patient’s G6PD levels should be tested to prevent primaquine-induced hemolysis as a phenolic metabolite, 5-hydroxyprimaquine (5-HPQ), mediates primaquine hemotoxicity by generating reactive oxygen species (ROS) within erythrocytes that overwhelm antioxidant defenses15,16. Supportive care should be given to the patient to prevent morbidity and mortality. In addition, inappropriate combinations and inadequate dosing of drugs should be avoided to prevent drug resistance and relapse (WHO, 2021). As there is no available malarial vaccine, chemoprophylaxis can be administered to travelers before visiting the endemic areas according to (WHO, 2021).\n\nOverall, this reported case has one limitation at the diagnostic level: the unavailability of the quantitative real-time PCR (q-PCR) test to confirm the identification of both the malaria species and to measure the level of the parasitemia, since q-PCR is more reliable in detecting mixed infection at levels of low parasitemia9. Although it would have been an added value in terms of confirmation, q-PCR unavailability did not affect the proper diagnosis. The accuracy in the PBS examination enabled the identification of both malaria species which is considered one of the strengths of this case. Another strength of this reported case is the timely, appropriate management plan. The patient responded well with the treatment without any side effects and the parasitemia was resolved in due time.\n\nTo conclude, malarial infection can be controlled with different measures taken at different levels: appropriate vector control measures; early case detection; timely management; entomological monitoring; population movement monitoring and surveillance; reporting of cases; and public health education strategies.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of patient’s clinical details was obtained from the patient.", "appendix": "References\n\nKamareddine L: The biological control of the malaria vector. Toxins (Basel). 2012; 4(9): 748–767. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntinori S, Galimberti L, Milazzo L, et al.: Biology of human malaria plasmodia including Plasmodium knowlesi. Mediterr J Hematol Infect Dis. 2012; 4(1): e2012013. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAshley EA, Phyo AP, Woodrow CJ: Malaria. Lancet. 2018; 391(10130): 1608–1621. PubMed Abstract \| Publisher Full Text\n\nAl Zahrani MH, Omar AI, Abdoon AMO, et al.: Cross-border movement, economic development and malaria elimination in the Kingdom of Saudi Arabia. BMC Med. 2018; 16(1): 98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGenton B, D'Acremont V, Rare L, et al.: Plasmodium vivax and mixed infections are associated with severe malaria in children: a prospective cohort study from Papua New Guinea. PLoS Med. 2008; 5(6): e127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan W, Zakai HA, Umm-E-Asma: Clinico-pathological studies of Plasmodium falciparum and Plasmodium vivax - malaria in India and Saudi Arabia. Acta Parasitol. 2014; 59(2): 206–12. PubMed Abstract \| Publisher Full Text\n\nAmer OS, Waly MI, Burhan IW, et al.: Epidemiological trends of malaria in the Western regions of Saudi Arabia: a cross sectional study. J Infect Dev Ctries. 2020; 14(11): 1332–1337. PubMed Abstract \| Publisher Full Text\n\nDarraj MA: Clinical Profile of Severe Plasmodium falciparum and P. vivax Malaria in Jazan Region, Saudi Arabia. Mal J Med Health Sci. 2020; 16(4): 73–80. Reference Source\n\nBin Dajem SM: Molecular investigation of mixed malaria infections in southwest Saudi Arabia. Saudi Med J. 2015; 36(2): 248–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHawash Y, Ismail K, Alsharif K, et al.: Malaria Prevalence in a Low Transmission Area, Jazan District of Southwestern Saudi Arabia. Korean J Parasitol. 2019; 57(3): 233–242. PubMed Abstract \| Free Full Text\n\nSoliman RH, Garcia-Aranda P, Elzagawy SM, et al.: Imported and autochthonous malaria in West Saudi Arabia: results from a reference hospital. Malar J. 2018; 17(1): 286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFontenille D, Lepers JP, Coluzzi M, et al.: Malaria transmission and vector biology on Sainte Marie Island, Madagascar. J Med Entomol. 1992; 29(2): 197–202. PubMed Abstract \| Publisher Full Text\n\nTalapko J, Škrlec I, Alebić T, et al.: Malaria: The Past and the Present. Microorganisms. 2019; 7(6): E179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNewton P, White N: Malaria: new developments in treatment and prevention. Annu Rev Med. 1999; 50: 179–92. PubMed Abstract \| Publisher Full Text\n\nRecht J, Ashley EA, White NJ: Use of primaquine and glucose-6-phosphate dehydrogenase deficiency testing: Divergent policies and practices in malaria endemic countries. PLoS Negl Trop Dis. 2018; 12(4): e0006230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBowman ZS, Morrow JD, Jollow DJ, et al.: Primaquine-induced hemolytic anemia: role of membrane lipid peroxidation and cytoskeletal protein alterations in the hemotoxicity of 5-hydroxyprimaquine. J Pharmacol Exp Ther. 2005; 314(2): 838–45. PubMed Abstract \| Publisher Full Text" }	[ { "id": "91522", "date": "01 Sep 2021", "name": "Mira El Chaar", "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 case study presented by the authors is well elaborated. The authors describe in detail the sequence of the clinical outcome of a patient who was admitted to the hospital with plasmodium species coinfections.\nThe Sequence of the case is well presented including the background, the method of detection, the result, and the discussion. The authors have tackled the limitation of this study in particular the absence of real-time PCR to study the parasitaemia level in the patient which is very important.\nThe article needs minor changes for indexing including the following:\nAll laboratory tests and markers could be presented in one table instead of 4 tables. If the authors prefer to keep it as such, table 4 should be deleted.\n\nThe authors should mention the year when the patient was admitted to the hospital in the result part, although it is included in figure 1.\n\nIt is worth mentioning in the discussion about the prevalence of coinfection of plasmodium species at the regional and international levels.\n\nThe authors should mention in the discussion if coinfection can increase the severity of the disease.\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": "92549", "date": "21 Sep 2021", "name": "Ali M. Gabali", "expertise": [ "Reviewer Expertise Immunology", "hematology and hematopathology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n`The paper reviewed presented a Mixed infection of Plasmodium vivax and Plasmodium falciparum in a tertiary hospital. The authors discussed thoroughly the clinical presentation of concomitant Plasmodium vivax and falciparum infection in this case and the clinical course and modality of therapy used. The case is addressing an important issue; the presentation and clinical course are largely well explained and appropriately used and the paper is well written. My conclusion is to approve the paper for indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-779
https://f1000research.com/articles/9-316/v1	30 Apr 20	{ "type": "Research Article", "title": "Augmented ustekinumab dosing is needed to achieve clinical response in patients with anti-TNF refractory pediatric Crohn’s disease: a retrospective chart review", "authors": [ "Phinga Do", "John Andersen", "Ashish Patel", "Gaith Semrin", "Luis Sifuentes-Dominguez", "Phuong Luu", "Bhaskar Gurram", "John Andersen", "Ashish Patel", "Gaith Semrin", "Luis Sifuentes-Dominguez", "Phuong Luu", "Bhaskar Gurram" ], "abstract": "Background: Ustekinumab is a monoclonal antibody that inhibits interleukins 12 and 23. It is approved for treatment of Crohn’s disease (CD) in adults; however, there is a paucity of data regarding its use in pediatric CD. We describe our experience using ustekinumab in anti-TNF refractory CD pediatric patients. Methods: We performed a retrospective chart review on pediatric patients with CD who were started on ustekinumab from January 2016 to November 2018. We collected patient’s clinical history, previous treatment history, surgeries related to CD, disease severity, as measured by abbrPCDAI, and endoscopic severity as recorded by SES-CD before and after ustekinumab. Results: We identified 10 patients with CD who were started on ustekinumab due to non-response to currently approved agents. Seven patients needed augmented maintenance dosing every 4-6 weeks to achieve clinical response or remission. Six of these seven patients had therapeutic drug monitoring during the course of treatment, with five patients showing subtherapeutic drug levels of <4.5 μg/mL while on standard maintenance dosing every 8 weeks, and four patients showing therapeutic drug levels of >4.5 μg/mL on augmented dosing interval. The remaining three patients were on standard maintenance dosing for the duration of treatment. Conclusion: In this retrospective chart review, 7 out of 10 patients with anti-TNF refractory pediatric-onset CD required augmented maintenance doses of ustekinumab to achieve clinical response or remission. A prospective study is needed to define appropriate ustekinumab dosing and interval in management of pediatric CD.", "keywords": [ "Ustekinumab", "Pediatric Crohn's disease", "anti-TNF-refractory Crohn's disease", "Inflammatory bowel disease", "Therapeutic drug monitoring", "Clinical response" ], "content": "Introduction\n\nTogether, ulcerative colitis and Crohn’s disease (CD) make up inflammatory bowel disease (IBD), an autoimmune-mediated process of unclear etiology. The global incidence of pediatric IBD has been rising rapidly, with the highest incidence of CD being in Europe at 23/100000 person years and North America at 15.2/100000 person years1–3. Earlier onset of IBD is associated with higher impact on growth and development, more aggressive disease course, and increased need for immunomodulators4. Anti-tumor necrosis factors (anti-TNFs) form the forefront of management of patients with CD who do not respond to steroids and immunomodulatory medications5.\n\nAmong pediatric patients with CD who are started on anti-TNF treatments, about 10–25% do not respond to it (primary non-responders)6. Of those who initially respond, loss of response and adverse effects limit duration of therapy. At 1, 3, and 5 years after therapy initiation, the probability of patients remaining on infliximab is only 0.87, 0.74, and 0.67, respectively (secondary non-responders)7. Thus, there is a significant need for novel therapies for management of CD.\n\nAmong the newer biologics approved for treatment of CD is ustekinumab, a human immunoglobulin G1 kappa monoclonal antibody that binds with high affinity to the p40 subunit of human interleukin (IL)-12 and IL23. Ustekinumab prevents IL12 and IL23 bioactivity by preventing their interaction with their cell surface receptor protein IL12Rb1. Through this mechanism of action, ustekinumab effectively neutralizes IL12 (Th1)- and IL23 (Th17)-mediated cellular responses. It has recently been approved for the treatment of moderate to severe active CD in adults8. However, data on usage of ustekinumab in management of pediatric Crohn’s disease is limited to small case series9–11. Here we describe our experience on using ustekinumab for management of TNF-refractory pediatric CD.\n\n\nMethods\n\nWe performed a retrospective chart review on 10 pediatric CD patients who failed anti-TNF therapy and were treated with ustekinumab between January 2016 and November 2018.\n\nThis study was approved by the Institutional Review Board (IRB) of Dallas Children’s Hospital (study #25338). Request for waiver of patient/guardian consent for this study was approved by the IRB.\n\nWe collected baseline demographic data, disease phenotype based on Paris Classification12, disease related complications, previous treatment history, and reason for changing therapy.\n\nTo assess clinical response to ustekinumab, we calculated the Abbreviated Pediatric Crohn’s Disease Activity Index (abbrPCDAI) prior to starting therapy, 2–3 months after therapy initiation, and at the last office visit before conclusion of the study13. When no office visits were available immediately prior to treatment initiation, telephone and email encounters were used to assess patients’ clinical symptoms to calculate abbrPCDAI. Where possible we also calculated the Simple Endoscopic Score for Crohn’s Disease (SES-CD) before and after treatment initiation14. Body Mass Index (BMI) before and after treatment was collected.\n\nLaboratory measurements, which include hematocrit, C-reactive protein (CRP), and albumin, were also collected before and after treatment initiation. We also looked at the trough ustekinumab levels where available in relation to dose and response to therapy.\n\nPatients are categorized as anti-TNF primary non-responders if there’s no clinical response during therapy induction, and secondary non-responders if there’s loss of response during maintenance phase15. Based on abbrPCDAI, clinical response is defined as ≥15 points reduction, and clinical remission is defined as <10. We define sustained clinical remission as abbrPCDAI of <10 with no subsequent elevation in AbbrPCDAI as of the last visit. Disease severity is categorized as follows: severe <25; moderate 16-25; mild <16. We use the following SES-CD cutoff to define disease severity: remission 0-2; mild 3-6; moderate 7-15; and severe >1616. Endoscopic response is defined as ≥50% decrease in SES-CD score compared to baseline17. Based on previous studies we used a target ustekinumab trough level of >4.5 µg/mL18.\n\n\nResults\n\nPatients’ age at initial diagnosis ranged from 2 to 14 years (median age of 9.5 years). Age at initiation of ustekinumab ranged from 9 to 19 years (median age of 14.5 years). Duration of disease ranged from 3 to 14 years (median duration of 6.5 years). Table 1 summarizes patients’ demographic, disease phenotype at diagnosis, extraintestinal manifestations, disease related surgeries, treatment history, and reasons for changing therapy to ustekinumab. Of note, all 10 patients in our cohort were refractory to anti-TNF therapy.\n\nEIM- extra intestinal manifestations.\n\nTable 2 summarizes the ustekinumab induction and maintenance dose used in these patients. For induction, the dosing varied among patients with 7 out of 10 receiving induction doses per current recommendations: patients with weight <55kg received either 6mg/kg or 260mg, 55-85kg received 390mg, and >85kg received 520mg. For the remaining three patients, one received 2 doses of 45mg every 4 weeks (Q4) for induction; the second patient was induced on two separate times, 1.5 years apart, he received 90mg the first induction and 390mg for the second; the third patient was induced with 90mg Q4 for 3 doses.\n\n* Ustekinumab was reintroduced to patient 6 two years later.\n\nFor maintenance, 6 of the 10 patients received 90mg every 8 weeks (Q8), while 3 patients received 90mg every 6 weeks (Q6), and 1 patient received 45mg Q8. One patient was on ustekinumab on two separate occasions, the first time 90mg Q4 for two doses, and the second time 90mg Q4, then Q8 once therapeutic level and remission were achieved. Subsequently, two patients required frequency escalation to Q4 weeks, and one of them went back to Q6 after she went into remission. One patient required escalation to 45mg Q5, and then back to 90mg Q8 when disease was controlled. Another patient was maintained on Q8 for 32 months before he relapsed and required increase in dosing frequency to Q6. Maintenance frequency was titrated based on clinical response and/or ustekinumab trough level. Duration of therapy ranges from 4–135 weeks (median 61 weeks).\n\nOf the seven patients who received augmented maintenance doses, all seven showed clinical response, as shown in Figure 1 (patients 1-7), and all but one patient achieved sustained clinical remission as assessed by abbrPCDAI. One patient achieved remission after 5 months of specific carbohydrate diet (SCD) and ustekinumab at Q8 maintenance. He remained in remission for the next 17 months, then developed fatigue and bloody stool when diet was liberalized. His symptoms did not respond to reintroduction of SCD. However, he went into clinical remission when ustekinumab maintenance interval was changed to Q6 weeks.\n\nAbbreviated-pediatric Crohn’s disease activity index in patients with (A) augmented ustekinumab dosing and (B) Q8 dosing; (C) simple endoscopic score-Crohn’s disease (SES-CD) pre and post-ustekinumab initiation.\n\nOnly 4 out of 10 patients had endoscopy before and after ustekinumab treatment. Of these, three patients showed endoscopic response and one showed worsening of SES-CD score (Figure 1C). While no patient showed mucosal remission, mucosal inflammation did improve from severe to mild, severe to moderate, and moderate to mild in three patients.\n\nLaboratory indices also improved in 6 out of 7 patients (Figure 2). The most significant and consistent improvements were seen in CRP and albumin (Figure 2C–F). BMI improved significantly in patients with pre-treatment BMI below the 2nd percentile, and either decreased or showed small numerical improvement in patients with pre-treatment BMI above the 15th percentile (Figure 2G,H).\n\nHematocrit in patients with (A) augmented dosing and (B) Q8 dosing; CRP in patients with (C) augmented dosing and (D) Q8 dosing; albumin in patients with (E) augmented dosing and (F) Q8 dosing; BMI in patients with (G) augmented dosing and (H) Q8 dosing.\n\nAmong the seven patients, five had ustekinumab trough level showing low or undetectable drug level when receiving medication at a 6 or 8 week intervals (Table 2). Subsequently, four patients had escalation in frequency to Q4 and either achieved remission or clinical improvement. Following this change in interval, repeat drug levels for three patients were all therapeutic at 8, 7.1, and >10 μg/mL. Subsequently one of these three patients’ maintenance interval was decreased to Q6, and by the time of this study’s conclusion, a repeat level has not been obtained. Frequency was increased to Q6 in another patient, resulting in clinical remission. One of the patients was empirically started on a maintenance dose of Q4 interval and had trough levels of >10 μg/mL at 4 weeks. Frequency was subsequently changed to Q8, but a repeat drug level was not obtained (Table 2).\n\nThree of the 10 patients were on standard ustekinumab dosing. One patient had symptomatic duodenal stricture and obstruction, resulting in abdominal pain, vomiting, and weight loss. He underwent endoscopic stricture dilation 2 months prior to initiation of ustekinumab. After ustekinumab was started, he required two subsequent dilations in a 2-month period, but had subsequently been in remission on high dose steroid. His hematocrit, CRP, and albumin all improved compared to levels prior to ustekinumab, while his BMI decreased slightly. The remaining two patients had disease worsening on ustekinumab, shown by serology and increasing abbrPCDAI. None of these three patients had their levels checked.\n\nComplications observed while on ustekinumab included infusion reactions, such as low grade fever, joint pain and vomiting within one week of infusion, and infections such as Clostridium difficile, influenza, and pneumonia. Of note, one patient developed perianal abscess within a few weeks of the first ustekinumab induction, requiring hospitalization and resulting in stopping therapy. Upon the second induction more than 1.5 years later, he again developed forearm abscess requiring hospitalization. However, his CD went into remission with ustekinumab. Work-up for immune deficiency was negative. He was later diagnosed with maturity-onset diabetes of the young.\n\n\nDiscussion\n\nHere we report 10 pediatric patients with CD refractory to currently approved medications including anti-TNFs, immunomodulators and some to vedolizumab, with seven showing a clinical response to ustekinumab treatment. The majority of our patients showed positive response to ustekinumab within the first 2–3 months of therapy and remission by the time this study was concluded. Four of these seven patients had endoscopic data pre and post ustekinumab, out of which three showed an improvement as measured by SES-CD. In general, SES-CD score showed higher level of disease activity than abbrPCDAI, which is likely due to poor correlation between these two indices19. Moreover, abbrPCDAI and SES-CD information were collected at different times in the treatment course, resulting in small differences in disease activities. CRP, albumin, and BMI showed the largest improvement, and hematocrit improved in all but two patients who responded to treatment.\n\nTo achieve clinical response and/or remission, 7 out of 10 patients needed augmented maintenance doses. Of note, one among these seven patients, one (patient 7) initially achieved remission on standard Q8 dosing and SCD for 17 months. He had a disease flare when family liberalized his diet and failed to improve when he went back on SCD. He had no ustekinumab trough level during disease remission, but the most recent level of 1.1 μg/mL coincided with disease exacerbation and increase in maintenance frequency to Q6 resulted in clinical remission. More data from subsequent follow ups is needed to determine if disease activity corresponds to dosing frequency and trough level.\n\nAmong the three patients on standard dosing for the entire duration of treatment, only one achieved remission. He required two endoscopic dilations for duodenal stricture within 4 months of starting ustekinumab, but thereafter remained in remission for the next 5 months. However, this was confounded by his family continuing 60mg prednisone daily for at least 4 months (2 months longer than prescribed). Unfortunately, there was no subsequent follow ups as he had transitioned to adult care. He had ileocolonic as well as symptomatic gastroduodenal CD, which is a relatively rare manifestation and only affects about 2% of CD patients20. There are currently no well- established treatment protocols for gastroduodenal CD, and despite treatments with corticosteroid, 6-MP, ASA, and anti-TNF agents, 31% of patients eventually require surgery21. We cannot conclude if patient 7’s clinical improvement was secondary to ustekinumab or corticosteroids.\n\nIn the six patients on whom therapeutic drug monitoring (TDM) was performed, we found subtherapeutic drug levels on Q6 and Q8 dosing intervals, which corresponded with poorly controlled disease activities and all these patients showed clinical response to changing the dosing interval. On the two patients who were on standard dosing interval (Q8 weeks), TDM was not performed and thus we cannot determine if treatment failure was due to subtherapeutic dosing or a primary non-response to ustekinumab. A larger, randomized trial is needed to confirm the role of ustekinumab TDM in pediatric CD patients. Battat et al showed that over 75% of adult CD patients needed Q4 week dosing for maintenance of clinical response. This study also demonstrated a positive association of biomarkers and endoscopic improvement with ustekinumab trough levels >4.5 μg/mL18. In addition, in a case series of three adult CD patients, Park et al demonstrated an ability to recapture response by dose escalation among patients who lost response to standard ustekinumab dosing regimen22. Thus, based on our experience and existing literature, we recommend proactively checking trough levels 4 weeks after maintenance therapy initiation to guide dosing frequency early in the treatment course or to consider reactively checking levels and augmenting maintenance dosing interval in patients with sub-optimal or poor response to standard dosing.\n\nSerious adverse effects were rare among our patients despite shorter dosing intervals. Only two patients developed recurrent infections and required hospitalization while on ustekinumab. Even though the patient who was hospitalized for abscesses also had other comorbidities such as acne, skin picking, psoriasis, and was previously hospitalized twice for recurrent abscesses on certolizumab, ustekinumab could not be ruled out as a cause of these infections. We did not observe any serious infections or cancers in the remaining eight patients, suggesting that ustekinumab is relatively well tolerated even at a higher frequency.\n\nOur study is limited by its small size and retrospective nature. Furthermore, induction and maintenance doses were not uniform among all patients. TDM was only performed on six patients, and follow-up trough for five out of those six patients have not been obtained after changes in dosing frequency.\n\nOnly six patients had pre and post-treatment endoscopy, and two of those patients had intestinal surgeries, which might have altered SES-CD scores. Even though abbrPCDAI and other laboratory workups were helpful to correlate disease activities, fecal calprotectin should be added to further assess inflammation.\n\n\nConclusion\n\nIn this retrospective chart review, 7 out of 10 patients with anti-TNF refractory pediatric-onset Crohn’s disease required augmented maintenance doses of ustekinumab to achieve clinical response or remission as measured by abbrPCDAI. The remaining three patients on standard maintenance doses either did not respond or had confounding factors affecting clinical response. Further large randomized studies with closer therapeutic drug monitoring are needed to assess the relationship between dosing interval, trough levels, and clinical response in the pediatric population. Longer follow up is also needed to assess response once ustekinumab has reached therapeutic level.\n\n\nData availability\n\nFigshare: Table 1. Baseline characteristics of patients included in the study, https://doi.org/10.6084/m9.figshare.1204864523.\n\nFigshare: Table 2. Ustekinumab dosing, interval, levels, duration of therapy, and complication, https://doi.org/10.6084/m9.figshare.1204863324.\n\nFigshare: Table 3 Clinical response of anti-TNF refractory pediatric Crohn Disease patients on ustekinumab.csv, https://doi.org/10.6084/m9.figshare.1201260025.\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 abstract of this work was presented at the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition Annual Meeting (October 17-19, 2019, Chicago, IL; abstract 101).\n\n\nReferences\n\nOng C, Aw MM, Liwanag MJ, et al.: Rapid rise in the incidence and clinical characteristics of pediatric inflammatory bowel disease in a South-East Asian cohort in Singapore, 1994-2015. J Dig Dis. 2018; 19(7): 395–403. PubMed Abstract \| Publisher Full Text\n\nSýkora J, Pomahačová R, Kreslová M, et al.: Current global trends in the incidence of pediatric-onset inflammatory bowel disease. World J Gastroenterol. 2018; 24(25): 2741–2763. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPozler O, Maly J, Bonova O, et al.: Incidence of Crohn disease in the Czech Republic in the years 1990 to 2001 and assessment of pediatric population with inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2006; 42(2): 186–9. PubMed Abstract \| Publisher Full Text\n\nPigneur B, Seksik P, Viola S, et al.: Natural history of Crohn’s disease: comparison between childhood- and adult-onset disease. Inflamm Bowel Dis. 2010; 16(6): 953–61. PubMed Abstract \| Publisher Full Text\n\nRuemmele FM, Veres G, Kolho KL, et al.: Consensus guidelines of ECCO/ESPGHAN on the medical management of pediatric Crohn’s disease. J Crohns Colitis. 2014; 8(10): 1179–1207. PubMed Abstract \| Publisher Full Text\n\nCameron F, Wilson M, Basheer N, et al.: Anti-TNF therapy for paediatric IBD: the Scottish national experience. Arch Dis Child. 2015; 100(4): 399–405. PubMed Abstract \| Publisher Full Text\n\nGrossi V, Lerer T, Griffiths A, et al.: Concomitant Use of Immunomodulators Affects the Durability of Infliximab Therapy in Children With Crohn’s Disease. Clin Gastroenterol Hepatol. 2015; 13(10): 1748–1756. PubMed Abstract \| Publisher Full Text\n\nFeagan BG, Sandborn WJ, Gasink C, et al.: Ustekinumab as Induction and Maintenance Therapy for Crohn’s Disease. N Engl J Med. 2016; 375(20): 1946–1960. PubMed Abstract \| Publisher Full Text\n\nBertrand V, El Haite A, Carré D: Efficiency of Ustekinumab in Crohn’s Disease with Severe Psoriasiform Rash Induced by Biotherapies in an Adolescent. Pediatr Dermatol. 2017; 34(4): e214–e215. PubMed Abstract \| Publisher Full Text\n\nRinawi F, Rosenbach Y, Assa A, et al.: Ustekinumab for Resistant Pediatric Crohn Disease. J Pediatr Gastroenterol Nutr. 2016; 62(4): e34–e35. PubMed Abstract \| Publisher Full Text\n\nBishop C, Simon H, Suskind D, et al.: Ustekinumab in Pediatric Crohn Disease Patients. J Pediatr Gastroenterol Nutr. 2016; 63(3): 348–51. PubMed Abstract \| Publisher Full Text\n\nLevine A, Griffiths A, Markowitz J, et al.: Pediatric modification of the Montreal classification for inflammatory bowel disease: the Paris classification. Inflamm Bowel Dis. 2011; 17(6): 1314–21. PubMed Abstract \| Publisher Full Text\n\nShepanski MA, Markowitz JE, Mamula P, et al.: Is an abbreviated Pediatric Crohn's Disease Activity Index better than the original? J Pediatr Gastroenterol Nutr. 2004; 39(1): 68–72. PubMed Abstract \| Publisher Full Text\n\nDaperno M, D'Haens G, Van Assche G, et al.: Development and validation of a new, simplified endoscopic activity score for Crohn's disease: the SES-CD. Gastrointest Endosc. 2004; 60(4): 505-12. PubMed Abstract \| Publisher Full Text\n\nRoda G, Jharap B, Neeraj N, et al.: Loss of Response to Anti-TNFs: Definition, Epidemiology, and Management. Clin Transl Gastroenterol. 2016; 7(1): e135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoutroumpakis E, Katsanos KH: Implementation of the simple endoscopic activity score in crohn's disease. Saudi J Gastroenterol. 2016; 22(3): 183–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerrante M, Colombel JF, Sandborn WJ, et al.: Validation of endoscopic activity scores in patients with Crohn's disease based on a post hoc analysis of data from SONIC. Gastroenterology. 2013; 145(5): 978–986.e5. PubMed Abstract \| Publisher Full Text\n\nBattat R, Kopylov U, Bessissow T, et al.: Association Between Ustekinumab Trough Concentrations and Clinical, Biomarker, and Endoscopic Outcomes in Patients With Crohn’s Disease. Clin Gastroenterol Hepatol. 2017; 15(9): 1427–1434.e2. PubMed Abstract \| Publisher Full Text\n\nTurner D, Levine A, Walters TD, et al.: Which PCDAI Version Best Reflects Intestinal Inflammation in Pediatric Crohn Disease? J Pediatr Gastroenterol Nutr. 2017; 64(2): 254–260. PubMed Abstract \| Publisher Full Text\n\nNugent FW, Richmond M, Park SK: Crohn’s disease of the duodenum. Gut. 1977; 18(2): 115–120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurray JJ, Schoetz DJ Jr, Nugent FW, et al.: Surgical management of Crohn's disease involving the duodenum. Am J Surg. 1984; 147(1): 58–65. PubMed Abstract \| Publisher Full Text\n\nPark S, Evans E, Sandborn WJ, et al.: Ustekinumab IV 6 mg/kg Loading Dose Re-induction Improves Clinical and Endoscopic Response in Crohn’s disease: A Case Series. Am J Gastroenterol. 2018; 113(4): 627–629. PubMed Abstract \| Publisher Full Text\n\nPhinga Do, Gurram B, Luu P, et al.: Table 1. Baseline characteristics of patients included in the study. figshare. 2020; Dataset. https://www.doi.org10.6084/m9.figshare.12048645.v1\n\nPhinga Do, Gurram B, Luu P, et al.: Table 2. Ustekinumab dosing, interval, levels, duration of therapy, and complication. figshare. 2020; Dataset. https://www.doi.org/10.6084/m9.figshare.12048633.v2\n\nPhinga Do, Gurram B, Luu P, et al.: Table 3 Clinical response of anti-TNF refractory pediatric Crohn Disease patients on ustekinumab.csv. figshare. 2020; Dataset. https://www.doi.org/10.6084/m9.figshare.12012600.v2" }	[ { "id": "62953", "date": "18 May 2020", "name": "Richard Kellermayer", "expertise": [ "Reviewer Expertise Inflammatory bowel disease", "Crohn's disease", "ulcerative colitis", "molecular genetics", "epigenetics", "nutrition", "developmental origins of disease", "recurrent Clostridioides difficile infection", "fecal transplantation", "microbiome", "microbial therapeutics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPhinga Do and colleagues, with the leadership of Bhaskar Gurram, give a nice summary on 10 pediatric Crohn’s disease patients, refractory to anti-TNF biologic therapy, who were treated with ustekinumab. Such real life observations on the off label use of new biologic agents in pediatric inflammatory bowel disease (IBD) patients are rather important. Their conclusions coincide with our subjective observation that pediatric IBD patients require a more intensified use of ustekinumab than the manufacturer’s guidance, in order to achieve drug levels that have been found to associate with improved outcomes in adult patients. There are a couple of recommendations, which may further improve the manuscript.\nIn the Methods section, please clarify which laboratory and what methodology was used (especially for ustekinumab therapeutic drug monitoring) for the laboratory measurements discussed in the paper.\n\nVery early onset (VEO) IBD is more and more considered a disease category of its own within IBD. Consider excluding the VEO-IBD (patient 1) case from this cohort, or highlight the case as a variant.\n\nExcluding the brief 4 week course of patient 6 from the table and discussion is recommended. The treatment course was too short to make any conclusions. The comment below the figure is important to keep, however, to clarify that patient 6 was exposed to ustekinumab 2 years prior to the treatment course examined.\n\nIn Table 1, or in a separate table, it would be useful to clarify the type of anti-TNF failure for each patient and each anti-TNF biologic, respectively. Primary non-response could be due to rapid antibody development or primary biological non-response. The same is true for secondary non-response (i.e. late development of antibodies, or immune pathway shift). It is fine to state unknown/clinical if level and antibody for the anti-TNF agent was not examined.\n\nIn the results, consider omitting the complicated description of dosing, since Table 2 describes that in detail.\n\nBoth figures are complicated, and the results those depict could be easily described and followed in writing within the Results section. Omitting the figures is recommended.\n\nThe clinical outcomes are difficult to follow based on the separation of the patients by augmented ustekinumab treatment versus not. Separating the patients by responders vs. non-responders, and examining ustekinumab dosing differences between the 2 groups is recommended. If I am not mistaken, and even if the patient 1 (VEO-IBD) case is excluded, there were 7 responders (6 on intensified ustekinumab therapy) and 2 non-responders (all on conventional ustekinumab treatment). These results, if compared by Chi squared testing, significantly favor (p=0.0233) treatment intensification in order to achieve clinical response.\n\nThere are parts of the discussion, which continue specific patient outcome descriptions. This should be moved to the Results section. The discussion should only draw general conclusions from what has been presented in the results and compare the findings to the existing literature.\n\nInclude in the discussion, more recent similar studies, such as the ones below, and highlight the novelties of the case series presented within this work compared to other pediatric cohorts:\n\nReal World Experience With Ustekinumab in Children and Young Adults at a Tertiary Care Pediatric Inflammatory Bowel Disease Center. Dayan JR et al. J Pediatr Gastroenterol Nutr. (2019)1 Ustekinumab for the treatment of refractory pediatric Crohn's disease: a single-center experience. Cohen A, Ahmed N, Sant'Anna A.. Intest Res. 2020 Apr 22.2\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6723", "date": "09 Aug 2021", "name": "phinga do", "role": "Author Response", "response": "In the Methods section, please clarify which laboratory and what methodology was used (especially for ustekinumab therapeutic drug monitoring) for the laboratory measurements discussed in the paper. All of the tests were done either by Miraca or Inform Diagnostics. Both these labs use Automated ELISA assay for simultaneous antibody and drug levels Very early onset (VEO) IBD is more and more considered a disease category of its own within IBD. Consider excluding the VEO-IBD (patient 1) case from this cohort, or highlight the case as a variant. Although some patients with VEOIBD might behave differently than conventional-onset IBD patients (onset>10 years of age), there is still significant overlap with their disease behavior and response to therapy. Moreover, although the patient 1 included in the current study had onset at 2 years of age, he was started on ustekinumab at 9 years age. Inflamm Bowel Dis 2021 Feb 16;27(3):295-302. doi: 10.1093/ibd/izaa080. Excluding the brief 4-week course of patient 6 from the table and discussion is recommended. The treatment course was too short to make any conclusions. The comment below the figure is important to keep, however, to clarify that patient 6 was exposed to ustekinumab 2 years prior to the treatment course examined. We will remove this part. Agree that it does not add much aside from noting that there’s prior exposure. In Table 1, or in a separate table, it would be useful to clarify the type of anti-TNF failure for each patient and each anti-TNF biologic, respectively. Primary non-response could be due to rapid antibody development or primary biological non-response. The same is true for secondary non-response (i.e. late development of antibodies, or immune pathway shift). It is fine to state unknown/clinical if level and antibody for the anti-TNF agent was not examined. Table 1 has been modified to include more specific reasons for anti-TNF failure. In the results, consider omitting the complicated description of dosing, since Table 2 describes that in detail. The results section has been edited to omit dosing descriptions. Both figures are complicated, and the results those depict could be easily described and followed in writing within the Results section. Omitting the figures is recommended. We believe that the figures compliment the text and some readers prefer just to look at the tables and pictures. They provide visual aid and show a clear trend of improvement for patients on augmented dosing. The clinical outcomes are difficult to follow based on the separation of the patients by augmented ustekinumab treatment versus not. Separating the patients by responders vs. non-responders and examining ustekinumab dosing differences between the 2 groups is recommended. If I am not mistaken, and even if the patient 1 (VEO-IBD) case is excluded, there were 7 responders (6 on intensified ustekinumab therapy) and 2 non-responders (all on conventional ustekinumab treatment). These results, if compared by Chi squared testing, significantly favor (p=0.0233) treatment intensification in order to achieve clinical response. We did organize it by responder vs non responder initially but found that organizing by augmented vs standard dosing shows a clearer trend. Moreover, what we want to point out to the readers through this paper is that most pediatric patients in our cohort needed higher dose than the standard dosing. In addition, we cannot say if patient 8 (the one with duodenal stricture) is an ustekinumab responder or not and will require 3 distinct categories (responder, non-responder, and indeterminant). All 7 responders were on augmented dose There are parts of the discussion, which continue specific patient outcome descriptions. This should be moved to the Results section. The discussion should only draw general conclusions from what has been presented in the results and compare the findings to the existing literature. The text has been reorganized. Include in the discussion, more recent similar studies, such as the ones below, and highlight the novelties of the case series presented within this work compared to other pediatric cohorts: Edited to include these 2 studies." } ] }, { "id": "62949", "date": "12 Apr 2021", "name": "Abdul Elkadri", "expertise": [ "Reviewer Expertise Pediatric inflammatory bowel Disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study reviews retrospectively the experience of one pediatric institution with ustekinumab in Crohn's disease patients. This is an understudied population with paucity of data. Interestingly, it showed good response in 7/10 patients, but also showed that there was a need for shortened intervals of dosing. This shortened interval of dosing was shown previously in one of the adult studies cited and suggests that there needs to be more studies to support this shortened interval. The review is limited by the lack of comprehensive endoscopic data for all patients, and by the lack of comprehensive data for therapeutic drug monitoring to try to prove the hypothesis that shortened intervals lead to remission and clinical response (with resulting increased drug levels). The retrospective nature of this paper makes the data limited in its ability to prove the hypothesis, but the data is still reviewed well and with details all available to show what the authors have stated. The population is somewhat heterogeneous in nature, limiting the ability to identify a subpopulation where the response is higher.\n\nDue to the small number of patients, the manuscript is by default limited to a descriptive review which the authors have done properly in this manuscript. The data needs to be published to expand the available literature on this medication in this population.\n\nThe current version of the manuscript is appropriate for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-316
https://f1000research.com/articles/9-708/v2	28 Sep 20	{ "type": "Research Article", "title": "Association between socioeconomic factors and soft drink consumption among adults in Cambodia: a cross-sectional study", "authors": [ "Wonga Laohasiriwong", "Sim Samphors", "Pall Chamroen", "Vong Pisey", "Rebecca S. Dewey", "Wonga Laohasiriwong", "Pall Chamroen", "Vong Pisey", "Rebecca S. Dewey" ], "abstract": "Background: Soft drinks are an attractive and popular drink, consumed by many people to fulfill their energy. However, soft drink consumption is an important risk factor for non-communicable diseases. This study aimed to investigate the association between socioeconomic factors and soft drink consumption among adults in Cambodia. Methods: A cross-sectional analytical study with multi-stage random sampling was used to select 749 respondents from 12 communes across 5 districts in Phnom Penh, the capital city of Cambodia. A structured questionnaire was used to assess socioeconomic factors and soft drink consumption. Data were analyzed using bivariate and multivariable logistic regression. Crude odds ratios and adjusted odds ratios (AORs) with 95% confident intervals (CI) were calculated to show the strength of associations. Results: The overall prevalence of soft drink consumption was 44.73% (95%CI: 41.16-48.30%) across the sample population. The final model of multiple logistic regression showed only four factors to be significantly associated with soft drink consumption: gender, age, family size and income. Respondents most likely to consume soft drinks were men (AOR: 1.49, 95%CI: 1.10-2.00; p=0.009), those aged 31-59 years (AOR: 1.93, 95%CI: 1.98-4.62; p<0.001), those in a household of less than 5 individuals (AOR: 1.38, 95%CI: 1.44-3.19; p=0.036), and those earning <300$/month (AOR: 1.59, 95%CI: 1.10-2.31; p=0.003). Conclusion: The prevalence of soft drink consumption in Cambodia is a major concern for public health. The predictive factors significantly associated with soft drink consumption identified by this study were gender, age, household size and income. Therefore, all stakeholders should contribute to social marketing and intervention focusing on reducing the consumption of soft drinks through advertising on social media, television and other popular media platforms. Furthermore, additional taxation of soft drinks should be considered.", "keywords": [ "Socio-economic", "adults", "multiple logistic regression", "Cambodia" ], "content": "Introduction\n\nNutritional status is a principle determinant of health. Assessment of nutritional status, dietary intake, and related factors are of worldwide importance to public health. Although recent studies reported that nutritional status has improved remarkably over the last few decades, the rates of people being classed as underweight or overweight remain high. In general, being overweight/obese or underweight severely affect an individual’s health and their quality of life, increase their risk of developing infectious diseases, reduce their ability to work, prolong hospitalization and increase the risk of illness and death. Thus, achieving and maintaining an ideal body weight is highly advisable1,2.\n\nThere are many factors influencing becoming overweight and obese. Lifestyle, cultural activities, socioeconomic factors, physical inactivity and diet are the main factors that have been found to have an association with being overweight and obese3–6. Many studies have reported that one of the main factors influencing becoming overweight and obese is inappropriate diet, especially soft drink consumption7–10.\n\nSoft drinks provide little nutritional value and are energy-dense11. Dietary patterns have changed rapidly throughout the world, especially in Western countries where consumption of soft drinks is popular12. However, this trend has been extended across Asian countries, including Cambodia, where soft drink consumption has replaced nutritional Asian foods such as fish, vegetables and other less energy-dense foods.\n\nA number of developing and developed countries in the world are facing what is described by the World Health Organization (WHO) as the double burden of malnutrition, the simultaneous impacts of undernutrition and overweightness/obesity13. Research has shown the prevalence of individuals being overweight to be significantly higher in urban compared to rural areas14. However, the true relationships between socioeconomic factors and soft drink consumption among adults in urban Cambodia is hitherto unknown.\n\nAs such, the present study aimed to determine the association between socioeconomic factors and soft drink consumption of adults in Phnom Penh, the urban metropolis of Cambodia, providing vital information for public health professionals and policy makers in order to prepare educational programs and interventional policies.\n\n\nMethods\n\nA structured questionnaire was administered from March, 2018 to July, 2018 at participant homes in participants’ free time using a cross-sectional multi-stage random sampling method in order to select 749 samples among 12 communes out of 5 districts in the Phnom Penh municipality of Cambodia.\n\nThe study design was reviewed by the Khon Kaen University ethics committee and was approved for human research (Reference No. HE582071). Data collection was conducted by administering the questionnaire in a face-to-face interview. All participants gave written informed consent to participate in the study. If the participant was unable to read the consent form, the researcher read it aloud for them.\n\nMulti-stage random sampling was used to select the samples in this study. Phnom Penh capital city was selected and 5 districts were randomly selected from the total of 12 districts of Phnom Penh capital city. Then 2 or 3 communes were randomly selected from each selected district, for a total of 12 communes to be selected. Each household in each commune was listed and assigned a number and a systematic random sampling procedure was used to select 748 households from the total 44,436 households. Finally, one member aged 18-59 of each household was randomly selected if the household had more than one member in this age category.\n\nThe inclusion criteria for the study were that participants over 18 years of age, and were able to understand the questionnaire. Exclusion criteria were any serious health problems (causing them to be bedbound), diarrhea at the time of data collection (defecating more than 3 times per day), pregnancy, mental illness, deformity and lower limb amputation.\n\nThe sample size for this study was calculated using an established formula15 and the estimated sample size was 749.\n\nThe outcome measure used in this study is soft drink consumption, for which a threshold of ≥3 times per week was chosen. This threshold was used as it has been previously associated with being overweight/obese, and having metabolic syndrome16–18.\n\nA structured questionnaire was developed based on the research questions and relevant literature. The questionnaire consisted of two parts: part 1, demographic and socioeconomic characteristics, including gender, age, marital status, educational attainment, occupation, number of family members and personal monthly income; part 2, lifestyle and behavior characteristics, including tobacco use, alcohol consumption, food habits, soft drink consumption, physical activity and sedentary behavior.\n\nAs the questionnaires were developed for data collection, a pre-test of the questionnaire was conducted in 30 participants from different areas from the target group. Reliability was calculated using Cronbach’s alpha coefficient, and was 0.857. Then the questionnaire was developed and tested for content validity by five experts. Finally, a forum of researchers edited and rearranged the questionnaire based on the results of the field test, with the aim to ensure the validity and reliability of the tool.\n\nDependent variable. Participants consuming soft drinks more than three times per week was the dependent variable used in this study. Participants were asked “How many times per week have you drunk a soft drink in the last month?” Participant answers were classified into two groups: those who drank soft drinks less than 3 times per week (“no” = 0) and 3 times per week of more (“yes” = 1).\n\nPredictor variables. Age, household income and expenditure, and the number of individuals in the household were coded as continuous variables. Categorical variables comprised gender (male or female), marital status (single, married or divorced), level of education (no formal education, primary, secondary, high school, associated degree, Bachelor’ degree, Master’s degree or higher), occupation (farmer, unemployed, NGO employee, self-employed, student, government officer, home maker, unskilled worker, or other) and the people they were cohabiting with (spouse, parents, relatives, none, friend, or other).\n\nData were imported to Stata version 13 (College Station, Texas, USA) for analysis. Continuous and categorical data were inspected using descriptive statistics to determine the frequencies and percentages (categorical variables) and means, medians and standard deviations (continuous variables) of each socio-economic and demographic characteristic collected. Bivariate logistic regression was used to estimate the association between each socioeconomic factor and the outcome measure of soft drink consumption. Crude odds ratios (CORs) were computed using 95% confidence intervals (CIs) and variables with significance of p<0.25 were entered into the final model. In this final model, multivariate logistic regression was used to estimate the association between socioeconomic factors and soft drink consumption. Adjusted odds ratios (AORs) were then computed using the 95% CI. Significance was considered at a threshold of p<0.05.\n\n\nResults\n\nA total of 749 participants from 5 districts and 12 communes were recruited into the study. The socio-demographic characteristics of respondents are summarized in Table 1. In total, 50.2% of participants were women and 49.80% were men, and the mean age of participants (± standard deviation) was 32.26±11 years. The marital status of participants was 53.94% married, 43.52% single and 2.54% divorced. The most frequent level of participants’ highest educational attainment was high school (31.91%), followed by Bachelor’s degree (26.44%) and primary school level (15.22%). The most frequent occupations were private company workers (28.97%), self-employed (21.36%) and students (20.16%), with only 0.67% of the participants being farmers and 0.93% unemployed. More than half of the participants (57.41%) had a household size of less than 5 members, and participants most frequently reported living with their spouse (47.93%), parents (25.37%) and relatives (12.95%). Monthly income ranged from US$40 to $5,100 with a mean (±SD) of US$495±686.7. Monthly expenditure ranged from US$20 to $3,750 USD with mean of US$288.5±394.1 (see Table 1).\n\nThe overall prevalence of soft drink consumption was 44.73% (95%CI: 41.16-48.30%). Educational attainment, gender, age, occupation, household composition, household size, and income were found to have associations with soft drink consumption (p<0.25) in the bivariate analysis (Table 2).\n\nThe final model found only four factors associated with soft drink consumption: gender, age, household size and income. Men (AOR: 1.49, 95%CI: 1.10-2.00; p = 0.009), those aged 31-59 years (AOR: 1.93, 95%CI: 1.98-4.62; p <0.001), those in smaller households (AOR: 1.38, 95%CI: 1.44-3.19; p = 0.036), and those earning <US$300/month (AOR: 1.59, 95%CI: 1.10-2.31; p = 0.003) were more likely to consume soft drinks at least three times a week (Table 3).\n\n\nDiscussion\n\nThis study found that the prevalence of soft drink consumption among Cambodian adults was 44.73% (95%CI: 41.16–48.30). Almost one-third of adults aged 18–59 consumed soft drinks more than 3 times per week in Cambodia. The trend of soft drink consumption has rapidly increasing over a period of the last few years19, during which time the Cambodian economy has increased twofold in economic growth rates. Globally, the consumption of soft drinks is higher in upper-middle income countries (0.8 servings per person per day) compared to the lower-middle income countries (0.59 servings per day)20. The findings of the present study are consistent with findings from Malaysia showing that 53.3% of individuals consume soft drinks three times per week or more21, with similar findings also in Saudi Arabia (40%) and Nigeria (42.8%). Daily consumption has been reported in developed countries such as the USA (40%)22 and the UK (20.4%)23 and similarly in South Africa (48.3%)24. This may be related to the culture of western and European countries preferring fast food and artificially sweetened soft drinks. Furthermore, there may be a tendency towards busier lifestyles with less emphasis on making time to cook. In the present study, the factors associated with soft drink consumption were found to be gender, age, household size and income.\n\nGender was found to be significantly associated with soft drink consumption. Men were more likely (AOR: 1.49; 95%CI:1.10-2.00) to consume soft drinks compared to their female counterparts. This finding is consistent with a study from the USA, which revealed that men were more likely to consume soft drinks than women22. A study in Australia also found the men are 2.11 times more likely to consume soft drinks compared to women11. In the context of Cambodia, it might be that men go out to work more often, requiring more energy and undertake activities where soft drinks are often consumed in order to counteract fatigue both during and after work.\n\nMany studies have found age to be significantly associated with soft drink consumption, and the present study’s results support this. Our finding was that 31–59-year-olds are more likely to consume soft drinks compared to 18–30-year-olds (AOR:1.93; 95%CI: 1.98-4.62). However, there are also published studies that contradict this finding11,21. The age range in the present study was 18–59-year-olds; therefore, the older age bracket (31 to 59) are likely to be in paid employment, and not yet in retirement. These individuals may be more likely to consume soft drinks to combat fatigue while working, and this may be more prevalent amongst older individuals within the subgroup.\n\nFinally, income was found to be significantly associated with soft drink consumption. Those who earned less than US$300 per month were more likely to consume soft drinks (AOR:1.59; 95%CI: 1.10-2.31) compared to those who earned more than US$300 per month. This finding is consistent with research from Singapore showing that high and medium earners consumed less soft drinks when compared to low earners11. Conversely, a study from Australia reported that households with higher annual incomes were more likely to consume soft drinks compared to households with lower annual incomes24.\n\n\nConclusion\n\nThe prevalence of soft drink consumption in Cambodia has become a recent concern for public health. Predictive factors found in this study that are significantly associated with higher soft drink consumption are being a man, aged 31–59 years, living in a household of less than 5 individuals, and having a monthly income of less than US$300.\n\nBased on our research findings, we recommend taking measures to reduce the frequency of soft drink consumption. All stakeholders within each ministry in Cambodia should promote and raise awareness of the impacts of soft drink consumption within the population. Specifically, the Ministry of Health should develop and launch a social marketing and intervention program focusing on reducing the consumption of soft drinks through advertising on social media, television and other means. Furthermore, the taxation of soft drinks should be considered.\n\n\nData availability\n\nFigshare: Underlying Data 1, https://doi.org/10.6084/m9.figshare.12612998.v125\n\nThis project contains the following underlying data:\n\n- Raw data from a survey the consumption of fast food and soft drinks in Phnom Penh municipality of Cambodia in 2019.\n\n- Code book for interpreting the data\n\nFigshare: Extended data, https://doi.org/10.6084/m9.figshare.12612992.v126\n\nThis project contains the following extended data:\n\n- Questionnaire in Khmer\n\n- Questionnaire in English\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAbdullah A: The double burden of undernutrition and overnutrition in developing countries: an update. Curr Obes Rep. 2015; 4(3): 337–49. PubMed Abstract \| Publisher Full Text\n\nWang Y, Lobstein T: Worldwide trends in childhood overweight and obesity. Int J Pediatr Obes. 2006; 1(1): 11–25. PubMed Abstract \| Publisher Full Text\n\nFazah A, Jacob C, Moussa E, et al.: Activity, inactivity and quality of life among Lebanese adolescents. Pediatr Int. 2010; 52(4): 573–8. PubMed Abstract \| Publisher Full Text\n\nHu F: Obesity epidemiology. Oxford University Press; 2008. Publisher Full Text\n\nPopkin BM, Adair LS, Ng SW: Global nutrition transition and the pandemic of obesity in developing countries. Nutr Rev. 2012; 70(1): 3–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Nuaim AA, Al-Nakeeb Y, Lyons M, et al.: The prevalence of physical activity and sedentary behaviours relative to obesity among adolescents from Al-Ahsa, Saudi Arabia: rural versus urban variations. J Nutr Metab. 2012; 2012: 417589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinkley JK, Eales J, Jekanowski M: The relation between dietary change and rising US obesity. Int J Obes Relat Metab Disord. 2000; 24(8): 1032. PubMed Abstract \| Publisher Full Text\n\nGarcia G, Sunil TS, Hinojosa P: The fast food and obesity link: consumption patterns and severity of obesity. Obes Surg. 2012; 22(5): 810–8. PubMed Abstract \| Publisher Full Text\n\nPereira MA, Kartashov AI, Ebbeling CB, et al.: Fast-food habits, weight gain, and insulin resistance (the CARDIA study): 15-year prospective analysis. Lancet. 2005; 365(9453): 36–42. PubMed Abstract \| Publisher Full Text\n\nDuffey KJ, Gordon-Larsen P, Jacobs DR, et al.: Differential associations of fast food and restaurant food consumption with 3-y change in body mass index: the Coronary Artery Risk Development in Young Adults Study. Am J Clin Nutr. 2007; 85(1): 201–8. PubMed Abstract \| Publisher Full Text\n\nScully M, Morley B, Niven P, et al.: Factors associated with high consumption of soft drinks among Australian secondary-school students. Public Health Nutr. 2017; 20(13): 2340–8. PubMed Abstract \| Publisher Full Text\n\nScully M, Morley B, Niven P, et al.: Factors associated with high consumption of soft drinks among Australian secondary-school students. Public Health Nutr. 2017; 20(13): 2340–8. PubMed Abstract \| Publisher Full Text\n\nKim SH, Hwang JY, Kim MK, et al.: Dietary factors related to body weight in adult Vietnamese in the rural area of Haiphong, Vietnam: the Korean Genome and Epidemiology Study (KoGES). Nutr Res Pract. 2010; 4(3): 235–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMendez MA, Monteiro CA, Popkin BM: Overweight exceeds underweight among women in most developing countries. Am J Clin Nutr. 2005; 81(3): 714–21. PubMed Abstract \| Publisher Full Text\n\nHsieh FY, Bloch DA, Larsen MD: A simple method of sample size calculation for linear and logistic regression. Stat Med. 1998; 17(14): 1623–34. PubMed Abstract \| Publisher Full Text\n\nDarebo T, Mesfin A, Gebremedhin S: Prevalence and factors associated with overweight and obesity among adults in Hawassa city, southern Ethiopia: a community based cross-sectional study. BMC Obes. 2019; 6(1): 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerreira-Pêgo C, Babio N, Bes-Rastrollo M, et al.: Frequent Consumption of Sugar- and Artificially Sweetened Beverages and Natural and Bottled Fruit Juices Is Associated with an Increased Risk of Metabolic Syndrome in a Mediterranean Population at High Cardiovascular Disease Risk. 2016; 146(8): 1528–36. Publisher Full Text\n\nKang Y, Kim J: Soft drink consumption is associated with increased incidence of the metabolic syndrome only in women. Br J Nutr. 2017; 117(2): 315–24. PubMed Abstract \| Publisher Full Text\n\nCasico: Market Analysis for fast food chain in Cambodia, Vietman, Loa and Myanmar. 2014. Reference Source\n\nSingh GM, Micha R, Khatibzadeh S, et al.: Global, Regional, and National Consumption of Sugar-Sweetened Beverages, Fruit Juices, and Milk: A Systematic Assessment of Beverage Intake in 187 Countries. PLoS One. 2015; 10(8): e0124845. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoh DA, Moy FM, Zaharan NL, et al.: Sugar-sweetened beverage intake and its associations with cardiometabolic risks among adolescents. Pediatr Obes. 2017; 12(1): e1–e5. PubMed Abstract \| Publisher Full Text\n\nMendy VL, Vargas R, Payton M, et al.: Association Between Consumption of Sugar-Sweetened Beverages and Sociodemographic Characteristics Among Mississippi Adults. Prev Chronic Dis. 2017; 14: E137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarrett P, Imamura F, Brage S, et al.: Sociodemographic, lifestyle and behavioural factors associated with consumption of sweetened beverages among adults in Cambridgeshire, UK: the Fenland Study. Public Health Nutr. 2017; 20(15): 2766–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPengpid S, Peltzer K: Prevalence and socio-behavioral factors associated with sugar-sweetened beverages consumption among 15 years and older persons in South Africa. Diabetes Metab Syndr Obes. 2019; 12: 937–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamphors S, Pisey V, Dewey RS: Underlying Data 1. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12612998.v1\n\nSamphors S, Pisey V, Dewey RS: Extended Data. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12612992.v1" }	[ { "id": "84775", "date": "17 Jun 2021", "name": "Prince Atorkey", "expertise": [ "Reviewer Expertise Health risk behaviours and mental health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of this study is to examine the association between socioeconomic factors and consumption of soft drinks among adults in consumption. The prevalence of soft drink consumption was 44.73%. Factors associated with soft drink consumption were male gender, age, family size and income. This is a good study but needs some improvements. See below some comments that need improvement.\nI am not sure what the authors mean by \"to fulfill their energy\" in the abstract. I suggest they reword.\n\n\"confident interval\" in the abstract should be confidence interval.\n\nThe second and third sentences in the introduction should be referenced.\n\nHow the random sampling was done at each stage should be specified.\n\nFor inclusion criteria, the authors stated \"..and were able to understand the questionnaire.\" Please specify the language the questionnaires were in.\n\nWhy was diarrhea at the time of data collection an exclusion criteria?\n\nIn table 1, should >300 and > 500 be <300 and <500 instead?\n\nThe practical implications of the findings should be stated.\n\nLimitations of the study should be stated.\n\nThe manuscript should be checked properly for grammar.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6972", "date": "09 Aug 2021", "name": "Samphors Sim", "role": "Author Response", "response": "It has already been reworded. It has already been edited. It has already been added. We use the lottery random sampling in every stage with random. Khmer language is the native language of participants. In case the participants are not able to read the Khmer language, they are excluded. Participants will not feel well for answering or responding the answer. It has been corrected. It has been added in the manuscript. It has been added in the manuscript. Checked." } ] } ]	2	https://f1000research.com/articles/9-708
https://f1000research.com/articles/10-775/v1	09 Aug 21	{ "type": "Opinion Article", "title": "The impact of COVID-19 and the NCAA’s (National Collegiate Athletic Association) season cancellation on sport support professionals", "authors": [ "Peter Economou", "Tori Glascock", "Mark Louie", "Peter Economou", "Mark Louie" ], "abstract": "The coronavirus (COVID-19) pandemic forced athletes to learn to navigate a world void of athletic competition and contend with the intricacies that life during a pandemic brings. Similarly, those that dedicate their lives to these athletes such as sport psychology practitioners, sport medicine personnel, athletic trainers, or academic advisors (i.e., sport support professionals) also experienced an abrupt ending to their routines, and in some cases their livelihoods. These professionals have been pushed to modify their art and find ways to engage their community from a distance (e.g., virtually, or physically from a social distance). Sport support professionals are experiencing a collective loss due to the COVID-19 pandemic. This paper addresses the impact of a global pandemic and the subsequent issues faced by sport support professionals across diverse disciplines, emphasizing the significance of these relationships and the necessary adjustments to manage the cessation of these relationships.", "keywords": [ "COVID-19", "sports psychology practitioners", "sports medicine", "academic advisors", "loss", "relationship" ], "content": "Introduction\n\nEven as a mindfulness practitioner, a global pandemic can challenge one’s practice and create copious amounts of emotions. This has been the roller coaster that many professionals have ridden during recent and unique times. While much of the current research has focused on our clients (i.e., athletes), this paper will focus on the impact the pandemic and subsequent cancellation of sport has had on the professionals involved primarily within the National Collegiate Athletic Association (NCAA) sports. To consider the global effect the pandemic had on a diverse range of professionals, we use the term sport support professionals to include sport psychology practitioners, sports medicine personnel, athletic trainers, and academic advisors. The COVID-19 pandemic has presented sport support professionals with a novel situation that has not had to be navigated before, and as such has never been studied. The coronavirus disease (COVID-19) prompted the cancellation of all sports during conference tournaments of the 2020 winter sports season (i.e., right at the start of “March Madness”, the annual NCAA basketball tournament) and the remainder of all NCAA seasons, thereafter, across all divisions. As we entered into the fall 2020 season, COVID-19 and the decision of how and whether to return to sport was politicized; generating a debate between healthcare providers, politicians, and civilians concerning the impact of the virus on athletes and whether it was safe to begin a new season. As of July 2021, there is no standard practice nationally surrounding decisions to return to sport amidst a pandemic and professionals are left to answer this moment’s determination, often made by an executive with limited scientific or medical knowledge. To put this article into context, let us look back to the initial impact of COVID-19 on sport.\n\nDuring the spring of 2020, thousands of student-athletes in the U.S. were left without the proper goodbye; and senior athletes had the inability to conclude their intercollegiate athletic careers in the traditional manner. Due to college campuses closing and lockdowns being implemented, student-athletes were forced to leave campus abruptly and quickly. These student-athletes were prematurely lurched into a new world void of sports and were left to navigate the intricacies that come with being a retired or non- student-athlete. Entering the fall of 2020, athletes were faced with new problems including living in a bubble without their loved ones, uncertainty, and indecision about the fate of their upcoming athletic year, and so much more. Finding themselves in a similar situation are the sport support professionals (i.e., healthcare providers, sport psychologists, athletic trainers, or academic advisors) who work with these athletes.\n\nResearch has examined clinical terminations (the ending of relationships between practitioner and patient) within the context of case studies (Bhatia & Gelso, 2017; Fragkiadaki & Strauss, 2012). The global pandemic in 2020 has caused terminations on a scale not seen before. Sport support professionals were experiencing the stress brought on by the pandemic just as others were, and it is compounded with the shared stress from all different directions (i.e., economic stressors, job uncertainty, etc.). While “termination” is a psychological term referring to the natural end of a therapeutic relationship, the natural end to a relationship with a student-athlete occurs for all sport support professionals. As with therapeutic providers, when termination occurs abruptly and not through the normal process, the emotional impact may be greater.\n\nWhile much of the supported research surrounding terminations and stress addresses the student-athlete experience (Lally, 2007; Putukian & Putukian, 2016), we instead focus on the experience of the sport support professional within the United States, including sport psychology practitioners, sports medicine personnel, athletic trainers, and academic advisors. These professionals form strong relationships with student-athletes and dedicate much of their craft to ensuring student-athlete wellbeing. Following the onset of COVID-19, these professionals were precluded from formal termination with their student-athletes in the traditional sense. The termination process includes reflection upon the relationship between the client and the professional, review of the current functioning and coping skills learned, application of these new skills in the real world, expectations related to potential future sessions, long-terms goals following the cessation of treatment, and sharing pride in the client’s progress (Norcross et al., 2017).\n\nCurrent research shows that sport support professionals build strong therapeutic bonds with student-athletes through their work together, and premature termination can affect these professionals in the future, while also causing feelings of abandonment, hurt, and disappointment (Alessandro et al., 2011). As such, we know these professionals experience loss of meaningful working relationships, which are often accompanied by anxieties and feelings of loss associated with the abrupt termination (Joyce, 2007). What is less understood is how these professionals navigated the abrupt end of their work with their student-athletes, and how they coped with the non-traditional termination during the COVID-19 pandemic.\n\n\nThe consequences of early termination\n\nTermination is defined as “an intentional process that occurs over time when a client has achieved most of the goals of treatment, and/or when psychotherapy must end for other reasons” (Vasquez et al., 2008, p. 653). The phrase, “termination within psychology”, refers to the final stages of the process in mental health treatment, and in the context of this paper, we consider termination as a process which will eventually occur within any substantial relationship as it concludes. While this is the formal process within the practice of psychological sciences, we believe this sharing occurs at varying levels across all significant relationships.\n\nFor the provider, terminations bring about feelings of anxiety, guilt, and loss (Joyce, 2007). Despite commonalities in the experiences of termination, abrupt termination disrupts the natural processes and denies the traditional goodbyes. The abrupt termination of treatment that materialized gives one cause to examine the psychological toll that the ending of these therapeutic relationships has had on the sport support professional as well as the student-athlete.\n\nThe impact of early termination on sport support professionals is ever-present and far-reaching. For instance, Alessandro and colleagues (2011) studied the psychology of relationships and how ending therapeutic relationships impacts support professionals’ future approaches to clients, their professional development, and personal well-being. Feelings of abandonment, hurt, and disappointment are common (Alessandro, 2011). Piselli et al. (2011) highlighted that the more personally invested the provider was in their client, the greater the emotional toll of the premature termination. This is particularly pertinent for sport support professionals working with student-athletes, as they often work with student-athletes for the entirety of their collegiate careers. Their ability to work effectively can be altered by powerful emotions (anxiety, guilt, and loss) that the provider struggles to cope with. It is important to note that there is a lack of literature examining the impact of abrupt termination, as well as termination resulting from a global pandemic. What we can infer from the literature is that the process of termination carries a heavy emotional burden.\n\n\nThe sport support professional\n\nThe sport support team behind every student-athlete is composed of sport psychologists, sports medicine personnel, athletic trainers, and academic advisors who forge relationships with the student-athlete as they work to develop goals and tasks to work towards, and develop and emotional bond (Petitpas et al., 1999). For sport psychology practitioners, the aim of their relationship with the student-athlete may be to create therapeutic goals for behavioral change. Goals for treatment and preventative care are often set by medical physicians. For athletic trainers, this may look like student-athlete compliance with an injury rehabilitation program. Academic advisors help athletes to create goals for academic success and long-term vocational support. These professionals work with student-athletes to help them balance mental and physical wellness and maintain a healthy lifestyle. Through working with student-athletes and facing obstacles together, a therapeutic relationship is forged that has deep meaning to the student-athlete and to the sport support professional. This dynamic role establishes a strong relationship with student-athletes, and as we know from therapeutic alliance research (Sexton et al., 2005), requires a formal process; a process which begins during the first meeting with the provider and hinges on connection, personalities, and levels of engagement (Secton et al., 2005). Regardless of whether the conclusion of the relationship occurs following graduation, transferring, retiring, or a global pandemic, both the professional and the student-athlete are affected. Providers are known to experience their own anxieties over termination (Joyce, 2007) and student-athletes have difficulty coping with the loss of support that follows the conclusion of relationships (McKnight et al., 2009). A common thread running through these relationships is that while supporting the student-athlete in various sectors, all of these professionals share a vested interest in the student-athlete’s collegiate success, and all allow for a mentorship relationship to form and a subsequent emotional bond (Petitpas et al., 1999). As an indispensable member of the sport support team, academic advisors ensure that student-athletes enjoy success in the classroom in addition to athletic competition. Since 1991, the NCAA has mandated that academic counseling services be a part of the student-athlete support system at all division one institutions, in an effort to ensure athletic success was not taking precedence over academic achievement (Meyer, 2005). Academic advisors help student-athletes utilize behavioral strategies which will help them succeed in the classroom. Academic advisors actively participate in all areas pertaining to academic and athletic evaluation, academic evaluation, support, and guidance (Meyer, 2005). As we consider the developmental nature of student-athletes within the NCAA context, the values impressed upon student-athletes often have a lifelong impact. A student-athlete will arrive at college with a strong athletic identity, but they may lack confidence in their identity as a student (Lally & Kerr, 2005). Academic advisors are tasked with motivating student-athletes to apply themselves in the classroom when they may not be intrinsically inclined to do so. Academic advising has been described as having overlapping responsibilities with counseling. (Kuhn et al., 2006). Kuhn and colleagues conclude that academic advisors, alongside academics, help with giving guidance for time management, interpersonal issues, career goals, and personal goals.\n\nStudent-athletes must also cope with the stress that accompanies the pressure to perform well in their sport. The convergence of athletic pressure, maintaining a social life, adjusting to college, and potentially contending with injury, can easily affect academic performance. Challenges arise from their involvement in athletics that require additional support (Watson & Kissinger, 2007). The pressure that accompanies athletic involvement converges with academic and social obligations, which in turn can cause overwhelming stress for the student-athlete. Sport support professionals serve as sounding boards, advocates, and confidants when student-athletes are most vulnerable. In many ways, these professionals are mentors, helping the student-athlete focus on growth and accomplishments whilst providing support. For reasons such as these, understanding how professionals have coped with the abrupt loss of such relationships is imperative to understanding how to protect their future well-being and careers.\n\n\nCoping\n\nThe COVID-19 pandemic brought about a collective loss felt by sport support professionals, both professionally and personally; demanding the use of coping skills to maneuver this new landscape. Coping skills can be effective in preventing burnout; a pervasive and debilitating state resulting from an unsustainable period of overwhelming stress (Staten & Lawson, 2017). Mental health care providers, sports medicine providers, and academic advisors are all at higher risk for burnout due to the tremendous stress and personal involvement of their fields (Hendrix et al., 2000; Mullen et al., 2018; Vredenburgh et al., 1999). All sport support professionals utilize different coping skills, salient to their individual skill sets. Athletic trainers and other sport support professionals represent a vulnerable group, at a high risk of career burnout, who could greatly benefit from improved understanding of effective coping strategies to effectively manage challenging situations (Hendrix et al., 2000).\n\nFindings from previous studies show there is typically a delayed trauma response following a life-changing event (McFarlane, 2004). After the SARS-Cov outbreak in Singapore in 2003, 27% of health care workers reported psychiatric symptoms (Lee et al., 2018). The ramifications and effects of the COVID-19 pandemic, and any associated delayed trauma, will likely be examined as time passes. Examining how sport support professionals have managed the months immediately following the crisis could provide knowledge applicable elsewhere. Understanding the coping strategies they used and how their support systems were utilized, as well as identify patterns and strategies that were most effective, could provide insight into how individuals can best cope with stressful situations more broadly.\n\n\nRole identity\n\nThe relationship between sport support professionals and the student-athlete creates a mentorship that is meaningful to both sides. A qualitative study of 55 Black female student-athletes in the Big 12 conference found that mentors were perceived as needing to be guides or role models, who have relational characteristics, and provide constructive criticism (Carter & Hart, 2010). The relationship between the mentor and the mentee is acknowledged as being beneficial to both sides: through providing the mentee with career and professional development assistance, and for the mentor who serves as a confidant androle model, thus grows and learns from the relationship as well (Jacobi, 1991). The loss of this salient relationship can cause psychological distress (Umberson & Torling, 1997).\n\nRole identity, the set of meanings applied to one’s self in a social situation, is also at stake following the loss of a relationship like the one between these professionals and student-athletes (Burke, 1991; Umberson & Torling, 1997). Major life events such as a career change, death of a loved one, starting a family, and global events, like a pandemic, threaten psychological well-being and role identity, and can lead to a role residual: parts of a previous role carried into one’s current roles and self-identity (Swann & Brown, 1990; Ebaugh, 1988). Losing a relationship entails losing information about the self (Umberson & Torling, 1997). Role identity is an important aspect of the self because it links the individual self and society, and this is threatened with the experience of stressful events (Callero, 1985). Salient role identities, the probability that a given identity will be invoked in social interaction, are an integral facet of how one defines oneself (Brenner et al., 2014). Self-esteem and evaluation of self are closely linked with salient role identities (Callero, 1985). COVID-19 has caused a disruption in the mentor-mentee relationship, leaving sport support professionals in a limbo state of being unable to perform their regular roles.\n\nSport support professionals have experienced significant loss as support figures to student-athletes due to COVID-19. Their role identity is at stake and the loss of relationships with the student-athletes they were working with puts them at greater risk of experiencing psychological distress. Research surrounding mental health is dedicated to finding ways to best support student-athletes during times of struggle including finding ways to destigmatize mental health for student-athletes, making mental health services more accessible by having various locations to go to for support close to the athletic facilities, and continuing to work to break down the barrier that exists for many student-athletes to seeking help. Our research team holds the opinion that research should be equally as invested in determining the best ways to help these professionals cope with unplanned change.\n\n\nConclusion\n\nThe research explored within this paper shows a general consensus on the feelings of anxiety, loss, grief, and sadness that a mental health care provider may feel after the termination of a therapeutic relationship. However, more research is needed to examine the effect that unplanned and sudden terminations have on the provider. The Center for Disease Control (CDC) highlighted that the COVID-19 pandemic presents a stressful situation, and anxiety and fear are common reactions to the current situation (Centers for Disease Control and Prevention, 2019). Unprecedented circumstances have resulted in trauma as well as moral dilemmas, relating to the challenges of maintaining a standard of care while transitioning to telehealth, (using digital technologies such as computers to access health care services remotely). This has the potential to increase the risk of mental health disorders such as post-traumatic stress disorder (PTSD), depression, anxiety disorders, and substance misuse in healthcare providers in the future (Greenberg et al., 2020). Healthcare providers, including mental healthcare workers, have experienced burnout during the COVID-19 pandemic due to a high workload and multiple psychosocial stressors (Sultana et al., 2020). Increased awareness of work-related stress, implementing mindfulness and self-care practices, and using technologies to address stress can help to mitigate burnout in this population (Sultana et al., 2020). Current research largely focuses on healthcare workers in hospital settings, but the findings can also be applied to mental healthcare providers serving the student-athlete population who are also considered essential workers. PTSD, losing sleep due to excessive worry, headaches, depression, anxiety, and general psychological stress are all effects of COVID-19 for this vulnerable population (Hall, 2020). Navigating a new virtual world in which medical appointments take place online, presents many challenges to these professionals and difficulties such as grappling with how to be inclusive of those who are less technologically savvy can weigh heavily on their minds. All sport support professionals in the U.S. have been impacted and while we, as mental healthcare providers, might write about this, it is also critical that we educate and provide support for our colleagues.\n\nThe COVID-19 pandemic affords us the opportunity to critically evaluate mental health care delivery systems which already exist in the United States and work towards equitable delivery of mental healthcare. When doing so, it is critical that we consider the needs of the sport support professional, as no sport support professional is immune to feelings of loss and grief in wake of the pandemic. Self-care is imperative during these stressful times. The APA Board of Professional Affairs Advisory Committee on Colleague Assistance conceptualizes the progressive downward spiral that can occur without successful management of stress through the stress-distress-impairment-improper behavior continuum (Wise et al., 2012). When stress is not managed effectively, professional functioning is impaired which can result in improper behavior. There is a broad consensus that implementing self-care strategies such as mindfulness, therapeutic lifestyle changes, and promoting physical health can help sport support professionals keep burnout at bay (Asuero et al., 2014). Just as sport support professionals are looking for ways to support the student-athletes who have been affected by abrupt season terminations, such as by providing increased access to mental health services (Fogaca, 2019; Kern et al., 2017), we should also seek ways to support their sport psychologist practitioners, sport medicine providers, and academic advisors who may be struggling with their own feelings of loss.\n\nWe hope that future direction of research around COVID-19 includes the sport support professionals noted in this paper. We hypothesize that latent effects will be researched as we surpass this first global pandemic of the twenty first century, and many of these studies will primarily focus on our clients. Future studies need also consider the effects on sport support professionals and guidelines to help future generations prepare for abrupt terminations occurring during their professional careers.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nPiselli A, Halgin RP, MacEwan GH: What went wrong? Therapists’ reflections on their role in premature termination. Psychother Res. 2011; 21(4): 400–415. PubMed Abstract \| Publisher Full Text\n\nAsuero AM, Queraltó JM, Pujol-Ribera E, et al.: Effectiveness of a Mindfulness Education Program in Primary Health Care Professionals: A Pragmatic Controlled Trial. J. Contin. Educ. Health Prof. 2014; 34: 4–12. PubMed Abstract \| Publisher Full Text\n\nBrenner PS, Serpe RT, Stryker S: The Causal Ordering of Prominence and Salience in Identity Theory: An Empirical Examination. Soc Psychol Q. 2014; 77(3): 231–252. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown G: Mind, body and sport: Understanding and supporting student-athlete mental wellness. Indianapolis, IN: National Collegiate Athletic Association; 2014.\n\nEbaugh HR: Becoming an ex the process of role exit. Chicago: Univ. of Chicago Press; 1998.\n\nFarber BA: Psychotherapists’ perceptions of stressful patient behavior. Professional Psychology: Research and Practice. 1983; 14(5): 697–705.\n\nFogaca JL: Combining Mental Health and Performance Interventions: Coping and Social Support for Student-Athletes. J Appl Sport Psychol. 2019; 33(1): 4–19. Publisher Full Text\n\nGreenberg N, et al.: Managing mental health challenges faced by healthcare workers during covid-19 pandemic. Br. Med. J. 2020; m1211: 368. PubMed Abstract \| Publisher Full Text\n\nHendrix AE, Acevedo EO, Hebert E: An examination of stress and burnout in certified athletic trainers at division I-a universities. J Athl Train. 2000; 35(2), 139–144. PubMed Abstract \| Free Full Text\n\nJacobi M: Mentoring and undergraduate academic success: A literature review. Am Educ Res Assoc. 1991; 61(4), 505–532. Publisher Full Text\n\nJoyce A: Termination in psychotherapy: a psychodynamic model of processes and outcomes. 1st ed: American Psychological Association; 2007.\n\nKern A, Heininger W, Klueh E, et al.: Athletes Connected: Results From a Pilot Project to Address Knowledge and Attitudes About Mental Health Among College Student-Athletes. J Clin Sport Psychol. 2017; 11(4): 324–336. Publisher Full Text\n\nKuhn T, Gordon VN, Webber J: The Advising and Counseling Continuum: Triggers for Referral. NACADA J. 2006; 26(1): 24–31. Publisher Full Text\n\nLevinson HL: Termination of psychotherapy: Some client issues. Social Casework. 1977; 58: 480–489.\n\nMcFarlane A: The contribution of epidemiology to the study of traumatic stress. Soc Psychiatry Psychiatr Epidemiol. 2004; 39: 874–882. PubMed Abstract \| Publisher Full Text\n\nMoreland JJ, Coxe KA, Yang J: Collegiate athlete’s mental health services utilization: A systematic review of conceptualizations, operationalizations, facilitators, and barriers. J Sport Health Sci. 2018; 7(1): 58–69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Collegiate Athletic Association: [NCAA] Compliance Manual.2010-2011. 2010-2011.\n\nNCAA Division I manual: Indianapolis, IN: National Collegiate Athletic Association.\n\nNorcross JC, Zimmerman BE, Greenberg RP, et al.: Do All Therapists Do That When Saying Goodbye? A Study of Commonalities in Termination Behaviors. Psychotherapy. 2017; 54(1): 66–75. PubMed Abstract \| Publisher Full Text\n\nPetitpas AJ, Giges B, Danish SJ: The Sport Psychologist-Athlete Relationship: Implications for Training. Sport Psychol. 1999; 13(3): 344–357. Publisher Full Text\n\nSultana A, Sharma R, Hossain MM, et al.: Burnout among healthcare providers during COVID-19: Challenges and evidence-based interventions. Indian J Med Ethics. 2020: 1–4. PubMed Abstract \| Publisher Full Text\n\nSwann WB Jr, Brown JD: From self to health: Self-verification and identity disruption. In: Sarason BR, Sarason IG, Pierce GR (Eds.), Wiley series on personality processes. Social support: An interactional view. John Wiley & Sons; 1990; (p. 150–172).\n\nVasquez MJT, Bingham RP, Barnett JE: Psychotherapy termination: clinical and ethical responsibilities. J Clin Psychol. 2008; 64(5): 653–665. PubMed Abstract \| Publisher Full Text\n\nWise EH, Hersh MA, Gibson CM: Ethics, Self-Care and Well-Being for Psychologists: Reenvisioning the Stress-Distress Continuum. Professional Psychol: Res Prac. 2012; 43(5): 487–494. Publisher Full Text\n\nYelsa E, Young D: Grief as an emotional reaction of athletes retired from competitive sport.ProQuest Dissertations Publishing; 1995." }	[ { "id": "102529", "date": "10 Feb 2022", "name": "Michael Christian Leitner", "expertise": [ "Reviewer Expertise Psychology", "Sportpsychology", "Neuropsychology" ], "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 deals with a highly relevant topic. The mental health of sport support professionals in the wake of the COVID-19 pandemic. Previous research has focused primarily on the athletes themselves. But not on sport support professionals. The authors aim to change that.\nAbstract:\nI am missing a clear conclusion. What do I specifically know more about after reading the article? What is specifically meant by \"experiencing a collective loss\"? What research could be done specifically to change this?\nIntroduction:\nThe five paragraphs (beginning with \"During the spring of 2020, thousands of student-athletes ...) to the end of the section are highly repetitive and should be streamlined and logically structured. This could be done, for example, according to the following scheme:\nResearch status to date and significance of the COVID-19 pandemic for athletes\n\nResearch status to date and significance of the COVID-19 pandemic for sports support professionals\n\"As with therapeutic providers, when termination occurs abruptly and not through the normal process, the emotional impact may be greater\" -> Is there empirical evidence on this? Without reference(s) this seems speculative to me.\nThe consequences of early termination\nThis part is very similar to what I have read before in the Introduction. Thus, I also recommend streamlining this part and implementing it into the Introduction if necessary or extending the existing part. In addition, I find the reported number of studies conducted on this topic in this chapter to be very low. Are there not more than 3 studies (that are newer than 2011) on this topic?\nThe sport support professional\nAgain, I recommend streamlining the content. You don't get the feeling of learning much in this section that hasn't been already discussed before. Similarly, the sources don't seem up to date to me. Are there no more recent studies on this?\nCoping\nThis part of the article raises very interesting questions. For example, what coping strategies might be important for sport support professionals or how sport support professionals dealt with the consequences of the pandemic in the first months and afterwards? I would elaborate this part of the article and back it up with current evidence.\nRole identity\nAgain, I read a lot of what has been touched on before. I would also shorten this part and implement it in previous parts of the work.\nConclusion\n\"The research explored within this paper shows a general consensus on the feelings of anxiety, loss, grief, and sadness that a mental health care provider may feel after the termination of a therapeutic relationship.\"\nThis assertion needs to be elaborated in more detail and precision in the preceding pages. I cannot agree with this as it stands after reading the article. If the present work is supposed to be an analysis of the current state of knowledge and give research recommendations, a comprehensive, structured, and up-to-date mapping of the state of research is missing.\nIn general, the Conclusion Section seems a little disorganized and \"crowded\". In very few sentences the authors deal with various topics, such as emotional consequences of the COVID-19 pandemic, trauma, telehealth, PTSD, and other clinical pathologies. Here, a logically comprehensible line of reasoning is missing. In a few sentences, many completely different topics are addressed but no specific potential answers and guidance for future empirical work is provided. Something I would expect from the work at hand.\nREVIEW CONCLUSION\nThe title of the manuscript does not keep its promise. What is the actual impact on sport support professionals? What was done recently to empirically answer these questions? As it stands, this article is a list of (mostly older) studies and I miss specific empirical background for future studies to be done on this topic.\nI recommend streamlining the article by concentrating on one or two specific topics behind the problematic situation of sport support professionals in the COVID-19 pandemic. Then the authors should present in much more detail potential solutions to this problem, elaborate the empirical background and back it up with more recent studies and work.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No", "responses": [] }, { "id": "253966", "date": "29 Mar 2024", "name": "Guilherme Grubertt", "expertise": [ "Reviewer Expertise Physical Activity and Sport", "Sport Psychology" ], "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, congratulations on your choice and on the time spent investing in this manuscript. However, there are still a lot of gaps that should be considered. Nevertheless, I added some contributions and References that may improve your research in the future.\n\nThe impacts of COVID-19 in various sports contexts are still being investigated. This subject is relevant nowadays. Regarding the context of student-athletes and their stakeholders, some manuscripts can help the writing of this manuscript (see References).\n\nAbstract Should contain all elements of a paper (it is missing the manuscript objective, methodology, and conclusions);\n\nIntroduction There is a lack of literature review and state of art. For example, what is already known about the subject? What is contradictory? What is the research gap? It's necessary to describe the problem of the study; It's necessary a brief review of literature relevant to the topic; It's necessary to explain how your study will challenge, expand, or improve existing knowledge;\n\nMaterials and Methods The study design must be presented; I believe this paper is an opinion article. Therefore, the methodology should be well described. For example, opinion articles allow researchers to contribute their point of view on controversial issues (controversial information is not clear), the interpretation of recent findings [Ref 1,2,3,4,5.6.7], the validity of commonly used methods, and so on. Opinion articles are based on data already available in the literature, to encourage constructive discussion by the scientific community.\nDiscussion What are the main findings of the manuscript? What are the potential strengths and weaknesses of the article? It is necessary to point out these aspects. There is a lot of repeated information between the introduction and other subheadings of the article. There should be a balance between the information presented in the introduction and the other subheadings.\n\nConclusion Mental health is an extremely important issue, especially after Covid-19.\n\nFuture studies are highlighted by the authors. This underlines the importance of the topic.\n\nThe conclusion must resume the main findings or the most relevant information about the manuscript. The conclusion should avoid references and new speculations like the last paragraph.The conclusion of an opinion article should emphasize the constructive discussion of contradictory information from the scientific literature.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No", "responses": [] } ]	1	https://f1000research.com/articles/10-775
https://f1000research.com/articles/9-1162/v1	21 Sep 20	{ "type": "Case Report", "title": "Case Report: Secondary bilateral parkinsonism and dystonia treated with dronabinol", "authors": [ "Natalia Szejko", "Florian Burger", "Victoria Sidoroff", "Gregor K. Wenning", "Florian Burger", "Victoria Sidoroff", "Gregor K. Wenning" ], "abstract": "Drug abuse may damage basal ganglia that are essential for planning and execution of movements. We report a 38-year old patient with ischemic lesions of the basal ganglia presenting with bilateral painful dystonia and parkinsonism caused by polyintoxication. Dronabinol resulted in improvement of pain and gait disturbance, suggesting a novel therapeutic strategy in these challenging patients.", "keywords": [ "dronabinol", "secondary parkinsonism", "dystonia", "intoxication", "ischemic stroke" ], "content": "Introduction\n\nDrug abuse is an important health issue, not only affecting the social surrounding of a patient but also the cerebral integrity. Besides a number of different somatic abnormalities provoked by an intoxication, certain drugs may damage areas of the brain, such as basal ganglia or cortex, which are essential for the movement onset and coordination1,2.\n\nIn this case report, we present a 38-year-old male patient with a long-lasting drug history, including the abuse of cocaine, cannabinoids, benzodiazepines, opiates, methadone and amphetamines. As a result of the multi-intoxication induced bilateral ischaemic lesions to the basal ganglia, the patient presented with secondary bilateral dystonia and parkinsonism. It is not certain whether the cause of ischemia could be related to any specific drug or combinations thereof. Cocaine and its metabolites are known to cause cerebral vasospasm that could lead to ischemic infarctions in the whole brain1,3,4 or can provoke haemorrhagic stroke2. Heroin causes ischemia more often in the globus pallidus5. Amphetamines are known as the second most frequent cause of ischemia after cocaine, especially in younger patients due to their vasoconstrictive effect1,6. Although it is not clear whether cannabinoids and its metabolites can cause cerebrovascular events, there is evidence that cannabis can increase the risk for haemorrhagic stroke1,7. Opioids may not have a direct toxic impact on the neurons, but ischaemic lesions or necrosis can be triggered by recurrence of drug-induced hypoxia1.\n\nThe aim of this case report is to show the possible use of dronabinol for a multi-intoxicated patient with ischemia of the basal ganglia in order to temper his pain and improve gait.\n\n\nCase report\n\nWe present a case of a 27-year-old Caucasian man who was admitted to the Intensive Care Unit (ICU) after multi-intoxication. Drug screening on admission identified the following substances: opiates, benzodiazepines, cannabinoids, crack cocaine, methadone and amphetamine. Due to the history of drug abuse he was unemployed. During the first days the patient was in coma, with Glasgow Coma Scale of 5 points. After slow amelioration of his status he presented with severe dysarthria, dysphagia, as well as bilateral parkinsonism and dystonia of his extremities. The neurological examination showed a bilateral positive Babinski sign, global rigidity in all extremities as well as symmetrical hyperreflexia. Furthermore, he suffered from high fever that resolved after treatment with benzodiazepines. Therefore, vegetative symptoms accompanying the patient on submission were interpreted as drug withdrawal syndrome. Both, CT and MRI showed bilateral hypoxemic infarction of the basal ganglia and boundary zone (Figure 1). Those changes could be the consequence of the mixed intoxication or could be attributed to only one harmful substance, especially crack cocaine or amphetamine. During the hospitalization he developed a tracheobronchitis and respiratory insufficiency due to acute respiratory distress syndrome (ARDS). Moreover, because of severe dysphagia he was nourished via percutaneous endoscopic gastrostomy. Further consequences of his primary condition included post ischemic epilepsy treated with levetiracetam (1000mg). After six months of intensive care, the patient was discharged from hospital.\n\nDuring the next five years the patient was treated with levodopa (titrated up to 800mg/day), apomorphine (up to 100 mg/day), selegiline (10mg/day) and baclofen (75mg/day). Further up-titration of levodopa was not possible because of subsequent side effects, such as hallucinations. Due to sleeping problems and agitation he was treated with trazodone (150mg/day) and quetiapine (250mg/day). The substitution dose of buprenorphine 20mg/day was tapered off constantly to keep the patient in a drug naive state. Nonetheless, he showed aggressive behaviour under the influence of alcohol and was admitted to psychiatric ward several times due to delusional disorder and recurrent addictive behaviours. After several months of psychiatric treatment, his addiction and hallucinations resolved. In spite of intense treatment, both parkinsonism as well as dystonia persisted for years and additional symptoms such as generalized pain as well as gait disturbances occurred.\n\nDuring the examination in our outpatient clinic, 11 years after the hospitalization in the ICU, the patient was still experiencing moderate dysarthria, bilateral dystonia of all extremities, bilateral akinetic-rigid parkinsonism, camptocormia and freezing. Due to those symptoms and increased anxiety as well as painful dystonia as well as unsuccessful treatment with evidence-based agents the patient was treated with dronabinol by his psychiatrist (capsules slowly up titrated to 20mg/day).\n\nAfter two months of dronabinol treatment, the patient reported subjective improvement of the dystonic pain and a moderate improvement of freezing of gait. We examined the patient at two timepoints: after two months and after two months of dronabinol therapy. Importantly, the medication used at both timepoints remained stable, which excludes confounding contribution of other agents. Results of clinical assessments after two and six months of dronabinol administration are shown in Table 1. The baseline Unified Parkinson’s Disease Rating Scale and Unified Dystonia Rating Scale results cannot be provided as the data were not collected. Although the patient reported the subjective improvement of his symptoms, this was not confirmed in the neurological examination or in the UPDRS and UDRS assessments. Particularly, there was no minimal clinically important differences for UPDRS nor UDRS, although there was an improvement in sleep quality according to ESS. It can therefore be concluded that dronabinol had major analgesic and calming effect and, as a consequence, also improved sleep and general performance. Although he self-reported gait amelioration, it is not clear whether the patient exhibited any motor improvement.\n\nUPDRS, Unified Parkinson’s Disease Rating Scale; H-Y, Modified Hoehn and Yahr Staging; PDSS-2, Parkinson Disease Sleep Scale-2; UDRS, Unified Dystonia Rating Scale; ESS, Epworth Sleepiness Scale; BDI, Beck Depression Inventory.\n\n\nDiscussion\n\nWhile there is some evidence that cannabis-based medicine (CBM) could improve both motor and non-motor symptoms in Parkinson’s disease (PD)8,9, the effectiveness of CBM in secondary parkinsonism as well as dystonia is unknown. Recently, Peball et al.10 reported the potential efficacy of nabilone for PD patients with disturbing non-motor symptoms, which appears to be driven by positive effects on anxious mood and night-time sleep problems. Therefore, CBM might be a useful therapeutic alternative for therapy resistant patients with pain accompanying movement disorders. In the case of our patient, his condition improved only subjectively, but this was not confirmed with objective neurological testing. Moreover, the use of cannabinoids in this case is controversial as the patient had a positive medical history for cannabis misuse. Additionally, long-term effectiveness should also be investigated with accuracy. Potential psychotic effects could be dangerous, therefore the dosage in our patient was slowly increased. Finally, no baseline assessments, prior to dronabinol administration, were available. Results of international randomized, double-blind controlled trials with CBM in PD might offer more scientific rationales for discussion of potential usefulness of CBM in other movement disorders.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nBüttner A: Review: The neuropathology of drug abuse. Neuropathol Appl Neurobiol. 2011; 37(2): 118–34. PubMed Abstract \| Publisher Full Text\n\nBüttner A: The neuropathology of drug abuse. Current Opinion in Behavioral Sciences. 2017; 13: 8–12. Publisher Full Text\n\nVidal SGM, Hornik A, Morgan C: Cocaine induced hippocampi infarction. BMJ Case Rep. 2012; 2012: bcr0320125998. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarrell CM, Cucu DF: Cocaine-Related Acute Spinal Cord Infarction. R I Med J (2013). 2018; 101(1): 28–9. PubMed Abstract\n\nVila N, Chamorro A: Ballistic movements due to ischemic infarcts after intravenous heroin overdose: report of two cases. Clin Neurol Neurosurg. 1997; 99(4): 259–62. PubMed Abstract \| Publisher Full Text\n\nLappin JM, Darke S, Farrell M: Stroke and methamphetamine use in young adults: a review. J Neurol Neurosurg Psychiatry. 2017; 88(12): 1079–91. PubMed Abstract \| Publisher Full Text\n\nShere A, Goyal H: Cannabis can augment thrombolytic properties of rtPA: Intracranial hemorrhage in a heavy cannabis user. Am J Emerg Med. 2017; 35(12): 1988.e1–1988.e2. PubMed Abstract \| Publisher Full Text\n\nBalash Y, Bar-Lev Schleider L, Korczyn AD, et al.: Medical Cannabis in Parkinson Disease: Real-Life Patients' Experience. Clin Neuropharmacol. 2017; 40(6): 268–72. PubMed Abstract \| Publisher Full Text\n\nPeres FF, Lima AC, Hallak JEC, et al.: Cannabidiol as a Promising Strategy to Treat and Prevent Movement Disorders? Front Pharmacol. 2018; 9: 482. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeball M, Krismer F, Knaus HG, et al.: Non-Motor Symptoms in Parkinson's Disease are Reduced by Nabilone. Ann Neurol. 2020. PubMed Abstract \| Publisher Full Text" }	[ { "id": "71804", "date": "05 Oct 2020", "name": "Jose Luis Lopez-Sendon Moreno", "expertise": [ "Reviewer Expertise Clinical research. Cannabinoids." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe an interesting case about a patient with secondary dystonia and parkinsonism and an aparent improvement with dronabinol. The case is relevant since it provides insight into the treatment of a disabling condition. Also, the use of cannabinoids in the setting of movement disorders is a promising field.\n\nThe case is well described but I have several comments to make:\n\nDo the authors have information on other similar cases treated with dronabinol in which no improvement (or perhaps secondary effects) were noted? If so, please comment in order to avoid \"publication bias\".\n\nIs the mechanism of action of the parkinsonism isquemic? perhaps the toxicity could be due to other mechanisms (toxic, inflamatory...) Could other \"over the counter\" substances contribute to the toxicity?\n\nThe authors comment on the lack of structured data on the neurological exploration before treatment. Gait improvement is based on the subjective experience alone. However, could this improvement be quantitatively reflected (Eg. was able to walk unaided, use of support, falls....). If not, then the abstract and conclusions could be overstated. Please correct.\n\nThe improvement was noted after two months. Please comment why the delay, since the effect should have happened earlier.\n\nSome information on the text should be clarified (was the patient six months in intensive care or was that the total time of hospitalization?). As well is repeated twice in the same sentence. Please correct. The examination was done after two months twice (in the text, not in the table), please correct.\n\nThere is some evidence (RCT and good reviews) in the use of CBM in mov disorders. Please consider adding further references.\n\nI believe that the case report should be of interest to the readers and should be indexed with some changes.\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": "6019", "date": "23 Oct 2020", "name": "Natalia Szejko", "role": "Author Response", "response": "Thank you so much for your valuable comments, we would like to address them below. We have also submitted the new version of the manuscript in the effort to address your comments. As for other cases of the movement disorders treated with cannabis based medicine, there is already extensive evidence in this area, reporting also about the side effects. We discuss it in the paper, but have included more references and more extensive description, as suggested. When it comes to the exact mechanism of ischemia in this case, it is not entirely clear. However, we suspect it was both toxic and secondary due to vasoconstriction. We have updated the information about the gait improvement, as the patient experienced less falls after treatment with dronabinol. We would like to comment on the time of assessments. The first follow-up was performed after two months and we do not consider it too long, taking into consideration previous reports, this is an adequate time of evaluation. We have checked the manuscript for the timing and updated it accordingly. Finally, we have included more references reporting about the use of CBM in treatment of movement disorders." } ] } ]	1	https://f1000research.com/articles/9-1162
https://f1000research.com/articles/10-773/v1	06 Aug 21	{ "type": "Research Article", "title": "Determinants of preterm birth in public hospitals in central Ethiopia: an unmatched case-control study", "authors": [ "Berhanu Senbeta Deriba", "Agumas Fentahun Ayalew", "Addis Adera Gebru", "Agumas Fentahun Ayalew", "Addis Adera Gebru" ], "abstract": "Background: Around 15 million babies are born prematurely in the world every year. The most common cause of neonatal death in Ethiopia is premature birth. To reduce the rate of preterm delivery by correcting modifiable or preventable causes, the availability of local data is important. Hence, this study aimed to identify the determinants of preterm birth among women who gave birth in public hospitals in central Ethiopia. Methods: An Institutional-based unmatched case-control study was conducted at public hospitals in central Ethiopia to select 170 cases and 340 controls. The collected data were entered into EPI INFO and transferred to SPSS for analysis. Tables, graphs, and proportions were used to present the results. Binary and multiple logistic regressions analysis were computed to identify determinants of preterm birth. Adjusted Odds Ratio (AOR), 95% Confidence Interval (CI), and a p-value < 0.05 were computed to determine the presence of an association between preterm birth and independent variables. Results: A total of 166 cases and 332 controls participated in the study, giving a response rate of 97.6%. Cigarette smoking (AOR=3.77, 95% CI=1.35,10.56), alcohol consumption (AOR=1.85, 95% CI=1.11,3.10), wanted but unplanned pregnancy (AOR=3,95% CI=1.68,5.34), neither wanted nor planned pregnancy(AOR=3.61% CI=1.62,8.06), lack of antenatal care (ANC) visits (AOR=4.13, 95% CI=1.95, 8.74), adverse birth outcomes (AOR=5.66, 95% CI=2.88,11.12), presence of a diagnosed illness (AOR=2.81, 95% CI=1.37, 5.76), presence of one or more of obstetrics complications(AOR=6.44, 95% CI=5.49, 3.35, 9), and hemoglobin level < 11g/dl (AOR=2.78, 95% CI=1.48, 5.22) were determinants of preterm birth. Conclusion:-In this study, cigarette smoking status, alcohol drinking status, pregnancy status, adverse birth outcomes, ANC visits, obstetric complications, presence of medical illness, and anemia were identified as determinants of preterm birth. It is important to encourage such women to attend ANC visits, stop smoking, and abstain from alcohol.", "keywords": [ "Central Ethiopia", "Determinants", "Hospitals", "Mothers", "Preterm birth" ], "content": "Abbreviations\n\nANC; antenatal care, HIV/AIDS; human immunodeficiency virus/acquired immune deficiency syndrome, IFA; iron folic acid, LBW; low birth weight, PIH; pregnancy induced hypertension, PTB; preterm birth, PROM; premature rupture of membrane, WHO; World Health Organization.\n\n\nIntroduction\n\nThe World Health Organization (WHO) defines preterm birth as childbirth that occurs before 37 weeks of gestation starting from the first day of the last menstrual period. Based on gestational age it can be classified as: Extremely preterm (<28 weeks), very preterm (28 to 31 weeks), and late preterm (32 to 37 weeks).1 Preterm birth can be also classified by its clinical presentations as spontaneous and iatrogenic preterm.2 From 130 million babies born annually, almost 15 million are preterm birth globally; more than one in ten newborns are born prematurely. Furthermore, more than one million children die due to complications of preterm birth every year. Preterm birth is the top cause of neonatal mortality and the second most common cause of mortality after pneumonia among children under five.3 Preterm birth also affects developed countries, for instance, USA is among the ten countries with the greatest numbers of preterm births.4 Worldwide, the incidence of preterm births is; 9.6% globally, 7.5% in developed countries, 12.5% in developing countries, 9.1% in Asia, 6.2% in Europe, 10.6% in North America, and 11.9% in Africa.5 In developing countries, more than 60% of preterm births occur in Africa and South Asia.5 The rate of premature birth in southeast Nigeria rose from 9.8% to 17.1%.6 The rate of preterm birth is 5% in Northern European countries and 18.1% in Malawi.7 The prevalence of preterm birth varied from place to place for instance it is 18.3% in Kenya,8 11.6.% in Debremarkos town, Ethiopia,9 31.4% in Mettu Karl Hospital, Ethiopia,10 34% in Tigray region, Ethiopia,11 35% in Dessie Referral Hospital,12 and 25.9% in Jimma, Ethiopia.13 Preterm birth causes 75% perinatal mortality and greater than 50% of long term morbidity.14 Prematurity is the second principal reason for death in children under five years and the single most significant direct cause of death in the critical first month of life. In spite of the decline in global child mortality rates, newborn death denotes a growing proportion of all death among children less than five years of age. The risk of dying from preterm birth complications is 10 times greater for babies born prematurely in low-income countries than for those born in high-income countries.15 Low educational status, advanced maternal age, chronic diseases, parity, lack of antenatal care visits, multiple pregnancies, urinary tract infection, prolonged premature rupture of membrane (PPROM), and pregnancy induced hypertension (PIH) are risk factors of preterm birth.8,9,16 Although preterm birth is common in Ethiopia, the determinants of preterm birth like drinking alcohol, medication intake, and cigarette smoking during pregnancy were not evaluated in previous studies. Although neither planned nor wanted pregnancies are common in Ethiopia, whether they cause preterm birth or not has not been studied. Long term research over the past several decades had failed to solve these problems. To identify the determinants of premature birth among the Ethiopian populations and try to minimize the rate of preterm delivery by correcting modifiable or preventable causes, the availability of local data is important. Thus, this study aimed to identify the determinants of preterm birth among women who gave birth at public hospitals in central Ethiopia.\n\n\nMethods\n\nAn institutional-based unmatched case-control study was conducted from 1st February to 30th June 2020 in public hospitals in North Shewa, central Ethiopia. The North Shewa zone has a total population of 1.6 million. From the total population, 49,667 were pregnant women according to the North Shewa Zone health office report.17 Fiche is the capital city of the zone and 114 kilometers away from Addis Ababa.18 It has five hospitals and 64 health centers. It also has 2,420 health professionals out of whom 213 are midwives.17 We have conducted this study in two general and three primary hospitals. All of the hospitals in this Zone provide maternal health services like family planning, ANC, delivery, and postnatal care to the surrounding communities. We selected all of the five hospitals present in the zone purposely given the extensive maternal health services provided.\n\nAll women 15-49 years old that gave birth at all public hospitals in the North Shewa zone during the study period were the source population of the study. All women 15-49 years of age that delivered their babies at public hospitals in the North Shewa zone during the data collection period and who were included in the study were the study population. Women who gave live newborns before 37 completed weeks of gestational age were cases (preterm) whereas women who gave live newborns at or after 37 completed weeks of gestational age were controls (non-preterm).7\n\nAll women who gave birth during the data collection period at selected hospitals and whose gestational age were known or estimated from the last menstrual period or by ultrasound were included in the study whereas, women with induced termination of pregnancy for medical reasons, women who gave birth before 28 weeks of gestation, and those who had stillbirths were excluded from the study. Cases were selected by taking all cases until the required number of cases were obtained and controls were selected using systematic random sampling, which was conducted in hospital before the discharge of the mother from the hospital after consent was obtained.\n\nWe have calculated the sample size by using two population proportion formula and through EPI INFO version 7 statistical software package with the assumptions of confidence interval 95% (Z𝛼/2=1.96), power 80% (Z𝛽= 0.84), case to control ratio 1:2, where P1 is the proportion of cases exposed and P2 is the proportion of controls exposed. The variables used for sample size calculations were reactive to STI, lack of formal education, lack of ANC care visit, maternal anemia as taken from previous similar articles.19–23 Sample size was calculated for all of the variables mentioned above and of all variables, the sample size calculated from anemia during pregnancy was the largest of all, where the proportion of cases exposed was 79%, proportion of controls exposed was 11.2%, and AOR=2.2, which gave a final sample size of 170 cases and 340 controls with a total of 510 after adding a 10% non-response rate.\n\nThe number of study participants was allocated proportionally to each hospital based on estimations obtained from the average of previous quarter delivery services by referring to delivery registration books at each hospital. Therefore, the sample of each hospital was calculated by multiplying the average number of pregnant women who delivered in each hospital per five months with the total sample size, dividing by a total number of pregnant women attending antenatal care units for five months for all hospitals which was obtained from delivery registration of the previous months. Cases were selected by taking all cases until the required sample size obtained and controls were selected using systematic random sampling [Table 1].\n\nThe data collection instrument was developed by reviewing the existing literature.20,24,25 The questionnaire was developed and arranged according to study objectives. The questionnaire was prepared in English and then translated to Afan Oromo and Amharic languages that are local languages of the area to improve understanding of both the data collectors and respondents, and translated back to the English version by a language expert.26 The reliability of the used questions was checked with Cronbach’s alpha, which was found to be 0.860. Two days of training was provided for data collectors and supervisors on the objective of the study, contents of the questionnaire, confidentiality, the right of respondents, and how to collect data. The questionnaire was pre-tested on 5% (17 cases and 34 controls) of the sample at Chencho Hospital which is outside the study area. To assess the reliability of data collection instruments and findings, data collectors and supervisors discussed the questionnaire so that; the tool was modified for any inconsistencies and ambiguity before actual data collection. Ten diploma midwives and five BSc Public Health professionals were selected for data collection and supervision respectively. Information such as socio-demographic, substance-use related, and obstetrics related factors was collected by face to face interviews by using pretested questionnaire during a post-delivery hospital stay of women in the first 24 hours or during discharge from hospitals in the post-natal care room of the hospital. Gestational age was obtained from ANC records which in turn was estimated from the last menstrual period or early ultrasound conducted during the first-trimester pregnancy of women. Information related to obstetric, fetal characteristics and diagnosed illness of mothers (renal diseases, diabetes mellitus (DM), and urinary tract infection (UTI)), and newborns were also obtained from ANC records.\n\nWe checked the data for completeness after data collection. The data was then coded, cleaned, and entered using EPI INFO version 7 and transferred to SPSS version 23 for cleaning and analysis. Tables, graphs, and proportions were used to present the results of this study. Bivariate and multi variables logistic regressions analysis were carried out to determine the presence of an association between preterm birth and independent variables. Bivariate analysis was carried out to determine a significant association between each predictor variable and preterm birth at a p-value < 0.25.27 Multivariate logistic regression was carried out to identify the determinants of preterm birth. The enter model selection method was used to identify variables that remained for the final model. The goodness of fit model (Hosmer and Lemshow) was used to select the best multivariate model and the p-value of the model fitness test was 0.850. Multicollinearity and confounding effects were checked by using standard error and Hosmer and Lemshow goodness of fit test. Finally, AOR with 95% CI and p-value < 0.05 were considered to indicate significant association.\n\nThe study protocol and methodology were approved by Salale University Ethical Review Committee on February 02, 2020 with the reference number SLU ERC/010/2020. A formal letter of cooperation was written to each hospital. Before starting the interview the respondents were informed the purpose of the study and how their data could be used. The respondents were told as their participation in the study is voluntary and they have right to withdraw from the study at any time. Written consent was obtained from every study participant for those aged ≥ 18 years and written assent was obtained from the guardians for those aged below 18 years for access to ANC records for review as well as participation in the face-to-face interview. The privacy and confidentiality of the study participants were also strictly protected. Data collectors were told to code the questionnaire and not to write the name of the study participants.\n\n\nResults\n\nA total of 498 women (166 cases and 332 controls) participated in this study making the response rate 97.6%. The age of study participants ranged 17-43 years for cases and 17-42 years for controls with the mean age of 27.77±5.5 for cases and 26.94±5.007 for controls. About one-third (56, 33.7%) of cases and around a quarter (78, 23.5%) of controls had no formal education [Table 2].\n\nNear to one-third (35, 31.8%) of cases and 39 (17.6%) of controls had a birth interval < 2 years. More than a quarter (32, 29.1%) of cases and 10 (4.5%) controls had a previous history of preterm birth. Near to half (51, 46.4%) of cases and 36 (10.8%) controls had a previous history of adverse birth outcomes [Table 3].\n\nFrom women who came to the hospitals for delivery services, 50 (30.1%) cases and 23 (6.9%) controls neither wanted nor planned their current pregnancies. Around a quarter (44, 26.5%) of cases and 21 (6.3%) controls had no ANC visit during their pregnancy. In total, 34 (20.5%) of cases and 26 (7.8%) controls had of cases and 26 (7.8%) controls had diagnosed medical illness (renal diseases, DM, and UTI) during their current pregnancies. In total, 104 (62.7%) cases and 95 (28.6%) controls had obstetrics complications (Premature rupture of membrane, pregnancy-induced hypertension, antepartum hemorrhage) during their pregnancy [Table 4].\n\nCigarette smoking during their pregnancy was reported by 16 (9.6%) cases and 8 (2.4%) controls. Furthermore, 29 (17.5%) cases and 21 (6.3%) controls, had husbands who smoked cigarettes over the course of the pregnancy. In total, 69 (41.6%) cases and 86 (25.9%) controls reported a history of alcohol consumption during their current pregnancies [Table 5].\n\nIn total, 12 (7.2%) neonates born from cases and 12 (3.3%) neonates born from controls were twins. Regarding preterm birth, 37.3% were induced and 62.7% were spontaneous preterm births [Figure 1]. More than three quarters (78.9 %) of neonates born from cases and 21.1% of neonates born from controls had low birth weight [Figure 2].\n\nWe conducted bivariate logistic regression for each independent variable. Multivariate analysis was done for all variables with p-value <0.25 in the bivariate logistic regression analysis after adjusting for covariates. The fitness of the model was also assessed. The result of multiple logistic regressions indicated that women who had a of history of cigarette smoking during their current pregnancies had four-folds higher odds of preterm birth compared to women who did have a history of cigarette smoking (AOR=3.77, 95% CI: 1.35,10.56, p-value:0.007). Women who had history of drinking alcohol during their pregnancies had two folds higher odds of preterm birth compared to their counterparts (AOR=1.85, 95% CI: 1.11, 3.10, p-value: 0.019). Women who wanted but did not plan their pregnancies had four times higher odds of preterm birth compared to those who wanted and planned their pregnancies (AOR=3.95% CI: 1.68, 5.34, p-value: 0.002). Women who neither wanted nor planned their current pregnancies had 3.61 folds higher odds of preterm birth compared to their counterparts (AOR=3.61% CI: 1.62, 8.06. p-value: 0.001). Women who had no ANC visit for their current pregnancies had four-fold higher odds of preterm birth compared to those who had ANC care follow-up (AOR=4.13, 95% CI: 1.95, 8.74, p-value:0.001). Women who had a previous history of adverse birth outcomes like stillbirth, low birth weight, and preterm birth had 5.66 times higher odds of preterm birth compared to those who had no previous history of adverse birth outcomes (AOR=5.66, 95% CI:2.88,11.12, p-value: 0.001). Women who had a diagnosed illness during their current pregnancies had three folds higher odds of preterm birth compared to their counterparts (AOR=2.81, 95% CI: 1.37, 5.76, p-value: 0.005). Women who had one or more obstetrics complications (Premature rupture of membrane, pregnancy-induced hypertension, Antepartum hemorrhage) during their current pregnancies had 6.44 folds higher odds of preterm birth compared to their counterparts (AOR=6.44, 95% CI: 5.49, 3.35, 9, p-value: 0.001). Women who had anemia had three folds higher odds of preterm birth compared to those who were not anemic (AOR=2.78, 95% CI: 1.48, 5.22, p-value: 0.001) [Table 6].\n\n** = statistically significant at p-value < 0.05, 1= reference\n\n\nDiscussion\n\nPreterm birth is the primary cause of neonatal deaths in Ethiopia, where information on the determinants of preterm birth is commonly recorded. This study aimed to identify the determinants of preterm birth at public hospitals in central Ethiopia. The chance of preterm birth was higher among mothers that drank alcohol, smoked cigarettes, had unplanned pregnancies, neither wanted and nor planned pregnancy, had previous history of adverse birth outcomes, had no ANC visit, were anemic, had co-morbidities, and had obstetrics complications during their current pregnancies.\n\nAnemia was significantly associated with the occurrence of preterm birth in this study. The finding is consistent with studies conducted in Debramarkos town, Ethiopia,9 and Teran, Iran.24 This is because anemia decreases the immunity of the mother and leads to genital tract infection which in turn causes PROM and then causes preterm birth.28\n\nIn this study, cigarette smoking during the pregnancy was an independent predictor for preterm birth. The finding is similar to other studies29–31 where cigarette smoking was positively associated with preterm birth. This might be due to the fact that from the 3000 chemicals present in cigarettes, nicotine, and carbon dioxide cause decrease blood flow to the utero-placenta and damage the placenta because of their dominant vasoconstriction nature.28 Cigarette smoking can also cause a systemic inflammatory response which in turn causes spontaneous preterm birth.1 Tobacco causes preterm birth by causing low birth weight and placental abruption.28\n\nWomen who drank alcohol during their pregnancy were at higher risk of delivering preterm birth compared to their counterparts. The finding is in agreement with a study from South Africa,32 and another study in which women who took more than three drinks per day were at high risk of delivering preterm birth.33 The possible reason might be due to unidentified factors that precipitate preterm birth.\n\nUnwanted and unplanned pregnancy was another independent predictor for preterm birth in this study. Moreover, wanted but unplanned pregnancies were also positively associated with preterm birth. One possible reason for this is the stress induced by the pregnancy, which can lead to depressions that in turn causes preterm birth.28,34,35 Moreover, the more women want and plan their pregnancies, the more they take care for their pregnancies by consuming a variety of foods, visiting health institutions for ANC follow-up, and limiting heavy work. It is currently suggested that mothers of unplanned pregnancies will neglect their child, which leads to preterm birth. In addition, women with unplanned pregnancies are more likely to drink alcohol and smoke cigarettes that in turn lead to preterm birth.36\n\nHaving a previous history of adverse birth outcomes (low birth weight, preterm birth, and stillbirth) was positively associated with preterm birth. The finding is in line with a study conducted in Amahara region, Ethiopia,37 Sidama zone, South Ethiopia,21 Jimma zone South-west, Ethiopia,38 a study conducted at Gonder Hospital,10 and a case-control study conducted in West Iran39 where a previous history of adverse birth outcomes was positively associated with preterm birth. This might be due to women with a previous history of negative birth outcomes (adverse birth outcomes) experiencing stressful conditions that lead to recurrences (re-occurrence of preterm birth).28\n\nThis study signified that women who had no ANC visit for their index pregnancies were at higher risk of delivering preterm birth compared to their counterparts. It is consistent with a study conducted in the Amhara region,40 Dabramarkos town,12 North-west Ethiopia, and Hossana town, Southwest Ethiopia.41 Women who do not have ANC follow-up cannot get nutritional counseling, ferrous sulfate supplementation, and are unaware of the danger signs of obstetrics complications that lead to adverse birth outcomes like preterm birth.\n\nWomen who had conditions like diabetes and hypertension during their current pregnancies had a higher incidence of preterm birth compared to their counterparts. It is similar to studies conducted in Debramarkos,41 and Aksum towns, Ethiopia.42 This might be due to the fact that diabetes and hypertension cause a reduction in nutrient and oxygen delivery to the fetus via the placenta which leads to pre-eclampsia which in turn causes preterm birth.43\n\nSimilarly, this study also found that women who had one or more obstetrics complications (PIH, PROM, multiple pregnancy, and polyhydramnios) were more likely to give birth prematurely than those who had no obstetrics complications. It is consistent with a study conducted at Debremarkos town, Ethiopia.9 This might be due to the fact that complications like pregnancy-induced hypertension and PROM can cause damage to the vasculature of the placenta which in turn causes preterm birth. Multiple pregnancies and polyhydramnios (high amount of amniotic fluid) can stretch the myometrium (muscle of uterus); and it induces the oxytocin receptors which results in labor, and delivery.7 As far as we know, this study is the first to find a significant association of preterm births with smoking cigarettes, drinking alcohol, wanted but, unplanned pregnancy, and neither wanted nor planned pregnancy in Ethiopia.\n\nThis study maybe susceptible to recall bias due to its retrospective nature and social desirability bias with regard to face to face interview\n\n\nConclusion\n\nIn this study, cigarette smoking; alcohol consumption during current pregnancies; wanted but, unplanned pregnancies; neither wanted, nor planned pregnancies; lack of ANC visit for their current pregnancies; previous history of adverse outcomes; obstetric complications; presence of comorbidities; and being anemic were identified as determinants of preterm birth in this study.\n\nOromia regional health bureau is recommended to disseminate health information via the media on the impact of identified determinants of preterm birth and how to mitigate the problems surrounding premature birth. North Shewa health facilities or health professionals are recommended to counsel pregnant women to visit ANC, provide counseling at ANC clinics, encourage pregnant women to stop cigarette smoking and alcohol drinking, provide modern family planning to females in reproductive age groups, early identification, and treatment of medical illness among pregnant women.\n\n\nData availability\n\nFigshare: Determinants of preterm birth in public hospitals in central Ethiopia: An unmatched case-control study. https://doi.org/10.6084/m9.figshare.13713172.v1.26\n\nThis project contains the following underlying data:\n\n- SPSS 4 preterm birth.4 analysis.sav\n\nFigshare: Determinants of preterm birth in Public hospitals in central Ethiopia: An unmatched case-control study. https://doi.org/10.6084/m9.figshare.13713172.v1.26\n\nThis project contains the following extended data:\n\n- English version questionnaire.docx\n\n- Afan Oromo version questionnaire.docx\n\n- Amharic version questionnaire.docx\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 acknowledge Salale University, all of the five hospitals, and the study participants for their unreserved contribution to the success of this study.\n\n\nReferences\n\nGoldenberg RL, Culhane JF, Iams JD, et al.: Epidemiology and causes of preterm birth. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSifer SD, Kedir BS, Demisse GA, et al.: Determinants of preterm birth in neonatal intensive care units at public hospitals in Sidama zone South East Ethiopia case control study. J Pediat Neonatal Care. 2019; 9(9). Publisher Full Text\n\nMekuriyaw AM, Mihret MS, Yismaw AE: Determinants of Preterm Birth among Women Who Gave Birth in Amhara Region Referral Hospitals, Northern Ethiopia, 2018: Institutional Based Case Control Study. Int J Pediatr. 2020; 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSifer S, Kedir B, Demisse G: Determinants of preterm birth in neonatal intensive care units at public hospitals in Sidama zone, South East Ethiopia; case control study. J Pediatr Neonatal Care. 2019; 9(6): 180–6. Publisher Full Text\n\nWoday A, Muluneh MD, Sherif S: Determinants of preterm birth among mothers who gave birth at public hospitals in the Amhara region, Ethiopia: A case-control study. PloS one. 2019; 14(11). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbaraya M, Seid SS, Ibro SA: Determinants of preterm birth at Jimma university medical center, Southwest Ethiopia. Pediatric Health Med Ther. 2018; 9: 101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhalajinia Z, Gholamreza J: Maternal risk factors associated with preterm delivery in Qom province of Iran in 2008. Sci Res Essays. 2012; 7(1): 51–4. Publisher Full Text\n\nChimhini G, Tshimanga M, Chikwasha V, et al.: Determinants of premature births in two central hospital Harare, Zimbabwe, 2011. Cent Afr J Med. 2013; 59(9-12): 49–57. PubMed Abstract\n\nDeriba BSA: Agumas Fentahun; Gebru, Addis Adera Determinants of preterm birth in Public Hospitals in central Ethiopia: An unmatched case-control study. figshare. Dataset. 2021.Publisher Full Text\n\nSperandei S: Understanding logistic regression analysis. Biochem Med. 2014; 24(1): 12–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOffiah I, O’Donoghue K, Kenny L: Clinical risk factors for preterm birth. Preterm Birth-Mother and Child 1st ed InTech. 2012: 73–94. Publisher Full Text\n\nTong VT, England LJ, Rockhill KM, et al.: Risks of Preterm Delivery and Small for Gestational Age Infants: Effects of Nondaily and Low-Intensity Daily Smoking During Pregnancy. Paediatr Perinat Epidemiol. 2017; 31(2): 144–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaskins A, Mukhopadhyay S, Pekow P, et al.: Smoking and risk of preterm birth among predominantly Puerto Rican women. Ann Epidemiol. 2008; 18(6): 440–6. PubMed Abstract \| Publisher Full Text\n\nLiu B, Xu G, Sun Y, et al.: Maternal cigarette smoking before and during pregnancy and the risk of preterm birth: A dose–response analysis of 25 million mother–infant pairs. PLoS Med. 2020; 17(8): e1003158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSania A, Brittain K, Phillips TK, et al.: Effect of alcohol consumption and psychosocial stressors on preterm and small-for-gestational-age births in HIV-infected women in South Africa: a cohort study. BMJ Open. 2017; 7(3): e014293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParazzini F, Chatenoud L, Surace M, et al.: Moderate alcohol drinking and risk of preterm birth. Eur J Clin Nutr. 2003; 57(10): 1345–9. PubMed Abstract \| Publisher Full Text\n\nBarton K, Redshaw M, Quigley MA, et al.: Unplanned pregnancy and subsequent psychological distress in partnered women: a cross-sectional study of the role of relationship quality and wider social support. BMC Pregnancy Childbirth. 2017; 17(1): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreyvaud K, editorParent and family outcomes following very preterm or very low birth weight birth: a review. Semin Fetal Neonatal Med. 2014: Elsevier. PubMed Abstract \| Publisher Full Text\n\nWellings K, Jones KG, Mercer CH, et al.: The prevalence of unplanned pregnancy and associated factors in Britain: findings from the third National Survey of Sexual Attitudes and Lifestyles (Natsal-3). Lancet. 2013; 382(9907): 1807–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoday A, Muluneh MD, Sherif S: Determinants of preterm birth among mothers who gave birth at public hospitals in the Amhara region, Ethiopia: A case-control study. PloS One. 2019; 14(11): e0225060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBekele I, Demeke T, Dugna K: Prevalence of preterm birth and its associated factors among mothers delivered in Jimma university specialized teaching and referral hospital, Jimma Zone, Oromia Regional State, South West Ethiopia. J Women’s Health Care. 2017; 6: 356. Publisher Full Text\n\nDerakhshi B, Esmailnasab N, Ghaderi E, et al.: Risk factor of preterm labor in the west of iran: a case-control study. Iran J Public Health. 2014; 43(4): 499. PubMed Abstract \| Free Full Text\n\nKarthik L, Kumar G, Keswani T, et al.: Protease inhibitors from marine actinobacteria as a potential source for antimalarial compound. PloS One. 2014; 9(3): e90972. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdo R, Endalemaw T, Tesso F: Prevalence and associated factors of adverse birth outcomes among women attended maternity ward at Negest Elene Mohammed Memorial General Hospital in Hosanna Town, SNNPR, Ethiopia. J Women’s Health Care. 2016; 5(4). Publisher Full Text\n\nAregawi G, Assefa N, Mesfin F, et al.: Preterm births and associated factors among mothers who gave birth in Axum and Adwa Town public hospitals, Northern Ethiopia, 2018. BMC Res Notes. 2019; 12(1): 640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButler AS, Behrman RE: Preterm birth: causes, consequences, and prevention. National academies press 2007. PubMed Abstract \| Publisher Full Text" }	[ { "id": "91513", "date": "13 Aug 2021", "name": "Eshetu E Chaka", "expertise": [ "Reviewer Expertise I am an Epidemiologist and I have much research published on maternal-child 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\nUnder the Methods section, the authors repeatedly wrote logistic regression. There are several types of logistic regression, such as binary logistic regression, multinomial logistic regression, ordinal logistic regression, etc. So it is better to write the specific logistic regression used for this study.\n\nThe authors mentioned that they have used the combined data collection technique (interview and record review) and the study population is those women who gave birth during the data collection period. However, under the ethical consideration section, they mentioned written assent was taken from the guardians for women who gave birth and age <18 years. 2.1. How was assent considered for women who gave birth and maybe married? 2.2. Assent could not be taken from the guardian but from those not eligible to give informed consent.\n\nUnder Data processing and analysis, the method and result are mixed. For example \"the goodness of fit model (Hosmer and Lemeshow) was used to select the best multivariate model and the p-value of the model fitness test was 0.850.\" Please separate them by taking p-value indicates about the goodness of fit and give your conclusion under the result section.\n\nThe authors plan to check multicollinearity using standard error. However, they did not say anything about whether collinearity is present or absent under the Results section. Why do you plan to use standard error in presence of other options like VIF and Tolerance?\n\nUnder Sample size determination, the authors mentioned assumption 95% as a confidence interval. It should be corrected as a 95% confidence level.\n\nSampling technique: write kth to sample controls using systematic random sampling.\n\nWhy do the authors use the term \"counterparts\" in comparison to two groups using the Odds ratio? Better to write the exact name of the comparison group.\n\nNo need to write what you have done under the Results section. Better to write what you have found under the Results section. So please move the following statement under the Methods section: \"We conducted bivariate logistic regression for each independent variable. Multivariate analysis was done for all variables with p-value <0.25 in the bivariate logistic regression analysis after adjusting for covariates. The fitness of the model was also assessed.\"\n\nIn some bivariate tables under the Results section, sparse data was observed for ethnicity, marital status, ANC visit, history of cigarette smoking, and history of previous abortion. How did the authors manage those variables with sparse data in multivariable binary logistic regression?\n\nIn Table 6, the COR and AOR direction change or reverse for variable family size and educational level.\n\nUnder the Discussion section, there are several inconsistent uses of terms like the risk of preterm and the incidence of preterm. As the study design of this study is a case-control study design, better to use odds of preterm in the interpretation of AOR rather than using incidence or risk.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7040", "date": "26 Aug 2021", "name": "Berhanu Senbeta Deriba", "role": "Author Response", "response": "Dear reviewer and editors We thank you for reviewing and providing us with important and valid comments that helped us to enrich our manuscript. Really I would like to appreciate and thank you for your valuable, constructive comments too. Based on your suggestions, I have incorporated the comments into the manuscript, and I have also provided a response to each comment as follows. 1. Under the Methods section, the authors repeatedly wrote logistic regression. There are several types of logistic regression, such as binary logistic regression, multinomial logistic regression, ordinal logistic regression, etc. So it is better to write the specific logistic regression used for this study. Authors’ response: We used binary logistic regression and if editor allow us we have incorporated the comment into the manuscript. 2. The authors mentioned that they have used the combined data collection technique (interview and record review) and the study population is those women who gave birth during the data collection period. However, under the ethical consideration section, they mentioned written assent was taken from the guardians for women who gave birth and age < 18 years. 2.1. How was assent considered for women who gave birth and may be married? 2.2. Assent was could not be taken from the guardian but from those not eligible to give informed consent. Authors’ response: Thank you it is a nice observation we have accepted the comment and corrected accordingly. Actually what we did was for mothers < 18 years old we took assent from attendants available with her during data collection (guardians, husband or any other relatives). 3. Under Data processing and analysis, the method and result are mixed. For example \"the goodness of fit model (Hosmer and Lemeshow) was used to select the best multivariate model and the p-value of the model fitness test was 0.850.\" Please separate them by taking p-value indicates about the goodness of fit and give your conclusion under the result section. Authors’ response: Thank you for your constructive comments we have accepted the comment and corrected accordingly. 4. The authors plan to check Multicollinearity using standard error. However, they did not say anything about whether collinearity is present or absent under the Results section. Why do you plan to use standard error in presence of other options like VIF and Tolerance? Authors’ response: We have used standard error, VIF, and Tolerance to check Multicollinearity for this study. The reason why we used (focused) standard error is that standard error is used to check Multicollinearity in logistic much better than others. If standard error is inflated, which means greater than two it indicates presence of Multicollinearity and if it is less than two, it indicates absence of Multicollinearity in logistic regression. No collinearity was found in our study. 5.Under Sample size determination, the authors mentioned assumption 95% as a confidence interval. It should be corrected as a 95% confidence level. Authors’ response: Thank you for your constructive comments we have accepted the comment and corrected accordingly. 6.Sampling technique: write kth to sample controls using systematic random sampling. Authors’ response: We have used Kth every 4th to select controls. 7. Why do the authors use the term \"counterparts\" in comparison to two groups using the Odds ratio? Better to write the exact name of the comparison group. Authors’ response: We thought counterpart can indicate the opposite of what was mentioned. Now we have corrected it accordingly. 8. No need to write what you have done under the Results section. Better to write what you have found under the Results section. So please move the following statement under the Methods section: \"We conducted bivariate logistic regression for each independent variable. Multivariate analysis was done for all variables with p-value. Authors’ response: We would like to appreciate your nice observation and corrected it accordingly. 9.In some bivariate tables under the Results section, sparse data was observed for ethnicity, marital status, ANC visit, history of cigarette smoking, and history of previous abortion. How did the authors manage those variables with sparse data in multivariable binary logistic regression? Authors’ response: In the face of sparse data, the performance of logistic regression is problematic. A typical issue in such a circumstance is the occurrence of large odds ratios (ORs) with very broad 95 percent confidence intervals (CI). We solved this issue in this study by employing the penalized logistic regression (PLR) approach. 10.In Table 6, the COR and AOR direction change or reverse for variable family size and educational level. .Authors’ response: Thank you we have corrected it accordingly. 11.Under the Discussion section, there are several inconsistent uses of terms like the risk of preterm and the incidence of preterm. As the study design of this study is a case-control study design, better to use odds of preterm in the interpretation of AOR rather than using incidence or risk. Authors’ response: We thank you and we have corrected it accordingly." } ] }, { "id": "247247", "date": "29 Mar 2024", "name": "Jenny Jung", "expertise": [ "Reviewer Expertise global health", "epidemiology", "maternal health", "non-communicable diseases", "health equity" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the invitation to peer-review this article 'Determinants of preterm birth in public hospitals in central Ethiopia: an unmatched case-control study\". The authors conducted an unmatched case-control study across five hospitals in central Ethiopia, and reported factors associated with having a preterm birth outcome.\nUpon review, I would like to raise key points from the peer-review. Analysis is well done, and the results have been presented clearly. My main concerns are with the justification interpretation/discussion of the findings. Additional information in the methods and justification of why certain approaches were used are required to strengthen the existing paper.\nJustification and addition to current literature\nThe background doesn't justify the need and value of this study. Authors need to address what existing literature is available, and what added value this study will bring. The determinants of preterm birth has been widely studied both in low- and middle-income country contexts and within Ethiopia. This raises concerns about the need for another study and what added value this paper will have to current literature. If novelty of the study cannot be stated, authors are advised to build on current knowledge which will require a thorough summary of what is known, where the gaps in literature are, and how this study aims to contribute to the evidence base.\nMethodology and reproducibility\nWhy did the authors choose an unmatched method instead of matched case-control study? Birth outcomes are largely determined by confounding factors such as age, parity, and pre-existing diseases. Authors are recommended to justify this approach in the methods section as well as discussion what additional limitations this approach should be considered under discussion section. Authors should provide further information regarding the hospitals and health center study sites. In particular, the number of beds and pregnancy services provided in the hospitals should be stated. Are they public or private facilities? The selection and variables used for sample size calculation need to be more clear in the methods section.\nReproducibility\nAn example of the data collection instrument (both developed in English and translated versions) should be provided in Supplementary Information to ensure replicability of this study. Why was the data collection instrument pre-tested outside the study area? There is no information about whether the population of women delivering outside the study area would be similar to the study area.\nAdditional comments\nLargely, the determinants found to be associated with preterm birth from this study seem to be preventable or manageable in nature. How does this impact clinical practice (preconception and antenatal care) and policy? The discussion and interpretation of the analysis presented in the paper cannot replicate a high-level summary of the results, with only a comparison of effect sizes reported in current literature. The discussion requires a thorough understanding and interpretation of what the findings add to existing literature. As mentioned previously, there is an abundance of literature on the determinants of preterm birth.\nOverall, I agree that this study would add some value to understanding determinants of preterm birth in Ethiopia if there are none in this specific region (as previous studies in other regions have already been conducted). However, majority of sections throughout the manuscript including background, methods, and discussion require significant revisions are recommended.\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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-773
https://f1000research.com/articles/10-757/v1	05 Aug 21	{ "type": "Research Article", "title": "Clinical characteristics and radiological domains among patients with recurrent strokes-a descriptive cross-sectional study from a tertiary care center in central Nepal", "authors": [ "Binod Bhattarai", "Shashi Bhushan Sah", "Shashi Bhushan Sah" ], "abstract": "Background: Stroke is a significant global health hazard that ripples continuum multi-spectral effects to the patients as well their caretakers.\n\nMethods: We studied 28 consecutive cohorts of patients with recurrent strokes managed in our centre within the last two years. Results: The most common recurrence stroke pattern was of that of hemorrhagic to hemorrhagic subtype observed in 50% of the patients. The most common anatomical region of involvement was cortical – cortical seen in 39.28% of our cohorts. The surgical intervention was required in 17.85% whereas 42.85% of them were managed conservatively. Paradoxically, 39.28% of patients left against medical advice. The receiver operating curve (ROC) predicting mode of management was highest (area under the curve (AUC) =0.635) for compliance to therapy followed by stroke territory (AUC=0.578), age (AUC=0.457) and motor grading (AUC=0.374). The receiver operating curve (ROC) for influencing decision to leave against medical advice was highest (area under the curve (AUC) =0.861) for motor score followed by sex (AUC=0.701) and age (AUC=0.564). The analysis of variance (ANOVA) study pertaining to the mode of management was significantly connoted by the motor score and the stroke territory only. The ANOVA study pertaining to the decision to leave against medical advice was significantly governed by the motor score, stroke territory, and sex respectively. The multivariate analysis for variables governing mode of management was significant for motor score and the stroke territory only. The multivariate analysis for variables governing leave against medical advice was significant for sex, motor score and the stroke territory. Conclusions: This study aims to appraise early dichotomization of high-risk patients for recurrent strokes to reduce the continuum of neurological events as well as to mitigate the financial aspects governing stroke care.", "keywords": [ "Recurrent", "stroke", "patterns", "outcome" ], "content": "Introduction\n\nStroke is a significant global health hazard1,2. It is a silent epidemic that ripples continuum multi-spectral effects to the patients as well their caretakers. Recurrent stroke is slowly coming out of its shell with the reported incidence of up to 22.5% within 5 years3. Patients with recurrent stroke have greater disability and poorer outcomes than those with the first stroke4. Amidst the current paradox between the monumental stoke economy and the dismal provisions for rehabilitative strategies; these cohorts are most often compelled to lead a poor quality of life becoming socially aloof.\n\nThe sound knowledge on the incidence and the patterns of recurrent stroke paves the way to format necessary steps in mitigating them as well as improvise newer reforms in combating them in the future. This study aims to foster the pertinent need for national stroke database studies pertaining to strokes.\n\n\nMethods\n\nConsecutive cohorts of patients with recurrent strokes managed in the Department of Neurosurgery in College of Medical Sciences, Chitwan, Nepal within the last two years (January 2019–January 2021) were enrolled in this descriptive cross-sectional study. The recurrent stroke was defined either as:\n\n• The stroke event occurring at the same anatomical location after 21 days of the index event or\n\n• Different stroke event at another anatomical region within 21 days of the index event5.\n\nCurrent smokers were defined as those who have smoked ≥1 cigarette per day for 6 months and have smoked in the last 28 days, whereas heavy drinkers were labeled as those who have consumed >2 and >1 standard drink per day (a glass of wine, a bottle of beer, or a shot of spirits, ∼10 to 12 g of ethanol) for men or and women respectively6. Measurement of treatment adherence (compliance) was performed by the self reported and pills count methods.\n\nThe anatomical regions of involvement with the recurrent events were further categorized into subgroups as:\n\n• Cortical and cortical\n\n• Cortical and basal ganglionic\n\n• Cortical and thalamic\n\n• Basal ganglion and basal ganglionic\n\n• Basal ganglionic and thalamic and\n\n• Thalamic and thalamic\n\nThe demographical and the clinico-radiological variables comprising of age, sex, presenting clinical motor score, medical compliance, patterns of the index and recurrent strokes, anatomical distribution of the strokes, the mode of management, and those who left against medical advice (LAMA) were thoroughly appraised and analyzed.\n\nThe ‘equipoise’ governed from the recurrent events occurring in the same patient within the same geographical minimized the bias on the outcome from other confounding factors such as alcohol intake, smoking status and other medical comorbidities in our cohort study.\n\n• Consecutive 28 patients presenting with recurrent strokes in our Neurosurgical unit\n\n• Failure of approval for participation in the study\n\n• Simultaneous multiple intracerebral strokes\n\n• Traumatic ICH\n\n• ICH secondary to vascular malformation, aneurysm, or cavernoma.\n\n• Transient ischemic attacks (TIA)\n\n• Patients with missing data pertaining to the study variables\n\nThe sample size required for adequate statistical elaboration was calculated according to the formula\n\nn = Z2 × p × q/d2 where\n\nZ=1.96 at 95% confidence interval,\n\np =7.4% prevalence of recurrent CVA5\n\nq =1–p and\n\nd=10% margin of error\n\nThe required sample size calculated was 26.32\n\nWe analyzed the records of 28 patients.\n\nFrequency distribution (counts and percentages) was undertaken for the studied variables of the cohorts included in our study. Data were recruited and analyzed using the SPSS version 16 software. Statistical analysis was done utilizing receiver operating curve (ROC) with area under curve (AUC) values, Analysis of variance (ANOVA) and multivariate logistic regression analysis among the pertinent variables applying mode of management and the decision to leave against medical advice as the final outcomes. P-value of <0.05 was considered significant. Patients who had missing data for variables were excluded from the analysis.\n\nAll procedures performed in studies involving human participants followed the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Institutional Review Committee (IRC) of the College of Medical Sciences (COMS-IRC-2021-35), Chitwan in Nepal.\n\nThe article was based on a review of anonymous clinical data. Therefore, no patient consent was required.\n\n\nResults\n\nIn our study, the prevalence of recurrent stroke was more common among male cohorts (M: F ratio of 1.54:1). The mean age of presentation of the studied population was 67.65 ±10.39 for males and 61.64 ±13.54 for females respectively.\n\nMedical compliance was observed in only 64.28% of the patients. The smoking and alcohol consumption habit was seen in 35.71% and 64.28% of patients respectively.\n\nThe mean motor score of the patient at presentation was 1.46 ±0.63.\n\nThe recurrence pattern was observed to be of hemorrhagic to hemorrhagic (H-H) in 14/28 (50%), ischemic to hemorrhagic (I-H) in 12/28 (42.85%), ischemic to ischemic (I-I) in 2/28(7.14%) of the cohorts.\n\nThe pattern of recurrent stroke pertaining to the anatomical distribution was cortical – cortical in 11/28 (39.28%), cortical –basal ganglia in 2/28 (7.14%), basal ganglion- basal ganglion in 9/28(32.14%), basal ganglion-thalamic in 3/28(10.71%), thalamic–thalamic in 2/28 (7.14%) and cortical-thalamic in 1/28 (3.57%) of our cohort study.\n\nThe surgical intervention was required in 5/28 (17.85%) whereas 12/28(42.85%) of them were managed conservatively. Paradoxically, 11/28(39.28%) of patients left against medical advice. The relevant findings of our study have been summarized in Table 1.\n\nThe receiver operating curve (ROC) of the study for predicting mode of management was highest (area under the curve (AUC) =0.635) for compliance to therapy followed by stroke territory (AUC=0.578), age (AUC=0.457) and motor grading (AUC=0.374) as shown in Figure 1.\n\nThe receiver operating curve (ROC) of the study for influencing decision to leave against medical advice was highest (area under the curve (AUC) =0.861) for motor score followed by sex (AUC=0.701) and age (AUC=0.564) as shown in Figure 2.\n\nThe ANOVA study pertaining to the mode of management was significantly connoted by the motor score and the stroke territory only as shown in Table 2.\n\nThe ANOVA study pertaining to the decision to leave against medical advice was governed by the motor score, stroke territory, and sex as shown in Table 3.\n\nThe multivariate analysis for variables governing mode of management was significant for motor score and the stroke territory as shown in Table 4.\n\nThe multivariate analysis for variables governing leave against medical advice was significant for sex, motor score and the stroke territory as shown in Table 5.\n\n\nDiscussion\n\nStroke is a significant global health hazard7. The continuum impact of the associated morbidity and mortality is mostly observed in low and middle-income nations6. With the concurrent increment in the lifespan of the population alongside adaptations of unhealthy lifestyle and living habits, the prevalence of stroke will certainly show an upward curve in the coming future7.\n\nThe reported incidence of recurrent stroke despite preventive measures is above 20%8. The risk of such adverse events is estimated at 5.4% and 11.3% during the first and the fifth years respectively, with an overall risk of 1.2/100/year for both hemorrhagic and the ischemic subtypes9,10.\n\nThe ‘‘cortical–cortical’’ is the most common pattern of anatomical involvement in recurrent hemorrhagic strokes. This has largely attributed secondary to cerebral amyloid angiopathy and coagulation disorders10. The similar anatomical pattern of involvement (39.28%) was seen in our study. The hemorrhagic-hemorrhagic patterns of the index and the recurrent stroke events were observed in 50% of the cohorts.\n\nHypertension is the strongest risk factor for both subtypes of recurrent stroke with an overall relative risk of almost 5.437,9. Similarly concurrent transient ischemic events and the radiological presence of chronic infarction have shown to prognosticate increased odds of recurrent ischemic strokes5. Such epiphenomenon have been shown to increase the mortality risk by almost 17 folds9. Increased age is also a risk variable for stroke recurrence with the estimated hazard ratio of 1.02/year9. The average age of patients in a study ranged from 54 to 66 years10. However, both age and sex were not found to be linked to harbinger subsequent IS10. Male genders have shown to have a higher cumulative risk for recurrent stroke11. Our study showed male gender preponderance with a ratio of 1.54:1. Age ≥65 years have been found to be an independent predictor of long-term mortality12. The average age of the patients in our cohort study was 65.45 years. In addition to having higher stroke risk, women have poorer post-stroke outcomes. However, in the literature, these differences in sex are not consistent12.\n\nSmoking habit has been attributed as a risk factor for ischaemic strokes only7. The smoking status was observed in 35.71% whereas the alcohol intake habit was seen in 68.28% of patients. Hypertension and diabetes mellitus are other documented independent risk factors. Moreover, hypertension at admission during index stroke events prognosticate the risk of early mortality12.\n\nOne study showed that only 45% of patients were aware of their hypertension, and approximately 30% of them were compliant with their medications7. Another study revealed that only 12% of patients with atrial fibrillation were receiving appropriate prophylactic anticoagulation therapy9. In our study, adherence to medication was found in 64.28% of patients.\n\nThe neurological improvement to compensate for carrying out activities of daily living is further compromised among patients with contralateral recurrence, further hampering their quality of life8.\n\nMortality among patients with recurrent strokes have been documented to be above 35% with a hazard ratio of 2.559. The all-cause mortality at five years pertaining to hemorrhagic strokes is almost twice comparing to the ischemic counterpart. The result mirrors the risk of mortality among patients with non-lacunar infarction comparing to that of the lacunar subtypes11.\n\nPatients with large vessel atherosclerosis and cardio-embolism have an early risk of stroke recurrence13.\n\nThe yearly estimates of recurrent stroke, death, and cardiovascular events were reported at 3.6%, 10.5%, and 6.7%, respectively in one study14. The 5-year rate event for MI is 41% for recurrent stroke comparing to only 2% after the first stroke. The cumulative event rates for major vascular events are 18% and 45% at 1 and 5 years respectively11.\n\nThe pattern of stroke recurrence mirrored that of the index events more for the ischemic subtypes, compared to their hemorrhagic counterparts (90% Vs. 56%)11.\n\nPatients managed in dedicated stroke units have shown to have improved outcome5. However; the expenditure pertaining to stroke care has been projected to cross 150 billion dollars by 203015. One of the salient findings in our study was the observation of almost 40% of patients who opted to leave against medical advice from the hospital. This reflects the poor perception about the disease and its impact upon the patient by their caretakers’ superadded by the financial burden associated with stroke care. This further reinforces the imminent need of implementing primary and secondary preventive measures to prevent ad mitigate such events.\n\nPlaque stability in the extracranial group, whereas the progression of stenosis in the intracranial group determines the risk of recurrence16. Among stroke with no determined cause, intracranial stenosis was often found at the time of recurrence16. Timely and appropriate screening of these high-risk patients helps to reduce the short and long-term multispectral neurological and financial burden among such patients as well as their caretakers.\n\n\nLimitations of the study\n\nThe true estimates of the recurrence may be underestimated owing to omission bias since our study reviewed medical records of admitted patients within the last two years only. We also have scarce data pertaining to the analysis of factors governing long-term morbidity and mortality. Finally, the results of our study may not mirror demographics from other topography.\n\n\nConclusion\n\nA provision for nationwide hospital and community-based stroke register is therefore of paramount importance to monitor the patterns and quality of primary and recurrent stroke care. The early dichotomization of high-risk patients for recurrent strokes is essential to reduce the continuum of neurological events as well as to mitigate the financial aspects governing stroke care. This is even more relevant in our context wherein there is a high stroke burden with paradoxical minimal stroke care and rehabilitative facilities.\n\n\nData availability\n\nFigshare. Clinical characteristics and radiological domains among patients with recurrent strokes-a descriptive cross-sectional study from a tertiary care center in central Nepal17. DOI: https://doi.org/10.6084/m9.figshare.14923071.v1\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC BY 4.0 Public domain dedication).", "appendix": "References\n\nGBD 2015 Neurological Disorders Collaborator Group: Global, regional, and national burden of neurological disorders during 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Neurol. 2017; 16(11): 877–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Projections of mortality and causes of death, 2015 and 2030. Health statistics and information systems. 2013; Accessed 21 June 2016.\n\nHan J, Mao W, Ni J, et al.: Rate and Determinants of Recurrence at 1 Year and 5 Years After Stroke in a Low-Income Population in Rural China. Front Neurol. 2020; 11: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamsa GP, Bian J, Lipscomb J, et al.: Epidemiology of recurrent cerebral infarction: a medicare claims-based comparison of first and recurrent strokes on 2-year survival and cost. Stroke. 1999; 30(2): 338–349. PubMed Abstract \| Publisher Full Text\n\nStahmeyer JT, Stubenrauch S, Geyer S, et al.: The Frequency and Timing of Recurrent Stroke: An Analysis of Routine Health Insurance Data. Dtsch Arztebl Int. 2019; 116(42): 711–717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhuo Y, Wu J, Qu Y, et al.: Clinical risk factors associated with recurrence of ischemic stroke within two years: A cohort study. Medicine (Baltimore). 2020; 99(26): e20830. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu M, Wu B, Wang WZ, et al.: Stroke in China: epidemiology, prevention, and management strategies. Lancet Neurol. 2007; 6(5): 456–64. PubMed Abstract \| Publisher Full Text\n\nJørgensen HS, Nakayama H, Reith J, et al.: Stroke recurrence: predictors, severity, and prognosis. The Copenhagen Stroke Study. Neurology. 1997; 48(4): 891–5. PubMed Abstract \| Publisher Full Text\n\nKhanevski AN, Bjerkreim AT, Novotny V, et al.: Recurrent ischemic stroke: Incidence, predictors, and impact on mortality. Acta Neurol Scand. 2019; 140(1): 3–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHanger HC, Wilkinson TJ, Fayez-Iskander N, et al.: The risk of recurrent stroke after intracerebral haemorrhage. J Neurol Neurosurg Psychiatry. 2007; 78(8): 836–840. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Y, Wright N, Guo Y, et al.: Mortality and recurrent vascular events after first incident stroke: a 9-year community-based study of 0·5 million Chinese adults. Lancet Glob Health. 2020; 8(4): e580–e590. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYan F, Yi Z, Hua Y, et al.: Predictors of mortality and recurrent stroke within five years of intracerebral hemorrhage. Neurol Res. 2018; 40(6): 466–472. PubMed Abstract \| Publisher Full Text\n\nElnady HM, Mohammed GF, Elhewag HK, et al.: Risk factors for early and late recurrent ischemic strokes. Egypt J Neurol Psychiatry Neurosurg. 2020; 56: 56. Publisher Full Text\n\nAndersen SD, Gorst-Rasmussen A, Lip GYH, et al.: Recurrent Stroke: The Value of the CHA2DS2VASc Score and the Essen Stroke Risk Score in a Nationwide Stroke Cohort. Stroke. 2015; 46(9): 2491–7. PubMed Abstract \| Publisher Full Text\n\nZhang J, Zhu P, Liu B, et al.: Time to recurrence after first-ever ischaemic stroke within 3 years and its risk factors in Chinese population: a prospective cohort study. BMJ Open. 2019; 9(12): e032087. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShin DH, Lee PH, Bang OY: Mechanisms of recurrence in subtypes of ischemic stroke: a hospital-based follow-up study. Arch Neurol. 2005; 62(8): 1232–7. PubMed Abstract \| Publisher Full Text\n\nBhattrai B: Clinical characteristics and radiological domains among patients with recurrent strokes-a descriptive cross-sectional study from a tertiary care center in central Nepal. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.14923071.v1" }	[ { "id": "92803", "date": "31 Aug 2021", "name": "Lekhjung Thapa", "expertise": [ "Reviewer Expertise Stroke" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good report highlighting characteristics of a group of stroke patients who presented to a neurosurgical unit of a tertiary care center with hemorrhagic stroke in 21 days, which the authors have considered as a recurrent stroke. Although the community prevalence of stroke report in Nepal is available1, the prevalence of recurrent stroke is still lacking. The authors have used data from an international study to calculate the sample size and have studied 28 patients with hemorrhagic stroke who had an ischemic or hemorrhagic stroke in the past 21 days. After reading the article, I found it very interesting that a very high proportion (40%) of the patients left against medical advice which is very common in developing countries because of various unavoidable reasons like a financial burden, and moreover, a grim prognosis associated with recurrent stroke. This information highlights not only the need for a stroke registry as the authors have highlighted but also for secondary stroke prevention awareness programs.2 Few suggestions for authors willing to conduct a similar study would be:\nIncorporation of important clinical variables that are known to be risk factors for recurrent stroke such as data on hypertension, metabolic syndrome, dietary habits, stress, etc.\n\nICH score would be a more meaningful way to communicate in cases of ICH instead of motor score\n\nCardio-embolism (Rheumatic Heart Disease) is a unique cause of recurrent stroke in developing countries like Nepal and a note on it would be better as there is no risk\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "91259", "date": "10 Nov 2021", "name": "Guoyi Gao", "expertise": [ "Reviewer Expertise Neurosurgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting report on recurrent stroke in one center in Nepal. I believe that many colleagues are keen to learn the current status of neurological diseases and the treatment in this Southern Asia area. Thus I suggest this manuscript is suitable for indexing after revision.\nThe inclusion criteria could be rewritten.\n\nIn the demographic variables, I did not find the preexisting condition of hypertension, as well as the antihypertensive treatment. The author could add this information to strengthen the evidence.\n\nThe author should not neglect that the major limitation of this study is the small sample, this could be described in the part of limitations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/10-757
https://f1000research.com/articles/10-753/v1	04 Aug 21	{ "type": "Systematic Review", "title": "Educational interventions for shared decision making and the role of patient agency: A Systematic Review", "authors": [ "Catherine Witkop", "Dario Torre", "Emily Harvey", "Lauren Maggio", "Dario Torre", "Emily Harvey", "Lauren Maggio" ], "abstract": "Background: Shared decision making is critical to patient-centered care and yet there is limited consensus on effective teaching approaches for training physicians in this domain. As a collaborative process in which the patient and physician co-create a decision, patient and relational agency may be important contributors and studies with patient-reported outcomes may identify successful approaches and determine gaps in pedagogy. The authors conducted a systematic review of educational interventions for shared decision making, focusing on patient-reported outcomes and consideration of agency. Methods: Ovid MEDLINE, Embase, and Web of Science were searched for studies describing educational interventions with patient-level outcomes published between January 2000 and January 2020. Articles were excluded if they were not in English, included only patient interventions, or reported only physician outcomes. Quality assessment was performed using the Medical Education Research Study Quality Instrument (MERSQI). Data about the educational methods used were extracted and included studies were assessed for quality. Thematic analysis was performed to identify the potential role of agency. Results: 26 articles were identified describing 17 unique studies. Educational interventions were diverse in duration and content, with multiple components. Three-quarters of studies used role play or simulated patients and 82% included tools to facilitate shared decisions. Although no articles explicitly discussed facilitating agency as a component of the intervention or as an outcome, one qualitative study demonstrated themes of patient and relational agency. Conclusions: Educational interventions included small group discussion, decision aids, role play, and simulated patients, and improved a range of patient outcomes, but our study included only studies including practicing physicians, limiting applicability to trainees and other health care providers. Interventions have not included explicit instructional design around agency, but qualitative analyses demonstrated interventions may facilitate agency and shared decision making. Future instructional strategies should consider the complexity inherent in co-constructing decisions.", "keywords": [ "shared decision making", "educational interventions", "agency", "cultural historical activity theory (CHAT)" ], "content": "Introduction\n\nShared decision making is critical to patient-centered care, and yet there is limited consensus on how to most effectively teach it to health professionals. Although numerous educational interventions have been developed, many have not been methodically studied nor described in the literature1,2. In a recent systematic review of published studies, Muller et al. found great variation in the interventions, outcome measures, and conclusions of the 41 included articles3. Because their analysis did not allow matching training components with outcomes, conclusions were limited for the most effective strategies, further, they noted that most of the published studies reported on provider-reported outcomes over observed or patient outcomes3. Because shared decision making is a collaborative process in which the patient and provider co-create a decision based on the patient’s preferences and values in addition to scientific evidence, we propose that in-depth analysis of studies with patient-reported outcomes may help us better identify successful approaches and determine gaps in pedagogy.\n\nFocusing on studies with patient outcomes may also allow for closer examination of the role of agency in shared clinical decisions. We suggest that shared decision making requires not only a patient with sufficient agency to participate in the collaborative process, but a clinician whose actions enhance relational agency in the encounter. One commonly cited definition of agency, by Giddens, is an individual acting intentionally, with the capacity to act on his or her intentions, and having the power to create a new event or intervene in an existing event4. According to Giddens, individual agency can be shaped, facilitated, or constrained by social structures. The clinical encounter is such a social structure, made up of the actions of the physician, the clinical and patient support tools, the culture, environment, and expectations. Relational agency has been defined as “the capacity to work with others to expand the object that one is working on by bringing to bear the sense-making of others”5. By examining relational agency of a physician and patient dyad, we can better understand how a shared decision can be accomplished.\n\nDespite the apparent importance of agency to shared decision making, it is unclear if educational interventions address the roles of patient and relational agency. Thus, we sought to answer the questions: How is shared decision making taught to physicians and, secondarily, in what ways, if any, is the concept of agency addressed in these educational efforts?\n\n\nMethods\n\nWe conducted a systematic review of studies evaluating educational interventions for physicians that aimed to increase shared decision making in the clinical encounter, as measured by patient-level outcomes. Our review followed guiding principles of conducting systematic reviews in medical education6 and is reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta-analyses7. (See PRISMA Checklist: https://zenodo.org/record/5029150#.YNTs6ndKicY)8.\n\nWe hand-searched a 2012 environmental scan of shared decision making training and its 2016 update1,2 and identified relevant articles. Leveraging these results, we conducted searches of Ovid MEDLINE, Embase, and Web of Science limited to studies published between January 2000 and January 2020. A medical librarian created search strategies for each database that utilized combinations of keywords and controlled vocabulary terms. (See extended data https://zenodo.org/record/5029150#.YNTs6ndKicY)8. Searches were limited to English-language publications and conducted on January 29, 2020. We also hand searched references of all included articles and key review articles. All citations were managed in EndNote, including the removal of duplicate citations.\n\nWe included studies that described educational interventions for physicians that included patient outcomes (objective health outcomes or patient-reported outcomes). Articles were excluded if they were not in English, included only patient interventions, or reported only physician outcomes.\n\nTwo authors (CW and LM) independently screened all titles and abstracts using Rayyan (https://www.rayyan.ai), a webtool optimized for data screening. Screening was based on the above inclusion/exclusion criteria. Upon completing the screening, LM and CW discussed any conflicts until consensus was reached. In instances in which CW and LM did not reach consensus, the full text was reviewed and if appropriate included.\n\nCW, EH, and LM independently extracted data from the included articles, using a form that was created based on our research question and examples from the literature. The data extraction form was operationalized in Excel. CW, a physician and doctoral student in a Health Professions Education program, extracted data from all full-text articles. LM and EH, both HPE researchers, each extracted data from half of the articles. Discrepancies were resolved by discussion. If multiple articles reported different findings from the same study, data extraction occurred from individual studies, but they were grouped together for future analysis. Upon completion of extraction, the authors met to reconcile their coding.\n\nData extracted included the context of the intervention, clinical area, physician-type targeted, theories/approaches used, educational intervention(s), instruments or measures used (delineated as patient and provider outcomes) and outcomes (including effect of intervention). We identified any discussion of agency in either the intervention specifically or in the analysis or discussion of results.\n\nBecause of the complexity of shared decision making, we drew on Cultural Historical Activity Theory (CHAT) as a framework. We propose that CHAT allows examination of interacting activity systems that can impact shared outcomes. An activity system in CHAT is made up of a subject (person engaged in the activity), tools (symbolic or physical objects that facilitate accomplishment of a defined objective), and object (purpose of the activity), as well as rules, community, and division of labor9. Any components of the intervention, results, or discussion that appeared to be aligned with elements of CHAT were extracted from each study.\n\nQuality assessment of the included studies was performed, with an understanding that these studies were diverse in methodology. All articles in the final review underwent evaluation using the Medical Education Research Study Quality Instrument (MERSQI)10. CW and LM independently scored each of the included studies. For studies with more than one article, one with quantitative patient outcomes was used for rating purposes. Any discrepancies were discussed and resolved by consensus. If disputes remained, EH served as an adjudicating reviewer. Reliability kappa coefficient scores for the MERSQI used in other systematic reviews of educational interventions have ranged from 0.72 to 0.9910,11. We utilized categories of quality from previous studies: 4.5 to 8.5 is considered low quality, 9.0 to 13.0 is moderate, and 13.5 to 18 is considered high quality12. Quality assessment was performed for studies with quantitative data, which made up a majority of the studies.\n\nWe tabulated the key characteristics of the included studies (e.g., intervention modality, learner and patient population, assessment instruments). To identify the presence of agency within the interventions, thematic analysis was performed on all studies with a focus on the intervention and results.\n\nWhile we remained open to all elements related to agency in our thematic analysis, we used CHAT as a framework for coding. Codes were created for each of the elements of activity systems and for tensions within and between activity systems. Codes were applied to findings and representative quotes for each study. A code for patient agency was also applied to intervention descriptions and results and authors’ interpretations of study findings. Codes were organized into related areas and used to identify themes.\n\n\nResults\n\nOur search yielded 2879 articles after duplicates were removed. After title/abstract review, we identified 34 for full-text review, of which we excluded 8. We also reviewed reference lists of 10 articles marked as key background/review articles and the 26 remaining articles. In total, we identified 26 articles13–38, describing 17 unique studies meeting the inclusion criteria for our review. Figure 1 diagrams our review process. In this section, we first report study characteristics and describe the educational interventions. We then turn attention to elements of shared decision making and agency which we have mapped to CHAT.\n\nLiterature search and study selection process for a systematic review of the literature, published between January 2000 and January 2020, on educational interventions for health professionals that aimed to increase shared decision making in the clinical encounter, as measured by patient-level outcomes. The study followed the PRISMA guidelines for conducting a systematic review.\n\nTable 1 includes characteristics of the included studies. From the included studies, 14 (82%) were randomized controlled trials (RCT), with the majority characterized as cluster RCTs. Six studies were conducted in Germany, four in the United States, three in the Netherlands, and the remainder in Canada (2), the United Kingdom (1), and Mexico (1). Most studies (n=12; 71%) were conducted with primary care physicians and the remainder were specialists or unspecified. All studies received external funding and were found to be of high quality based on MERSQI scores ranging from 13.5 to 18.\n\nAbbreviations: MERSQI indicates Medical Education Research Study Quality Instrument; cRCT, cluster randomized-controlled trial; qual, qualitative analysis; RCT, randomized-controlled trial\n\nEducational interventions described in the studies were diverse in duration and content (See Table 2). All interventions involved more than one method to deliver the intervention, including a mix of: lecture/video/web-based presentation, group discussion, role play or consultations with simulated patients, self-reflection, feedback on performance (either direct observation or in visit with simulated patient), and tools for interaction (for the physician, the patient, or both). All studies used some form of lecture, video, or web-based didactics. Group discussion was included in 6 (35%) studies, feedback on performance in 11 (65%), and 5 (29%) studies explicitly included self-reflection.\n\nAbbreviations: SDM indicates shared decision making; LV, lecture or video; GD, group discussion; RP, role play; SR, self-reflection; FP, feedback on performance; TI, tool for interaction; WM, written material; O, other; SP, simulated patient; COMRADE, Combined Outcome Measure for Risk Communication and treatment Decision making Effectiveness; OPTION, Observing Patient Involvement Scale; RIAS, Roter Interaction Analysis System; FAPI, Questionnaire on Doctor-Patient Interaction; SWD, Satisfaction With Decision scale; DCS, Decisional Conflict Scale; PHQ-D, Patient Health Questionnaire-Depression; CVD, coronary vascular disease; SDM-Q, Shared Decision Making Questionnaire; CPS, Control Preference Scale; DRS, Decisional Regret Scale; SF-12, 12-item Short Form-Survey; QOL, quality of life; PSA, prostate specific antigen; CVR, cardiovascular risk; BP, blood pressure; HTN, hypertension; PPS, Patient Perception Scale; ASK, AskShareKnow; MAPPIN’SDM, Multifocal Approach to the ‘Sharing’ in Shared Decision Making; SDMmass, SDM Meeting its concept's ASSumptions\n\nThe majority of studies (n=13; 76%) utilized role play or simulations within their educational interventions, although the approach differed between studies. We describe two of the interventions as examples of disparate approaches with different study populations, however, it should be noted that they are not representative of all interventions. One of the earliest studies in this review was a cluster RCT that utilized two workshops with didactics that focused on shared decision-making competencies13–16. A second group received a similar intervention focused only on risk communication skills and a third group received both interventions (combined interventions). The primary care physicians then participated in consultations with simulated patients and received feedback on their performance in at least one of the consultations with simulated patients13–16.\n\nAnother study compared radiation oncologists before and after participating in two plenary sessions (3-hours each), group discussion, trained patient simulators, and three videotaped real patient consultations with a 1-hour feedback session on each20. Training included activities to emphasize components of shared decision making and outcomes related to communication of the physician participants before and after training20.\n\nMost studies (n= 14; 82%) included tools that the physician and/or patient could utilize to assist with decision making. For example, decision aids were paper-based booklets or web-based applications that provide information about health options and were designed to help patients clarify their personal preferences and values. Tools were directed at physicians in 14 (82%) studies and at patients and physicians in 6 (35%) studies; the majority were designed to be used during the clinical interaction.\n\nAlthough there were significant differences in how outcomes were assessed (Table 2), a few measures were utilized in multiple studies. The observing patient involvement in decision making (OPTION) scale or an adapted version (e.g., OPTION 5), was used in five studies16,17,26,31,34,37,38. The extent to which providers were observed involving patients in shared decision making was considered an outcome in all these studies39.\n\nThe Roter scale, from the Roter Interaction Analysis System (RIAS) is another measure utilized in three studies17,20,23. RIAS is a validated tool for coding medical dialogue, notable for its ability to code multiple speakers and in its flexibility in coding subcategories and content-specific topics of interest40. Examples of outcomes included numbers of utterances that represent physician support and encouragement of shared decisions and patient involvement.\n\nOutcomes for each of the studies are described in Table 2. The majority of studies demonstrated improvement in measures of shared decision making that were used. However, because of the wide variation in outcome measures used between studies, comparing effectiveness of interventions between studies is a challenge. We highlight a few of the outcomes. For example, studies that utilized the OPTION scale16,17,26,31,34,37,38 all demonstrated improvement in patient involvement to some extent. Those that used the Roter scale demonstrated improved shared decision making behaviors and patient participation with the intervention17,20,23.\n\nStudies in which role play was part of the intervention generally demonstrated an improvement in patient participation during the clinical encounter as reported by physician or observed by external rater16–18,20,23,24,31,35,37,38. Of these studies, one had a discrepancy between the patients’ perception of shared decision making and that reported by the physician or observed35. One study demonstrated a discrepancy between the physicians’ subjective assessment of and evidence for shared decision making on transcripts21.\n\nWe now describe the concepts of agency as identified in the included studies. Although none of the studies included facilitating agency or impacting relational agency as an explicit element of an educational intervention to teach shared decision making, aggregation of codes for CHAT and agency enabled us to identify concepts related to relational agency. To illustrate, we describe one study18,19 by Bieber et al. which had the most robust collection of codes associated with agency and CHAT. We offer this study as the clearest application of relational agency in studies of educational interventions for shared decision making process.\n\nThis study describes a group of German outpatient physicians caring for patients with fibromyalgia syndrome. Patients in this study reported they often encountered resistance from within the medical community and \"often spend all their strength on struggling, quarrelling and asserting themselves during consultations and tend to use 'war' and 'legal' metaphors in their descriptions of medical encounters”18. Physicians in the study also reported negative feelings, sometimes making them want to discontinue treating the patient18. With a CHAT lens, these descriptions evoke images of two interacting activity systems in which the objects are not aligned, and tensions have developed. The patient and the physicians had varying degrees of agency, and their existing interaction did not promote shared decisions.\n\nThe study intervention included twelve 90-minute sessions with the goal of training providers to develop a working alliance with their patients. Role plays allowed providers to practice dealing with challenging situations and inviting the patient to participate in decision making18. There was also a patient intervention, a computer-based information tool which was designed to lead to behavior change within the patient. Qualitative analysis of recorded consultations, semi-standardized interviews, and questionnaires all supported the finding that there were differences in patients’ reported quality of the physician-patient interaction in the intervention group.\n\nApplying the CHAT lens, we see the study intervention provided tools for both the patient and physician to apply within their activity systems, and the object became aligned (“working alliance”); the intervention impacted community (providers and patients shared a common object) and rules (the patient was now involved in decision-making). The tensions seemed to resolve; more importantly, there were longer term effects, possibly indicating expansive learning, a concept within CHAT that refers to cycles of interacting activity systems leading to learning and behavior change.\n\n\nDiscussion\n\nWe identified and reviewed a broad range of educational interventions around shared decision making. As in previous reviews1–3, all included studies described multicomponent interventions and it is difficult to draw conclusions about effectiveness of any individual component. However, by focusing on patient outcomes, we reveal common elements among many of the studies, including use of tools such as decision aids, small group discussions, and role play or practice with simulated patients, which may be critical for shared decision making. None of the included studies explicitly addressed patient or relational agency.\n\nBased on our analysis, role play and practice with simulated patients were key elements of effective interventions. Like many educational interventions aimed at improving physician communication skills, role play, and the use of simulated patients provide opportunities for physicians to practice those competencies. An umbrella review that examined effective educational programs for teaching physicians’ communication skills found that the interventions were effective if they were at least one day long, learner-centered (practical), and focused on skills practice41. Among specific strategies, role play with simulated or real patients, feedback and small group discussions were found to be effective41. The authors of the umbrella review reported that interventions were often poorly described and varied greatly, most lacked theoretical underpinnings, and relative efficacy was difficult to ascertain because many components were combined41. One of the included reviews specifically examined the use of simulated patients and/or role play in 23 studies and concluded that more studies report improvement in communication skills when practice has taken place; the single study that compared role play with simulated patients showed no difference in extent of improvement42. Although limitations included multicomponent interventions, variable outcomes, small study size, and potential selection bias in some studies, the three studies considered higher quality demonstrated better outcomes when simulated patients and/or role play were utilized42.\n\nAlthough the multicomponent nature of the included interventions in this review prevents us from teasing out the specific impact of role play and/or simulated patients, the studies that included these components demonstrated improvements in shared decision making outcomes. In addition to the importance of communication competencies in making shared decisions, we propose that practice and feedback might also provide the social context required to develop and maintain physician behaviors that bolster patient engagement and partnership in the complex shared decision making process. Although relational agency was not mentioned explicitly, these interactive interventions may enhance the physician’s comfort with relational agency. Role play, in particular, may be well suited to facilitate relational agency. As Lane and Rollnick wrote in their 2007 study: “. . . trainees can get additional knowledge by experiencing the role of the patient”42.\n\nWe did not identify any studies that explicitly addressed patient or relational agency, either in the design of the intervention or in reported outcomes. The study by Bieber et al. allowed us to retrospectively consider how relational agency may be emphasized in educational interventions and how CHAT could be utilized in the analysis of an educational intervention18,19. But we must be clear that this was our interpretation of a study and not the authors’ explicit intent. Given our finding that the majority of studies included role play or simulated patients and considering that these approaches most logically allow the introduction and development of the concept of relational agency between patient and physicians, we would recommend consideration of this area for future study. Street et al. addressed the issue of how clinician-patient communication can directly and indirectly impact health outcomes, by empowering patients to be active agents in their health: “Clinicians can facilitate patient involvement in the decision making process by helping patients actively seek information, clarify treatment goals, and express concerns and feelings”43. Applying the CHAT framework to studies of shared decision making interventions will allow investigators to examine how well the intervention addresses tensions in the simulated clinical encounter and how physicians learn to engage patients in decisions, potentially leading to expansive learning over time.\n\nConsideration of relational agency may be important to successful shared decision making and should be included in development of future educational interventions. Focused on relational agency, Edwards and D’Arcy studied young bilingual pupils teaching a foreign language to student teachers. Similar to shared decision making between a physician and patient, “teaching was . . . seen as joint action between pupils and student teachers on the shared object of the learners’ communicative competence, using some fairly traditional pedagogic tools”44. The investigators also found that the sessions became more interactive and responsive; the authors stated: “. . . the concept of relational agency, embedded within the idea of a classroom as an open-ended learning zone is a useful way forward. Its particular strength lies in the emphasis it places on the need for learners to develop the capacity to both seek and give support in joint action on the object of activity to expand mutual understandings of the object”44. We can envision a similar application to shared decision making, in which role play and simulated patients give the physician learners an opportunity to learn from the interaction with the “patient”; similarly, over time, physicians and patients learn from each other how to reach the shared object of shared decision making. Expansive learning can begin with an educational intervention that has development of relational agency between patient and physician as a central theme.\n\nThere do not appear to be well-accepted instruments or tools to evaluate or measure patient or relational agency; therefore, studies that address the role of agency in shared decision making should include a qualitative component. The use of qualitative analysis within the Bieber study design provided a rich example of the potential impact of the intervention on patient agency18. Our use of CHAT in this review demonstrates its potential as a future framework to analyze the role of agency in studies of shared decision making and to suggest potential areas for future research. Specifically, the tensions between each of the components of an activity system shed light on the complexity of shared decision making. Furthermore, the more complex tensions between two activity systems are also worth investigating.\n\nIn order to characterize studies of shared decision-making educational interventions and identify those that address patient agency, we included only studies that reported patient outcomes. One benefit of our approach, besides answering our research questions, was that many of our studies scored high on quality ratings. Other potential reasons for the higher MERSQI ratings are that the majority of the studies were RCTs and, in particular, many of the included studies used previously validated instruments to report on outcomes, such as the OPTION and Roter scales.\n\nHowever, by focusing on actual patient outcomes, our approach excluded articles describing medical students or other trainees who do not see patients independently; all of the interventions involved physicians who cared for patients independently (i.e., attending physicians, fellows, and residents). Another limitation was that, while the majority of studies were RCTs, making them methodologically strong educational studies, they often did not include qualitative analyses that may have provided more insight into the role of agency. Finally, despite our best efforts to conduct a comprehensive search, it is possible we inadvertently missed articles.\n\n\nConclusions\n\nEducational interventions to teach shared decision making to physicians commonly include decision aids, role play, and/or simulated patients, and have had a positive impact on a range of patient outcomes. Explicit instructional design around individual and relational agency is absent from educational interventions for shared decision making. Using CHAT can highlight the role of agency and identify tensions that could be included in the design and development of training programs. Future research and development of instructional strategies should consider the complexity inherent in co-constructing decisions.\n\nEducators should consider utilizing multicomponent interventions to teach shared decision making, including role play and/or simulated patients and should ensure health professionals are familiar and can effectively engage patients using decision aids. Although formal educational programs around facilitating patient agency have not been developed, clinicians should look for opportunities to encourage patients to actively seek information and should inquire about patients’ preferences and treatment goals.\n\n\nData availability statement\n\nZenodo dataset: Educational interventions for shared decision making and the role of patient agency: A Systematic Review.\n\nDOI: 10.5281/zenodo.50291508\n\nThe project contains the following extended data:\n\n• Complete Search StrategiesWitkop.pdf\n\n• PRISMA checklist\n\nData are available under the terms of the Creative Commons Attribution 4.0 United States", "appendix": "Acknowledgments\n\nThe authors would like to thank Rhonda Allard for her invaluable assistance with the literature search.\n\n\nDisclaimer\n\nThe contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views, assertions, opinions, or policies of the Uniformed Services University of the Health Sciences (USUHS), the Department of Defense (DoD), or the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government.\n\n\nReferences\n\nLégaré F, Politi MC, Drolet R, et al.: Training health professionals in shared decision-making: An international environmental scan. Patient Educ Couns. 2012; 88(2): 159–169. PubMed Abstract \| Publisher Full Text\n\nDiouf NT, Menear M, Robitaille H, et al.: Training health professionals in shared decision making: Update of an international environmental scan. Patient Educ Couns. 2016; 99(11): 1753–58. PubMed Abstract \| Publisher Full Text\n\nMüller E, Strukava A, Scholl I, et al.: Strategies to evaluate healthcare provider trainings in shared decision-making (SDM): a systematic review of evaluation studies. BMJ Open. 2019; 9(6): e026488. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiddens A: The Constitution of Society. Berkeley, CA: University of California Press; 1984; [accessed: 17 May 2021]. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBieber C, Nicolai J, Gschwendtner K, et al.: How Does a Shared Decision-Making (SDM) Intervention for Oncologists Affect Participation Style and Preference Matching in Patients with Breast and Colon Cancer? J Cancer Educ. 2018; 33(3): 708–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTai-Seale M, Elwyn G, Wilson CJ, et al.: Enhancing shared decision making through carefully designed interventions that target patient and provider behavior. Health Aff (Millwood). 2016; 35(4): 605–12. PubMed Abstract \| Publisher Full Text\n\nDillon EC, Stults CD, Wilson C, et al.: An evaluation of two interventions to enhance patient-physician communication using the observer OPTION5 measure of shared decision making. Patient Educ Couns. 2017; 100(10): 1910–17. PubMed Abstract \| Publisher Full Text\n\nGeiger F, Liethmann K, Reitz D, et al.: Efficacy of the doktormitSDM training module in supporting shared decision making - Results from a multicenter double-blind randomized controlled trial. Patient Educ Couns. 2017; 100(12): 2331–8. PubMed Abstract \| Publisher Full Text\n\nKasper J, Liethmann K, Heesen C, et al.: Training doctors briefly and in situ to involve their patients in making medical decisions—Preliminary testing of a newly developed module. Health Expect. 2017; 20(6): 1254–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanders AR, Bensing JM, Essed MA, et al.: Does training general practitioners result in more shared decision making during consultations? Patient Educ Couns. 2017; 100(3): 563–74. PubMed Abstract \| Publisher Full Text\n\nHenselmans I, van Laarhoven HWM, van Maarschalkerweerd P, et al.: Effect of a Skills Training for Oncologists and a Patient Communication Aid on Shared Decision Making About Palliative Systemic Treatment: A Randomized Clinical Trial. Oncologist. 2020; 25(3): e578–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElwyn G, Hutchings H, Edwards A, et al.: The OPTION Scale: measuring the extent that clinicians involve patients in decision-making tasks. Health Expect. 2005; 8(1): 34–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoter D, Larson S: The Roter interaction analysis system (RIAS): utility and flexibility for analysis of medical interactions. Patient Educ Couns. 2002; 46(4): 243–51. PubMed Abstract \| Publisher Full Text\n\nBerkhof M, van Rijssen HJ, Schellart AJM, et al.: Effective training strategies for teaching communication skills to physicians: An overview of systematic reviews. Patient Educ Couns. 2011; 84(2): 152–62. PubMed Abstract \| Publisher Full Text\n\nLane C, Rollnick S: The use of simulated patients and role-play in communication skills training: A review of the literature to August 2005. Patient Educ Couns. 2007; 67(1–2): 13–20. PubMed Abstract \| Publisher Full Text\n\nStreet RL Jr, Makoul G, Arora NK, et al.: How does communication heal? Pathways linking clinician-patient communication to health outcomes. Patient Educ Couns. 2009; 74(3): 295–301. PubMed Abstract \| Publisher Full Text\n\nEdwards A, D’Arcy C: Relational agency and disposition in sociocultural account of learning to teach. Educ Rev. 2004; 56(2): 147–55. Publisher Full Text" }	[ { "id": "97489", "date": "10 Nov 2021", "name": "Jeroen M. Hendriks", "expertise": [ "Reviewer Expertise Cardiac arrhythmias", "novel models of care delivery", "patient involvement" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paper exploring educational interventions for shared decision making and the role of patient agency. The topic is crucial in temporary models of care delivery, given the role of the patient is changing including active involvement in the decision making. The manuscript is well written, however I have the following comments:\nGiven the role of agency in this research, the concept has been scarcely explained. Please further elaborate on this concept and its role in clinical practice in the manuscript.\n\nCo-design of these interventions is important. According to international practice guidelines for some conditions, patients should be active members of the treatment team. How does this align with the findings of your research? Please elaborate on this in the manuscript.\n\nLimitations RE: this research have not been reported?\n\nThe educational interventions reviewed are important indeed, but the implementation in practice is next level. In line with this and the current findings, the discussion is lacking implications for practice. Please add this to the discussion section.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "292342", "date": "03 Jul 2024", "name": "Tim Dornan", "expertise": [ "Reviewer Expertise Medical education", "patient-centred care", "education research", "critically-informed qualitative research", "Activity Theory" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 recommend this article unreservedly for the attention of readers. The topic is important at the present time. The review is up to date. The large amount of work involved in doing the review was conducted rigorously. The article provides a valuable overview of how others have educated physicians for shared decision-making. I commend the authors for theorising their work; and Activity Theory (AT) lends itself well to the topic. I particularly commend the way the authors have presented relational agency in Results and considered it also in Discussion. Since F1000 will (helpfully) publish this review alongside the article, I would not want the authors to revise the article. I hope the critical comments that follow (written from a physician/Activity Theorist’s perspective) may add value to the authors’ work. In other words, my response is dialectic: it uses a perspective that differs a little from the authors’ to (I hope) advance the field beyond this publication.\nActivity theory has an extreme constructionist epistemology: ie it regards knowledge as subjective and provisional. This research has constructionist leanings, for which I commend the authors, but epistemological tensions remain. These are exemplified by using systematicity as an arbiter of rigour (PRISMA diagram), and objectively (quantitatively) judging the quality of articles. It uses atheoretical thematic analysis to explore agency, which assumes that words in published articles can be interpreted at face value, when AT would guide researchers towards theoretically informed interpretation of discourse. Blunden’s recent book entitled ‘Activity theory’ (Ref 1), citing the seminal research of Engeström and colleagues (who analyse language in exemplary ways), reviews meanings of the term ‘agency’ that exist within other scholars’ AT-informed research. Blunden's review might help future AT researchers theorise their qualitative analyses, and make qualitative analyses of agency more rigorous. I commend the authors for moving beyond Giddens’ individualistic conceptualisation of agency and for highlighting the informative work of Bieber and colleagues. Their discussion of Bieber and colleagues’ work highlights a classical ‘contradiction’ between the activity systems of physicians and patients in the care of fibromyalgia, which might lend itself to close analysis of language. Future research informed by AT might advance the field by locating agency even more strongly within actions that are essentially communal, rather than in dyads of individuals interacting (nicely discussed by Blunden).\nFar from questioning the validity of the work, these comments are intended to encourage the authors and others to be even bolder in taking constructionist stances. This article exposes a ‘fault line’ (what Kuhn might have called a ‘paradigmatic fissure’) in medical education research. The authors have ‘nudged’ research into relational agency from the objective world of the RCT towards the world of social complexity. I commend others to follow their cue and, if using AT, to do so with even greater constructionist courage.\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\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.) Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/10-753
https://f1000research.com/articles/10-750/v1	04 Aug 21	{ "type": "Method Article", "title": "Strategies for cellular deconvolution in human brain RNA sequencing data", "authors": [ "Olukayode A. Sosina", "Matthew N. Tran", "Kristen R. Maynard", "Ran Tao", "Margaret A. Taub", "Keri Martinowich", "Stephen A. Semick", "Bryan C. Quach", "Daniel R. Weinberger", "Thomas Hyde", "Dana B. Hancock", "Joel E. Kleinman", "Jeffrey T. Leek", "Andrew E. Jaffe", "Olukayode A. Sosina", "Matthew N. Tran", "Kristen R. Maynard", "Ran Tao", "Margaret A. Taub", "Keri Martinowich", "Stephen A. Semick", "Bryan C. Quach", "Daniel R. Weinberger", "Thomas Hyde", "Dana B. Hancock", "Joel E. Kleinman", "Jeffrey T. Leek" ], "abstract": "Background: Statistical deconvolution strategies have emerged over the past decade to estimate the proportion of various cell populations in homogenate tissue sources like brain using gene expression data. However, no study has been undertaken to assess the extent to which expression-based and DNAm-based cell type composition estimates agree. Results: Using estimated neuronal fractions from DNAm data, from the same brain region (i.e., matched) as our bulk RNA-Seq dataset, as proxies for the true unobserved cell-type fractions (i.e., as the gold standard), we assessed the accuracy (RMSE) and concordance (R2) of four reference-based deconvolution algorithms: Houseman, CIBERSORT, non-negative least squares (NNLS)/MIND, and MuSiC. We did this for two cell-type populations - neurons and non-neurons/glia - using matched single nuclei RNA-Seq and mismatched single cell RNA-Seq reference datasets. With the mismatched single cell RNA-Seq reference dataset, Houseman, MuSiC, and NNLS produced concordant (high correlation; Houseman R2 = 0.51, 95% CI [0.39, 0.65]; MuSiC R2 = 0.56, 95% CI [0.43, 0.69]; NNLS R2 = 0.54, 95% CI [0.32, 0.68]) but biased (high RMSE, >0.35) neuronal fraction estimates. CIBERSORT produced more discordant (moderate correlation; R2 = 0.25, 95% CI [0.15, 0.38]) neuronal fraction estimates, but with less bias (low RSME, 0.09). Using the matched single nuclei RNA-Seq reference dataset did not eliminate bias (MuSiC RMSE = 0.17). Conclusions: Our results together suggest that many existing RNA deconvolution algorithms estimate the RNA composition of homogenate tissue, e.g. the amount of RNA attributable to each cell type, and not the cellular composition, which relates to the underlying fraction of cells.", "keywords": [ "Statistical deconvolution", "DNA methylation", "RNA-seq", "single nucleus RNA-seq" ], "content": "Introduction\n\nHomogenate tissues like brain and blood contain a mixture of cell types which can each have unique genomic profiles, and these mixtures of cell types, termed “cellular composition”, can vary across samples (Jaffe and Irizarry 2014). The importance of considering cellular composition within heterogeneous tissue sources has been highlighted in epigenetics research over the past several years (Houseman et al. 2012; Montaño et al. 2013; Jaffe and Irizarry 2014), as, generally, failure to account for cellular composition when analyzing heterogeneous tissue sources can increase both false positives and negatives (Michels et al. 2013). Previous work has identified widespread epigenetic differences between neurons and glia using DNA methylation (DNAm) data (Montaño et al. 2013; Guintivano et al. 2013), and false positives may arise when there are cellular composition differences associated with dissection variability, disease, normal development or any other outcome of interest. For example, loss of neurons (or glia) because of disease may cause spurious loci associations with illness that stem solely from differing cellular compositions between disease states, or cell-type specific biological differences may exist that become more difficult to detect in the presence of unaffected cell types.\n\nStatistical algorithms estimate the relative or absolute amounts of each cell type in the homogenate tissue data. These so called “cellular deconvolution” algorithms have been especially popular using DNAm data (Houseman et al. 2014) as DNAm levels are constrained between 0 and 1 and are binary within single cells (i.e. individual CpGs are either methylated or unmethylated). These deconvolution algorithms can be classified into two general types, termed “referenced-based” and “reference-free” (Houseman et al. 2014; Avila Cobos et al. 2018). Reference-free approaches only require as input an estimate of the number of potential cell types in a particular dataset (which can be non-trivial), and return latent components that preferentially capture cellular heterogeneity that can be adjusted for in differential methylation analysis (Houseman et al. 2014; Jaffe and Irizarry 2014; Rahmani et al. 2016). However, these approaches do not return fractions of cells and may capture potential batch effects in addition to cellular composition. Conversely, reference-based approaches require cell type-specific genomic profiles for each cell type of interest as an input and return the relative fraction of each input cell type for each queried bulk sample (Houseman et al. 2012), akin to an in silico cell counter. This class of algorithms therefore requires the generation of potentially many pure cell populations, which are typically generated from flow cytometry for applications to DNAm data from bulk tissue.\n\nWhile DNAm data can generate accurate absolute cell fractions in homogenate brain tissue (Amanda J. Price et al. 2019; Guintivano et al. 2013; Montaño et al. 2013), there are several important considerations limiting more widespread application. First, RNA and gene expression profiling has been much more popular in postmortem brain studies, with more samples profiled with RNA sequencing (RNA-seq) than DNAm microarrays or sequencing. Secondly, the two cell classes typically used by DNAm-deconvolution algorithms are likely too broad to identify more subtle differences in dissection variability and potential stereological differences (van Haren et al. 2011; Nelson et al. 1998). While recent work has extended the number of cell populations that can be isolated by antibodies to separate neurons into their excitatory and inhibitory subclasses and oligodendrocytes from other glia (Kozlenkov et al. 2018), there are likely very few additional cell types that are possible to isolate using nuclear antibodies for DNAm samples. Researchers have therefore turned to using cell type-specific RNA microarray and sequencing datasets to adapt these reference-based deconvolution algorithms to homogenate RNA-seq samples (Avila Cobos et al. 2018; Newman et al. 2015; Mohammadi et al. 2017; Gong and Szustakowski 2013; Baron et al. 2016; Wang et al. 2020; Shen-Orr et al. 2010; Abbas et al. 2009; Wang et al. 2018; Burke et al. 2020; Collado-Torres et al. 2019). The majority of these studies have focused on tissues other than the brain, which can be freshly obtained and dissociated into individual cells for single cell RNA-seq (scRNA-seq) or be sorted into specific cell populations using flow cytometry for cell type-specific expression profiling. For example, the popular CIBERSORT approach (Newman et al. 2015) was designed for blood gene expression microarray data, but has been adapted to RNA-seq datasets in other tissues. Several of the above algorithms have been designed, adapted or implemented for brain tissue, including linear regression followed by quadratic programming using the Houseman algorithm (Houseman et al. 2012; Collado-Torres et al. 2019; Burke et al. 2020), NNLS (Wang et al. 2018), the support vector machine-based CIBERSORT (Fromer et al. 2016; Hoffman et al. 2017), the empirical Bayes method MIND (Wang et al. 2020), and MuSiC, which combines a recursive tree based approach with weighted NNLS for cell type proportion estimation (Wang et al. 2019).\n\nHowever, few of these approaches have validated that the resulting composition estimates are accurate, i.e., are absolutely similar to the true underlying composition, particularly in brain tissue. No approach to our knowledge has quantified the consequences of parameter and algorithm choices when only non-ideal reference data is available (e.g., mismatched tissue type, species, sequencing protocol, etc.), which occurs in almost all applications. Many reference datasets have been constructed from purified cell type-specific RNA-seq data from mouse (Xu et al. 2014), or RNA-seq data from sorted or dissociated nuclei in humans (Lake et al. 2016, 2018; Velmeshev et al. 2019; Hodge et al. 2019; Mathys et al. 2019), and not whole cells, which are typically profiled in homogenate sequencing studies. Gene expression levels are also quantitative within individual cells (and not binary like in DNAm data) and the necessity of absolute expression levels for absolute composition quantification has largely been overlooked.\n\nHere we directly evaluated the absolute accuracy of several popular RNA-seq-based deconvolution strategies using several different reference datasets including a bulk/homogenate dataset with paired DNAm and RNA-seq data from the nucleus accumbens (NAc) from 200+ deceased individuals (Markunas et al. 2019). We used the DNAm data to estimate absolute neuronal fractions for each sample and evaluated absolute RNA-based deconvolution accuracy, using DNAm based neuronal fractions as proxies for the true unobserved fractions, across a variety of scenarios. We first evaluated the effects of using deep single cell RNA-seq (scRNA-seq) from healthy fresh human tissue obtained from surgically resected temporal cortex (Darmanis et al. 2015). This dataset likely produces the most comparable RNA-seq profiles to frozen bulk postmortem tissue, since whole cells were profiled, and 90% of RNA is cytosolic in the cortex (Jaffe et al. 2015). However, this dataset was derived from cells in a cortical brain region. We next produced snRNA-seq data from postmortem human NAc to use as a reference dataset, which results in potentially less comparable nuclear reference profiles but comes from a more comparable brain region. We lastly used cyclic-ouroboros single-molecule fluorescence in situ hybridization (osmFISH) imaging data from the somatosensory cortex region in mouse (Codeluppi et al. 2018) to derive important parameters in popular deconvolution algorithms. Together, our results demonstrate that many algorithms are not accurate, even when estimating only two cell classes (neurons and glia), and we offer several strategies to assess and improve accuracy that can be applied across multiple datasets and cell types.\n\n\nResults\n\nWe motivate this work with a large human postmortem brain genomic dataset from the NAc, a brain region containing functionally distinct cell types critical in reward-processing and addiction (Koob and Volkow 2016; Nestler 2005). Genomic data from this region has been underrepresented in postmortem human brain sequencing studies, which have primarily focused on the frontal cortex (Jaffe et al. 2018; Wang et al. 2018; Gandal et al. 2018) but its underrepresentation allows us to more comprehensively evaluate the accuracy of cellular deconvolution using potentially imperfect and/or mismatched reference datasets (described below). We dissected homogenate NAc tissue from the ventral striatum (anterior to the optic chiasm) across 223 adult donors and concurrently extracted DNA and RNA from the exact same tissue aliquot (see Methods), which allows for directly comparable cellular composition in each fraction. We profiled genome-wide DNAm with the Illumina Infinium MethylationEPIC microarray and performed reference-based deconvolution to estimate the fraction of neurons in each sample (see Methods). We have previously demonstrated the absolute accuracy of the Houseman deconvolution algorithm (Houseman et al. 2012) in postmortem human brain DNAm data (Guintivano et al. 2013; Amanda J. Price et al. 2019); here we found very high correlation (ρ = −0.949, Figure S1, Extended data (Sosina et al. 2021)) between the neuronal fraction and the first principal component (PC) of the entire DNAm profile (32.3% of variance explained), which we have shown to be an accurate surrogate of composition in frontal cortex (Jaffe et al. 2016) and blood (Jaffe and Irizarry 2014). The corresponding RNA was sequenced using the Illumina sequencing with RiboZero Gold library preparations (see Methods). This “gold standard” dataset, therefore, has DNAm-derived neuronal composition values and RNA-seq data from 223 samples to explore the accuracy and concordance of many popular cellular deconvolution algorithms.\n\nWe first assessed the accuracy and concordance of four reference-based deconvolution algorithms: Houseman, CIBERSORT, NNLS/MIND, and MuSiC for two cell populations - neurons and non-neurons/glia - in our NAc RNA-seq dataset using recommended default settings (see Methods). We initially used single-cell RNA-seq (scRNA-seq) data from the temporal cortex of eight adult donors obtained during surgical resection generated and described in Darmanis et al. (2015) as the cell type-specific reference profiles for these algorithms. Importantly, these reference data were generated from fresh tissue, which preserved the integrity of the cells and corresponding cytosolic RNA, the predominant fraction of total RNA from brain (Jaffe et al. 2015) profiled in homogenate tissue. Furthermore, these reference profiles provide coverage of entire transcripts (as opposed to only the 3' ends) using Fluidigm C1 sequencing. Therefore, these expression profiles should be more comparable to bulk brain sequencing studies, with the caveat that the reference dataset was obtained from a different brain region (temporal cortex versus NAc and from living subjects as opposed to postmortem subjects).\n\nWe used measures of root mean square error (RMSE) to assess accuracy and squared Pearson correlation coefficients (R2) to assess concordance for each algorithm's estimated neuronal fraction compared to the DNAm-based neuronal fractions (Figure 1). RMSE quantifies the degree of bias, i.e., how much our cell type estimates (RNA composition estimates) deviate from the inferred absolute cell type fractions, with smaller values corresponding to the inferred cellular composition and RNA composition being more similar. R2 quantifies the amount of information our estimates contain about how the inferred absolute cell type fractions vary in the population being studied, i.e., how much inferred variability of the unobserved cell type fractions, across individuals, is captured by our composition estimates. This is important since higher R2 values will lead to better elimination of cell-type composition effects in downstream analyses. Houseman (Figure 1A), MuSiC (Figure 1B), and NNLS produced concordant (high correlation; Houseman R2 = 0.51, 95% CI [0.39, 0.65]; MuSiC R2 = 0.56, 95% CI [0.43, 0.69]; NNLS R2 = 0.54, 95% CI [0.32, 0.68]) but biased (high RMSE, ≥0.35) neuronal fraction estimates. CIBERSORT produced more discordant (moderate correlation; R2 = 0.25, 95% CI [0.15, 0.38]) neuronal fraction estimates (Figure 1C), but with less bias (low RSME, 0.09). We found that CIBERSORT, compared to either MuSiC or the Houseman RNA approach, was the most accurate. However, its estimates had the lowest correlation (R2) with estimates based on DNAm data. In comparing the R2 metric across the three approaches, we found that MuSiC had the highest correlation. Hence, it provides the most information about the inferred variability of the cell type proportions based on DNAm data. However, its estimates showed the greatest divergence, relative to other results, when compared to the inferred estimates from DNAm. These results suggest that all four of these approaches overestimated the inferred proportion of neurons in bulk brain tissue, even under the simplest application to deconvoluting two distinct cell populations. However, it was unclear how much algorithm parameters and reference dataset differences (in regard to technology and brain region) contributed to the performance of these methods.\n\nScatter plots showing the estimated neuronal proportions across the 223 individuals using the Houseman approach for DNAm reference vs neuronal proportions estimated using (A) the Houseman approach with scRNA reference, (B) MuSiC with default settings and scRNA reference data, and (C) CIBERSORT with scRNA reference data.\n\nMany of the above deconvolution strategies have several parameters whose adjustment could reduce the observed bias (i.e. maximize accuracy) and increase the concordance between these neuronal fractions. The MuSiC algorithm particularly has an interpretable “cell size” (see Methods) parameter used in the deconvolution process. Different cell types could have more or less absolute RNA abundance, for example if they were larger or smaller, or if they were more or less transcriptionally active. We hypothesized that the overestimation of neuronal fractions resulted from neurons being larger and more transcriptionally active. However, this “cell size” parameter, regularly defined by the algorithm as the average expression level for a given cell type summed across genes, is estimated directly from the reference cell type-specific RNA-seq profiles by default (see Methods). However, some scRNA-seq (or snRNA-seq) library preparation and sequencing strategies, like the Fluidigm C1 system, may normalize cDNA libraries to the same concentration prior to sequencing, which will remove potential variability in RNA abundances across cell types. We therefore sought to use external data to better estimate these cell size parameters (Table 1) and assessed the resulting effects on cellular deconvolution accuracy.\n\nFirst, we used external ouroboros single-molecule fluorescence in situ hybridization (osmFISH) data from mouse somatosensory cortex (Codeluppi et al. 2018) to construct two different types of cell size parameters for the MuSiC algorithm (as data from NAc did not exist). We extracted the estimates of both cell size (via their provided segmentations) and total RNA abundance (via the sum of all gene fluorescence signal) aggregated across neuronal and non-neuronal cell types. We subsequently utilized these estimates as proxies for cell size in human RNA-seq data when deconvoluting neuronal fractions. In these data, comparing neurons to non-neurons, neurons were both larger (123 vs 91 μm2, 95% CI [28 μm2, 36 μm2]) and had more total RNA (199 vs 180 intensity, 95% CI [11, 25]) as we observed in the estimated cell size in the MuSiC algorithm using the Darmanis dataset (18,925 vs 12,880 normalized counts). We did not observe any improvement in the concordance (osmFISH cell area R2 = 0.55, 95% CI [0.43, 0.69] ; osmFISH totalRNA R2 = 0.54, 95% CI [0.42, 0.68]) or accuracy (osmFISH cell area RMSE = 0.39; osmFISH totalRNA RMSE = 0.43) of the estimated cell type fractions when we compared our results from default settings to those based on applying cell size proxies using mouse data (Figure S2 A-B, Extended data (Sosina et al. 2021)). These results may not be particularly surprising, given the numerous differences between mouse and human morphology, and the different brain regions profiled.\n\nWe then generated snRNA-seq dataset from two postmortem NAc donors and 4,169 total nuclei to produce more comparable cell type-specific cell size (see Methods) parameters and reference expression profiles (see Methods) (Tran et al. 2020). First, we used the NAc reference dataset at the single nucleus level and ran the MuSiC algorithm with default settings, which used both NAc-based cell sizes and expression profiles, to deconvolute neuronal fractions (Figure 2A). We confirmed that, on average, neurons had more total RNA than non-neurons using this NAc snRNA-seq dataset (103 vs. 72 unique molecular identifiers [UMIs] per gene). Furthermore, while there was a high correlation among neuron-specific gene expression effects across the NAc and temporal cortex (Darmanis et al. 2015) reference profile datasets, we observed genes with different magnitudes of effects based on differential expression results between neuronal and non-neuronal cell types (Figure S3, Extended data (Sosina et al. 2021)). When using both the NAc-based cell size and gene expression reference profiles, we observed a substantial improvement in both the concordance and the RMSE for the estimated neuronal fractions compared to using the temporal cortex dataset only. However, the estimates were still biased, and this bias increased as the neuronal fraction across individuals increased, suggesting that the NAc-based cell sizes together with the estimated abundance may be incorrectly characterizing the true underlying neuronal expression level for these individuals. Eliminating the cell size parameter resulted in similarly reduced concordances in both the temporal cortex and the NAc reference datasets, but increased accuracy only using the NAc reference dataset (Table 2). This implies that the underlying broad cellular composition was well captured by the gene “abundance” information for a matched brain region.\n\nScatter plots comparing the estimated neuronal proportion obtained for each individual using the Houseman approach with DNAm reference dataset vs neuronal proportions obtained using (A) MuSiC with default settings and a snRNA-seq NAc reference dataset, (B) MuSiC based on a snRNA-seq NAc reference dataset with cell sizes for each cell type estimated using osmFISH cell area (mouse), and (C) MuSiC based on a snRNA-seq NAc reference dataset with cell sizes for each cell type estimated using osmFISH total RNA abundance (mouse) per cell type.\n\nWe then combined different estimates of cell size parameters (NAc snRNA-seq versus osmFISH) and gene expression reference profiles (NAc snRNA-seq versus temporal cortex scRNA-seq) and assessed the effects on deconvolution accuracy in bulk NAc RNA-seq data. When running MuSiC using the estimates of cell size based on osmFISH data with the NAc expression reference profiles, we observed further improvements in the bias of the estimated cell type fractions but saw a minimal difference in the concordance (Figure 2B and C). Surprisingly, when we used only the Darmanis cell type-specific expression levels, the best (least biased and most concordant) deconvolution results were produced using cell sizes estimated from NAc snRNA-seq data, with improvements in both the concordance and the RMSE (Figure 3A). Specifically, when we compared the R2 and the RMSE estimates to those observed under the default setting for the Darmanis reference with the mismatched brain region, we see a small (6% relative change, 95% CI [0.18%, 11.67%]) increase for the concordance and a substantial (78% relative change, 95% CI [77%, 79%]) decrease for the RMSE. We further refined the NAc cell size estimates using sets of the top 25 and 50 cell type discriminating genes (see Methods), which slightly improved our estimates of the absolute cell type fractions (Figure 3B and C). Both the concordance (7% relative change, 95% CI [0.16%, 12.74%]) and RMSE (86% relative change, 95% CI [84%, 87%]) improved even more when compared to the default approach using a mismatched reference dataset. Across all approaches, the most accurate (least biased) result occurred when we used cell sizes estimated from Darmanis scRNA-seq data and gene expression from NAc snRNA-seq data, while the most concordant results were observed when we used NAc snRNA-seq data exclusively.\n\nScatter plots comparing the neuronal fraction estimated for each individual using DNAm data and the Houseman method vs neuronal fractions based on scRNA-seq data and estimated using MuSiC with (A) cell-size estimated using all genes expressed in the NAc snRNA-seq reference dataset, (B) cell-size estimated using the top 50 cell type discriminating genes in the NAc snRNA-seq reference dataset, and (C) cell-size estimated using the top 25 cell type discriminating genes in the NAc snRNA-seq reference dataset.\n\nIn summary, when we used a region-matched appropriate dataset - NAc snRNA-seq data - as the reference, or to derive estimates of the cell size, we observed that estimates of the cell type proportions generally improved (Table 2, Figure 2A and Figure 3A-C). In settings where we had a mismatched reference dataset (e.g., mismatched on brain region or species), incorporating estimated cell sizes obtained from the matched brain region (NAc) provided the best result in metrics for both concordance and accuracy, and we slightly improved these metrics when we refined the gene sets used to estimate the cell sizes.\n\n\nDiscussion\n\nStatistical deconvolution strategies have emerged over the past decade to estimate the proportion of various cell populations in homogenate tissue sources like blood and brain from both gene expression and DNAm data. Our results together suggest that many existing RNA deconvolution algorithms estimate the RNA composition of homogenate tissue, e.g. the amount of RNA attributable to each cell type, and not the cellular composition, which relates to the underlying fraction of cells. This was evident by the consistent overestimation of larger and more transcriptionally active neuronal cells. We have identified that incorporating cell size parameters into RNA-based deconvolution algorithms can successfully recover cellular fractions in homogenate brain RNA-seq data. We have lastly shown that using both cell sizes and cell type-specific gene expression profiles from brain regions other than the target/user-provided bulk tissue RNA-seq dataset consistently resulted in overestimating neuronal fractions. We have developed an extension of the MuSiC framework (Wang et al. 2019) that allows for the incorporation of independent cell size estimates, and have further provided cell size estimates for human brain (shown in Table 1) as a part of the package: https://github.com/xuranw/MuSiC.\n\nCharacterizing cellular heterogeneity is especially important in human brain, where the underlying cell types can have diverse functions and disease associations that could be missed in studies of bulk tissue (Michels et al. 2013). Here we show that RNA-based deconvolution for just two cell populations - neurons and non-neurons - largely fails to estimate the underlying cellular composition of bulk human brain tissue across a variety of algorithms and strategies. We quantified the diverse range of neuronal fractions estimated by several popular algorithms to better understand the effects of reference cell type-specific expression profiles and differences in cell size and/or activity profiles on deconvolution. We specifically examined the common scenario of performing RNA deconvolution using cell type-specific reference datasets that can be fundamentally different from user-provided homogenate tissue target datasets, for example differing in profiled brain region, sequencing technology and/or cellular compartment. These problems are likely magnified in human brain tissue compared to suspended cells like blood, where deconvolution strategies are more easily validated against true cell fractions obtained by routine complete cell counts (Newman et al. 2015). We also note that while the effect of replacing the cell sizes is a general shift of all the points in the scatter plot which in turn changes the RMSE estimates, our goal in this paper is way to develop an approach to estimate the right shift needed to correct the biased estimates. Our results show that using the same dataset set to estimate both cell-size and abundance can lead to biased estimates. We lastly emphasize caution when performing RNA-based deconvolution using many cell types (i.e., more finely partitioned cell classes) without having the ability to validate cell counts on at least a subset of samples.\n\nWe therefore offer several recommendations for performing RNA-based deconvolution in bulk human brain gene expression data, particularly when aiming to identify cellular, and not RNA, composition.\n\n1. Providing estimates of cell size for each reference cell type improves the concordance and reduces bias when performing RNA deconvolution to estimate cellular fractions. Biologically-motivated and valid external estimates of cell-size improve the accuracy of the estimated cell type fractions, even when gene expression profiles for reference cell populations are obtained from other brain regions (Figure 3). The exact biological interpretation of these estimated cell sizes, particularly when estimated across species, is arguably unclear, but likely relates to correcting for absolute RNA abundance and differences in transcriptional activity between cell populations. Regardless of the method used for deriving cell sizes, neurons consistently had more RNA than glia. We note that our recommended strategies for estimating cell size have only been assessed for broad classes of cell types, and further work is needed to validate extensions to more stratified subclasses of cells.\n\n2. The concordance and bias improvements using full-length single cell sequencing from a different brain region (temporal cortex), rather than single nuclei RNA-seq from the target brain region (NAc) highlighted the importance of comparability between reference gene expression profiles and the homogenate tissue expression levels. While previous reports have identified high correlation between nuclear and cytosolic gene expression levels in both bulk (Amanda J. Price et al. 2019) and single cell (Bakken et al. 2018; Hodge et al. 2019) resolution, comparable absolute (and not relative) expression levels are seemingly important for the accuracy of these RNA-based cellular deconvolution algorithms. There further is an experimental design tradeoff between profiling more nuclei (1000s) using 3' technologies like 10x Genomics Chromium Single Cell Gene Expression compared to profiling fewer nuclei (or cells, 100s) using full-length sequencing technologies like SMART-seq if researchers wish to generate their own reference profiles.\n\n3. Using reference cell type-specific expression profiles from comparable brain regions as the bulk RNA-seq target dataset is important and can especially greatly increase the concordance of these RNA deconvolution strategies with neuronal fractions.\n\nThe choice of maximizing accuracy (by minimizing bias) versus increasing concordance in assessing these algorithms is an important consideration, particularly when generating custom expression reference profiles is prohibitive (Table 2). These two objectives largely relate to whether the goal of RNA deconvolution is to obtain correct estimates of absolute cell fractions (and maximize accuracy) or to identify/leverage differences in cell/RNA fractions (and maximize concordance) as a basis to obtain cell-type specific inference for a given gene. Estimation of RNA fractions (by maximizing concordance) may be sufficient to control for potential confounding due to composition differences between outcome groups (Michels et al. 2013). We note this can also be accomplished using “reference-free” deconvolution (Houseman et al. 2014) or through the estimation of potentially sparse principal components (Rahmani et al. 2016; Jaffe and Irizarry 2014) that control for relative differences in cellular composition. However, estimation of cellular fractions (and maximizing accuracy) is arguably more useful, both for assessing human brain tissue dissection during data generation and to identify cell type-specific effects when using these cellular fractions in downstream differential expression analyses (Zheng et al. 2018).\n\nTogether, our results demonstrate that many RNA deconvolution algorithms do not produce accurate cellular fractions when estimating only two cell classes (neurons and non-neurons). We offer several strategies and corresponding software to assess and improve accuracy that can be applied across multiple datasets and cell types.\n\n\nMethods\n\nBrain donation protocols were approved by IRB and oversight bodies. Legal next-of-kin gave informed consent to brain donation according to protocols Maryland Department of Health MDHMH# 12-24 (MD), National Institute of Mental Health [NIMH]# 90-M-014 (for brain donations in DC and VA), and Western Institutional Review Board [WIRB] # 1126332 (for brain donations in MD, WMU, UND), respectively. Subsequent research on human postmortem brain tissue - including the RNA sequencing performed here - is not classified as Human Subjects Research (since postmortem subjects are not classified as Human Subjects, per the Department of Health and Human Services in DHHS 45 CFR §46.102(e)(1):).\n\nData generation and processing were described extensively in Markunas et al. (2019). Briefly, the nucleus accumbens (NAc) was dissected under visual guidance using a hand-held dental drill. Samples were obtained from the ventral striatum, anterior to the optic chiasm, at the level where the NAcforms a bridge between the putamen and the head of the caudate. DNA and RNA were concurrently extracted from dissected tissue using the Qiagen AllPrep DNA/RNA Mini Kit (Cat No./ID: 80204).\n\nNAc DNA was profiled with the Infinium MethylationEPIC microarray (catalog #: WG-317-1003) using the manufacturer's protocol. Raw idat files were processed and normalized using the minfi Bioconductor package (Aryee et al. 2014) using stratified quantile normalization (Touleimat and Tost 2012). Resulting neuronal fractions were estimated using the minfi estimate CellCounts function (Jaffe and Irizarry 2014) using sorted reference data from the DLPFC for neurons and non-neurons (Guintivano et al. 2013; Andrew E. Jaffe 2017) using the Houseman algorithm (Houseman et al. 2012).\n\nNAc RNA was subjected to RNA-seq library preparations using the Illumina RiboZero Gold kits (catalog #: 20020599) and sequenced using 2×100 bp paired end reads on an Illumina HiSeq 3000.\n\nDarmanis: SRP057196 (Darmanis et al. 2015).\n\nscRNA-seq data for 58,037 genes and 556 cells were obtained for brain samples across eight individuals, as described previously (Darmanis et al. 2015). We filtered this dataset by removing cells based on embryonic samples and retaining cells from one of the following five cell types: neuronal, oligodendrocyte progenitor cells (OPC), astrocytes, oligodendrocytes, and microglia. We also removed genes that had no expression for all cells in the reference dataset or did not show any expression in the bulk dataset (i.e., mean and variance zero). In total, we used 265 cells for this reference and 24,048 genes to estimate the cell type proportions for the 223 samples with bulk NAc data.\n\nWe performed single-nucleus RNA-seq (snRNA-seq) on nucleus accumbens (NAc) tissue from two donors using 10× Genomics Single Cell Gene Expression V3 technology (PN-1000075) as previously described (Tran et al. 2020). Briefly, nuclei were isolated using a “Frankenstein” nuclei isolation protocol developed by Martelotto et al. for frozen tissues (2017; Lacar et al. 2016; Lake et al. 2016; Habib et al. 2016; Hu et al. 2017). Briefly, ~40 mg of frozen NAc tissue was homogenized in chilled Nuclei EZ Lysis Buffer (MilliporeSigma) in a glass dounce with ~15 strokes per pestle. Homogenate was filtered using a 70 um-strainer mesh and centrifuged at 500×g for 5 minutes at 4°C in a benchtop centrifuge. Nuclei were resuspended in the EZ lysis buffer, centrifuged again, and equilibrated to nuclei wash/resuspension buffer (1× PBS, 1% BSA, 0.2 U/uL RNase Inhibitor). Nuclei were washed and centrifuged in this nuclei wash/resuspension buffer three times, before labeling with DAPI (10 ug/mL). The sample was then filtered through a 35 um-cell strainer and sorted on a BD FACS Aria II Flow Cytometer (Becton Dickinson) at the Johns Hopkins University Sidney Kimmel Comprehensive Cancer Center (SKCCC) Flow Cytometry Core. Gating criteria were hierarchically selected for whole, singlet nuclei (by forward/side scatter), then for G0/G1 nuclei (by DAPI fluorescence). A null sort was additionally performed from the same preparation to ensure nuclei input was free of debris. Approximately 8,500 single nuclei were sorted directly into 25.1uL of reverse transcription reagents from the 10× Genomics Single Cell 3’ Reagents kit (PN-1000075, without enzyme). Libraries were prepared according to manufacturer’s instructions (10× Genomics) and sequenced on the Next-seq (Illumina) at the Johns Hopkins University Transcriptomics and Deep Sequencing Core.\n\nWe processed the sequencing data with the 10× Genomics’ Cell Ranger pipeline, aligning to the human reference genome GRCh38, with a reconfigured GTF such that intronic alignments were additionally counted given the nuclear context, to generate UMI/feature-barcode matrices. We used R package Seurat (Satija et al. 2015) for raw feature-barcode quality control, dimensionality reduction (PCA), choosing the top 30 PCs as the optimal dimensions for clustering. We performed graph-based clustering with the default Louvain approach, taking a computed K-nearest neighbors graph as input, which were then annotated with well-established cell type markers for nuclear type identity (Mathys et al. 2019). We also used Seurat’s implementation of non-linear dimensionality reduction techniques, t-SNE and UMAP, simply for visualization of the high-dimensional structure in the data, which complemented the clustering results (Figure S4, Extended data (Sosina et al. 2021)). With the five broad cell type annotations (neurons, oligodendrocytes, oligodendrocyte precursors, astrocytes, and microglia) of nuclear clusters, we identified unbiased cluster-driving genes (with Seurat’s ‘FindAllMarkers()’ function, using the Wilcoxon rank-sum test), that were upregulated in each cell type/cluster, compared to all other nuclei. Using the same set of 24,048 genes, we have 4,169 high-quality nuclei in this reference, evenly distributed across donors. The top 50- and top 25-per-cell-type gene sets had 247 and 125 genes, respectively, which included many cell type marker genes used for annotation.\n\nHOUSEMAN\n\nThis algorithm (Houseman et al. 2012) uses a linearly constrained quadratic optimization approach with additional non-negative constraints on the parameters. The linear constraint does not require that the sum of all coefficients equal one. This allows the possibility of unknown cell types in case the specification is not comprehensive. It was implemented using the minfi R Bioconductor package (Aryee et al. 2014).\n\nMuSiC\n\nThe MuSiC (Wang et al. 2019) approach models the relationship between the relative abundance of gene g in the bulk RNA-seq data and the mean expression level of the same gene in the reference dataset for a given individual. The relationship is provided below\n\nWhere k = 1,...,K is the index of the cell types, pk is the proportion of cells from cell type k, and θgk is the relative abundance for the gth gene with respect to the kth cell type. Sk is the cell size parameter and is defined as the average number of total mRNA molecules for cell type k. By default, Sk is estimated automatically by MuSiC. For the deconvolution method comparisons that assessed cell size impact on neuronal cell type proportion estimation, Sk was derived from one of multiple data sources (Table 1) using 1) default settings 2) osmFISH or 3) the average number of total mRNA molecules for cell type k using only the top 25 or 50 most discriminating genes per cell type. We defined “most discriminating” as genes with the smallest p-values and fold change >0.25, relative to other cell types. All estimation was carried out using the MuSiC package in R v3.6.3.\n\nCIBERSORT\n\nCIBERSORT uses a machine learning approach called nu-support vector regression (Newman et al. 2015; Schölkopf et al., 2000) and requires at least two input datasets to work. The first is a signature matrix that identifies the set of genes that are informative for the deconvolution procedure. The second is a bulk RNA-seq dataset to estimate cell type proportions.\n\nThe signature matrix depends on the tissue of interest. We generated a custom signature matrix. Using the Darmanis reference dataset, we generated both a reference sample file (gene-by-cell matrix) and a phenotype classes file (cell type-by-dummy variable identifying the cell type for each cell) and used the default setting (CIBERSORT) to obtain a custom signature gene expression matrix. The specified false discovery rate (FDR) threshold used to include genes in the signature matrix was 0.30 (i.e. q = 0.30, default). Using this signature matrix, we then performed deconvolution on our bulk NAc RNA-seq data. As suggested in the documentation for CIBERSORT, we disabled quantile normalization for our RNA-seq data.\n\nNNLS/MIND\n\nThis is a simple linear regression with non-negativity constraints on the parameter estimates. The estimated fractions are then the value of each parameter estimate divided by the sum of all parameter estimates across cell types. MIND uses NNLS to estimate cell type fractions.\n\n\nData availability\n\nGene Expression Omnibus: Genome-wide RNA-sequencing differences in nucleus accumbens of smokers vs. nonsmokers, Accession number GSE171936: https://identifiers.org/geo:GSE171936.\n\nZenodo: Strategies for cellular deconvolution in human brain RNA sequencing data. https://doi.org/10.5281/zenodo.4899937 (Sosina et al. 2021).\n\nThis project contains the following extended data:\n\n- sosina_extended_sf1.docx (Figure S1, DNAm estimated neuronal fractions vs PC1)\n\n- sosina_extended_sf2.docx (Figure S2, Deconvolution in bulk NAc data using gene expression profiles from the temporal cortex with cell size estimates derived using mouse samples)\n\n- sosina_extended_sf3.docx (Figure S3, Neuronal enrichment of gene expression in scRNA-seq from temporal cortex and snRNA-seq from nucleus accumbens)\n\n- sosina_extended_sf4.docx (Figure S4, t-distributed stochastic neighbor embedding (t-SNE) of single-nucleus RNA-seq data from the two postmortem NAc samples, representing the 4,169 high-quality nuclei after processing)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nAnalysis code available from: https://github.com/ksosina/Strategies-for-cellular-deconvolution-in-human-brain-RNA-sequencing-data.\n\nArchived analysis code at time of publication: https://doi.org/10.5281/zenodo.5009842 (ksosina, 2021).\n\nLicense: CC0 1.0 Universal.", "appendix": "Acknowledgements\n\nThe authors thank the authors of MuSiC; X. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "99217", "date": "02 Feb 2022", "name": "Kathryn Roeder", "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\nSosina et al. compared the performance of methods to perform cellular deconvolution of bulk gene expression from brains. They question the performance of popular methods when they use reference samples from mismatched brain regions and ignore cell size. Differential cell size leads to a fundamental difference between the fraction of cells of each type, versus the fraction of transcripts originating from each cell type. The authors seem to take the view that the former quantity is generally more appropriate. In their study they use paired bulk DNA methylation (DNAm) and gene expression data from the same subjects, so that they can estimate cell fraction fairly accurately from the DNAm. This quantity is used as ground truth. The paper presents its message clearly and provides a comprehensive inquiry of the question at hand. Portions of the text would benefit from revision to improve the writing style. Below are some comments that may improve the manuscript:\n\nIn the discussion, the authors say “We also note that while the effect of replacing the cell sizes is a general shift of all the points in the scatter plot which in turn changes the RMSE estimates, our goal in this paper is way to develop an approach to estimate the right shift needed to correct the biased estimates”. This statement is awkwardly phrased, and this is troubling because this is a key point of the paper. Apparently, their point is that RMSE is affected by location shift, and using cell size to correct for this location shift may not help much in downstream analyses.\n\nThe authors need to more clearly differentiate between downstream applications where the true cell type fraction is required and others where it is actually better to estimate the fraction of transcripts originating from each cell type. The authors mention that “Estimation of RNA fractions may be sufficient to control for potential confounding due to composition differences between outcome groups”. But they should go further to note that for some downstream analyses that aim to estimate cell-type-specific expression (e.g., MIND and bMIND) (Bakken et al. (20181)) it does not make much difference to estimate the transcript or cellular fraction. The authors do say, “However, estimation of cellular fractions (and maximizing accuracy) is arguably more useful, both for assessing human brain tissue dissection during data generation and to identify cell type-specific effects when using these cellular fractions in downstream differential expression analyses (Zheng et al. 2018).” This statement relies very heavily on the cited reference, but we find very little difference between controlling for cell fractions versus RNA fractions in that report. Indeed the difference amounts to adding an interaction of fractions and outcome to the regression, and adding/subtracting a constant for the fractions may not matter much to the key inferences. Simulations/analyses are needed to convince readers that RMSE is an important metric, and using cell size to correct for the mean fraction matters in downstream analysis.\n\nThe authors used paired DNAm and RNA-seq data, and the Houseman’s approach estimated neuronal fraction as the gold standard. Patrick et al.(20202) measured cellular fractions from 70 individuals. With DNAm measured from those individuals and the reference and Houseman’s approach implemented in the minfi package, we observed R2 = 0.008 and RMSE = 0.08. This contradicts the claim that they have a “gold standard”, and some justification is required.\n\nBakken et al. (20181) used well-matched snRNA-seq and scRNA-seq datasets from the visual cortex to compare cell type detection. They estimated that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. This contradicts the claim in the paper “90% of RNA is cytosolic in the cortex.”\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "146615", "date": "22 Aug 2022", "name": "Arjun Bhattacharya", "expertise": [ "Reviewer Expertise Statistical genetics", "bioinformatics", "transcriptomics", "brain traits", "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\nIn this manuscript, the authors present a thorough benchmarking of cell-type deconvolution methods and motivate new datasets to benchmark these methods. Their findings emphasize a very important fact: that cell-type deconvolutions methods are often not suitable to estimate cell-type proportions, but rather the proportion of total RNA produced by different cell types.\nGenerally, I have no major comments/critiques of the paper. I believe this is a very thorough examination of cell-type deconvolution methods, with a specific conclusion that is important when deconvoluted proportions are included in the downstream analyses. I would suggest a citation for the linseed method (Zaitsev et al., 20191) would be helpful, as this question of cell size vs. RNA produced is discussed in this methods paper. Most important, the addition of the \"gold standard\" dataset is very important, as many existing datasets that are used to benchmark these methods are outdated.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-750
https://f1000research.com/articles/10-410/v1	21 May 21	{ "type": "Research Article", "title": "Simple statistical insights into the COVID-19 data of Saudi Arabia: figures prior to vaccination campaign", "authors": [ "Omar S. El-Masry" ], "abstract": "Background: COVID-19, the disease caused by the newly emerging coronavirus, SARS-COV2, is still a major health burden worldwide as it continues to spread rapidly in many countries after being contained for a while. The aim of the study was to analyze the official current disease estimates in the Kingdom of Saudi Arabia to anticipate future risks and needs. Methods: Publicly available COVID-19 data published by the Saudi Ministry of Health were analyzed to extract statistical estimates of the disease. These include monthly case fatality rates, death rates/1000, comparison of death figures and regression analysis. Results: The number of confirmed, recovered and deaths surged in the middle of the outbreak (June and July). The case fatality rates reported later in September-November were the highest despite the decline in the number of confirmed cases. The death rates/1000 were higher during the middle of the outbreak, where the highest numbers of deaths were recorded. The number of recovered cases was the highest as well during this time. Regression analysis showed that the number of deaths was related to that of confirmed cases, especially during the peak time. On the other hand, the number of recovered cases was related to that of confirmed cases at the beginning of the outbreak. Conclusion: Statistical estimates of COVID-19 fatalities provide simple figures to understand the disease progression pattern and the health care management success in disease containment. However, the absolute numbers should never be disregarded to reflect on the real situation.", "keywords": [ "COVID-19", "Case fatality rate", "Death rate", "outbreak" ], "content": "Introduction\n\nCOVID-19, the disease caused by the newly merging coronavirus (SARS-COV2) is a major health concern worldwide currently (Zhang et al., 2020). The pandemic is unprecedented in recent history and a lot of restrictions were imposed across the globe in an attempt to contain the spread of the infection hoping to reduce the number of carriers and, consequently, the fatality rates. This, in turn will relieve the pressure on health authorities. The outbreak has spread rapidly from its start point in the mainland of China to more than 200 countries, according to World Health Organization figures (Zhou et al., 2020). The large number of patients who need medical care, especially those with an immediate requirement of intensive care and hospitalization resulted in overwhelming the financial, manpower and the managerial facets of healthcare centers and represented a huge burden on states (Zhang et al., 2020).\n\nData available so far indicates a remarkable quick spread of the virus, suggesting a high infectivity in comparison to SARS-COV of the same genus (Zhou et al., 2020). Regarding the virus spread, SARS-COV2, which causes COVID-19, can be transmitted via direct contact with infected surfaces or airborne droplets. Vertical transmission was not proven so far, therefore, transmission of the virus to babies from mothers is unlikely, according to the available evidence (Zhu et al., 2020). It was also reported that SARS-COV2 has a higher morbidity and mortality, and higher fatality rates than SARS-COV. At this end, COVID-19 patients suffer from severe suppression of cellular-mediated immune response to the virus as evident by the low count of CD3+, CD4+, and CD8+ cells (Chen et al., 2020). In this respect, SARS-COV, downregulates interferon (IFN) regulatory factor 3 (IRF-3), which ameliorates the antiviral activity mediated by IFN α and β axis; this scenario might also contribute to the suppression of antiviral activity in COVID-19 patients (Kuri and Weber, 2010). In a similar respect, early reports this year indicated that SARS-COV2 is detectable in clinical samples of non-survivors throughout the clinical course of the disease (Weiss and Murdoch, 2020). Moreover, an American study postulated that ethnicity and gender differences might have a potential influence on hospitalization and mortality of COVID-19 patients (Price-Haywood et al., 2020).\n\nThe aim of the current study was to provide up to date simple statistical estimates and insights into the official numbers published by the Saudi Ministry of Health and establish an overview to understand the epidemiological data of COVID-19 in the Kingdom of Saudi Arabia. This might help to set future action plans to improve the overall outcomes of disease management.\n\n\nMethods\n\nThis is a data-based study in which the official data on COVID-19 disease that were published by the Saudi Ministry of Health on COVID-19 dashboard were analyzed. Data are available from: https://covid19.moh.gov.sa/. The data published on the dashboard are presented as graphs showing the numbers of confirmed cases, recovered cases and deaths day by day. The numbers are updated on a daily basis.\n\nThe following data were extracted from the dashboard for this study: minimum and maximum numbers for confirmed cases of COVID-19, recovered cases of COVID-19, and deaths relating to COVID-19 reported for March-November 2020 The total number of people living in Saudi Arabia was determined according to the estimates of the United Nations that were published on the Worldometer website: https://www.worldometers.info/.\n\nThe total number of deaths in each month and case fatality rates were compared between the months in 2020, and death rates/1000 of the population were calculated. Regression analysis was also performed to underline the relationship between different figures reported for each month including the relationships between: (1) the number of confirmed cases (daily reported infections in each month) and the number of the recovered cases (daily reported recoveries in each month), and (2) the number of confirmed cases and the number of monthly reported COVID-19-related deaths (daily reported deaths in each month).\n\nStatistical presentations and calculations were performed using Graphpad Prism (version 7). The data were tested for normality and the Kruskal Wallis test was employed for comparison between groups, followed by Dunn’s multiple comparison test. The difference was considered statistically significant at p ≤ 0.05.\n\n\nResults\n\nTable 1 shows the minimum and maximum numbers recorded for COVID-19 confirmed cases, recovered cases and deaths for each month of 2020. The maximum numbers of confirmed cases were recorded during June, while the maximum recovered cases and deaths were recorded in July. There was an increase in the number of confirmed cases from March-June, and then the numbers started to decrease during July-November. The low number recorded in the first one or two months might be associated with the total number of performed tests, where the capacity of health authorities worldwide may not have been fully prepared for such a surprising pandemic.\n\nTable 2 shows the statistical comparison between the numbers of daily deaths recorded in each month (March-November). As the data were not following the Gaussian distribution, the Kruskal Wallis test was performed, followed by Dunn’s multiple comparison test, which indicated that the numbers of deaths recorded in May-November were significantly higher than the numbers recorded in March (p < 0.0001; except for May, p < 0.05). The difference between the numbers of deaths recorded in April and May was not significant, whilst the numbers recorded in June-October were significantly higher than April and May figures (p < 0.0001), except for the comparison between May and October, the difference was not significant, perhaps due to the reduction in number of deaths in October and November. Also, the numbers of deaths in October and November were significantly lower than that reported in June-August indicating the gradual reduction in the total number of deaths. In addition, there was no significant difference between, June, July, August and September figures, which might explain that the average reduction in the death numbers in September was not the desirable one as compared to the reduction observed in October and November. In other words, despite the decline in the number of confirmed cases in September in comparison to June-August period, the decline in the reported deaths in this month was not significantly different from death figures in June-August.\n\nThe case fatality rate is defined as the total number of deaths from a particular cause (COVID-19, in this case)/total number of cases x 100. The case fatality rates in each month are presented in Table 3. The lowest numbers of confirmed cases were reported throughout March and case fatality rate was higher than the rate reported for May (0.64 vs. 0.56%), perhaps due to the surprising outbreak of the pandemic and lack of the necessary preparations to confront such situations. Likewise, the rate in April was higher than that of May (0.7). Also, despite the declining figures of confirmed disease and death cases until November following the surge in June and July, the case fatality rates in September-November were the highest (4.71%, 4.98%, and 4.88%, respectively), higher than June and July, wherein the highest numbers of confirmed cases and deaths were reported. This might suggest the poor prognosis in the patient group of non-survivors, or the increased virulence of the virus, but we have to bear in mind the large number of cases in June and July in comparison to the following months. In the same context, the death rates/1000 of the population (Table 3) were also calculated. The results suggested that the highest death rates/1000 were recorded in June, July and August (0.03). Obviously, the lowest figures of this parameter were seen in March and April. In addition, the figures of September-November were less than that of June and July, despite the highest case fatality rates observed in these months.\n\n* Case fatality rate = Total number of deaths from Covid-19/total number of confirmed cases100.\n\n The Saudi population number was estimated at 34813871 at mid-year according to the UN estimates.\n\nThe relationship between the number of confirmed cases and the number of deaths in each month was investigated by regression analysis (Figure 1). Using this type of analysis we can understand how the change in the number of confirmed cases of COVID-19 is related to the number of deaths. The analysis showed that the slope was significantly non-zero (this means that the relationship between the tested variables was represented as a line with an apparent steepness, where the number of deaths (y) changes according to the change in the number of confirmed cases (x); simply the line is not horizontal) for March, though the association relationship was weak (R = 0.218; p value = 0.009). The slope was close to zero for April, May, and September (R = 0.12, 0.01, and 0.01; p value = 0.06, 0.51, and 0.49), which means that the change in the number of confirmed cases in these months was not associated with the change in the number of deaths. Regarding the figures of regression analysis in June and July, the best fit graph showed a stronger significant association between the number of confirmed cases and deaths (R = 0.5, and 0.68; p value = < 0.0001) as compared to the figures in the other months. August results showed a weaker positive association and a non-zero slope of the best fit line (R = 0.21 and p value = 0.008). The slope was close to zero for October indicating a lack of the relationship, while there was a weak association for November figures (R = 0.206 and p value = 0.012).\n\nRegression analysis showed that the number of deaths could be weakly related to the number of confirmed cases in March (A), August (F), and November (I). The relationship was significantly strong in June (D) and July (E).\n\nFigure 2 shows the regression analysis results in March-November for the assessment of the relationship between the number of the confirmed cases and recovered cases. The results showed that the number of the recovered cases of COVID-19 patients in March, April, May and August could be explained by the number of confirmed cases (R = 0.22, 0.54, 0.2, and 0.2; p values < 0.008, 0.0001, 0.01, and 0.01). The strongest relationship between the number of confirmed cases and the number of the recovered cases was then observed during April, where 50% of the recovery figures could be explained by the confirmed cases figures. The slope was close to zero for June, July and September analysis, which means that the number of the recovered cases are not related to the numbers of the confirmed cases, although June and July witnessed the highest number of the recovered cases (R = 0.009, 0.014, and 0.08; p value = 0.52 and 0.12). However, these results are consistent with the high death figures observed in June, July and September. There was a significant relationship between the numbers of the recovered and the confirmed cases in October (R = 0.224 and p value = 0.007). The figures for November showed that the relationship was not significant.\n\nRegression analysis showed that the number of deaths could be significantly related to the number of confirmed cases in March (A), April (B) and May (C). A weaker association was reported for August (F) and October (H).\n\n\nDiscussion\n\nAs the recently emerging pandemic of COVID-19 continues to attract attention all over the world with uncertainty about when the disease will vanish and cease new waves, the need continues to grow to assess the capacity of health care authorities to cope with the current challenge and be ready for all possibilities. In this respect, the assessment of the past and present situation data is important for accurate need assessment for future preparation. Therefore, the main aim of the current study was to analyze the data to conclude some statistical figures that could help to understand how effective the management of the situation was, and what should we do for better clinical management outcomes of the COVID-19 crisis. The results showed that the numbers of confirmed deaths reached the peak during June and July, with the highest death rates/1000 of the population. Also, despite the decrease in the number of the confirmed deaths in September-November, the case fatality rates were the highest throughout the period from March-November, which also showed a small number of confirmed cases that might have resulted in the high case fatality rate. The world is still overwhelmed with the pandemic; however, the situation seems to be under control in Saudi Arabia, despite the high case fatality rates recorded in September-November. Case fatality could be affected with multiple factors, such as patients’ age, comorbidities or virus virulence and immune response efficacy in patients. To this end, other data, such as patients’ demographics, comorbidities, medical history, autopsy reports and detailed lab records and clinical follow up data should be available for a more comprehensive figure. In this regard, it was reported that up to 81% of COVID-19 patients might suffer only from a mild disease, which does not require hospitalization (Price-Haywood et al., 2020). Having said that, the other 19%, which is a proportion large enough to overwhelm the health care sector, might suffer from severe symptoms that impose hospitalization and here is where mortality rates could increase as a result of poor clinical management.\n\nIt was also recently reported that the average case fatality rate of COVID-19 is less than 5% (Sun et al., 2020). This estimate suggests that the case fatality rates reported in the current study are within the global figures. The surprising hit of the pandemic and/or the virulence of the virus strain and its quick spread needed some time for the governments to confine the situation. In this regard, many comorbidities were linked to the increased severity and mortality of COVID-19 patients, including diabetes mellitus (Kumar et al., 2020). It is of importance to denote that diabetes mellitus is prevalent in Saudi Arabia (Alotaibi et al., 2017). However, more data should be available on COVID-19 casualties before assuming any link between the disease and COVID-19 mortality figures presented herein. It might be also of importance to mention that the case fatality rates of COVID-19 are lower than that of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Sun et al., 2020), but we have to bear in mind that the number of cases of COVID-19 patients is much higher than that recorded for the other two counterparts. Similar case fatality rates were reported for both China and Italy (~2.3) at the beginning of the pandemic, with comorbidities that might have contributed significantly to mortality in elderly patients (Porcheddu et al., 2020). These figures are almost similar to the average case fatalities reported in the period (March-September) in Saudi Arabia. In line with the current study, Boretti compared some figures of the outbreak between Saudi Arabia and the United Kingdom in the middle of the outbreak; Boretti reported that the number of cases per million, the number of deaths per million and the number of newly daily deaths per million were much less in Saudi Arabia than those in the United Kingdom (Boretti, 2020).\n\nIn conclusion, the results presented in the current study suggested that the fatality rates of COVID-19 mortality and their association with the number of confirmed cases and the recovery figures should be always seen in the same frame with the absolute figures to reflect the real situation to the policy makers for better needs assessment, future planning and prompt response in crisis time. Amongst the limitations of the current study, and maybe other related published articles, are the lack of information on cases and deaths age groups, comorbidities, ethnicity, and medical history that might have affected disease progression and mortality rates. The availability of such information could help to clarify risk factors and those predisposed for a severe disease clinical course. Also, we have to bear in mind that the number of COVID-19 cases are much higher than that of the disease counterparts SARS and MERS when we look at the fatality rates.\n\n\nData availability\n\nAll data presented herein were extracted by analyzing the publicly available data on COVID-19 that were uploaded to the COVID-19 data portal established by the Saudi Ministry of Health: https://covid19.moh.gov.sa/.", "appendix": "References\n\nAlotaibi A, Perry L, Gholizadeh L, et al.: Incidence and prevalence rates of diabetes mellitus in Saudi Arabia: An overview. J Epidemiol Glob Health. 2017; 7: 211–218. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoretti A: COVID-19 fatality rate for Saudi Arabia, updated 3 June 2020. J Glob Antimicrob Resist. 2020; 22: 845–846. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen F, Liu ZS, Zhang FR, et al.: [First case of severe childhood novel coronavirus pneumonia in China]. Zhonghua Er Ke Za Zhi. 2020; 58: E005. PubMed Abstract \| Publisher Full Text\n\nKumar A, Arora A, Sharma P, et al.: Is diabetes mellitus associated with mortality and severity of COVID-19? A meta-analysis. Diabetes Metab Syndr. 2020; 14: 535–545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuri T, Weber F: Interferon interplay helps tissue cells to cope with SARS-coronavirus infection. Virulence. 2010; 1: 273–275. PubMed Abstract \| Publisher Full Text\n\nPorcheddu R, Serra C, Kelvin D, et al.: Similarity in Case Fatality Rates (CFR) of COVID-19/SARS-COV-2 in Italy and China. J Infect Dev Ctries. 2020; 14: 125–128. PubMed Abstract \| Publisher Full Text\n\nPrice-Haywood EG, Burton J, Fort D, et al.: Hospitalization and Mortality among Black Patients and White Patients with Covid-19. N Engl J Med. 2020; 382: 2534–2543. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun P, Qie S, Liu Z, et al.: Clinical characteristics of hospitalized patients with SARS-CoV-2 infection: A single arm meta-analysis. J Med Virol. 2020; 92: 612–617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeiss P, Murdoch DR: Clinical course and mortality risk of severe COVID-19. Lancet. 2020; 395: 1014–1015. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang L, Yan X, Fan Q, et al.: D-dimer levels on admission to predict in-hospital mortality in patients with Covid-19. J Thromb Haemost. 2020; 18: 1324–1329. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou MY, Xie XL, Peng YG, et al.: From SARS to COVID-19: What we have learned about children infected with COVID-19. Int J Infect Dis. 2020; 96: 710–714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu H, Wang L, Fang C, et al.: Clinical analysis of 10 neonates born to mothers with 2019-nCoV pneumonia. Transl Pediatr. 2020; 9: 51–60. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "88338", "date": "15 Jul 2021", "name": "Yahia Ali Kaabi", "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 titled “Simple statistical insights into the COVID-19 data of Saudi Arabia: figures prior to vaccination campaign” is a good attempt to brief the disease figures in Saudi Arabia by providing a statistical summary of death and recovery rates, as well as the number of confirmed cases throughout the past year before starting vaccination.\nThe article rationale is good and the author summarized the official figures of COVID-19 in Saudi Arabia that were published by the Ministry of Health in a simple way for the readership. This simple presentation of COVID-19 data is needed to understand the graph of the outbreak and what health authorities did to contain the outbreak. Availability of such figures is also needed to help health authorities anticipate their future needs and put plans for future actions and precautionary measures.\nThe introduction provided a sufficient background on Coronavirus and the study aim was clearly mentioned at the end of this section. However, I would suggest adding one paragraph to provide an account on the current situation worldwide and in Saudi Arabia.\nThe methods section was concise, but clear and data source and all statistical analysis were stated and justified by the author.\nThe results were also clearly presented; in particular the detailed comparison of death figures in each month was important when correlating to the number of new incidents/monthly. In addition, comparison between local figures and international figures highlighted the similarity between Saudi Arabia and many other international figures, which was important to show how far or close are we from international statistics. It would be good if the author could also explain how death rate/1000 is calculated below table 3, as done for case fatality rate. In Figure 2, the legend should be corrected to describe the recovered cases not deaths.\nI would also suggest adding one paragraph to the discussion section to report on the figures after vaccination campaign till now, just to provide a simple comparison between the situations before and after vaccination to assess the success of vaccination in containing the mortality effects of COVID-19 in the country.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6968", "date": "27 Jul 2021", "name": "Omar S. El-Masry", "role": "Author Response", "response": "In response to the valuable comment on the introduction section, I think adding more information on the current situation in the introduction could also require a change in the paper’s title, which mainly focuses on the figures prior to the vaccination campaign in the Kingdom. However, the current figures will be briefly discussed in the discussion section. Thank you for your valuable comment on the results section, the equation for death rate/1000 was added below table 3 and Figure 2 legend was corrected as indicated, thank you. The following paragraph was added to the discussion section as recommended; I focused on the case fatality rate, which reflects directly on the number of confirmed cases and the number of deaths, death rate /1000 was not calculated as the number of Saudi population in 2021 is different than that in 2020 therefore comparison is not significant. In order to give a brief account on the figures post-vaccination campaign, the case fatality rates were calculated from the officially published figures in the first 6 months in 2021 (January-June). The number of newly recorded cases escalated from ~5000 cases in January to ~37000 in June. This means that following flattening of the curve at the end of 2020, there was an ascending increase in the number of cases again after the start of the vaccination campaign. However, the number of cases is still much less than the numbers recorded in the same months in 2020 before vaccination. The case fatality rate in January 2021 was 0.03 (3%), whilst that calculated for February-June was 0.01 (1%), which is similar to that recorded for June 2020; however, there was a clear reduction in the number of confirmed cases and deaths in 2021. Whether the reduction in confirmed cases and deaths numbers is due to vaccination or other factors, this is not confirmed due to lack of information about the number of vaccinated people who contracted the infection nor those who died after being vaccinated. Therefore, we have to wait for the release of all official figures and information before we make a final conclusion about the efficacy of vaccination. An updated version was submitted for your kind review." } ] } ]	1	https://f1000research.com/articles/10-410
https://f1000research.com/articles/10-747/v1	04 Aug 21	{ "type": "Systematic Review", "title": "Brain-derived neurotrophic factor, nerve growth factor, and high sensitivity C-reactive protein levels in urine in overactive bladder patients: a meta-analysis", "authors": [ "Edwin Utomo", "Farhat .", "Melvin Nova Gunawanto Barus", "Mohd. Rhiza Z. Tala", "Edwin Utomo", "Farhat .", "Melvin Nova Gunawanto Barus" ], "abstract": "Background: Overactive bladder (OAB) is a clinical syndrome characterized by a combination of symptoms including urgency, frequency, and nocturia, with or without urinary incontinence. Overactive bladder has a high prevalence especially in those of an older age and women, with diagnosis depending on the patient’s symptoms. This study aims to assess brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and high sensitivity C-reactive protein (HSCRP) in urine as biomarkers in OAB. Methods: Studies were searched from Pubmed, Science Direct, Wiley Online Library, and Google Scholar. All studies assessing BDNF, NGF, and HSCRP in urine in OAB patients were included. The standardized mean difference (SMD) and 95% confidence intervals (CI) were then calculated. Results: A total of 85 studies were included with a total of 11,483 subjects (6,885 OAB patients and 4,598 controls). Based on data analysis results, urinary NGF/Creatinine (NGF/Cr) and NGF level in OAB patients were significantly higher than control (SMD = 1.00, 95%CI = 0.80-1.20, P<0.00001; and SMD = 1.11, 95%CI = 0.79-1.43, P<0.00001). NGF/Cr level was found higher in OAB with incontinence (OAB wet) compared with OAB without incontinence (OAB dry) (SMD = 0.41, 95%CI = 0.23-0.60, P<0.0001), and decreased after treatment (SMD = 0.76, 95%CI = 0.49-1.03, P<0.00001). Urinary BDNF/Cr level was significantly higher in OAB patients compared with controls (SMD = 1.97, 95%CI = 1.14-2.79, P<0.00001), and also decreased significantly after treatment (SMD = 0.75, 95%CI = 0.42-1.08, P<0.00001). The level of HSCRP was significantly higher in OAB patients when compared with controls (SMD = 0.38, 95%CI = 0.12-0.64, P<0.004). Conclusions: The level of BDNF/Cr, NGF/Cr, NGF, and HSCRP in urine were found higher in OAB compared with controls, which means they may be used as a biomarkers for OAB.", "keywords": [ "brain-derived neurotrophic factor", "nerve growth factor", "high sensitivity C-reactive protein", "overactive bladder" ], "content": "Introduction\n\nOveractive bladder (OAB) is defined by the International Continence Society as a syndrome characterized with symptoms such as urgency, frequency, and nocturia, with or without urinary incontinence.1 OAB can affect daily activities and social functions such as work, physical activitiy, and sleep. OAB prevalence was found to be higher in older patients and women.1–3 Chronic low-grade inflammation is related to old age, which is a risk factor for various morbidities and mortality.4 The pro-inflammatory state in aging is believed to cause molecular and structural damage contributing to bladder aging and OAB.5 In women, menopause is associated with increased OAB symptoms due to decreased levels of oestrogen after menopause, which has an important role in lower urinary tract function.6\n\nOAB diagnosis currently depends on the patient’s clinical symptoms rather than molecular identification from samples, such as urine, blood, or other biological samples, which are easy to acquire and analyse. While patient examination includes urodynamic test, symptom questionnaire, and urination diary, which has low objectivity and accuracy.7,8\n\nBrain-derived neurotrophic factor (BDNF), along with nerve growth factor (NGF), is a neurotrophic factor, which is a group of proteins with functions in neuron growth, survival, and differentiation. Neurotrophins are able to bind and activate high-affinity tropomyosin-receptor kinase receptor and low-affinity p75 neurotrophin receptor (p75NTR). Increases of NGF levels have been found in inflammation, while BDNF secretion is induced by NGF in inflamed tissue. NGF also plays a role as a peripheral mediator in inflammation.9,10 High sensitivity C-reactive protein (HSCRP) able to detect CRP levels with sensitivity range of 0.01 to 10 mg/L, and HSCRP levels can help to measure low-grade systemic inflammation.11 We perform meta-analysis to assess NGF, BDNF, and HSCRP levels in OAB patients based on previous studies.\n\n\nMethods\n\nThis study was a meta-analysis study. Inclusion criteria were as follows: studies with OAB patients; studies which measured BDNF, NGF, and HSCRP levels in urine; studies that had data available for size effect analysis (mean, standard deviation, and sample size); and studies available in Bahasa Indonesia or English. Exclusion criteria were review articles, studies in animals, and studies in children. Studies in children were excluded because OAB prevalence was found higher in older patients.\n\nLiterature was searched using Pubmed, Science Direct, Wiley Online Library, and Google Scholar. Keywords used for literature searching were ((BDNF OR NGF) OR HSCRP) AND overactive bladder. Information sources were searched until 31 November 2020.\n\nThe literature searching and data extraction were performed by at least two reviewers independently.\n\nData variables that were extracted from study samples were as follows: first author name; year of publication; sample size; and BDNF/Creatinine (BDNF/Cr), NGF/Creatinine (NGF/Cr), and HSCRP levels in urine. BDNF/Cr, NGF/Cr and HSCRP levels were extracted in mean and standard deviation.\n\nData were analysed using standardized mean difference (SMD) and 95% confidence interval (CI) using Review Manager 5.3 software. A P value of less than 0.05 indicated significant statistical data. Heterogeneity was estimated using I2 and Q test. I2 above 50% and P value less than 0.05 indicated significant heterogeneity between studies. If I2 was more than 50%, a random effects model was used. If I2 was less than 50%, a fixed effects model was used instead.\n\nPublication bias was evaluated using funnel plot.\n\n\nResults\n\nA total of 85 studies7,12–95 were included, with 11,483 subjects (6,885 OAB and 4,598 without OAB) from 969 studies searched from Pubmed, Science Direct, Wiley Online Library, and Google Scholar. Literature searching results can be seen in Figure 1.\n\n\nComparison of NGF/Cr levels between OAB patients and controls\n\nBased on data analysis results, NGF/Cr levels in urine was found significantly higher in OAB patients compared to controls (SMD = 1.00, 95% CI = 0.80-1.20, P < 0.00001). The heterogeneity test showed significant heterogeneity (P < 0.00001, I2 = 89%), so a random effects model was used to analyse data. The results of the forest plot can be seen in Figure 2.\n\n\nComparison of urinary NGF levels between OAB patients and controls\n\nUrinary NGF levels were found significantly higher in OAB patients when compared with controls (SMD = 1.11, 95% CI = 0.79-1.43, P < 0.00001). Heterogeneity was found (P < 0.00001, I2 = 91%); therefore, a random effects model was used to analyse data. The results of the forest plot can be seen in Figure 3.\n\n\nComparison of BDNF/Cr levels between OAB patients and controls\n\nOAB patients have significantly higher levels of BDNF/Cr compared to controls (SMD = 1.97, 95% CI = 1.14-2.79, P < 0.00001). Heterogeneity was found (P < 0.00001, I2 = 97%), and a random effects model was used to analyse data. The results of the forest plot can be seen in Figure 4.\n\n\nComparison of HSCRP levels between OAB patients and controls\n\nHSCRP levels were found to be higher in OAB patients compared with controls (SMD = 0.38, 95% CI = 0.12-0.64, P < 0.004). There was significant heterogeneity (P < 0.00001; I2 = 93%), so a random effects model was used in data analysis. The results of the forest plot can be seen in Figure 5.\n\n\nComparison of NGF/Cr levels between OAB wet patients and OAB dry patients\n\nLevels of NGF/Cr were found significantly higher in patients with OAB with incontinence (OAB wet) compared with OAB without incontinence (OAB dry) (SMD = 0.41, 95% CI = 0.23-0.60, P < 0.0001). A random effects model was used in data analysis because there was a significant heterogeneity (P < 0.02; I2 = 50%). The results of the forest plot can be seen in Figure 6.\n\n\nDifferences in NGF/Cr levels in OAB patients before and after treatment\n\nNGF/Cr levels in urine in OAB patients were significantly decreased after treatment (SMD = 0.76, 95% CI = 0.49-1.03, P < 0.00001). Heterogeneity was found (P < 0.00001; I2 = 88%), and a random effects model was used to analyse the data. The results of the forest plot can be seen in Figure 7.\n\n\nDifferences in BDNF/Cr levels in OAB patients before and after treatment\n\nThe levels of BDNF/Cr in OAB patients’ urine were significantly decreased after treatment (SMD = 0.75, 95% CI = 0.42-1.08, P < 0.00001). A random effects model was used in data analysis because significant heterogeneity was found (P < 0.00001; I2 = 80%). The results of the forest plot can be seen in Figure 8.\n\n\nSubgroup analysis\n\nSubgroup analysis was performed based on subject gender and age. Based on subgroup analysis results, there was a significant difference of NGF/Cr levels in urine in both male (SMD = 0.88, 95% CI = 0.60-1.17, P < 0.00001) and female groups (SMD = 1.06, 95% CI = 0.70-1.11, P < 0.00001) compared with controls. A significant difference of NGF/Cr levels was also found in the >50 year old (SMD = 0.91, 95% CI = 0.71-1.11, P < 0.00001) and the <50 year old groups (SMD = 1.47, 95% CI = 0.77-2.18, P < 0.00001) compared with controls.\n\nThere was a significant difference of BDNF/Cr levels in urine in both the male (SMD =1.78, 95% CI = 0.95-2.61, P < 0.0001) and female groups (SMD = 2.11, 95% CI = 0.63-3.59, P = 0.005). A significant difference was also significantly in both the >50 year old (SMD = 2.35, 95% CI = 0.89-3.80, P = 0.002) and the <50 year old groups (SMD = 1.46, 95% CI = 0.95-1.96, P < 0.00001).\n\nThere was also a significant difference of HSCRP levels in both the male (SMD = 0.63, 95% CI = 0.12-1.13, P = 0.01) and female groups (SMD = 0.24, 95% CI = 0.14-0.33, P < 0.00001). A significant difference was also found in both the >50 year old (SMD = 0.48, 95% CI = 0.11-0.84, P = 0.01) and <50 year old groups (SMD = 0.24, 95% CI = 0.13-0.35, P < 0.0001). Subgroup analysis results can also be seen in Table 1.\n\n\nPublication bias\n\nThere was no publication bias by funnel plot (Figure 9).\n\n\nDiscussions\n\nBased on our analysis, NGF/Cr and NGF levels in urine were found higher significantly in OAB patients compared with controls. Higher NGF/Cr levels were also found in OAB wet patients compared with OAB dry patients. This result is related to higher detrusor muscle activity in OAB wet patients compared with OAB dry.96,97 NGF/Cr levels were decreased after various treatments, such as lifestyle management and antimuscarinic. This means NGF/Cr levels could be used to assess therapeutic effects of OAB treatment. An increase of NGF levels occurs in inflammatory conditions or organ dysfunction which has a role in regulation of sensory sensitivity. NGF could sensitise C afferent fibres, which cause urgency and hyperactivation of bladder reflex, resulting in frequency and urgency incontinence.98,99 When a cut-off point of 26.32 pg/mg was used, NGF/Cr has a sensitivity and specificity of 93.3% and 64.0%.7\n\nIn our study, BDNF/Cr levels in urine was found significantly higher in OAB patients compared with controls. An increase of BDNF levels is induced by increased NGF synthesis in inflamed tissue.9 Our study showed that BDNF/Cr levels decreased after various treatments, so BDNF/Cr levels could be used to assess therapeutic effect of OAB treatment. Previous research by Antunes-Lopes et al in 201318 showed that BDNF/Cr levels were higher in OAB patients compared with controls (628.1 ± 590.5 vs 110.4 ± 159.5; P < 0.001), and the levels decreased after treatment with lifestyle modification (432.5 ± 598.0; P < 0.033) or treatment with antimuscarinic (146.6 ± 264.9; P < 0.001).\n\nBased on our data analysis, HSCRP levels was found significantly higher in OAB patients compared with controls. This result shows a connection between inflammation and OAB. Previous research by Kupelian et al in 2012100 showed an association between increases in CRP levels and OAB in males and females. OAB prevalence was also increased with higher CRP levels in that study. An increase in CRP levels could trigger endothelial dysfunction and atherosclerosis. Ischemia caused by atherosclerosis could lead to bladder dysfunction.101 This inflammation and bladder dysfunction then causes the release of various inflammatory mediators, such as NGF and BDNF, which cause an increase in afferent nerve activities resulting in OAB symptoms.102\n\nThe limitation of our study is the significant heterogeneity on our analysis. This could be caused by variations in methods and populations of included studies.\n\nIn conclusion, urinary NGF/Cr, BDNF/Cr, and HSCRP may be used as biomarker in OAB diagnosis and to assess therapeutic effects of OAB treatments. However, there are still low number of HSCRP studies. More studies on these biomarkers and HSCRP assessment in OAB should be performed in the future.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: PRISMA checklist for ‘Brain-derived neurotrophic factor, nerve growth factor, and high sensitivity C-reactive protein levels in urine in overactive bladder patients: a meta-analysis’, https://doi.org/10.17605/OSF.IO/R9XAK.103\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\nZhu J, Hu X, Dong X, et al.: Associations Between Risk Factors and Overactive Bladder. Female Pelvic Med Reconstr Surg. 2019; 25(3): 238–246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeron E, Weintraub AY, Mastrolia SA, et al.: Overactive Bladder Syndrome: Evaluation and Management. Curr Urol. 2008; 11(3): 117–125. 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Neurourol Urodyn. 2015; 35(8): 944–948. PubMed Abstract \| Publisher Full Text\n\nCiftci S, Ozkurkeugil C, Yilmaz H, et al.: Urinary nerve growth factor and a variable solifenacin dosage in patients with an overactive bladder. Int Urogynecol J. 2015; 27(2): 275–280. PubMed Abstract \| Publisher Full Text\n\nKuo HC, Liu HT, Chancellor MB: Urinary Nerve Growth Factor Is a Better Biomarker Than Detrusor Wall Thickness for the Assessment of Overactive Bladder With Incontinence. Neurourol Urodyn. 2010; 29(3): 482–487. PubMed Abstract \| Publisher Full Text\n\nAntunes-Lopes T, Pinto R, Carvalho-Barros S, et al.: Urinary Neurotrophins – Potential Biomarkers of Overactive Bladder. J Urol. 2011; 185(4S): 780–781. PubMed Abstract \| Publisher Full Text\n\nShalom DF, Pillalamarri N, Xue X, et al.: Sacral nerve stimulation reduces elevated urinary nerve growth factor levels in women with symptomatic detrusor overactivity. Am J Obstet Gynecol. 2014; 211(5): 561.e1–561.e5. PubMed Abstract \| Publisher Full Text\n\nLiu HT, Wang YS, Kuo HC: Nerve Growth Factor Levels are Increased in Urine but Not Urothelium in Patients With Detrusor Overactivity. Tzu Chi Medical Journal. 2010; 22(4): 165–170. Publisher Full Text\n\nAntunes-Lopes T, Pinto R, Carvalho-Barros S, et al.: High Urinary levels of nerve growth factor and brain-derived neurotrophic factor in women with overactive bladder syndrome normalize after lifestyle intervention and antimuscarinic therapy. Europ Urol Supp. 2012; 11(1): e993–e993a. Publisher Full Text\n\nYeniel AO, Ergenoglu AM, Meseri R, et al.: Is overactive bladder microvasculature disease a component of systemic atheroscleorosis? Neurourol Urodyn. 2017; 37(4): 1372–1379. PubMed Abstract \| Publisher Full Text\n\nAntunes-Lopes T, Ferreira A, Sérgio CB, et al.: Mirabegron does not Decrease Urinary Neurotrophins Levels in Overactive Bladder Patients Despite Symptomatic Improvement. J Urol. 2015; 193(4S): e576. Publisher Full Text\n\nLiu H, Jiang Y, Kuo H: Increased Suburothelial Nerve Fiber and Purinergic P2X3 Receptor Expressions in Patients with Idiopathic Detrusor Overactivity and their Relationship with Botulinum Toxin a Therapeutic Outcome. Neurol Urodynamics. 2013: 1–2.\n\nVijaya G, Gallo P, Derpapas A, et al.: Increased Nerve Growth Factor in Overactive Bladder: Is it Caused by Infection? Int Urogynecol J. 2011; 22: S187–S188.\n\nVijaya G, Cartwright R, Digesu G, et al.: Can Urinary Nerve Growth Factor (NGF) Replace Urodynamics to Diagnose Lower Urinary Tracts Symptoms? Int Urogynecol J. 2011; 22: S20–S21.\n\nJiang Y, Jhang J, Lee P, et al.: Potential Urinary Biomarkers to Diagnosis Interstitial Cystitis and Overactive Bladder. Neurourology and Urodynamics. 2019: S129–S131.\n\nKim SW, Im YJ, Choi HC, et al.: Urinary nerve growth factor correlates with the severity of urgency and pain. Int Urogynecol J. 2014; 25(11): 1561–1567. PubMed Abstract \| Publisher Full Text\n\nBasok EK, Basok BI, Pelit ES, et al.: Data do not support the use of elevated urinary nerve growth factor and fractalkine levels as diagnostic and prognostic biomarkers for women with overactive bladder. Bladder. 2015; 1(1): 1–7. Publisher Full Text\n\nLiu HT, Kuo HC: Urinary Nerve Growth Factor Level Is a Potential Biomarker of Overactive Bladder and Detrusor Overactivity. Incont Pelvic Floor Dysfunct. 2008; 2(1): 27–29.\n\nLee HN: Role of Urinary Nerve Growth Factor as a Specific Biomarker for Diagnosing and Monitoring Overactive Bladder. J Urol. 2011; 185(4): e321. Publisher Full Text\n\nChia-Yen C: Urinary Nerve Growth Factor Level can be Potential Biomarker for Diagnosis of Overactive Bladder. J Urol. 2008; 179(4): 535.\n\nBhide A, Gopalan V, Cartwright R, et al.: Urinary brain-derived neurotrophic factor (BDNF) level does not correlate with detrusor overactivity. Europ Urol Suppl. 2013; 12(1): e672. Publisher Full Text\n\nAntunes-Lopes T, Pinto R, Carvalho-Barros S, et al.: Brain-Derived Neurotropic Factor: A New Potential Biomaker of Overactive Bladder Syndrome. Urology. 2011; 78(3): S411.\n\nAntunes-Lopes T, Pinto R, Barros S, et al.: BDNF: A Potential Biomarker of Overactive Bladder. Neurourology Urodynamics. 2012; 31(6): 804.\n\nLiu HT, Chen CY, Kuo HC: Urinary nerve growth factor in women with overactive bladder syndrome. BJU Int. 2010; 107(5): 799–803. PubMed Abstract \| Publisher Full Text\n\nKuo HC, Liu HT, Tyagi P, et al.: Urinary Nerve Growth Factor Levels in Urinary Tract Diseases With or Without Frequency Urgency Symptoms. LUTS. 2010; 2: 88–94. PubMed Abstract \| Publisher Full Text\n\nYamauchi H, Akino H, Ito H, et al.: Urinary Prostaglandin E2 was Increased in Patients With Suprapontine Brain Diseases, and Associated With Overactive Bladder Syndrome. Urology. 2010; 76(5): 1267.e13–1267e19. Publisher Full Text\n\nJacobs BL, Smaldone MC, Tyagi V, et al.: Increased Urine Levels of Nerve Growth Factor in Patients with Neurogenic Overactive Bladder and Interstitial Cystitis/Painful Bladder Syndrome. J Urol. 2009; 181(4): 19.\n\nCarbone A, Palleschi G, Paraseani R, et al.: The Evaluation of Nerve Growth Factor Levels in Patients with and without Detrusor Overactivity. Europ Urol. 2003; 2(1): 73.\n\nCho KJ, Kim HS, Koh JS, et al.: Changes in urinary nerve growth factor and prostaglandin E2 in women with overactive bladder after anticholinergis. Int Urogynecol J. 2013; 24: 325–330. PubMed Abstract \| Publisher Full Text\n\nKim JC, Park EY, Seo SI, et al.: Nerve Growth Factor and Prostaglandins in the Urine of Female Patients With Overactive Bladder. J Urol. 2006; 175(5): 1773–1776. PubMed Abstract \| Publisher Full Text\n\nLatjindung N: Korelasi Kadar HS-CRP (High Sensitivity – C Reactive Protein) dan Kadar BDNF (Brain-derived Neurotrophic Factor) Urin Wanita Menopause dengan Overactive Bladder.2019.\n\nDhanu RS: Hubungan Kadar Brain-derived Neurotrophic Factor (BDNF) dan Glial Cell Line Derived Neurotrophic Factor (GDNF) Urin Terhadap Derajat Keparahan Overactive Bladder pada Paramedis Wanita di RSUP H. Adam Malik Medan. 2019.\n\nLiu RT, Chung MS, Lee WC, et al.: Prevalence of overactive bladder and associated risk factors in 1359 patients with type 2 diabetes. Urology. 2011; 78(5): 1040–1045. PubMed Abstract \| Publisher Full Text\n\nKim JC, Park EY, Hong SH, et al.: Changes of urinary nerve growth factor and prostaglandins in male patients with overactive bladder symptom. Int J Urol. 2005; 12: 875–880. PubMed Abstract \| Publisher Full Text\n\nKrivoborodov G, Tur E, Kolesanova E, et al.: Urine nerve growth factor in patients with idiopathic detrusor overactivity and in patients with overactive bladder without detrusor overactivity. Urologiia. 2015; 3: 19–22.\n\nKrebs J, Pavlicek D, Stoyanov J, et al.: Nerve growth factor does not seem to be a biomarker for neurogenic lower urinary tract dysfunction after spinal cord injury. Urodynamics. 2016; 36(3): 659–662. PubMed Abstract \| Publisher Full Text\n\nRichter HE, Moalli P, Amundsen CL, et al.: Urinary Biomarkers in Women with Refractory Urgency Urinary Incontinence Randomized to Sacral Neuromodulation versus OnabotulinumtoxinA Compared to Controls. J Urol. 2017; 198(6): 1487–1495. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMirkin Y, Bedretdinova D, Eizenakh I, et al.: Intravesical administration of resiniferatoxin for treatment OAB with increased bladder sensation. Europ Urol. 2012; 11(1): e693–e693a.\n\nYokoyama T, Kumon H, Nagai A: Correlation of Urinary Nerve Growth Factor Level With Pathogenesis of Overactive Bladder. Neurourol Urodyn. 2008; 27(5): 417–420. PubMed Abstract \| Publisher Full Text\n\nChen S, Wu S, Kuo H: Urinary Biomarkers in Patients with Detrusor Underactivity with and without Bladder Function Recovery. Neurourol Urodyn. 2017; 36(3): 497–498.\n\nYoon H, Yoo SS, Hong JP, et al.: Urinary Nerve Growth Factor in Oab and Cystitis. Neurourol Urodyn. 2010; 29(2): 251.\n\nNorthington G, Pennycuff J, Schutte S, et al.: Urinary Neurotrophic Peptides in Postmenopausal Age-matched Women with and wihtout Overactive Bladder. Neurourol Urodyn. 2014; 33(2): 232–233.\n\nPennycuff J, Schutte S, Karp D, et al.: Urinary Nerve Growth Factor and Symptoms in Women with Overactive Bladder. Neurourol Urodyn. 2014; 33(2): 237.\n\nStewart J, Khavari R, Chan R, et al.: Increased in Females with Anatomic Bladder Outlet Obstruction. Neurourol Urodyn. 2014; 33(2): 192–193.\n\nKim JC, Kim JH, Lee JY, et al.: Changes of Urinary Nerve Growth Factor and Prostaglandin E2 after Anticholinergic Treatment in Female Patients with Overactive Bladder. Neurourol Urodyn. 2008; 27(7): 628.\n\nLee HS, Kang JH, Cho WJ, et al.: Correlation of Urinary Nerve Growth Factor Level with Bladder Outlet Obstruction. Neurourol Urodyn. 2011; 30(6): 1142–1143.\n\nTrivedi S, Patnaik P, Ramole Y, et al.: Role of Serum and Urinary Biomarkers in Evaluation and Management of Patients With Overactive Bladder. Clin Med Insights: Urol. 2019; 12: 1–6. Publisher Full Text\n\nAydoğmuş Y, Sunay M, Arslan H, et al.: Acupuncture versus Solifenacin for Treatment of Overactive Bladder and Its Correlation with Urine Nerve Growth Factor Levels: A Randomized, Placebo-Controlled Clinical Trial. Urol Int. 2014; 93(4): 437–443. PubMed Abstract \| Publisher Full Text\n\nLiu HT, Kuo HC: Urinary nerve growth factor levels are elevated in patients with overactive bladder and do not significantly increase with bladder distention. Neurourol Urodyn. 2009; 28(1): 78–81. PubMed Abstract \| Publisher Full Text\n\nLee SY, Lee SW, Ahn JS, et al.: Change of urinary nerve growth factor levels after 12 weeks antimuscarinic therapy in patients with overactive bladder. Europ Urol Suppl. 2012; 11(1): e990–e990a.\n\nMatsuo T, Miyata Y, Nakamura T, et al.: Prosultiamine for treatment of lower urinary tract dysfuntion accompanied by human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis. Int J Urol. 2017; 25(1): 54–60. PubMed Abstract \| Publisher Full Text\n\nJiang YH, Liu HT, Kuo HC: Derived Neurotrophic Factor in Patients with Interstitial Cystitis/Bladder Pain Syndrome Treated with Hyaluronic Acid. PLoS One. 2014; 9(3): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu HT, Tyagi P, Chancellor MB, et al.: Urinary nerve growth factor level is increased in patients with interstitial cystitis/bladder pain syndrome and decreased in responders to treatment. BJU Int. 2009; 104: 1476–1481. PubMed Abstract \| Publisher Full Text\n\nLiu HT, Kuo HC: Increased Urine and Serum Nerve Growth Factor Levels in Interstitial Cystitis Suggest Chronic Inflammation Is Involved in the Pathogenesis of Disease. PLoS One. 2012; 7(9): 1–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinto R, Lopes T, Barros S, et al.: Urinary Neurotrophic Factors in Bladder Pain Syndrome/Interstitial Cystitis. J Urol. 2012; 187(4S): e333–e334.\n\nTonyali S, Ates D, Akbiyik F, et al.: Urine nerve growth factor (NGF) level, bladder nerve staining and symptom/problem scores in patients with interstitial cystitis. Adv Clin Exp Med. 2018; 27(2): 159–163. PubMed Abstract \| Publisher Full Text\n\nChung MS, Chuang YC, Lee JJ, et al.: Prevalence and associated risk factors of nocturia and subsequent mortality in 1301 patients with type 2 diabetes. Int Urol Nephrol. 2014; 46(7): 1269–1275. PubMed Abstract \| Publisher Full Text\n\nLu Z, Gao Y, Tan A, et al.: Increased High-Sensitivity C-Reactive Protein Predicts A High Risk of Lower Urinary Tract Symptoms in Chinese Male: Results From the Fangchenggang Area Male Health and Examination Survey. Prostate. 2012; 72: 193–200. PubMed Abstract \| Publisher Full Text\n\nPinto R, Lopes T, Frias B, et al.: Trigonal Injection of Botulinum Toxin A in Patients with Refractory Bladder Pain Syndrome/Interstitial Cystitis. Euro Urol. 2010; 58(3): 360–365. PubMed Abstract \| Publisher Full Text\n\nLiu HT, Chancellor MB, Kuo HC: Urinary Nerve Growth Factor Levels are Elevated in Patients with Detrusor Overactivity and Decreased in Responders to Detrusor Botulinum Toxin-A Injection. Eur Urol. 2009; 56(4): 700–707. PubMed Abstract \| Publisher Full Text\n\nChen S, Wen H, Zhou B, et al.: Therapeutic effect of solifenacin combined with tamsulosin on beingn prostatic hyperplasia with overactive bladder. Int J Clin Exp Med. 2018; 11(12): 13820–13826.\n\nPinto RA, Frias B, Lopes T, et al.: Intra-trigonal Injection of Botulinum Toxin A in Patients with Refractory Bladder Pain Syndrome Decreases Urinary Neurotrophins and Improves Lower Urinary Tract Symptoms. J Urol. 2010; 183(4): e648.\n\nCarbone A, Parascani R, Fileni A, et al.: Higher levels of detrusorial NGF and of its high affinity receptor in overactive bladder patients: What is their significance? Urodinamica. 2003; 13(4): 274–275.\n\nChermansky CJ, Kadow BT, Kashyap M, et al.: MicroRNAs as potential biomarkers to predict the risk of urinary retention following intradetrusor onabotulinumtoxin—A injection. Neurourol Urodyn. 2017; 37(1): 99–105. PubMed Abstract \| Publisher Full Text\n\nYoon H, Shim BS, Chung WS: Urinary NGF in OAB after Anticholinergic Treatment. World J Urol. 2017; 35(1): 158–159.\n\nLiu HT, Chancellor MB, Kuo HC: Urinary nerve growth factor level could be a biomarker in the differential diagnosis of mixed urinary incontinence in women. BJU Int. 2008; 102(10): 1440–1444. PubMed Abstract \| Publisher Full Text\n\nLiu HT, Liu AB, Chancellor MB, et al.: Urinary nerve growth factor level is correlated with the severity of neurological impairment in patients with cerebrovascular accident. BJU Int. 2009; 104(8): 1158–1162. PubMed Abstract \| Publisher Full Text\n\nAntunes-Lopes T, Coelho A, Pinto R, et al.: Urinary Neurotrophin Levels Increase in Women With Stress Urinary Incontinence After a Midurethral Sling Procedure. Urology. 2017; 99: 49–56. PubMed Abstract \| Publisher Full Text\n\nTseng Y, Chung S, Liu H, et al.: The levels of serum C-reactive protein and urinary nerve growth factor in patients with OAB and IC/BPS. Urology. 2010; 76(3): S25.\n\nRobinson D, Tubaro A, Kimberg M, et al.: The effect of solifenacin on urinary nerve growth factor (UNGF) and brain-derived neurotrophic factor (BDNF) in women with overactive bladder and detrusor overactivity: results from the shrink study. Int Urogynecol J. 2014; 25(S1): S60–S61.\n\nHsiao S, Chang T, Lin H: Comparisons of clinical outcome and urodynamic effects in female overactive bladder patients after 3-month versus 6-month solifenacin treatment: a randomized prospective study. Neurourol Urodyn. 2016; 35(S4): S358–S359.\n\nRodríguez A, Garcia A, Francés A, et al.: Urinary Excretion of NGF (Nerve Growth Factor) and Bdnf (Brain-derived Neurotrophic Factor) in Women with Idiopathic Detrusor Overactivity. Neurourol Urodyn. 2014; 33: 924.\n\nLiu RT, Chung MS, Chuang YC, et al.: The Presence of Overactive Bladder Wet Increased the Risk and Severity of Erectile Dysfunction in Men with Type 2 Diabetes. J Sex Med. 2012; 9(7): 1913–1922. PubMed Abstract \| Publisher Full Text\n\nMossa AH, Cammisotto PG, Shamout S, et al.: Imbalance of nerve growth factor metabolism in aging women with overactive bladder syndrome. World J Urol. 2020: 1–9. PubMed Abstract \| Publisher Full Text\n\nAl-Ghazo MA, Ghalayini IF, Al-Azab R, et al.: Urodynamic Detrusor Overactivity in Patients with Overactive Bladder Symptoms. Int Neuroruol J. 2011; 15(1): 48–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChung SD, Liao CH, Chen YC, et al.: Urgency Severity Scale Could Predict Urodynamic Detrusor Overactivity in Patients With Overactive Bladder Syndrome. Neurourol Urodyn. 2011; 30(7): 1300–1304. PubMed Abstract \| Publisher Full Text\n\nQiao LY: Neurotrophin signaling and visceral hypersensitivity. Front Biol. 2014; 9(3): 216–224. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeth JH, Sahai A, Khan MS, et al.: Nerve growth factor (NGF): a potential urinary biomarker for overactive bladder syndrome (OAB)?. BJU Int. 2013; 111(3): 372–380. PubMed Abstract \| Publisher Full Text\n\nKupelian V, Rosean RC, Roehrborn CG, et al.: Association of overactive bladder and C-reactive protein levels. Results from the Boston Area Community Health (BACH) Survey. BJU Int. 2012; 110(3): 401–407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndersson KE, Boedtkjer DB, Forman A: The link between vascular dysfunction, bladder ischemia, and aging bladder dysfunction. Ther Adv Urol. 2017; 9(1): 11–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshimura N, Ogawa T, Miyazato M, et al.: Neural Mechanisms Underlying Lower Urinary Tract Dysfunction. Korean J Urol. 2014; 55(2): 81–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUtomo E, Farhat, Barus MNG, Tala MRZ: PRISMA checklist for ‘Brain-derived neurotrophic factor, nerve growth factor, and high sensitivity C-reactive protein levels in urine in overactive bladder patients: a meta-analysis.2021, July 19. Publisher Full Text" }	[ { "id": "91226", "date": "24 Aug 2021", "name": "Pradeep Tyagi", "expertise": [ "Reviewer Expertise Urine biomarkers and pathology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have performed a meta-analysis of reports on urine analysis of NGF, BDNF, and CRP.\n\nThe following concerns were noted:\nAuthors should cite PMID: 281025521 and discuss how their findings reconcile with earlier meta-analyses.\n\nAuthors should discuss the limitation of studies that used the enzyme-linked immunosorbent assay (ELISA), the Promega NGF Emax Immunoassay, to quantify NGF in urine. There have been concerns with the specificity of the assay for NGF as it may have contributed to some false-positive findings due to cross-reactivity with other urinary components, such as immunoglobulin G. Kit was withdrawn from the market in 20142.\n\nAlso, the authors should discuss the rationale for creatinine normalization of neurotrophins (NGF and BDNF) secreted by the bladder of OAB patients into the urine. Do urine levels of NGF or BDNF increase or decrease with the increase in urine creatinine concentration?\n\nHas anybody reported a correlation or regression coefficient between urine levels of NGF/BDNF and urine levels of creatinine to justify creatinine normalization? It appears the urology community just copied the use of creatinine normalization for NGF and BDNF in OAB for systemic diseases without any proper scientific justification.\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? No", "responses": [] }, { "id": "98386", "date": "08 Dec 2021", "name": "Volkan Sen", "expertise": [ "Reviewer Expertise Urology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors evaluated some inflammatory factor levels in urine in overactive bladder patients. I have some concerns about the manuscript;\nAuthors must take a PROSPERO number for meta-analysis before the study.\n\nWhich guideline was used for meta-analysis? Authors must state this information and explain step by step . Authors cannot conclude that 'these factors can be used as a biomarker for OAB.'\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] } ]	1	https://f1000research.com/articles/10-747
https://f1000research.com/articles/8-1652/v1	17 Sep 19	{ "type": "Case Report", "title": "Case Report: Kikuchi-Fujimoto Disease: A case of supraclavicular lymphadenopathy", "authors": [ "Shiza Sarfraz", "Hamza Rafique", "Hassam Ali", "Syed Zawahir Hassan", "Hamza Rafique", "Hassam Ali" ], "abstract": "Kikuchi-Fujimoto Disease (KFD), also known as histiocytic necrotizing lymphadenitis, is a rare cause of cervical lymphadenopathy. Patients usually present with localized lymphadenopathy, fever and fatigue. Because of the poorly understood etiology, it can be mistaken for an infectious disease or even malignance. Here we discuss a case of KFD that initially presented with left sided cervical lymphadenopathy that later progressed to left supraclavicular lymph nodes. Due to its characteristic overlap with other disorders like tuberculous lymphadenitis and lymphoma, KFD remains an arduous diagnosis for physicians. Therefore, one should be made aware of symptoms that can lead to misdiagnosis in patients.", "keywords": [ "Lymphadenitis", "Cervical lymphadenopathy", "Tuberculosis", "Lymphoma" ], "content": "Introduction\n\nKikuchi-Fujimoto Disease (KFD) is known to occur both in the juvenile and adult population. The first case of reported Kikuchi Fujimoto disease was in Japan in 1972 and since then this disease has been described worldwide, with most cases reported in Asia1,2. Kikuchi-Fujimoto Disease typically follows a benign and self-limited course, characterized by cervical lymphadenopathy (most common). Less frequently, other symptoms might also be present like nausea, weight loss, night sweats and fatigue1. Generally, KFD is diagnosed via excisional lymph node biopsy and histopathological analysis. KFD shares many characteristics with other causes of lymphadenopathy including lymphoma, inflammatory disorders, autoimmune conditions, and infectious causes of lymphadenopathy like tuberculosis infection; therefore, it is important consider KFD in cases of persistent lymphadenopathy and must be differentiated from these conditions1,3. Treatment is mostly symptomatic with antipyretics, non-steroidal anti-inflammatory drugs (NSAIDS) or on rare occasions, steroids. KFD is associated with spontaneous recovery in 1–4 months4,5.\n\n\nCase presentation\n\nA 25y/o South East Asian male medical student presented in our outpatient department in January 2018 with left-sided cervical lymphadenopathy. The patient reported small bulges along the left side of his neck for one month. Associated symptoms included one month of low-grade fever and fatigue. There was no history of night sweats or reported weight loss. A course of antibiotics two weeks earlier did not improve his symptoms. On presentation, the patient was hemodynamically stable with a temperature of 100.1°C, heart rate of 98 beats/min, respiratory rate was 18 breaths/min and blood pressure was 115/80 mm/hg. On physical examination there was diffuse left cervical and supraclavicular lymphadenopathy. Lymph nodes were rubbery, soft and mobile. There were no changes in hands, eyes or ears. His nose and throat examination were normal. On auscultation of the chest, breath sounds were normal bilaterally and normal heart sounds where present. The abdominal examination was also normal. Initial lab investigations included complete blood count with total and differential leukocyte count, metabolic profile, erythrocyte sedimentation rate (ESR) and lactate dehydrogenase (LDH). This was to rule out any possibility of lymphadenitis, or neoplastic disorder. On laboratory examination there was an increase in lymphocytes and an increase in inflammatory markers including ESR and LDH (Table 1).\n\nA provisional diagnosis of tuberculous lymphadenitis was made based on his occupation. Further investigations were ordered to determine the size and extent of the lymphadenopathy. These included ultrasonography of the neck and abdomen, to visualize any hidden lymphadenopathy that might have been missed during the initial physical examination; chest x-ray, to rule out any active tuberculosis; and interferon-gamma release assay.\n\nOn ultrasonography (Figure 1), the patient showed enlarged multiple discrete left cervical and supraclavicular lymph nodes measuring up to 16×10mm. The rest of the ultrasound report did not show any abnormalities. Chest x-ray was normal and interferon-gamma release assay was not conclusive.\n\nTo ensure a definitive diagnosis, surgery with lymph node excision and biopsy was performed. An excisional lymph node biopsy from the anterior cervical chain was performed and on histopathological analysis it showed necrotizing lymphadenitis with partial alteration of structure by clusters of histiocytic and interspersed nuclear debris. In preserved areas, lymphoid follicles with pale staining germinal centers were also seen. No evidence of tuberculous granulomas or malignancy was found. Stains for acid fast bacteria were also negative.\n\nIt was decided that no antibiotics should be given to the patient at this time and watchful waiting was advised. For fever, 500mg paracetamol twice daily was prescribed for one week only. The patient was followed up twice a month in our outpatient clinic to monitor any spread of the lymphadenopathy. The disease course was uneventful. The patient was not given any further medication and watchful waiting was continued. Within two months the lymphadenopathy decreased dramatically, and the patient reported no fever. It completely disappeared in four months.\n\n\nDiscussion\n\nThe actual cause of KFD is still unknown but it has been proposed to have infectious and immunological etiologies1. This disease is thought to be a hyperimmune response to infectious, physical or chemical agents. Some of the unidentified agents may include toxoplasmosis, Brucella, Bartonella henslae, Yersinia enterolitica, human herpes virus, Ebstein Bar virus, parainfluenza, paramyxovirus, parvovirus B19, cytomegalovirus and human immunodeficiency virus1,3–5. However, serological and molecular studies have been unable to identify a single specific pathogen. Due to this reason, KFD diagnosis is markedly limited to invasive procedures like excisional biopsy (to observe cellular changes) and not just physical examination and history.\n\nPrevalence of Kikuchi disease has been seen highest amongst the Japanese population and people from East Asia but more recently this disease has been reported all over the world2. Our case is from South East Asia, Pakistan.\n\nTypically young adults (aged 20–30) are affected, but it does not seem to spare any age group as cases have been reported in the pediatric population as well, which can be seen in the reports of Byun JH6. However, the case report by Byun JH shows that, when Kikuchi disease occurs in children, it often involves the central nervous system leading to meningitis and encephalitis.\n\nAs reported by Deaver et al.7, clinical course of this disease has some specific and non-specific features with the specific one being unilateral cervical lymphadenopathy. Although lymphadenopathy is commonly found in cervical lymph nodes other groups such as the axillary and mediastinal lymph nodes may also be involved. Unexplained fever and night sweats are also among the common clinical presentations8. Our patient also presented with all the above listed common complaints. Less common complaints include headache, fatigue, arthralgia, myalgia, night sweats, weight loss, rash and abdominal pain8. Our patient experienced none of them except fatigue. Although rare, patient may present with the involvement of central nervous system and peripheral nervous system9.\n\nConfirmation of diagnosis is done by lymph node biopsy and histopathological analysis which shows distorted nodal architecture. The nodules are mostly necrotic and have debris from nuclear fragmentations due to cellular apoptosis. These necrotic foci are either isolated or clumped together. In addition, there is presence of proliferating histiocytic, T lymphocytes (CD8) and immunoblasts7. The minimum criteria for KFD diagnosis is presence of aggregated histiocytic with occasional crescent-shaped nuclei, plasmacytoid histiocytic, and scattered karyorrhexis7. The biopsy results of our patient were quite similar, making KFD our primary diagnosis. Due to similar clinical characteristics, KFD is often mistaken for lymphoma, tuberculosis, systemic lupus erythematosus and even metastatic adenocarcinoma. Therefore, any physician who comes across a case of lymphadenopathy, should keep KFD in mind when consider differential diagnoses. KFD is self-limiting and resolution occurs is one to four months. There are no specific drugs for KFD and usual treatment is symptomatic, consisting of antipyretics and analgesics.\n\n\nConclusions\n\nWe describe a case of Kikuchi-Fujimoto Disease, a self-limiting necrotizing lymphadenitis that started with cervical lymph node swelling but progressed to left supraclavicular lymph node involvement, which makes it unique. Recognition of this disease is important as it can mimic lymphoma or even metastatic adenocarcinoma. Early diagnosis and treatment can help avoid unnecessary testing and improper treatments among patients. Correctly recognizing the symptoms of KFD can also save one from the emotional stress of misdiagnosis.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nChiu CF, Chow KC, Lin TY, et al.: Virus infection in patients with histiocytic necrotizing lymphadenitis in Taiwan. Detection of Epstein-Barr virus, type I human T-cell lymphotropic virus, and parvovirus B19. Am J Clin Pathol. 2000; 113(6): 774–81. 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\n\nHudnall SD, Chen T, Amr S, et al.: Detection of human herpesvirus DNA in Kikuchi-Fujimoto disease and reactive lymphoid hyperplasia. Int J Clin Exp Pathol. 2008; 1(4): 362–368. PubMed Abstract \| Free Full Text\n\nHuh J, Kang GH, Gong G, et al.: Kaposi's sarcoma-associated herpesvirus in Kikuchi's disease. Hum Pathol. 1998; 29(10): 1091–1096. PubMed Abstract \| Publisher Full Text\n\nYena A, Fearneyhough P, Raimer SS, et al.: EBV-associated Kikuchi's histiocytic necrotizing lymphadenitis with cutaneous manifestations. J Am Acad Dermatol. 1997; 36(2 Pt 2): 342–346. PubMed Abstract \| Publisher Full Text\n\nByun JH, Park SE, Nam SO, et al.: Three children of meningoencephalitis with Kikuchi necrotizing lymphadenitis. Brain Dev. 2018; 40(3): 251–255. PubMed Abstract \| Publisher Full Text\n\nDeaver D, Horna P, Cualing H, et al.: Pathogenesis, diagnosis, and management of Kikuchi-Fujimoto disease. Cancer Control. 2014; 21(4): 313–21. PubMed Abstract \| Publisher Full Text\n\nDumas G, Prendki V, Haroche J, et al.: Kikuchi-Fujimoto disease: retrospective study of 91 cases and review of the literature. Medicine (Baltimore). 2014; 93(24): 372–382. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShahid S, Alam SH, Hadley I: An Unusual Presentation of Kikuchi-Fujimoto Disease with Recurrent Subdural Effusion. Cureus. 2018; 10(3): e2302. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "56268", "date": "07 Nov 2019", "name": "Karl O. A. Yu", "expertise": [ "Reviewer Expertise infectious disease", "immunology", "pediatrics", "host/pathogen 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\nSarfaz et al. report a rare (but probably it is not-so-rare) case of Kikuchi-Fujimoto lymphadenitis in a young adult. (disclosure: I just co-authored a recent case series on KFD).\n\nI am glad for this report to go out, as I suspect, based on my anecdotal experience, that this is not as rare as people think it is, but is probably under-diagnosed/under-reported. Seeing more reports as this in the literature may change conventional opinion.\nI have a few suggestions to improve the manuscript:\nThe authors mention that testing for tuberculosis was done. I would suggest clarification as to why the IGRA was inconclusive. Was there a high background, or a failed mitogen testing. If the former -- this may be evidence for baseline inflammation (principally, IFN-gamma/Th1 inflammation) in the patient's blood at that time -- this is potentially important. If the latter, this may be due to shipping issues, or evidence for transient anergy or other immunosuppression.\n\nThe authors say stains for acid fast bacilli were done and were negative. Was there a culture?\n\nWas a PPD checked?\n\nThe authors state that histopathological confirmation of the diagnosis was made. Are microphotographs available for the reader themselves?\n\nThe authors state that the patient has \"high lymphocytes.\" The patient's absolute lymphocyte count (2000 / uL) is NOT high.\n\n\"Epstein-Barr\" is misspelled.\n\nWhile not necessarily needed, since the authors did talk about the different infections associated with KFD, it would be enlightening to see what infectious workup was done for this patient, apart from an IGRA and an AFB stain.\n\nWith the suspicion of tuberculous meningitis, please clarify if anti-tuberculous therapy was started.\n\nThe authors also state that the case from from \"South East Asia, Pakistan.\" I know that many Pakistanis and Indians are taught that their country is in southeast Asia -- but that is not the usual convention outside of Pakistan or India. Southeast Asia is conventionally the 10 states of ASEAN plus East Timor and Papua New Guinea. I would suggest the authors use \"south Asia\", or just say \"Pakistan\" to avoid any ambiguity.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6977", "date": "13 Aug 2021", "name": "Shiza Sarfraz", "role": "Author Response", "response": "IGRA being inconclusive secondary to delays in lab transport. IGRA was done there for PPD was deferred. Lymphocytic percentage in is in fact increased per table one per the pathology lab. Anti tuberculosis therapy was delayed till definitive diagnosis." } ] }, { "id": "62892", "date": "12 May 2020", "name": "Syed Hamza Bin Waqar", "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\nNeeds to improve the verbal/grammatical check.. as malignancy, not malignance.\n\nRun a grammar check thoroughly; check for repetition.\n\nTo rule out neoplastic disorder, you need to get an excisional biopsy - not perform blood tests (case presentation p1).\n\nAdd any serological tests like toxo, hiv, csd, cmv-ebv etc. that you might have done to rule out infectious causes of lymphadenopathy apart from IGRA which the authors have mentioned already.\n\nAdd any rheumatological tests like ANA etc. that might have been done to rule it out before proceeding to excisional biopsy.\n\nAny rash that the pt might have/haven't noticed should be mentioned.\n\nHow does the involvement of supraclav lad make the case unique?\nFinal Comments: Authors have done a great job in summarizing KFD and have beautifully presented the case. With only a little alteration in the case presentation by adding serological markers will make the case look more complete - although it's not necessary.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1652
https://f1000research.com/articles/9-1233/v1	14 Oct 20	{ "type": "Research Article", "title": "Perceived stress and generalized anxiety in the Indian population due to lockdown during the COVID-19 pandemic: a cross-sectional study", "authors": [ "Naina Wakode", "Santosh Wakode", "John Santoshi", "Naina Wakode", "John Santoshi" ], "abstract": "Background: Research on the psychosocial toll of the COVID-19 pandemic is being conducted in various countries. This study aimed to examine stress levels and causal stressors for perceived stress and generalized anxiety in the Indian population related to the lockdown during the COVID-19 pandemic. Methods: A total of 300 adults were invited to participate in the online study via snowball and virtual snowball sampling. They were requested to complete electronic survey forms for assessing perceived stress and anxiety, and questions related to psychosocial stressors. Frequency and percentage were used for categorical variables. One-way ANOVA test was applied to compare responses based on gender, level of education, employment, and place of residence. A p-value of <0.05 was considered statistically significant. Result: In total, 257 out of the 300 invited, responded and completed the survey. Men accounted for 58% (n=149) of the respondents. Overall, 84% (n=217) of participants had moderate to severe levels of perceived stress and 88% (n=228) had moderate to severe levels of anxiety. Women, as well as those not employed, reported significantly higher perceived stress and anxiety, urban residents reported higher perceived stress, while level of education had no difference in terms of perceived stress as well as anxiety. Fear of contracting COVID-19 was the highest stressor followed by difficulties in executing a routine exercise schedule and worry about the future. Conclusion: The psychosocial impact of the nationwide lockdown on the Indian population has been high. Vulnerable groups for increased stress and anxiety include women, younger ages, and the unemployed. The stressors recognized include fear of contracting COVID-19, inability to execute a routine exercise schedule and worry about the future.", "keywords": [ "COVID-19", "lockdown", "perceived stress", "anxiety", "stressors", "India", "PSS-10", "GAD-7" ], "content": "Introduction\n\nSince the beginning of 2020, humanity has been confronted with a pandemic caused by the severe acute respiratory syndrome coronavirus-2 that causes coronavirus disease 2019 (COVID-19)1. The government of India declared a 21-day nationwide ‘lockdown’ from 25th March 2020, which was subsequently extended in phases till 31st May 2020, to break the cycle of spread of infection. The lockdown was in tune with the initiatives taken by many countries across the globe against this pandemic2,3.\n\n‘Lockdown’ is an emergency protocol and is a means of preventing the public from moving from one place to the other. This led to shutting down of all activities except those considered ‘essential services’, which included healthcare, police, sanitation, grocery shops, petrol stations and fire stations. All educational institutions, offices, factories, shopping malls, religious places, and public transport, including buses, railways and aeroplanes, were completely shut down, and sports, religious ceremonies, family functions and all outdoor activities were strictly prohibited.\n\nWhile isolation and lockdown are recognized as effective strategies of social distancing to stop the spread of COVID-19, the reduced access to family, friends, and other social support systems causes loneliness, increasing mental health issues like anxiety and depression4–6.\n\nResearchers, in the past and during this present crisis, have tried to address the psychological stress in healthcare providers7–9 and the general population2,3,10. The present study, conducted during the fourth phase of nationwide lockdown, from 18th to 31st May 2020, attempts to examine levels of perceived stress and generalized anxiety disorders and causal stressors among the Indian population related to the COVID-19 pandemic and consequent lockdown.\n\n\nMethods\n\nWe conducted an online survey wherein 300 participants were invited via snowball and virtual snowball sampling; the sample size was decided on the basis of logistics and time availability for the study. The study was approved by the Institutional Ethics Committee of the ABV Government Medical College, Vidisha, MP, India (reference no. 19F/IEC/ABV GMC/Vidisha/2020).\n\nA link to the electronic survey forms (Extended data11) was posted on Facebook, and was sent via WhatsApp by the authors to multiple contacts, including colleagues and acquaintances that were from a wide section of society. Consent to participate was implied if the participant completed the questionnaire.\n\nInclusion criteria of participants were: a) aged >18 years; b) have an internet connection and Facebook or WhatsApp installed on their mobile phone. Those unwilling to participate or did not provide consent and those <18 years of age were excluded because the psychometric measures utilized in the study were designed for adults only.\n\nData was collected from 18th to 25th May 2020.\n\nThe survey questionnaire, based on the perceived stress scale (PSS-10)11 and Generalized Anxiety Disorder (GAD-7)12 instruments, explored the psychosocial stressors among the respondents. For each potential stressor, the frequency of occurrence was classified as never, almost never, sometimes, often, and very often, and these were scored as 0, 1, 2, 3 and 4, respectively. These instruments have been used in previous studies on this subject7,10,12.\n\nThe data collected were tabulated and analysed using Microsoft Excel 2016 with data analysis add-in. Frequency and percentage were used for categorical variables. One-way ANOVA test was applied to compare responses based on gender, age, level of education, and place of residence. A p-value of <0.05 was considered statistically significant.\n\nWe used the STROBE cross sectional checklist when writing our report13.\n\n\nResults\n\nA total of 257, out of the 300 participants who were sent the survey, responded and completed the survey. Men constituted 58% (n=149) of the respondents. Overall, 84% (n=217) of participants had moderate to severe levels of perceived stress and 88% (n=228) had moderate to severe levels of anxiety (Figure 1).\n\nTable 1 shows the PSS-10 and GAD-7 scores of the study participants as stratified by gender, age, level of education, and place of residence. Women as well as those not employed reported significantly higher perceived stress and anxiety, urban residents reported higher perceived stress while the level of education had no difference in terms of perceived stress as well as anxiety. The psychosocial impact of the lockdown due to the COVID-19 pandemic is shown in Table 2. Fear of contracting COVID-19 was the highest stressor followed by difficulties in executing routine exercise schedule and worry about the future.\n\n\nDiscussion\n\nThe levels of stress and anxiety reported in the present study are similar to those reported by researchers from other countries2,3,5,14. The present study is in agreement with previous studies from other parts of the world where women and those with lower incomes are prone to higher levels of stress and anxiety2,3,5,15,16; this was in contrast to a study from Pakistan where men reported a higher degree of stress during the current crisis17. This could be attributed to cultural factors, which need further evaluation for clearer understanding.\n\nIn the present study, older respondents reported lower levels of stress. This could suggest the struggle and hardships of daily life which the younger generation is under18; also, the younger generation tends to obtain a large amount of information from social media, which can easily trigger stress3,10. We found significant difference in the levels of perceived stress reported between urban and rural residents, while no such difference was noted in generalised anxiety scores.\n\nIn the present study, we found no difference in the levels of stress when considering the level of education of the respondents. Vallejo et al.19 found those with a lower level of education to be reporting higher stress. Other studies found that those who were highly educated had a higher risk of depression; it is presumed that highly educated and professional people are forced to stay at home and delve into other aspects of family life leading to higher levels of perceived stress5,10.\n\nWhen considering the psychosocial impact of COVID-19, fear of contracting COVID-19 was the highest stressor, which was consistent with other studies17,20. This was followed by difficulties in executing your routine exercise schedule and worry about the future (Table 2).\n\nThis being a cross-sectional study, the selection of participants was non-random, and it is impossible to make unbiased estimates from snowball samples so the results of this study need to be interpreted with due caution. However, this was the best available method of data collection in the current circumstances. The study was also limited by the lack of other socio-demographic and cross-cultural comparison groups.\n\n\nConclusions\n\nThe psychosocial impact of the nationwide lockdown on the Indian population has been high. The vulnerable groups for stress and anxiety include women, those of a younger age, and the unemployed. The stressors recognized include fear of contracting COVID-19, inability to execute routine exercise schedule and worry about the future.\n\n\nData availability\n\nFigshare: Raw data PSS_GAD Psychosocial impact of lockdown.csv, https://doi.org/10.6084/m9.figshare.12860060.v211.\n\nFigshare: Raw data PSS_GAD Psychosocial impact of lockdown.csv, https://doi.org/10.6084/m9.figshare.12860060.v211.\n\nThis project contains the following extended data:\n\n- Online questionnaire.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nEbeid FSE: COVID-19 effect on clinical research: Single-site risk management experience. Perspect Clin Res. 2020; 11: 106–110.\n\nHoresh D, Lev-Ari RK, Hasson-Ohayon I: Risk factors for psychological distress during the COVID-19 pandemic in Israel: Loneliness, age, gender, and health status play an important role. Br J Health Psychol. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQiu J, Shen B, Zhao M, et al.: A nationwide survey of psychological distress among Chinese people in the COVID-19 epidemic: implications and policy recommendations. Gen Psychiatr. 2020; 33(2): e100213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLau H, Khosrawipour V, Kocbach P, et al.: The positive impact of lockdown in Wuhan on containing the COVID-19 outbreak in China. J Travel Med. 2020; 27(3): taaa037. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Y, Di Y, Ye J, et al.: Study on the public psychological states and its related factors during the outbreak of coronavirus disease 2019 (COVID-19) in some regions of China. Psychol Health Med. 2020; 1–10. PubMed Abstract \| Publisher Full Text\n\nZhou X, Snoswell CL, Harding LE, et al.: The Role of Telehealth in Reducing the Mental Health Burden from COVID-19. Telemed J E Health. 2020 ; 26(4): 377–379. PubMed Abstract \| Publisher Full Text\n\nAl-Rabiaah A, Temsah MH, Al-Eyadhy AA, et al.: Middle East Respiratory Syndrome-Corona Virus (MERS-CoV) associated stress among medical students at a university teaching hospital in Saudi Arabia. J Infect Public Health. 2020; 13(5): 687–691. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNanjundaswamy MH, Pathak H, Chaturvedi SK: Perceived stress and anxiety during COVID-19 among psychiatry trainees. Asian J Psychiatr. 2020; 54: 102282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPodder I, Agarwal K, Datta S: Comparative analysis of perceived stress in dermatologists and other physicians during national lock-down and COVID-19 pandemic with exploration of possible risk factors: A web-based cross-sectional study from Eastern India. Dermatol Ther. 2020; e13788. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPedrozo-Pupo JC, Pedrozo-Cortés MJ, Campo-Arias A: Perceived stress associated with COVID-19 epidemic in Colombia: an online survey. Cad Saude Publica. 2020; 36(5): e00090520. PubMed Abstract \| Publisher Full Text\n\nWakode N, Wakode S, Santoshi J: Raw data PSS_GAD Psychosocial impact of lockdown.csv.figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12860060.v2\n\nShah M, Hasan S, Malik S, et al.: Perceived stress, sources and severity of stress among medical undergraduates in a Pakistani medical school. BMC Med Educ. 2010; 10: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Elm E, Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol. 2008; 61(4): 344–9. PubMed Abstract \| Publisher Full Text\n\nXiang YT, Yang Y, Li W, et al.: Timely mental health care for the 2019 novel coronavirus outbreak is urgently needed. Lancet Psychiatry. 2020,; 7(3): 228–229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIqbal S, Gupta S, Venkatarao E: Stress, anxiety and depression among medical undergraduate students and their socio-demographic correlates. Indian J Med Res. 2015; 141(3): 354–357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlein EM, Brähler E, Dreier M, et al.: The German version of the Perceived Stress Scale - psychometric characteristics in a representative German community sample. BMC Psychiatry. 2016; 16: 159. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalkhi F, Nasir A, Zehra A, et al.: Psychological and Behavioral Response to the Coronavirus (COVID-19) Pandemic. Cureus. 2020; 12(5): e7923. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAneshensel CS: Toward Explaining Mental Health Disparities. J Health Soc Behav. 2009; 50(4): 377–394. PubMed Abstract \| Publisher Full Text\n\nVallejo MA, Vallejo-Slocker L, Fernández-Abascal EG, et al.: Determining Factors for Stress Perception Assessed with the Perceived Stress Scale (PSS-4) in Spanish and Other European Samples. Front Psychol. 2018; 9: 37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDubey S, Biswas P, Ghosh R, et al.: Psychosocial impact of COVID-19. Diabetes Metab Syndr. 2020; 14(5): 779–788. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "79580", "date": "10 Mar 2021", "name": "Krishna Prasad Muliyala", "expertise": [ "Reviewer Expertise Psychiatry", "mental health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease mention what GAD measures under the section of data collection and survey. Whereas, PSS measures perception of stress over the last month. GAD measures anxiety for the last 2 weeks.\nWhat was the mean age of the respondents?\nHow were the items for the questions in Table 2 selected?\nWhat is the time frame for these questions?\nWhich parts of India did the respondents belong to? The positivity rates and lockdowns may have had differential impact.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "6573", "date": "05 May 2021", "name": "Santosh Wakode", "role": "Author Response", "response": "We thank the reviewer for the valuable comments. GAD measures anxiety for the past 2 weeks. The mean age of the respondents was 25 years. The items for the questions in Table 2 were selected based on previous studies on this subject. (reference 12). The time frame for the questions was the first week of 4th phase of nationwide lock down. The respondents were mainly from central, north and western India." } ] } ]	1	https://f1000research.com/articles/9-1233
https://f1000research.com/articles/9-1282/v1	29 Oct 20	{ "type": "Study Protocol", "title": "Development of a framework for the implementation of electronic interventions in mental health: a protocol for a meta-synthesis of systematic reviews", "authors": [ "David Villarreal-Zegarra", "Christoper A. Alarcon-Ruiz", "G.J. Melendez-Torres", "Roberto Torres-Puente", "Juan Ambrosio-Melgarejo", "Alejandra B. Romero-Cabrera", "Ana Lindo-Cavero", "Jeff Huarcaya-Victoria", "David Villarreal-Zegarra", "G.J. Melendez-Torres", "Roberto Torres-Puente", "Juan Ambrosio-Melgarejo", "Alejandra B. Romero-Cabrera", "Ana Lindo-Cavero", "Jeff Huarcaya-Victoria" ], "abstract": "Background: During the COVID-19 pandemic, it has been necessary to incorporate technologies in the care of mental health problems. But there have been difficulties in the application of technology-based interventions in mental health. Some quantitative systematic reviews don’t allow us to fully identify and properly describe this subject. In order to answer the question \"how do electronic interventions apply in mental health and what makes the application of any of these interventions work\", this study will carry out an overview of systematic reviews, which will make it possible to develop a theoretical framework on the implementation of electronic care in mental health problems. Methods: We will search MEDLINE, EBM Reviews, PsycINFO, EMBASE, SCOPUS, CINAHL Complete, and Web of Science databases from 1st January 2015 to September 2020, with no language restriction. We will follow a qualitative method approach and include systematic reviews that assess primary studies relating to adults with common mental health problems using any type of mobile mental health intervention that includes a synchronic component and communication with a mental health professional. For the analysis, we will make a meta-synthesis of the systematic reviews, using an emergent grounded theory approach to synthesize the information, prioritizing the systematic reviews with the lowest risk of bias in the AMSTAR-2 tool. The meta-synthesis will be based on interpreting, integrating, and inferring the evaluation elements to understand better the e-health implementation process for patients with mental health problems. Finally, we will present the overall assessment in a Summary of Qualitative Findings table. Conclusion: Our results will allow a better understanding of the facilitator and limitations in implementing e-health interventions for mental health problems.", "keywords": [ "Telemedicine", "Remote Consultation", "Online Systems", "Internet-Based Intervention", "Mental Health", "Mental Disorders", "Systematic Reviews as Topic", "Qualitative Research" ], "content": "Introduction\n\nIn the wake of the COVID-19 pandemic and after the actions taken by governments (such as social isolation), many mental health problems have increased in patients with COVID-19, patients with psychiatric symptoms, health personnel, and the general population1,2. As a result, greater interest has been taken in addressing mental health issues during the pandemic2,3. An example of this are the studies that identify anxiety disorders, depression, post-traumatic stress disorder, and stress as the most frequent health problems2–4. To deal with these mental health problems and considering the current context (remote attention), it has been necessary to incorporate the use of technologies in the care5,6 These technologies have been very well received and have served to complement or improve the effectiveness of treatments for various chronic diseases, reducing gaps in access to care and providing specialized care in inaccessible places7. In addition, these interventions using technologies show great promise in the care of mental health problems8–10, highlighting the possibilities of care using technologies in health systems where resources are limited11.\n\nWith the undeniable contribution of the use of technologies in mental health care, it has been important to document the aspects related to the application of interventions using technologies, since despite the effectiveness that these interventions can have, it is known that there have been difficulties in the application of interventions using technology12. Aspects such as adaptability, cost, complexity, external policies and incentives, compatibility or general fit between the e-health intervention and the organization, etc.13 are important to consider in order to be clear about how and what works in these interventions and consider its complexity. To try to answer this there are many studies which have gathered information and synthesized it. An example of this are the systematic reviews and meta-analyses of interventions in mental health care using technologies (the latter also allowed conclusions to be drawn about the effectiveness of the interventions used)14,15. However, there are certain aspects which this type of review (centered on quantitative studies) does not allow to identify16. That is why in order to answer the question \"how do electronic interventions apply in mental health and what makes the application of any of these interventions work\"; this study will carry out an overview of systematic reviews, which will make it possible to develop a theoretical framework on the implementation of electronic care in mental health problems.\n\n\nMethods\n\nThis protocol adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines17 A completed PRISMA-P checklist can be found in the Reporting guidelines section18. We used the SPIDER framework to develop the review question16:\n\nSample: Adults with depression (or major depressive disorder), anxiety (or generalized anxiety disorder), stress (or trauma-related disorders), and/or general mental health problems (unspecified).\n\nPhenomenon of interest: Any type of mobile mental health intervention that includes a synchronic component, communication with a mental health professional (psychiatrist, psychologist, etc.) or a health professional trained in mental health. These interventions include, among others, remote consultation, interactive application, video chats, calls, etc.\n\nDesign: Systematic review.\n\nEvaluation: We will include all types of outcomes of interest assessed by implementation studies, economic, qualitative, quantitative and others. For example, a) Health effectiveness outcomes: Depression, anxiety and/or stress symptoms, adherence to treatment, etc; b) Patient outcomes: Quality of life, satisfaction, etc; c) Economic outcomes; d) Damage or adverse effects. Also, we will include all kinds of statistical measures of the effects, if available: Relative risks, odds ratio, risk difference, mean difference and/or number needed to treat.\n\nResearch type: Quantitative studies, qualitative studies, mixed methods.\n\nInclusion criteria: Systematic reviews that report on inclusion/exclusion criteria, conducted an adequate search, synthesized the included studies, assessed the quality of the included studies, and presented sufficient detail on the individual included studies19. Systematic reviews that included primary studies as a unit of analysis focused on a research question. Systematic reviews that were published in the last five years (since January 1, 2015) without language restrictions. We are including this time frame in order to include only the last updated systematic reviews. Reviews must include primary studies relating to adults with common mental health problems: a) Adults with depression (or major depressive disorder), anxiety (or generalized anxiety disorder), stress (or trauma-related disorders) and/or general mental health problems (unspecified); b) Adults attending an outpatient mental health consultation.\n\nExclusion criteria: Narrative reviews, scoping reviews, primary studies, opinion/editorial manuscripts, letters to the editor, and reviews of mobile health interventions repositories (i.e. apps stores). Studies in which some of these subjects has participated will also be excluded: a) Adults with some other mental health problem; b) Healthy adults without mental health problems; c) Adults receiving emergency/crisis psychiatric care; d) Interventions that lack a synchronic component (that lack real time information exchange using technologies).\n\nIncluded databases will be MEDLINE (Ovid), EBM Reviews (Ovid), PsycINFO (Ovid), EMBASE (Elsevier), SCOPUS, CINAHL Complete (EBSCOhost), and Web of Science databases, including Science Citation Index Expanded, Social Sciences Citation Index and Conference Proceedings Citation Index (Clarivate Analytics). Articles published in the last five years (after January 1st, 2015) will be included and no language restrictions will be imposed. We will include systematic reviews that report a review of literature using a research strategy in at least two different databases. Later, all references of included studies will be reviewed, and they will be evaluated looking for any additional systematic review that meets the inclusion criteria.\n\nMain search terms to be used are \"telemedicine\" AND \"mental health, anxiety, depression or stress\" AND “systematic reviews”. The full search strategy for each database is available in the Extended data section18.\n\nData management: The results of the database search will be managed using the Rayyan QCRI free online application to manage the references (eliminate duplicates, and review titles and summaries)20. Full-text review and data extraction will be done using an Excel template (Extended data18).\n\nSelection process: There will be two distinct selection stages. First, a review of the title and abstract and the second a review of the full text. The reviews will be carried out based on pairs of reviewers, who will divide the total number of records. At each stage, there will be a first part where the reviewers will calibrate the accuracy of their reviews and a second part where the actual review will take place. To complete the calibration part, they will make calibration rounds until the discrepancy is less than or equal to 5% of the assigned records. If the percentage of the discrepancy is exceeded, a new round will be performed. Subsequently pairs of reviewers will divide the total of the records identified, and records will be screened independently and in duplicate. Discrepancies will be discussed within pairs with a third reviewer if needed.\n\nIn the review by title and abstract, first the calibration will be done, 50 records were selected from the total of records found as a result of the search strategy. Each pair had to make an independent review by title and abstract, selecting the studies that met the inclusion criteria. In the selection stage itself, the number of total records will be divided among all pairs of reviewers.\n\nIn the full-text review, first, a calibration of 10 records that will select in the previous phase will be performed. Only articles selected by title and abstract will review in full text. The articles excluded in the full-text review stage will be listed and a reason for exclusion will be given for each one of them.\n\nData collection process: For each eligible study, data will be extracted independently and in duplicate on pre-designed data extraction forms. In the event of discrepancies, the reviewers will discuss whether the extracted data should be retained and decide to resolve the discrepancy. If the discrepancy remains, a third reviewer will evaluate any unresolved disagreement between the reviewers on the data extraction and will decide on the value extracted that is in dispute.\n\nAn extraction form will be created for the included systematic reviews. Information will be collected on the author and date of the study, characteristics of the participants, main objective, research questions, inclusion criteria for the systematic review, search date, study selection process, quality assessment (if any), main findings and limitations. Also, the full text of the included article will be extracted along with the tables and supplementary material, to perform the qualitative analysis of the text.\n\nOur study seeks to make a meta-synthesis of the systematic reviews, using a qualitative strategy to synthesize the information and answer our research question. Therefore, we do not look for a specific result such as effectiveness, cost-effectiveness or other similar ones. Instead, we are interested in identifying the full text of all studies that answer our research question, to be able to perform a grounded theory analysis with an emergent approach. Priority will be given in the analysis of those studies with the lowest risk of bias assessed.\n\nTo assess the quality of the included systematic reviews we will use the \"A Measurement Tool to Assess Systematic Reviews-2\" (AMSTAR 2), which has sixteen domains. Seven of these domains are considered critical: 1) protocol registered before the start of the review, 2) adequacy of the literature search, 3) justification for the exclusion of individual studies, 4) risk of bias of individual studies included in the review, 5) adequacy of meta-analytic methods, 6) consideration of the risk of bias in interpreting the results of the review, and 7) assessment of the presence and likely impact of publication bias21.\n\nAMSTAR-2 classifies the quality of systematic reviews into four different categories: high (none or one non-critical weakness), moderate (more than one non-critical weakness), low (one critical weakness with or without non-critical weaknesses), and very low (more than one critical weakness with or without non-critical weaknesses). The quality assessment of each systematic review will be rated by two researchers independently. In case of difference in the overall quality of the systematic reviews, the AMSTAR-2 criteria will be discussed among the researchers to reach a consensus.\n\nThe analysis of the data will be carried out using a grounded theory approach with an emergent approach22,23. The reviewers will follow the three steps established by Thomas and Harden24. First, the extracted data are freely coded. The reviewers read the full texts of the included articles and code each text fragment that provides information to answer the research question. Second, the codified data are organized and grouped based on the descriptive aspects. The reviewers will review the codes generated in the previous step and group them into codes that are like each other and that allow the description of a part of the research question. Third, analytical concepts will generate different groups describing aspects generated in the previous step. The meta-synthesis will be based on interpreting, integrating and, inferring the evaluation elements that would allow a better understanding of the e-health implementation process from all the studies included24. In addition to generate hypotheses supported by the results of the included studies, which will be generated through discussion and consensus among the reviewers24.\n\nWhen included studies had been selected, they will be ranked based on the AMSTAR-2 score, with the highest quality studies being assessed first. We will assess all included studies, down to the criterion of theoretical saturation. All qualitative analyses will be performed with Atlas.ti v.8 software.\n\nIt will be evaluated the approach of Confidence in the Evidence from Reviews of Qualitative research (CERQual) which has four components (Methodological Limitations, Relevance, Coherence and Adequacy data), and thus contribute to an overall assessment for each systematic review to determine the level of confidence (high, moderate, low, or very low) and present the overall assessment in a Summary of Qualitative Findings (SoQF) table25,26.\n\nWe will present the systematic review findings and submit the manuscript to a relevant peer-reviewed journal.\n\n\nStudy status\n\nThis systematic review is currently in the first stage (title and abstract screening) in the selection process. The protocol of this systematic review was submitted to PROSPERO registry on 18th August, 2020 (CRD42020203811).\n\n\nData availability\n\nNo data are associated with this article.\n\nOpen Science Framework: PRISMA-P checklist for ‘Development of a framework for the implementation of electronic interventions in mental health: A protocol for a meta-synthesis of systematic reviews’, https://doi.org/10.17605/OSF.IO/3UH4N18. (Registered on 20th October 2020: osf.io/tf4b6.)\n\nThis project contains the following extended data:\n\n- Full text protocol\n\n- Full search strategy for MEDLINE, EBM Reviews, PsycINFO, EMBASE, SCOPUS, CINAHL Complete, and Web of Science databases\n\n- Excel template for data extraction\n\nOpen Science Framework: PRISMA-P checklist for ‘Development of a framework for the implementation of electronic interventions in mental health: A protocol for a meta-synthesis of systematic reviews’, https://doi.org/10.17605/OSF.IO/3UH4N18. (Registered on 20th October 2020: osf.io/tf4b6.)\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 acknowledge Jackeline Garcia and Juan Barrera, for helping with the initial phases of the information search.\n\n\nReferences\n\nVindegaard N, Benros ME: COVID-19 pandemic and mental health consequences: Systematic review of the current evidence. Brain Behav Immun. 2020; 89: 531–542. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTalevi D, Socci V, Carai M, et al.: Mental health outcomes of the CoViD-19 pandemic. Riv Psichiatr. 2020; 55(3): 137–44. PubMed Abstract \| Publisher Full Text\n\nHuarcaya-Victoria J: Consideraciones sobre la salud mental en la pandemia de COVID-19. Rev Peru Med Exp Salud Publica. 2020; 37(2): 327–34. PubMed Abstract \| Publisher Full Text\n\nXiong J, Lipsitz O, Nasri F, et al.: Impact of COVID-19 pandemic on mental health in the general population: A systematic review. J Affect Disord. 2020; 277: 55–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSullivan AB, Kane A, Roth AJ, et al.: The COVID-19 Crisis: A Mental Health Perspective and Response Using Telemedicine. J Patient Exp. 2020; 7(3): 295–301. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaxhihamza K, Arsova S, Bajraktarov S, et al.: Patient Satisfaction with Use of Telemedicine in University Clinic of Psychiatry: Skopje, North Macedonia During COVID-19 Pandemic. Telemed J E Health. 2020. PubMed Abstract \| Publisher Full Text\n\nRuiz EF, Proaño A, Ponce OJ, et al.: [Mobile health for public health in Peru: lessons learned]. Rev Peru Med Exp Salud Publica. 2015; 32(2): 364–72. PubMed Abstract\n\nMiranda JJ, Moscoso MG, Toyama M, et al.: Role of mHealth in overcoming the occurrence of post-stroke depression. Acta Neurol Scand. 2018; 137(1): 12–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrews G, Cuijpers P, Craske MG, et al.: Computer Therapy for the Anxiety and Depressive Disorders Is Effective, Acceptable and Practical Health Care: A Meta-Analysis. PLoS One. 2010; 5(10): e13196. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurvey C, Fortney J: The Use of Telemedicine and Mobile Technology to Promote Population Health and Population Management for Psychiatric Disorders. Curr Psychiatry Rep. 2017; 19(11): 88. PubMed Abstract \| Publisher Full Text\n\nKaonga NN, Morgan J: Common themes and emerging trends for the use of technology to support mental health and psychosocial well-being in limited resource settings: A review of the literature. Psychiatry Res. 2019; 281: 112594. PubMed Abstract \| Publisher Full Text\n\nMeurk C, Leung J, Hall W, et al.: Establishing and Governing e-Mental Health Care in Australia: A Systematic Review of Challenges and A Call For Policy-Focussed Research. J Med Internet Res. 2016; 18(1): e10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoss J, Stevenson F, Lau R, et al.: Factors that influence the implementation of e-health: a systematic review of systematic reviews (an update). Implement Sci. 2016; 11(1): 146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGriffiths KM, Farrer L, Christensen H: The efficacy of internet interventions for depression and anxiety disorders: a review of randomised controlled trials. Med J Aust. 2010; 192(S11): S4–S11. PubMed Abstract\n\nSenanayake B, Wickramasinghe SI, Chatfield MD, et al.: Effectiveness of text messaging interventions for the management of depression: A systematic review and meta-analysis. J Telemed Telecare. 2019; 25(9): 513–523. PubMed Abstract \| Publisher Full Text\n\nCooke A, Smith D, Booth A: Beyond PICO: The SPIDER Tool for Qualitative Evidence Synthesis. Qual Health Res. 2012; 22(10): 1435–43. PubMed Abstract \| Publisher Full Text\n\nMoher D, Shamseer L, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015; 4(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVillarreal-Zegarra D, Alarcon-Ruiz CA, Melendez-Torres G, et al.: Development of a framework for the implementation of electronic interventions in mental health: A protocol for a meta-synthesis of systematic reviews. 2020. http://www.doi.org/10.17605/OSF.IO/3UH4N\n\nDissemination CfRa. University of York: Centre for Reviews and Dissemination. [updated 3 June, 2015]. Reference Source\n\nOuzzani M, Hammady H, Fedorowicz Z, et al.: Rayyan—a web and mobile app for systematic reviews. Syst Rev. 2016; 5(1): 210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShea BJ, Reeves BC, Wells G, et al.: AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. BMJ. 2017; 358: j4008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuetterman TC, Babchuk WA, Howell Smith MC, et al.: Contemporary Approaches to Mixed Methods–Grounded Theory Research: A Field-Based Analysis. J Mix Methods Res. 2019; 13(2): 179–95. Publisher Full Text\n\nThorne S, Jensen L, Kearney MH, et al.: Qualitative metasynthesis: reflections on methodological orientation and ideological agenda. Qual Health Res. 2004; 14(10): 1342–65. PubMed Abstract \| Publisher Full Text\n\nThomas J, Harden A: Methods for the thematic synthesis of qualitative research in systematic reviews. BMC Med Res Methodol. 2008; 8: 45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGroup TG-CP: A practical guide for decision makers in health and social care and other users. Reference Source\n\nLewin S, Booth A, Glenton C, et al.: Applying GRADE-CERQual to qualitative evidence synthesis findings: introduction to the series. Implement Sci. 2018; 13(Suppl 1): 2. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "78252", "date": "11 Feb 2021", "name": "Amy Grove", "expertise": [ "Reviewer Expertise HTA and implementation 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\nAbstract:\n\"Some quantitative systematic\" can you indicate how many rather than using the generic some.\n\nWould the research question be better phrased as 2 questions? or a primary and secondary question? i) how question?; ii) what makes question?\n\nPlease define what approach you will use \"We will follow a qualitative method approach\".\n\nAre \"incorporate technologies\", \"implementation of electronic care\", \"mobile mental health intervention that includes a synchronic component and communication with a mental health professional\", \"e-health implementation process\" and, \"synchronic component (that lack real time information exchange using technologies).\" all the same thing? I would suggest you select one terminology and use it consistently.\n\nIntroduction:\n\nPlease remove \"To deal with these mental health problems\". This is not appropriate terminology.\n\nYou talk about \"These technologies\", can you provide examples?\n\nYou say \"(centered on quantitative studies) does not allow to identify\". Describe what it does not allow; what is missing from existing reviews?\nMethods:\n\nThe heading, \"SPIDER question\", needs to be replaced with \"research question\". The acronym may not be understood by all.\n\nRegarding the sample - do people have to have a certified diagnosis of the condition? What about undiagnosed groups?\n\nEvaluation:\nAre you assessing effectiveness? or just implementation of a technology that has shown to be effective elsewhere?\n\nIf you are following a \"qualitative method approach\" why are you including outcomes of \"all kinds of statistical measures of the effects\"? What will you do with this information?\n\nHow do you define \"conducted an adequate search\"? If you are using a criteria then please list it.\n\nPlease provided references for \"only the last updated systematic reviews\".\n\nThe study selection process is overly detailed and repetitive.\n\nYou say \"An extraction form will be created for the included systematic reviews.\", but above you imply it is already done and included in \"an Excel template (Extended data)\".\n\n\"Therefore, we do not look for a specific result such as effectiveness, cost-effectiveness or other similar ones.\" Then why are you searching for data?\n\n\"perform a grounded theory analysis with an emergent approach\" - you said earlier \"using an emergent grounded theory approach\", what is the difference? If no difference, then please use consistent terms.\n\n\"Priority will be given in the analysis of those studies with the lowest risk of bias assessed.\" - Explain why this is the case In data synthesis you do not state how many \"reviewers will follow the three steps\".\n\nIn the abstract you say you will \"develop a theoretical framework\" but you do not give detail of how this will be done in the manuscript.\n\nPlease also proof read for typographical errors and appropriate use of English.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "6881", "date": "03 Aug 2021", "name": "Christoper A. Alarcon-Ruiz", "role": "Author Response", "response": "Abstract: 1. \"Some quantitative systematic\" can you indicate how many rather than using the generic some. Reply: We specified this concern. Abstract: “Quantitative systematic reviews such as meta-analysis” 2. Would the research question be better phrased as 2 questions? or a primary and secondary question? i) how question?; ii) what makes question? Reply: It was modified in the abstract: “Thus, our study has two main objectives: a) \"to determine what evidence is available for synchronous e-mental health implementation\"; and b) \"to develop a framework informed by a realistic analysis for the implementation of synchronous e-mental health\".” 3. Please define what approach you will use \"We will follow a qualitative method approach\". Reply: It was modified in the abstract: “A systematic review with narrative description and a realist synthesis will be conducted. Primary studies related to adults with common mental health problems using any type of mobile mental health intervention that includes a synchronous component and communication with a mental health professional will be included. For the analysis, we will make a realistic synthesis of the systematic reviews, using a grounded theory approach with an emergent approach to synthesize the information, prioritizing the systematic reviews with a lower risk of bias in the AMSTAR-2 tool. The realist synthesis will be based on the interpretation, integration, and inference of the evaluated elements and the generation of hypotheses to better understand the implementation process of synchronic e-mental health.” 4. Are \"incorporate technologies\", \"implementation of electronic care\", \"mobile mental health intervention that includes a synchronic component and communication with a mental health professional\", \"e-health implementation process\" and, \"synchronic component (that lack real-time information exchange using technologies).\" all the same thing? I would suggest you select one terminology and use it consistently. Reply: We’ll use the term “e-mental health” to refer to mental health care delivery through technology. And we’ll use “synchronous e-mental health” to refer to the delivery of mental health care through technology in real-time. Introduction: 5. Please remove \"To deal with these mental health problems\". This is not appropriate terminology. Reply: We changed it for “As a result, it has been...” 6. You talk about \"These technologies\", can you provide examples? Reply: We specified some examples: “such as internet and mobile devices ” 7. You say \"(centered on quantitative studies) does not allow to identify\". Describe what it does not allow; what is missing from existing reviews? Reply: Does not provide information about experience, meaning, and perspective, which is important when addressing implementation issues. “However, qualitative studies are necessary because of their methodology (approach to answer questions about experience, meaning, and perspective) which is useful to address issues of implementation.” Methods: 8. The heading, \"SPIDER question\", needs to be replaced with \"research question\". The acronym may not be understood by all. Reply: The heading has been modified. 9. Regarding the sample - do people have to have a certified diagnosis of the condition? What about undiagnosed groups? Reply: It was modified in the methods: “Sample: Adults with depression (or major depressive disorder), anxiety (or generalized anxiety disorder), stress (or trauma-related disorders), and/or general mental health problems (unspecified). Participants may be diagnosed through clinical interviews or categorized based on screening assessments (self-reported scales).” Evaluation: 10. Are you assessing effectiveness? or just implementation of a technology that has shown to be effective elsewhere? Reply: It was modified in the method: “All types of outcomes of interest assessed by implementation, economic, qualitative, quantitative, and other studies will be included. For example, a) Health effectiveness outcomes: Depression, anxiety and/or stress symptoms, adherence to treatment, etc.; b) Patient outcomes: Quality of life, satisfaction, etc.; c) Economic outcomes; d) Damage or adverse effects. Our study seeks to perform a realist synthesis of the evidence, so we focused on different outcomes to use them as input that allow s us to evaluate the implementation of synchronous e-mental health. Therefore, we do not perform a quantitative synthesis in any case (i.e., a meta-analysis of effectiveness).” 11. If you are following a \"qualitative method approach\" why are you including outcomes of \"all kinds of statistical measures of the effects\"? What will you do with this information? Reply: We agree with the reviewer. The quantitative information was eliminated. It was modified in the method: “Our study seeks to perform a realist synthesis of the evidence, so we focused on different outcomes to use them as input. Therefore, we do not perform a quantitative synthesis in any case (i.e., a meta-analysis of effectiveness).” 12. How do you define \"conducted an adequate search\"? If you are using criteria then please list them. Reply: We agree with your observation. We have specified and listed the criteria we consider to include a systematic review “Systematic reviews using at least two databases that synthesized, assessed the quality, and presented sufficient detail of the primary included studies; and reports on inclusion/exclusion criteria” 13. Please provided references for \"only the last updated systematic reviews\". Reply: It was specified in the text: “We include this time frame to include only the latest systematic reviews since in the field of e-health, the launch of new technologies makes scientific development dynamic. “ 14. The study selection process is overly detailed and repetitive. Reply: We synthesized the study selection: “In the review by title and abstract, calibration will be done first, 50 records were selected from the total number of records found as a result of the search strategy. In the full-text review, calibration will first be performed on 10 records that will be selected in the previous stage. The articles excluded in the full-text review stage will be listed, and a reason for exclusion will be given for each of them.” 15. You say, \"An extraction form will be created for the included systematic reviews.\", but above, you imply it is already done and included in \"an Excel template (Extended data)\". Reply: It was modified in the subsection of data items: “An extraction form was created in an excel template for the included systematic reviews (https://osf.io/7esgz/).” 16. \"Therefore, we do not look for a specific result such as effectiveness, cost-effectiveness or other similar ones.\" Then why are you searching for data? Reply: It was modified in the method: “Our study seeks to perform a realist synthesis of the evidence, so we focused on different outcomes to use them as input. Therefore, we do not perform a quantitative synthesis in any case (i.e., a meta-analysis of effectiveness).” The section where we state that we will collect specific quantitative data, such as effect size, has been removed. Studies with efficacy, cost-effectiveness, or other similar outcomes are included because they will help answer our question. 17. \"perform a grounded theory analysis with an emergent approach\" - you said earlier, \"using an emergent grounded theory approach\", what is the difference? If no difference, then please use consistent terms. Reply: It was specified: “Our study aims to conduct a systematic review of systematic reviews and a realistic synthesis of systematic reviews, using a qualitative strategy to synthesize the information and answer our research question. Therefore, we do not look for specific results such as effectiveness, cost-effectiveness, or other similar ones. Instead, we are interested in identifying the full text of all the studies that answer our research question so that we can make a realistic synthesis using a grounded theory model with an emergent approach. The realistic synthesis will be based on the interpretation, integration, and inference of the evaluated elements and the generation of hypotheses to better understand the studied phenomenon (implementation of synchronous e-mental health) (20, 21).” 18. \"Priority will be given in the analysis of those studies with the lowest risk of bias assessed.\" - Explain why this is the case Reply: It was specified: “Priority will be given in the analysis of those studies with the lowest risk of bias assessed. If possible, a realist synthesis will be performed only with those systematic reviews with high quality, to assess whether the results change by selecting only those studies with high quality (low risk of bias).” 19. In data synthesis, you do not state how many \"reviewers will follow the three steps\". Reply: We agree with your observation. We have specified the number of reviewers involved in this process. “Two reviewers will follow the three steps established by Thomas and Harden for qualitative syntheses will be followed.” 20. In the abstract, you say you will \"develop a theoretical framework\" but you do not give detail of how this will be done in the manuscript. Reply: The authors have identified that the meta-synthesis analysis method was not the most appropriate for the review, as the description is more in line with a realistic synthesis analysis. It was specified in the section on data synthesis. “The develop a framework informed by a realist analysis for the implementation of synchronous e-mental health will be carried out using a grounded theory approach with an emergent approach (23). The realist synthesis will be based on interpreting, integrating and, inferring the evaluation elements that would allow a better understanding of the synchronic e-mental health implementation process from all the studies included (24). To answer the question \"what makes the implementation of these interventions work\", hypotheses supported by the results of the included studies will be developed and generated through discussion and consensus among researchers (24). Two reviewers will follow the three steps established by Thomas and Harden for qualitative syntheses will be followed (25). First, the extracted data are freely coded. The reviewers read the full texts of the included articles and code each text fragment that provides information to answer the research question. Second, the codified data are organized and grouped based on the descriptive aspects. The reviewers will review the codes generated in the previous step and group them into codes that are like each other and that allow the description of a part of the research question. Thirdly, the analytical concepts generated in the previous step will be grouped in a way that they are related to each other. The elements that are related to each other will be assumed to be part of a hypothesis that would help to answer the research aim. The sum of all these hypotheses will be referred to as a framework that will attempt to explain the results using a context-linked causality approach represented as context + mechanism = outcome (26). ” 21. Please also proofread for typographical errors and appropriate use of English. Reply: We accept your observation. We have reviewed and corrected typographical errors and the adequate use of English." } ] }, { "id": "85471", "date": "18 Jun 2021", "name": "Roger Hilfiker", "expertise": [ "Reviewer Expertise Systematic Reviews" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript reports on a protocol for a systematic review with a meta-synthesis of systematic reviews.\n\nThe authors describe a qualitative approach for the analysis of the data with three steps, i.e. coding of each text fragment that provides answers to the research questions, organizing and grouping the codified data, and grouping the aspects.\n\nI have some specific comments:\n\nResearch question: Please clarify how you will use the data to answer the question on \"what makes the application of any of these interventions work\".\n\nData synthesis: The sentence \"In addition to generate hypotheses supported by the results of the included studies, which will be generated through discussion and consensus among the reviewers\" is not clear to me.\n\nThe authors described the extraction of quantitative data on effectiveness. Please clarify whether these quantitative data are analyzed with the qualitative method or whether quantitative methods are used.\n\nPlease describe how the research question on the \"what makes the application work\" will be answered.\n\nConfidence in cumulative evidence: Wouldn't the research question \"what makes the application of any of these interventions work\" require a different evaluation of the confidence of the evidence, for example an approach such as GRADE?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "6882", "date": "03 Aug 2021", "name": "Christoper A. Alarcon-Ruiz", "role": "Author Response", "response": "1. Research question: Please clarify how you will use the data to answer the question on \"what makes the application of any of these interventions work\". Reply: It was specified in the subsection of Data synthesis “The develop a framework informed by a realist analysis for the implementation of synchronous e-mental health will be carried out using a grounded theory approach with an emergent approach (23). The realist synthesis will be based on interpreting, integrating and, inferring the evaluation elements that would allow a better understanding of the synchronous e-mental health implementation process from all the studies included (24). To answer the question \"what makes the implementation of these interventions work\", hypotheses supported by the results of the included studies will be developed and generated through discussion and consensus among researchers (24). Two reviewers will follow the three steps established by Thomas and Harden for qualitative syntheses will be followed (25). First, the extracted data are freely coded. The reviewers read the full-texts of the included articles and code each text fragment that provides information to answer the research question. Second, the codified data are organized and grouped based on the descriptive aspects. The reviewers will review the codes generated in the previous step and group them into codes that are like each other and that allow the description of a part of the research question. Thirdly, the analytical concepts generated in the previous step will be grouped in a way that they are related to each other. The elements that are related to each other will be assumed to be part of a hypothesis that would help to answer the research aim. The sum of all these hypotheses will be referred to as a framework which will attempt to explain the results using a context-linked causality approach represented as context + mechanism = outcome (26).” 2. Data synthesis: The sentence \"In addition to generate hypotheses supported by the results of the included studies, which will be generated through discussion and consensus among the reviewers\" is not clear to me. Reply: We agree with the reviewer, this has been modified in the previous comment. 3. The authors described the extraction of quantitative data on effectiveness. Please clarify whether these quantitative data are analyzed with the qualitative method or whether quantitative methods are used. Reply: The collection of quantitative data was eliminated, as specified in the method section: “Our study seeks to perform a realistic synthesis of the evidence, so we focused on different outcomes to use them as input. Therefore, we do not perform a quantitative synthesis in any case (i.e., a meta-analysis of effectiveness).” 4. Please describe how the research question on the \"what makes the application work\" will be answered. Reply: A better description of the research objective was made based on the first reviewer. “Thus, our study has two main objectives: a) \"to determine what evidence is available for synchronous e-mental health implementation\"; and b) \"to develop a framework informed by a realist analysis for the implementation of synchronous e-mental health\".” The process of how to achieve these objectives was answered in the first comment. 5. Confidence in cumulative evidence: Wouldn't the research question \"what makes the application of any of these interventions work\" require a different evaluation of the confidence of the evidence, for example an approach such as GRADE? Reply: we will evaluate each key finding using CERQual to assess the methodological limitations, coherence, adequacy, and relevance of findings. “It will be evaluated the approach of Confidence in the Evidence from Reviews of Qualitative research (CERQual) which has four components (Methodological Limitations, Relevance, Coherence and Adequacy data), will be evaluated to contribute to an overall assessment for each hypothesis resulting from the realist synthesis to determine the level of confidence (high, moderate, low, or very low) and present the overall assessment in a Summary of Qualitative Findings (SoQF) table (27, 28).”" } ] } ]	1	https://f1000research.com/articles/9-1282
https://f1000research.com/articles/6-2082/v1	04 Dec 17	{ "type": "Research Article", "title": "Characterization of cellulose-degrading microbiota from the eastern subterranean termite and soil", "authors": [ "Xianfa Xie", "Alonzo B. Anderson", "Latoya J. Wran", "Myrna G. Serrano", "Gregory A. Buck", "Alonzo B. Anderson", "Latoya J. Wran", "Myrna G. Serrano", "Gregory A. Buck" ], "abstract": "Background: While there have been a lot of studies on the termite gut microbiota, there has been very little research directly on the cellulose-degrading microbiota in termites or their soil environment. This study addresses this problem by profiling cellulose-degrading bacteria and archaea in the selective cellulose cultures of two samples of the eastern subterranean termite (Reticulitermes flavipes) and one soil sample collected at the same location as one of the termite samples. Methods: All the cultures were examined for cell concentration and remaining cellulose after the culture was completed. The 16S rRNA pyrotag sequencing method was used to identify the prokaryotic microbiota for the three cultures and one termite colony without culture. The MOTHUR, SSU-ALIGN, RDPTools, phyloseq, and other R packages were used for sequence and statistical analyses. Results: Biochemical analyses of the cultures suggested high efficiency of cellulose degradation. Comparative analyses between the cultured and uncultured termite gut microbiota revealed a significant difference. Proteobacteria and Firmicutes were found to be the two most abundant phyla of cellulose-degrading bacteria from the three cultures, but different classes within each phylum dominated the different samples. Shared and sample-specific cellulose-degrading taxa, including a core set of taxa across all the cultures, were identified. Conclusions: Our study demonstrates the importance of using selective cellulose culture to study the cellulose-degrading microbial community. It also revealed that the cellulose-degrading microbiota in the eastern subterranean termite is significantly influenced by the microbiota in the surrounding soil environment. Biochemical analyses showed that the microbial communities enriched from all the selective cultures were efficient in degrading cellulose, and a core set of bacteria have been identified as targets for further functional analyses.", "keywords": [ "cellulose degradation", "termite", "soil", "microbiome", "16S", "metagenomic" ], "content": "Introduction\n\nHow plant materials are decomposed or digested is a fundamental question for the study of nutrient cycling in the ecosystem as well as animal physiology. Plants as the primary producers on earth have been the major or even exclusive source of energy for many animals, including herbivorous insects like termites. Most of the plant materials in the ecosystem that have not been consumed by animals are ultimately decomposed in soil to provide nutrients and energy for a variety of other organisms. It has been well established that microbes play essential roles in such processes.\n\nCellulose is the most abundant plant material on earth, produced by plants from natural habitats like forests, prairies, and grasslands, as well as agricultural and other man-made habitats like lawns in residential, commercial, or public areas. Understanding how cellulose is degraded is not only critical for understanding the nutrient cycling in both natural and human-altered ecosystems but also essential for the study of animal metabolism and physiology. Furthermore, detailed understanding of the cellulose degradation process and the major players involved in the process would help develop the cost-effective next-generation biofuel technology based on cellulose rather than starch or sugar as commonly used in the current technology.\n\nTermites are a group of insects that have evolved efficient strategy to utilize cellulose-rich plant materials. There has been a growing interest in studying the microbiota in the digestive system of termites, including fungus-farming termites, particularly over the last decade or so1–10. However, most of the existing studies have focused on the profiling of whole microbiota in termite guts, rather than those taxonomic groups specifically involved in the degradation of cellulose.\n\nSoil, particularly the topsoil, is where most such cellulose degradation process takes place in the natural and human-modified ecosystems. Some termite species, like the subterranean termites, spend significant amount of their time living underground in soil. While there have been some studies on the source of microbiota in termite digestive system7,11–13, the debate about whether the microbiota is vertically inherited or largely shaped by the environment is still unresolved. So far, there seems to be a dearth of study on whether there is any connection between the cellulose-degrading microbiota in the termite digestive system and those in the soil.\n\nIn this study, we tried to elucidate the above questions by focusing on cellulose-degrading prokaryotes, particularly bacteria, from both termite and soil samples. The study of two colonies of the eastern subterranean termite species, Reticulitermes flavipes, and one soil sample collected at the same site as one of the termite colonies allowed not only comparison between termite colonies, but also between termite and soil samples from the same location. Through selective cultures, the abilities of the microbial communities from the different samples to degrade cellulose were biochemically tested, and the enriched cultures were characterized using the 16S rRNA genetic marker to identify the cellulose-degrading bacteria and archaea from the two termite and one soil samples. The diversity of cellulose-enriched prokaryotic microbiota was compared with the whole gut microbiota for one termite sample, which highlighted the value of using selective culture to study cellulose-degrading microbial communities.\n\n\nMethods\n\nTo test the variation between termite colonies, two colonies of the eastern subterranean termite species, R. flavipes, were collected about 1 km apart in central Virginia. Colony 1 (Termite_EW) was collected from inside a partially decomposed log on the ground in the forest (East Wood, EW; N: 37.238854, W: -77.414578) on the campus of the Virginia State University, while Colony 2 (Termite_AX) was from a log by the Appomattox River (AX; N: 37.231483, W: -77.418612) at Petersburg, Virginia. For each colony, both adult workers and soldiers were collected. The biological classification of the termites was verified with morphological characters, particularly those of the soldiers. However, only workers were used for subsequent microbiota analyses in order to eliminate any variation between castes8 as a confounding factor. In order to test the relationship between termite and soil microbiomes, a soil sample was also collected right underneath the log where the termite Colony 1 was collected.\n\nFor each termite sample, the hindguts of 30 adult workers were dissected and ground to release gut microbes in 1.5 ml of sterile saline water. 100 μl of the evenly mixed microbial cell suspension solution was then used to inoculate 10 ml of selective medium, while the rest was kept for later use. The selective medium was prepared by dissolving 1.25 grams of NaNO3, 1 gram of KH2PO4, 0.1 gram each of MgSO4 and NaCl, and 0.05 gram of CaCl2•6H20 in 500 ml of Type I water, which was then autoclaved and added 2 grams of cellulose. For the soil sample, 200 mg of soil was measured and then suspended in 10 ml of sterile saline water in a 15 ml tube. After the settlement of soil particles at the bottom of the tube, 100 μl of the microbial cell suspension in the top layer was used to inoculate 10 ml of the same selective medium as described above. The inoculated cellulose selective media were then cultured in an incubator at 30°C for 10 days.\n\nImmediately after the culture was completed, the cell density in each culture was measured by the absorbance at 600 nm using the SmartSpec Plus spectrometer, which was then used to calculate the cell concentration. The cellulose remaining in the medium was measured following an established method14.\n\n1.5 ml of each culture was used for DNA extraction using a standard phenol method. To study the microbial diversity in termite gut without the selective culture, the gut microbial resuspension from the Termite_EW colony was also directly used for DNA extraction using the same method. The size distribution, purity, and concentration of the genomic DNAs extracted from each sample were examined using 1% agarose gel electrophoresis, NanoDrop 2000 spectrophotometer, and Qubit 2.0 fluorometer following the standard protocols.\n\nA fragment of 16S rRNA marker covering the V1–V3 region was amplified from the genomic DNA extracted from each of the four samples as described above. Each reaction tube contains 1 unit of Taq polymerase, 2.5 μl of 10x Taq Buffer, 1 μl of 10mM Mg2+, 0.5 μl of dNTPs (all the above reagents from Fisher Scientific), 1 μl of the forward primer (10 mM), 1 μl of the reverse primer (10mM), 1 μl of template DNA, and 18 μl of ddH2O. The PCR amplification was carried out in an Eppendorf Mastercycler™ Nexus Thermal Cycler with the following stages: Initial denaturation at 95°C for 5 minutes, followed by 35 cycles each of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, and finished with 72°C for 5 minutes for extension.\n\nAfter verifying the success of the PCR reactions with gel electrophoresis, the PCR products were purified using the NucleoSpin Extraction Kits (Clontech Laboratories) following the recommended procedure. The purity and yield of the purified products were then analyzed using gel electrophoresis, NanoDrop 2000, and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).\n\nGS FLX Titanium Rapid Library Preparation Kit was used to ligate the RL Adaptors on either side of the existing PCR amplicons. The libraries for the four different samples were normalized, and the sequencing was completed using 454 GS FLX Titanium sequencing technology at the Virginia Commonwealth University. The raw reads were quality-filtered and removed of barcode and primer sequences, which were then summarized using the MOTHUR program v.1.39.515. The majority of the remaining sequences were shown to be at least 400bp in length (Table 2). Subsequently, only sequences of 400bp or longer were used for the analyses in order to improve the quality of sequence alignments and the accuracy of the analyses.\n\nThe sequences from the four samples were first aligned to the 16S rRNA reference sequences16 using structure-based alignment method Infernal v.1.1.217 as implemented in the SSU-ALIGN program v.0.1.1. To facilitate the comparison among samples, the alignments from all four datasets were masked with the default, pre-calculated mask, reformatted using a custom Perl script, and then analyzed using the unsupervised method through the locally installed RDPTools program v.2.0.218. For the unsupervised method, replicate sequences were first removed, then a pairwise distance matrix was calculated, based on which the sequences were clustered and an OTU table was generated. The representative sequence for each OTU was obtained and a BIOM file created, which was then added the classification and sample data. A tree was built from the representative sequences using the FastTree program v.2.1.1019. The sequences from the four datasets were also classified using the supervised method implemented in the RDPTools program, which compared each sequence against the reference database to identify the taxonomic group each sequence represents.\n\nThe results from the above analyses were then further analyzed and visualized in R v3.3.3 using the phyloseq v.1.19.120, vegan v.2.4-321, phangorn v.2.2.022, and GUniFrac v.1.023 packages. To facilitate the comparison among the samples across different taxonomic levels, the results from the RDPTools analysis with the supervised method were further processed using the KronaTools program v.2.724, which created pie charts that can be interactively viewed in a web browser.\n\nThe taxonomic classification using the RDPTools supervised method was compared among the four datasets in order to identify the shared and sample-specific cellulose-degrading microbes from the three selective cultures and the taxa only found in the uncultured termite gut sample. The results were visualized in R v.3.3.3. with the limma package v.3.30.1325.\n\n\nResults\n\nThe enrichment of the cellulose-degrading microbes from two termite gut colonies and one soil sample was achieved through the selective liquid medium culture with only cellulose and basic salts. As shown in Table 1, all the three cultures reached very similar concentrations between 2.38 × 108 to 2.75 × 108 cells/ml after being cultured under the same condition. The percentage of cellulose that has been degraded in each of the three cultures is 64.37%, 62.55%, and 55.28% for Termite_AX, Termite_EW, and Soil_EW cultures respectively.\n\nThe original datasets contains sequence reads that have been quality filtered and removed of barcode and primer sequences.\n\nAfter quality filtering and the removal of barcode and primer sequences, 54,554, 32,990, 34,939, and 51,595 sequence reads were obtained from the uncultured Termite_EW sample, the cultured Termite_EW sample, the cultured Termite_AX sample, and the cultured Soil_EW sample, respectively. The data summary using the MOTHUR program shows that at least three quarters of the reads in each sample is over 400bp. Consequently, only sequences of at least 400bp from each sample were used in subsequent analysis in order to improve the alignment quality and analysis accuracy, which yields 48, 563, 25,745, 27,095, and 42,578 reads for each of the above four samples, respectively. After the removal of identical sequences, the number of unique sequences in each sample is 48,277, 20,280, 22,320, and 34,134, respectively (Table 2).\n\nAcross all the four different samples, including the three cellulose cultures and the one uncultured termite gut microbial sample, at least 99.98% of all the prokaryotes identified were bacteria, which have become the focus of subsequent analyses.\n\nAs shown in Figure 1, at the phylum level, the bacterial flora in the uncultured Termite_EW population is much higher than each of the three selective cellulose cultures. Even for the phyla found in both uncultured and cultured samples, the relative proportion of each phylum is different, showing the enrichment of certain phyla compared to others. For example, while Firmicutes is the most abundant group in the uncultured termite gut community, it represents only the second most abundant group in cellulose-degrading microbes while the Proteobacteria are the most abundant in all the three cellulose cultures.\n\nThe two most abundant phyla of cellulose-degrading bacteria were further analyzed in details and compared to those in the uncultured termite sample. Supplementary Figure 1 shows that while Delta-proteobacteria are the most abundant group of Proteobacteria in the uncultured termite sample, the Gamma- and Beta-proteobacteria are the most abundant in the cellulose cultures. However, while Beta-proteobacteria dominated the cellulose-degrading Proteobacteria in the Termite_AX culture, the Gamma-proteobacteria was the most dominant group of the same phylum in both the termite and soil samples collected at a different (EW) site. For Firmicutes, while Clostridia is the most dominant group in the uncultured sample, it was almost nonexistent in all the three cellulose selective cultures, which contained Bacilli as the almost exclusive group of Firmicutes (Supplementary Figure 2).\n\nThe bacterial community composition at the class level for each sample was summarized in Supplementary Figure 3, while Supplementary Figure 4 shows the abundance of each class of at least 1% in abundance in each sample (not including the unclassified classes). Again, at the class level, the uncultured termite sample is much more diverse and dramatically different from all the three cultures in composition, and while the termite and soil cultures from the same location (EW) were very similar in the composition of cellulose-degrading bacteria, both of them are very different from the other termite gut microbial culture (AX).\n\nThe charts in Figure 2 show the relative abundance of each taxonomic group at different levels from the phylum to the genus. As immediately apparent from the comparison, the uncultured termite gut sample contained the highest level of diversity with more balanced representation of different taxa, and as expected, the cellulose-degrading bacteria from the selective cultures represent only a proportion of the biodiversity in the whole gut microbiota. Again, the similarity of termite and soil cultures from the same location was clear while the other termite cellulose culture is significantly different in composition and dramatically reduced in diversity.\n\nA. Uncultured Termite_EW sample; B. Cultured Termite_EW sample; C. Cultured Termite_AX sample; D. Cultured Soil_EW sample.\n\nTo quantify the alpha diversity within each sample, seven different measures including Observed, Chao1, ACE, Shannon, Simpson, InvSimpon, and Fisher diversities were calculated. The results, particularly the Shannon and Simpson indices, show that again the uncultured termite sample contains the highest level of bacterial diversity and the termite and soil samples from the same location (EW) are very similar in alpha diversity (Figure 3). All the measures also consistently show that the Termite_AX cellulose culture contains the lowest diversity of bacteria.\n\nTo mitigate the effect of varying numbers of sequence reads across samples on the comparison of diversity, all the four datasets were rarefied to the lowest number of sequence reads in any sample. The subsequent principal component analysis still shows the termite and soil samples from the same EW location were very similar to each other, while each of the other two samples (the Termite_AX culture and the uncultured Termite_EW) were very different (Supplementary Figure 5). The same pattern is observed in clustering analysis (Figure 4), which also shows the three cellulose cultures are overall more similar to each other than they are to the uncultured termite sample.\n\nThe principal coordinate analysis (PCoA) based on weighted UniFrac values using the results from the RDP unsupervised method and the phyloseq (Figure 5) and GUniFrac (Supplementary Figure 6) packages revealed a very similar pattern in terms of the similarity between the cultured termite and soil samples from the same location and their significant differences from each of the other two samples.\n\nTo examine the distribution of taxa among the cellulose selective cultures, a tree plot was created for the 20 most abundant OTUs with each taxon’s abundance in each sample. As shown on the tree (Supplementary Figure 7), Burkholderia and Dyella are the two most abundant genera, each with a number of lineages probably representing different species. However, not only these two genera are phylogenetically very distant from each other, their distributions were also almost opposite to each other. While Dyella was predominantly found in the termite and soil samples from the same location (EW), Burkholderia was mostly found in the other termite sample (AX). In fact, some Burkholderia lineages were exclusively found in the latter. Some other important genera in terms of abundance included Citrobacter and Microbispora, which were largely found in the soil sample, and Fontibacillus, which was found exclusively in the termite sample from the same location.\n\nNext, we tried to identify the shared as well as sample-specific cellulose-degrading taxa among the three cultured samples, while comparing to the taxa found in the uncultured termite sample at the same time. As shown in Figure 6, while each of the three selective cultures contained its own specific taxonomic groups, a significant proportion of the taxa at the genus level were shared between at least two samples, including 18 genera shared between the two termite cultures and 20 shared between the soil and termite from the EW location. In particular, there were 13 genera shared among all the three different cellulose cultures, defining a core set of cellulose-degrading bacteria. The taxonomic classification of these taxa was listed in details in Supplementary Table 1. However, the relative abundance of each of these taxa is different in different samples, as partially revealed in Supplementary Figure 7.\n\nThe number at the lower right corner indicates the number of genera found in the uncultured Termite_EW sample but not in any of the three cultured samples.\n\nThe percentage of archaeal sequence reads found in each sample was 0.004%, 0.008%, 0.002, and 0.02% in the uncultured Termite_EW, the Termite_EW culture, the Termite_AX culture, and the Soil_EW culture, respectively. The uncultured Termite_EW sample contained two reads, one classified as Crenarchaeota and the other Pacearchaeota, the Termite_EW cellulose culture also contained two sequence reads both classified as Crenarchaeota, and the Termite_AX culture contained only one read of archaea classified as Euryarchaeota. In contrast, the highest level of abundance and diversity was found in the Soil_EW culture, which contained 7 reads in both Crenarchaeota and Euryarchaeota phyla of archaea (Supplementary Table 2).\n\n\nDiscussion\n\nIdentifying the cellulose degrading microbes is not only important for the understanding of the nutrient cycling in the ecosystem, particularly in soil, but also for the understanding of food digestion in animals, including termites. It will also help to develop the next-generation bioethanol technology based on the vastly abundant cellulosic plant materials, although this requires additional technological advances as the authors are fully aware. The present study tried to characterize the cellulose degradation related prokaryotes from termites and soil samples, and the culture condition has allowed the identification of a variety of bacteria, including the aerobes and facultative anaerobes, which may prove to be more useful for next-generation bioethanol production under similar conditions.\n\nPrevious studies of whole gut microbiota in the lower termite species Reticulitermes speratus2 and the wood-feeding higher termite species Nasutitermes corniger11 found the dominance of Spirochaetes, particularly Treponema. Our study, however, shows that Proteobacteria are the most dominant group in cellulose degradation. Similarly, previous studies suggested Firmicutes, particularly Clostridia, as the other most abundant bacterial group in termite gut community. The uncultured termite gut in our study indeed showed Firmicutes to be the most dominant in the whole microbiota, but in all the three cellulose selective cultures they consistently appeared to be secondary in abundance (Figure 1), and class-level analysis (Supplementary Figure 2) revealed it was not Clostridia, but Bacilli, to be the dominant and almost exclusive group of Firmicutes involved in cellulose degradation. Altogether, it suggests that while direct termite gut microbiota studies are useful in characterizing the whole microbial community, one should be very careful in using the result to interpret the roles of various bacteria in the cellulose degradation process itself.\n\nWithin the most dominant group of bacteria in cellulose cultures, different classes of Proteobacteria dominated the different cultures, which surprisingly depended not on the type of sample (soil versus termite) but on the location of the collection site. As shown in Supplementary Figure 1, Gamma-proteobacteria dominated the cellulose cultures of the soil and termite samples collected at the same location while Beta-proteobacteria is the most dominant group in the cellulose culture of the other termite sample collected at a different location. The dominance of Gamma-proteobacteria and Bacilli at the class level in very similar proportion are major contributors to the overall similarity in cellulose-degrading bacterial community in the termite and soil samples collected at the East Wood site as revealed by the various analyses in our study.\n\nAt the genus level, 88% of the sequence reads from the cellulose culture of the Termite_AX sample belonged to one genus, Burkholderia, though several other genera also existed in the community. This genus contains members that are human pathogens, plant pathogens, plant symbionts, as well as fungal symbionts26. They are common soil inhabitants but their distribution is strongly affected by soil pH27,28. A number of studies also demonstrated that members of this genus have also formed symbiotic relationships with many insect species29,30, including a very tight association with the bean bug Riptortus pedestris31. A related study suggested the symbiotic Burkholderia was environmentally transmitted to the host R. pedestris32. These studies, together with the finding from our study that termite cellulose-degrading microbiota is strongly influenced by that of soil, suggest members of Burkholderia in the Termite_AX sample might have come from the soil, while their absence in the termite and soil samples of the East Wood site may be explained by different pH of the soil and historical processes. This genus has also been recognized to have tremendous biotechnological potential to produce a large variety of commercially important hydrolytic enzymes and bioactive substances26,33,34.\n\nIn the two cellulose cultures of the termite and soil samples from the East Wood site, Dyella emerged as the most dominant group at the genus level (with 36% and 28% relative abundance in the two samples, respectively). Various species of this genus, in the class of Gamma-proteobacteria, have been isolated from soil in a variety of habitats35–40. Members of this genus were also discovered from the lower termite species R. speratus41, as well as wood-feeding huhu beetle Prionoplus reticularis42. The discovery of Dyella species in a variety of soil habitats, termite gut, and wood-feeding beetles strongly suggest their involvement in cellulosic plant material degradation. In fact, both Dyella and Burkholderia have been shown to be metabolically active in the wood-feeding huhu beetle42.\n\nThe other major genera of bacteria found in the termite/soil samples from the East Wood site include Paenibacillus, Ammoniphilus, Citrobacter, and Fontibacillus. While Paenibacillus (25%) and Fontibacillus (8%) were very abundant in the Termite_EW culture, Ammoniphilus (26%) and Citrobacter (14%) were very prominent in the Soil_EW culture with Paenibacillus (5%) taking a minor role. Members of the genus Paenibacillus have been isolated from both soil43–45 and termite gut46,47. Studies have shown that species produce a variety of enzymes involved in lignocellulose degradation48–52. Fontibacillus has been discovered in soil53,54 but not in termite before, the discovery of this genus in cellulose culture of termite from the East Wood site represents a new finding. Citrobacter has been discovered in soil before55 but their role in cellulose degradation was previously unknown. Existing study on Ammoniphilus is even rarer, so the discovery of this genus in the soil sample and its involvement in cellulose degradation probably was very interesting and deserving further detailed studies.\n\nOur study also identified a core set of bacteria shared by all three cellulose cultures, which includes thirteen members at the genus level (Figure 6, Supplementary Table 1). However, the abundances of individual taxa in the three cultures vary significantly. While the abundance of each taxon in each of the three samples might have been affected by the competition and bias in the PCR process to some degree, their presence across all three cellulose cultures per se might be significant. Though, one should be aware that not all the members of the core cellulose-degrading microbiota are equally important, and nor are all of them more important than habitat-specific cellulose-degrading taxa.\n\nThe biochemical analyses of the cellulose cultures, however, showed the community of microbes in each of the cultures had worked very efficiently to break down cellulose. The community composition, as well as the major taxa in each sample and shared taxa among the samples, may be useful to develop cellulose-based production of biofuel or other valuable molecules. Upon further studies, the genes/enzymes extracted from these microbes could be used for genetic engineering or directly used for cellulose treatment, a key step in processing cellulose for a variety of biotechnological developments.\n\nThe design of this project made possible comparisons in various ways, including uncultured vs. cultured termite gut microbiomes, comparison between different termite colonies, as well as between termite and soil samples collected at the same location. The uncultured termite gut sample contained much higher bacterial diversity than the same sample that had been selectively cultured with cellulose medium, or any cellulose selective culture. This is consistent with the expectation that only a subset of the microbes in the termite gut community actually breaking down cellulosic plant materials. It also highlights the importance to use cellulose selective cultures to identify the cellulose-degrading microbes and separate them from other cohabitants.\n\nAmong all the three selective cultures, the soil sample had the highest level of diversity in cellulose-degrading bacteria, higher than both termite gut microbial cultures. This suggests that soil in the natural habitats, as a much larger ecosystem than the termite digestive system, serves as a reservoir of cellulose-degrading bacteria for decomposition and nutrient cycling. This should not be surprising given the vastness, historical continuity, and complexity of the natural forest ecosystems.\n\nThe cluster dendrogram, PCA, PCoA analyses all revealed two interesting patterns: (i) the two termite colonies collected at different locations harbored very different communities of cellulose-degrading bacteria; (ii) the termite and soil samples collected from the same location contained very similar compositions of cellulose-degrading bacterial communities. In short, the cellulose-degrading bacterial flora in termite gut was much more similar to that in the soil from the same location than to the other termite colony from a different location. This suggests that the microbiota composition, at least that of cellulose-degrading bacteria, in the gut of the eastern subterranean termite is strongly influenced by its living environment, i.e., soil.\n\nHowever, previous studies have offered different hypotheses on this question. A comparative study of the microbiota in two higher termite species, the dung-feeding Amitermes wheeleri and the wood-feeding N. corniger, using a combination of metagenomic and metatranscriptomic analysis suggested the microbial community composition and functional divergence were consistent with the dietary differences of the two species11. But a follow-up study of multiple genera of both higher and lower termites in Australia and North America7 inferred that vertical inheritance was the primary force shaping termite gut microbiomes based on the observed similarity between the termite phylogeny and the clustering pattern of their gut microbiomes from the analysis when not taking OTU abundance into account. However, when the OTU abundance was taken into consideration, the study did show the correlation between termite phylogeny and their microbiome similarity was reduced, suggesting the role of diet in shaping the termite microbiomes. A separate study13, however, suggested diet was clearly the primary determinant of bacterial community structure in the guts of higher termites. A related study of six species of higher termites feeding on wood, litter, humus, or soil revealed that the gut microbial community structure was correlated with the corresponding habitat56. A third study12 demonstrated that the bacterial communities changed throughout the life stages of higher termite Nasutitermes arborum as a result of dietary changes. The study also suggested that wood-degrading symbionts were gradually introduced in later developmental stages. The present study provides strong evidence that the cellulose-degrading bacterial community in the termite gut has largely been shaped by the one in the soil where subterranean termites live in, though the process to build this functional microbial community may have been gradual.\n\n\nConclusions\n\nOur study characterized the whole microbiota from a colony of the eastern subterranean termite (R. flavipes) and cellulose-enriched microbiota from the same termite colony, from the soil sample collected at the same site, and a second termite colony collected at a different site. The study demonstrates that the cellulose-enriched microbiota is significantly different from the whole microbiota in the termite digestive system, and indicates the importance to use selective cellulose culture to study the cellulose-degrading microbial community. Comparative analyses of the termite colony with the soil sample collected at the same site and the second termite colony from a different site reveals the termite and soil samples from the same location are much more similar to each other for cellulose-degrading bacteria, revealing the interrelation between the two. Biochemical analyses revealed that the microbial communities in all the three cultures were efficient in degrading cellulose. A core set of cellulose-degrading bacteria has been identified among the three cultured samples, which deserves further and detailed functional study.\n\n\nData availability\n\nThe 16S rRNA gene marker sequences generated from and used in this study have been deposited with NCBI under accession numbers SAMN07414062 to SAMN07414065.\n\nThe raw data for the biochemical analysis of remaining cellulose after the culture is available on OSF: http://doi.org/10.17605/OSF.IO/RZCBX57\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe research reported in this publication was partially supported by a U.S. Department of Defense grant (Award No. W911NF-14-1-0170, to Xianfa Xie), a U.S. Department of Education grant (Award No. P382G090018, to the Virginia State University), and a U.S. National Science Foundation grant (Award No. HRD-1036286, to the Virginia State University).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nSequencing was performed in the Genomics Core of the Nucleic Acids Research Facilities at the Virginia Commonwealth University.\n\n\nSupplementary material\n\nSupplementary Figure 1: Composition of Proteobacteria at the class level in the four samples.\n\nClick here to access the data.\n\nSupplementary Figure 2: Composition of Firmicutes at the class level in the four samples.\n\nClick here to access the data.\n\nSupplementary Figure 3: Comparison of the four samples at the class level.\n\nClick here to access the data.\n\nSupplementary Figure 4: The abundance of each class of at least 1% in each sample. The figure does not include the unclassified classes.\n\nClick here to access the data.\n\nSupplementary Figure 5: Principal component analysis (PCA) of the four samples.\n\nClick here to access the data.\n\nSupplementary Figure 6: Principal coordinate analysis (PCoA) using the phyloseq package based on weighted UniFrac values.\n\nClick here to access the data.\n\nSupplementary Figure 7: Distribution of the 20 most abundant cellulose-degrading OTUs in each of the cultured samples.\n\nClick here to access the data.\n\nSupplementary Table 1: Bacterial taxa shared among all three cellulose cultures. The numbers indicate the number of reads found in each sample.\n\nClick here to access the data.\n\nSupplementary Table 2: Archaean taxa in all samples. The number indicates the number of read(s) for each taxon.\n\nClick here to access the data.\n\n\nReferences\n\nBreznak JA: Phylogenetic diversity and physiology of termite gut spirochetes. Integr Comp Biol. 2002; 42(2): 313–318. 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Data Source" }	[ { "id": "40167", "date": "05 Nov 2018", "name": "Swapna Priya Rajarapu", "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\nOverview: The authors test the hypothesis that there is a relationship between the cellulose degrading bacteria in soil and ground dwelling termites such as eastern subterranean termite, Reticulitermes flavipes. The authors study the cellulose degrading bacterial profiles in two termite colonies and the soil samples corresponding to one of the colony. Microbiota groups present in the samples were identified using 16S rRNA bacterial marker. Results supported the hypothesis that there is a tight correlation between the cellulose degrading bacteria in soil and termite gut. Also, there was a significant difference between the bacterial profiles between two different termite colonies. Overall comments: Authors fail to describe the rationale for the experimental design. Particularly, the reason for sampling soil from only one termite colony but not two is not explained well. If the differences between the two termite colonies are attributed to the difference in the soil where the colony exists, then sampling the soil sample from the second colony would have provided clear evidence. Moreover, the experimental design is not balanced. Why wasn’t the termite-AX colony uncultured gut extract sequenced with 16S rRNA? Would the authors expect the uncultured termite guts from both the colonies cluster together? Authors also did not refer to the R. flavipes bacterial studies performed earlier in the following 2 citations 1 - 2 .\nA comparison of the microbial groups to these studies would be insightful as the termites used in the above mentioned studies used lab reared colonies. By addressing the above concerns the article will be complete and much stronger.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7719", "date": "10 Jun 2022", "name": "Xianfa Xie", "role": "Author Response", "response": "Thanks for your comments. Sorry if the experimental design was not very clear to you in the original manuscript. An explicit explanation of the experimental design has been added to the revised manuscript, together with a new figure (Figure 1) to explain this design. We have also included the two references you suggested in the revision. Hopefully this has addressed your original concerns." } ] }, { "id": "40805", "date": "12 Dec 2018", "name": "Xing-Feng Huang", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study has offered some potential values on characterization of cellulose-degrading microbiota from termite guts and soil.\nHowever, it lacks the basics of experimental design. Two termites were selected, but only one soil sample was included, and also only one termite gut sample was included. The major issue was no replicates (biological and technical) in this study. All results, discussion, and conclusion were from one sample which does not make much sense in a scientific publication. I doubt about how much the conclusion was reliable based on 1 data point from each sample.\n\nTherefore, I do not think this manuscript can be indexed as a research paper.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6183", "date": "03 Aug 2021", "name": "Xianfa Xie", "role": "Author Response", "response": "Thanks for your comments. Sorry if the experimental design was not very clear to you in the original manuscript. An explicit explanation of the experimental design has been added to the revised manuscript, together with a new figure (Figure 1) to explain this design. Hopefully this has addressed your original concern." } ] } ]	1	https://f1000research.com/articles/6-2082
https://f1000research.com/articles/10-417/v1	25 May 21	{ "type": "Research Article", "title": "Anesthesia modality does not affect clinical outcomes of intra-arterial vasodilator treatment in patients with symptomatic cerebral vasospasms", "authors": [ "Corinne Fischer", "Sonja Vulcu", "Johannes Goldberg", "Franca Wagner", "Belén Rodriguez", "Nicole Söll", "Pasquale Mordasini", "Matthias Haenggi", "Joerg C. Schefold", "Andreas Raabe", "Werner J. Z'Graggen", "Sonja Vulcu", "Johannes Goldberg", "Franca Wagner", "Belén Rodriguez", "Nicole Söll", "Pasquale Mordasini", "Matthias Haenggi", "Joerg C. Schefold", "Andreas Raabe", "Werner J. Z'Graggen" ], "abstract": "Background: Delayed cerebral ischemia and cerebral vasospasm remain the leading causes of poor outcome in survivors of aneurysmal subarachnoid hemorrhage. Refractory cerebral vasospasms can be treated with endovascular vasodilator therapy, which can either be performed in conscious sedation or general anesthesia. The aim of this study is to compare the effect of the anesthesia modality on long-term clinical outcomes in patients undergoing endovascular vasodilator therapy due to cerebral vasospasm and hypoperfusion. Methods: Modified Rankin Scale (mRS) scores were retrospectively analyzed at time of discharge from the hospital and six months after aneurysmal subarachnoid hemorrhage. Additionally, National Institutes of Health Stroke Scale (NIHSS) was assessed 24 hours before, immediately before, immediately after, and 24 hours after endovascular vasodilator therapy, and at discharge and six months. Interventional parameters such as duration of intervention, choice and dosage of vasodilator and number of arteries treated were also recorded. Results: A total of 98 patients were included in this analysis and separated into patients who had interventions in conscious sedation, general anesthesia and a mix of both. Neither mRS at discharge nor at six months showed a significant difference for functionally independent outcomes (mRS 0-2) between groups. NIHSS before endovascular vasodilator therapy was significantly higher in patients receiving interventions in general anesthesia but did not differ anymore between groups six months after the initial bleed. Conclusion: This study did not observe a difference in outcome whether patients underwent endovascular vasodilator therapy in general anesthesia or conscious sedation for refractory cerebral vasospasms. Hence, the choice should be made for each patient individually.", "keywords": [ "aneurysmal subarachnoid hemorrhage", "Nimodipine", "Papaverine", "delayed cerebral ischemia", "general anesthesia", "conscious sedation", "functional outcome", "hypoperfusion" ], "content": "Introduction\n\nCerebral vasospasms (CVS) and delayed cerebral ischemia still remain among the leading causes of morbidity and mortality in survivors of aneurysmal subarachnoid hemorrhage (aSAH). Up to 40% of aSAH patients experience symptomatic CVS, resulting in disability in up to 50% thereof.1 CVS, a narrowing of cerebral arteries thought to be caused by blood breakdown products, mostly develop between 5 to 14 days after aSAH.2 So far, there is no therapy known, which was shown in randomized trials to improve cerebral perfusion and thus to avoid brain ischemia and infarction in symptomatic patients. Commonly used rescue treatments for symptomatic CVS include induced hypertension, and in refractory CVS angioplasty or intra-arterial application of vasodilators, e.g. nimodipine or papaverine.1,3,4 Both have been shown in case series to improve neurological outcome in said patients.5–7\n\nIn recent years, several studies investigated the best method of anesthesia for endovascular treatment in acute ischemic stroke.8–20 While initially in mostly retrospective studies, data showed conscious sedation (CS) to be superior,8,9 a recent meta-analysis showed no significant difference in outcomes for CS and general anesthesia (GA)17 if only randomized controlled trials were considered.15,16,18 To the best of our knowledge, no studies comparing CS and GA in endovascular treatments for refractory CVS after aSAH have been performed.\n\nThe aim of this study is to compare six-month outcomes for choice of sedation in patients treated with endovascular vasodilators for CVS after aSAH.\n\n\nMethods\n\nThis is a single-center retrospective case-control study analyzing clinical outcomes in patients with symptomatic CVS after aSAH treated with endovascular vasodilators at the University Hospital Bern, Bern, Switzerland.\n\nThe University Hospital Bern conducts a prospective database for patients treated with aSAH. This database was retrospectively searched for patients hospitalized between September 2011 and October 2019. Only patients aged >18 and <85 years were included. Inclusion criteria were: 1) aSAH of all severities (World Federation of Neurosurgeons (WFNS) score I–V), 2) secured aneurysm either by endovascular or surgical treatment, 3) refractory CVS treated by intra-arterial admission of either nimodipine and/or papaverine. Exclusion criteria were: 1) incomplete data, 2) loss of follow up, 3) continuous intra-arterial nimodipine treatment, 4) re-rupture of aneurysm during the hospital stay.\n\nPatients were divided into three treatment groups: patients who underwent treatment with endovascular vasodilators in CS only (“CS”), in GA only (“GA”), or patients who received intra-arterial treatments in CS and GA (“both”).\n\nAll data was acquired from patient records and the institutional electronic Patient Data Management System (CentricityTM Critical Care, General Electric Company, GE Healthcare, United States of America). Vital signs are automatically recorded and the bedside team additionally enters clinical scores and administered drugs into the system.\n\nThe primary endpoint of this study was functional outcome at six months, analyzed by the modified Rankin Scale (mRS). Secondary outcome parameters included mRS at discharge and National Institutes of Health Stroke Scale (NIHSS) assessed 24 hours before the (first) intra-arterial vasodilator treatment (t1), directly before (t2), directly after (last) treatment (t3), 24 hours after (last) treatment (t4) and consecutively at discharge from the hospital (t5) and after six months (t6). Further parameters consisted of interventional parameters such as duration of intervention, choice of vasodilator (nimodipine or papaverine), number of treated arteries and vasodilator dosage.\n\nPatient characteristics such as age, sex, aneurysm location and treatment and Barrow Neurological Institute (BNI), Fisher, Hunt & Hess and WFNS scores were obtained from institutional patient records.\n\nThe statistical analysis was performed using SPSS Statistics 21.0 (IBM, Armonk, NY, USA). The Shapiro-Wilk normality test was used to test for normal distribution.\n\nUnivariate Analysis of Variance (ANOVA) test was used to compare “CS”, “GA” and “both” groups for differences in age. Differences in sex and aneurysm treatment were explored with Pearson Chi Squared analysis.\n\nFor mRS at discharge and six months, outcomes were divided in functionally independent (mRS 0-2) and functionally dependent (mRS 3-6). A Chi Squared test was used to test for significant group differences between CS, GA and both. An additional subgroup analysis was performed using a Chi Squared test with “CS” and “GA” groups divided into single versus multiple interventions, resulting in five groups (“single CS”, “single GA”, “multiple CS”, “multiple GA”, “both”).\n\nFor the analysis of NIHSS, a 3 × 6 analysis of variance (ANOVA) for repeated measures with post hoc Bonferroni correction for multiple comparisons was conducted. The factors were (i) treatment (“CS”, “GA” and “both”) and (ii) time (t1 – t6).\n\nInterventional parameters were analyzed for each intervention separately and therefore compared between those performed in CS and GA. For the duration of the intervention, a Welch's two sample t-test was performed. The choice of vasodilator was analyzed by Pearson Chi Squared test. Vasodilator dosage as well as number of treated arteries were analyzed with an independent samples t-test.\n\nData are presented as mean with standard deviation (SD) in brackets and in figures as mean with +1 SD as error bars. A p-value of p < 0.05 was considered statistically significant.\n\nThis study was carried out in accordance with the recommendations of the local ethics committee (Kantonale Ethikkommission Bern, Switzerland). All subjects gave written general consent in accordance with the Declaration of Helsinki. The protocol was approved by the local ethics committee (Kantonale Ethikkommission Bern, Switzerland).\n\n\nResults\n\nIn total, 109 patients with refractory CVS treated by intra-arterial admission of either nimodipine and/or papaverine between September 2011 and October 2019 at the University Hospital Bern, Bern, Switzerland were included. Of those, 11 patients had to be excluded. Reasons for exclusion were incomplete data (n = 2), re-rupture of aneurysm during the hospital stay (n = 2), continuous intra-arterial nimodipine treatment (n = 1) and loss of follow-up at six months (n = 6). The final study population consisted of 98 patients, 23 patients in the “CS” group, 53 patients in the “GA” group and 22 patients in the “both” group. In the “CS” group, 16 patients received a single intervention (“single CS”) and seven patients received up to five interventions (“multiple CS”). In the GA group, 26 patients received one intervention (“single GA”) and 27 patients received 2-10 treatments (“multiple GA”). As per definition, all patients in the “both” group received more than one and up to 10 interventions.\n\nTable 1 shows patient characteristics of the three anesthesia groups. Overall mean age was 54.7 years (range 24-81). All groups showed higher percentages of female patients. Age, sex, aneurysm treatment modality did not significantly differ between the three groups.\n\n# univariate ANOVA.\n\n+ Chi-squared test.\n\nmRS at six months is displayed in Figure 1a. There was a tendency for a slightly higher percentage of functionally independent patients (mRS 0-2) in the “CS” group (78.3%) at six months. However, this difference did not prove to be statistically significant (p = 0.109). The subgroup analysis comparing single and multiple intra-arterial interventions separately for each anesthesia modality is displayed in Table 2. This analysis also revealed no significant difference in functional outcome at six months between “single CS”, “single GA”, “multiple CS”, “multiple GA” and “both” groups (p = 0.089).\n\nNumbers represent the percentages for each mRS category per group. a) “CS”, “GA” and “both” at six-month follow up. b) “CS”, “GA” and “both” at discharge from hospital.\n\n+ Chi-squared test.\n\nFigure 1b shows mRS at discharge from hospital. This analysis displays no significant difference between “CS”, “GA” and “both” groups (p = 0.056). The subgroup analysis for single and multiple interventions separately also revealed no statistical significance (p = 0.156), as listed in Table 2.\n\nThe NIHSS time course analysis is presented in Figure 2. ANOVA for repeated measures displayed a significant interaction of “timetreatment” (p = 0.008) and of “time” (p < 0.001). Post-hoc analysis revealed that significant group differences only occur when comparing “GA” to the two other groups. All of these significant differences were between t1 and t5, meaning between 24 hours before (first) intervention and discharge from the hospital. At the six-month follow up appointment, “CS”, “GA” and “both” did not differ significantly regarding NIHSS.\n\nPatients were treated in conscious sedation (“CS”), general anesthesia (“GA”) or a combination of both (“both”). Each bar represents a different group. Each cluster represents a different time point. Time points are 24 hours before (first) intra-arterial vasodilator intervention (t1), immediately before (t2), immediately after (t3), 24 hours after (last) intervention (t4), at discharge from the hospital (t5) and at six-month follow up (t6). Data are presented as mean + 1 Standard Deviation (SD) as error bars. Significant inter-group differences are highlighted with asterisks. One asterisk represents p < 0.05, two asterisks represent p < 0.01 and three asterisks represent p < 0.001.\n\nInterventional parameters are displayed in Table 3. Overall, a total of 237 intra-arterial vasodilator treatments were performed, 65 of which were performed in CS and 172 in GA. Mean duration of intervention was significantly longer if performed in GA (p = 0.002). A total of four interventions had to be excluded from further analysis because of missing data about medication (n = 3) and number of treated arteries (n = 1). Neither choice and dosage of intra-arterial vasodilator, nor number of treated arteries showed significant differences between groups.\n\n+ Welch’s two sample t-test.\n\nx Chi-squared test.\n\n# Independent samples t-test.\n\n\nDiscussion\n\nThis retrospective study found no significant differences in functionally independent outcomes (mRS 0-2) six months after aSAH in patients who were treated with intra-arterial vasodilators in CS, GA or a combination of both. While NIHSS was significantly higher in patients undergoing endovascular therapy in GA compared to patients of the “CS” or “both” group in the time window 24 hours before intervention up to discharge from the hospital, this difference was no longer found at six months.\n\nTo the best of our knowledge, the effect of anesthesia modality (CS versus GA) on functional outcome has not yet been studied for intra-arterial admission of either nimodipine and/or papaverine in patients with refractory CVS after aSAH. Albeit, there have been several research papers published regarding anesthesia in aneurysm treatment.21–23 Most articles describe both CS and GA to be generally safe for treatment of unruptured aneurysms or aSAH with no clear recommendation for either one.\n\nAdditionally, multiple studies have had similar research questions in relation to the choice of anesthesia during endovascular therapy of acute ischemic stroke. Abou-Chebl et al. (2010) showed in their analysis of the “North American SOLITAIRE Stent-Retriever Acute Stroke” (NASA) registry, that patients treated in GA experienced poorer neurologic outcome at 90 days and higher mortality rates than patients treated in CS.8 Berkhemer et al. (2016) reported similar results in a post-hoc analysis of a prospective trial.15 Correspondingly, a recent analysis of the “Endovascular Therapy Following Imaging Evaluation for Ischemic Stroke 3” (DEFUSE 3) trial by Powers et al. (2019) showed higher rates of functional independence (mRS 0-2) and a lower NIHSS score at 24 hours for patients treated in CS. At discharge they did not find a statistically significant difference in NIHSS scores anymore.11\n\nIn contrast, most recent randomized controlled trials found a non-inferiority of GA when compared to CS. Hendén et al. (2017) reported no difference in outcome at three months or NIHSS after 24 hours in their Anesthesia During Stroke (AnStroke) Trial.16 Schönenberger et al. (2016) and Simonsen et al. (2018) report, that GA produced better 3-month outcomes, with the former even finding this result to be significant.18,19 Finally, a recent meta-analysis determined no significant difference between GA and CS if only randomized controlled trials were considered.17\n\nOur results are in line with these recent randomized controlled trials published for endovascular treatment in acute ischemic stroke. Similar to Schönenberger et al. (2016), Hendén et al. (2017), Simonsen et al. (2018) and Kim et al. (2019), we also found no significant difference in functional independency (mRS 0-2) at discharge or six months.16–19\n\nSimilar considerations regarding choice of anesthesia hold true in intra-arterial vasodilator therapy after aSAH and treatment for acute ischemic stroke alike. Disadvantages of GA may be a delay in treatment, hemodynamic changes and complications associated with intubation such as an increased risk for pneumonia.8 Disadvantages of CS may be procedural discomfort for patients, more difficult interventions because of patients’ movements, emergency conversion to GA or increased risk for aspiration.8 Many of the conceived disadvantages of GA have also been analyzed in prospective studies concerning ischemic stroke treatment. For example, Berkhemer et al. (2016) as well as Hendén et al. (2017) found no treatment delay in the GA group.15,16 Schönenberger et al. (2016) reported no difference in feasibility, safety and intra-interventional complication between the groups.18 They did however discover more postprocedural complications after GA such as delayed extubation and pneumonia. Different authors mention the possibility of blood pressure drops and decreased cerebral blood flow as possible additional complications of GA, which could potentially worsen outcomes because of an increase of the ischemic area.15 Others argue that GA on its own has a neuroprotective effect by lowering the neuronal oxygen need.24,25 Overall, the similar functional outcome in our study as well as in the above cited studies in ischemic stroke suggest that these factors are of minor relevance.\n\nThe major limitations of this study are its retrospective design and the single-center approach. A potential bias could lie in the choice of sedation for treatment. Even before intra-arterial vasodilator treatment, patients who would go on to receive treatments in GA showed significantly higher NIHSS scores when compared to the “CS” and “both” groups. This indicates that patients in clinically and neurologically worse conditions were more likely to be treated in GA. However, this bias was not reflected in our results, as we did not find a significant difference in mRS scores between the anesthesia groups at discharge or at six months and also no significant difference in NIHSS scores at six months. The “GA” group were therefore in an initially worse state but still managed to reach similar outcomes in the long-term clinical course. This suggests to an even greater degree, that GA will not negatively affect long term outcome in these patients.\n\nFurthermore, some subgroups consisted of a small number of patients. The results of this study will have to be replicated by a larger prospective trial in the future.\n\n\nConclusion\n\nOur preliminary results indicate that choice of anesthesia method does not negatively affect six-month outcome in aSAH patients who undergo intra-arterial vasodilator treatment for CVS. Treating physicians should therefore decide between CS and GA individually based on patient characteristics and circumstances.\n\n\nData availability\n\nDryad: Functional Outcome after intraarterial vasodilator therapy in CS vs GA, https://doi.org/10.5061/dryad.g4f4qrfq5.26\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\nBaggott CD, Aagaard-Kienitz B: Cerebral vasospasm. Neurosurg Clin N Am. 2014; 25: 497–528. PubMed Abstract \| Publisher Full Text\n\nvan Gijn J, Kerr RS, Rinkel GJ: Subarachnoid haemorrhage. Lancet. 2007; 369: 306–18. PubMed Abstract \| Publisher Full Text\n\nCho WS, Kang HS, Kim JE, et al.: Intra-arterial nimodipine infusion for cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage. Interv Neuroradiol. 2011; 17: 169–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLabeyrie MA, Gaugain S, Boulouis G, et al.: Distal balloon angioplasty of cerebral vasospasm decreases the risk of delayed cerebral infarction. Am J Neuroradiol. 2019; 40: 1342–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDabus G, Nogueira RG: Current Options for the Management of Aneurysmal Subarachnoid Hemorrhage-Induced Cerebral Vasospasm: A Comprehensive Review of the Literature. Interv Neurol. 2014; 2: 30–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndereggen L, Beck J, Z’Graggen WJ, et al.: Feasibility and safety of repeat instant endovascular interventions in patients with refractory cerebral vasospasms. Am J Neuroradiol. 2017; 38: 561–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHänggi D, Turowski B, Beseoglu K, et al.: Intra-arterial nimodipine for severe cerebral vasospasm after aneurysmal subarachnoid hemorrhage: Influence on clinical course and cerebral perfusion. Am J Neuroradiol. 2008; 29: 1053–60. PubMed Abstract \| Publisher Full Text\n\nAbou-Chebl A, Lin R, Hussain MS, et al.: Conscious Sedation Versus General Anesthesia During Endovascular Therapy for Acute Anterior Circulation Stroke. Stroke. 2010; 41: 1175–9. PubMed Abstract \| Publisher Full Text\n\nJumaa MA, Zhang F, Ruiz-Ares G, et al.: Comparison of safety and clinical and radiographic outcomes in endovascular acute stroke therapy for proximal middle cerebral artery occlusion with intubation and general anesthesia versus the nonintubated state. Stroke. 2010; 41: 1180–4. PubMed Abstract \| Publisher Full Text\n\nNichols C, Carrozzella J, Yeatts S, et al.: Is periprocedural sedation during acute stroke therapy associated with poorer functional outcomes? J Neurointerv Surg. 2010; 2: 67–70. PubMed Abstract \| Publisher Full Text\n\nPowers CJ, Dornbos D, Mlynash M, et al.: Thrombectomy with conscious sedation compared with general anesthesia: A DEFUSE 3 analysis. Am J Neuroradiol. 2019; 40: 1001–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakahashi C, Liang CW, Liebeskind DS, et al.: To tube or not to tube? The role of intubation during stroke thrombectomy. Front. Neurol. 2014; 5 AUG. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTalke PO, Sharma D, Heyer EJ, et al.: Republished: Society for neuroscience in anesthesiology and critical care expert consensus statement: Anesthetic management of endovascular treatment for acute ischemic Stroke. Stroke. 2014; 45. PubMed Abstract \| Publisher Full Text\n\nMolina CA, Selim MH: General or local anesthesia during endovascular procedures: Sailing quiet in the darkness or fast under a daylight storm. Stroke. 2010; 41: 2720–1. PubMed Abstract \| Publisher Full Text\n\nBerkhemer OA, Van Den Berg LA, Fransen PSS, et al.: The effect of anesthetic management during intra-Arterial therapy for acute stroke in MR CLEAN. Neurology. 2016; 87: 656–64. PubMed Abstract \| Publisher Full Text\n\nHendén PL, Rentzos A, Karlsson JE, et al.: General Anesthesia Versus Conscious Sedation for Endovascular Treatment of Acute Ischemic Stroke: The AnStroke Trial (Anesthesia during Stroke). Stroke. 2017; 48: 1601–7. PubMed Abstract \| Publisher Full Text\n\nKim C, Kim S-E, Jeon JP: Influence of Anesthesia Type on Outcomes after Endovascular Treatment in Acute Ischemic Stroke: Meta-Analysis. Neurointervention. 2019; 14: 17–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchönenberger S, Uhlmann L, Hacke W, et al.: Effect of conscious sedation vs general anesthesia on early neurological improvement among patients with ischemic stroke undergoing endovascular thrombectomy: A randomized clinical trial. JAMA - J Am Med Assoc. 2016; 316: 1986–96. PubMed Abstract \| Publisher Full Text\n\nSimonsen CZ, Yoo AJ, Sørensen LH, et al.: Effect of general anesthesia and conscious sedation during endovascular therapy on infarct growth and clinical outcomes in acute ischemic stroke a randomized clinical trial. JAMA Neurol. 2018; 75: 470–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFroehler MT, Fifi JT, Majid A, et al.: Anesthesia for endovascular treatment of acute ischemic stroke. Neurology. 2012; 79: S167 LP–S173. PubMed Abstract \| Publisher Full Text\n\nKan P, Jahshan S, Yashar P, et al.: Feasibility, safety, and periprocedural complications associated with endovascular treatment of selected ruptured aneurysms under conscious sedation and local anesthesia. Neurosurgery. 2013; 72: 216–20. PubMed Abstract \| Publisher Full Text\n\nLee CZ, Young WL: Anesthesia for Endovascular Neurosurgery and Interventional Neuroradiology. Anesthesiol. Clin. 2012; 30: 127–147. PubMed Abstract \| Publisher Full Text\n\nOgilvy CS, Yang X, Jamil OA, et al.: Neurointerventional procedures for unruptured intracranial aneurysms under procedural sedation and local anesthesia: A large-volume, single-center experience - Clinical article. J Neurosurg. 2011; 114: 120–8. PubMed Abstract \| Publisher Full Text\n\nHoffmann U, Sheng H, Ayata C, et al.: Anesthesia in Experimental Stroke Research. Transl Stroke Res. 2016; 7: 358–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSivasankar C, Stiefel M, Miano TA, et al.: Anesthetic variation and potential impact of anesthetics used during endovascular management of acute ischemic stroke. J Neurointerv Surg. 2016; 8: 1101–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFischer, et al.: Functional Outcome after intraarterial vasodilator therapy in CS vs GA. Dryad [dataset]. 2021. Publisher Full Text" }	[ { "id": "88125", "date": "13 Jul 2021", "name": "Roland Roelz", "expertise": [ "Reviewer Expertise Neurosurgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting preliminary study on the anesthesia modality for endovascular rescue therapies in patients with aneurysmal subarachnoid hemorrhage (aSAH). This single-center retrospective study included 98 aSAH patients (admitted in an 8-year period) selected for endovascular vasospasm therapy. Circa half of these patients were treated in general anesthesia, 25% were treated in conscious sedation and 25% had multiple interventions and both anesthesia modalities were applied. No differences in outcome (6-months mRS) were observed between these groups.\nCarefully interpreted and fully aware of the limitations associated with the study design and data collection, this study may indicate towards safety and non-inferiority of both general anesthesia and conscious sedation for endovascular interventions after aSAH. These findings are in keeping with the pertinent literature on anesthesiologic management of endovascular interventions for stroke.\nI thank the authors for sharing their first experience with conscious sedation as an alternative to general anesthesia for endovascular vasospasm therapy. I encourage investigating the clinical algorithm that triggers the choice for either method. 50% of patients treated in general anesthesia had admission WFNS grades 4 or 5 (i.e. poor grade aSAH) compared to 30% of patients selected for conscious sedation. A fact that may very well explain the presumed tendency towards better outcomes of the latter group. The rate of delayed infarction should have been reported. Given that safety of conscious sedation seems to apply to endovascular vasospasm therapy, future studies are warranted. They should follow a prospective and randomized design. Potential benefits of conscious sedation (i.e. more rapid return to neurological assessability after intervention, less complications of mechanical ventilation etc.) might be of high clinical relevance and could improve outcome of patients who develop clinical vasospasm.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6959", "date": "02 Aug 2021", "name": "Corinne Fischer", "role": "Author Response", "response": "The following text was added to the methods section of the manuscript: “Choice of anesthesia modality was made by the treating physician on an individual basis. However, according to institutional guidelines, GA was preferred in patients with impaired consciousness (GCS <=8) or insufficient swallowing.”" } ] }, { "id": "88709", "date": "13 Jul 2021", "name": "Giovanna Brandi", "expertise": [ "Reviewer Expertise Neurocritical care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a retrospective study.\nHow were the NIHSS extracted from the patients files? Is NIHSS performed routinely? If the patient was already intubated, how was NIHSS evaluated?\nWhile aneurysma location as well as Fisher score seem to be similar, WFNS/ H&H seem to differ between CS + GA (e.g. 52% vs 28% WFNS 1). Is there a statistical difference? If so, how do you explain the lack of association thereof with the outcome?\nPatients with high WFNS + Fisher scores commonly remain intubated in case of unsuccessful sedation-reduction trial. How many patients were already under GA before intra-arterial vasodilatation. Is there difference in delay between non-intubated patients to CS or GA intra-arterial vasodilatation?\nWhen mRS at 6 months is compared between CS and GA only (excluding both) irrespective of if they received a singular or multiple CS/GA statistical significance is reached. How do you explain this finding?\n\nIs the work 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": "6960", "date": "02 Aug 2021", "name": "Corinne Fischer", "role": "Author Response", "response": "In our institution, NIHSS is routinely assessed in stroke patients at least once a day as well as before and after interventions. In intubated patients, the NIHSS is determined after depth of sedation is decreased as much as clinically possible. The NIHSS assessment is performed according to the recommendation of the National Institute of Neurological Disorders and Stroke (https://www.ninds.nih.gov/sites/default/files/NIH_Stroke_Scale_Booklet.pdf). Most notably, intubated patients get a default score of 1 for verbally assessed categories such as orientation if they are unable to speak or write. Additionally, aphasia is assessed through writing. Dysarthria is not assessable in the intubated patient. The p-values for WFNS, Hunt&Hess, BNI and Fisher scores were added to Table 1. Only the WFNS and Hunt&Hess scores differed significantly between the groups. However, as it has been shown in two earlier publications cited below, WFNS 5 scores at admission are often over-estimated due to accompanying factors e.g. early seizures. In addition, if factors such as intubation, ventilation, sedation, muscle relaxation or insufficient pain stimuli inhibit said motor response, the WFNS grading can be over-estimated while the outcome is not necessarily impacted. Similar effects can alter Hunt&Hess gradings. In contrast the radiologically determined scores (BNI and Fisher) are not influenced by the above factors, which can at least partially explain that these two sores are not significantly different while the clinical ones are. (Fung et al., 2016; Fung et al., 2015). In addition, more severely ill patients corresponding to patients with higher WFNS and Hunt&Hess scores are more probable to undergo intra-arterial vasodilatator therapy in general anesthesia (see also below.) Twenty-two patients were already ventilated before the first intra-arterial vasodilator treatment (this corresponds to 37% of all patients who were intubated for the first intra-arterial vasodilator treatment). Unfortunately, due to the retrospective study design, the additional delay of intra-arterial vasodilatation therapy due to intubation was not assessable within the obtained documentation. Considering only mRS at 6 months and reducing the analysis to patients who were only treated with one modality (CS or GA), the outcome differed significantly between the two groups (Fisher exact p = 0.044). In our opinion this analysis has to be interpreted with caution as the number of patients undergoing intra-arterial vasodilator treatment in CS who showed an unfavorable outcome is very small (n = 5). Furthermore, if the above mentioned patients who remained intubated after the initial bleeding are excluded from this analysis, the result is no longer statistically significant (p = 1.000). This indicates that the difference in outcome of this additional analysis is probably mainly due to the initial disease severity and not due to the anesthesia modality chosen for intra-arterial vasodilator treatment." } ] } ]	1	https://f1000research.com/articles/10-417
https://f1000research.com/articles/10-723/v1	02 Aug 21	{ "type": "Case Report", "title": "Case Report: Pulmonary sarcomatoid carcinoma in a female patient from Nepal", "authors": [ "Anirudra Devkota", "Amrit Paudel", "Simit Sapkota", "Subash Pandit", "Aashish Baniya", "Amrit Paudel", "Simit Sapkota", "Subash Pandit", "Aashish Baniya" ], "abstract": "Sarcomatoid carcinoma of the lung is an uncommon subtype of non-small-cell lung cancer (NSCLC). Even in the early stages, pulmonary sarcomatoid carcinoma (PSC) has a dismal prognosis when compared to other kinds of NSCLC with a mean survival of 9–12 months and a five-year survival rate of around 20%. We present the case of a 68-year-old woman with a two-month history of shortness of breath and cough. Initial computed tomography (CT) scan showed features of interstitial lung disease with chronic obstructive airway changes. After 34 months, the patient’s condition worsened with newer complaints of sore throat and hemoptysis. A repeat CT scan showed a ∼49x38x59mm size lesion in the superior segment of the left lower lobe. A core needle biopsy was performed, which revealed tumor cells consisting of irregular tubules and sarcomatoid components. The patient was started on chemotherapy. Unfortunately, she succumbed to her disease. Our case highlights the aggressiveness of PSC.", "keywords": [ "aggressive", "carcinoma", "chemotherapy", "diagnosis", "pulmonary sarcomatoid." ], "content": "Introduction\n\nSarcomatoid carcinoma (SC) is a rare primary malignant tumor consisting of both carcinomatous and sarcomatous components. It can affect various organs and body parts including skin, bone, urinary tract, breast, pancreas, liver, and glands (Shen et al., 2013). Pulmonary SC (PSC) accounts for less than 1% of all lung cancers and is known to behave aggressively (Szkorupa et al., 2015; Travis et al., 2015). It is classified as pleomorphic carcinoma, giant cell carcinoma, spindle cell carcinoma, carcinosarcoma, and pulmonary blastoma. The mean age of diagnosis is 65-75 years and is more common in males, with smoking being the most common risk factor. Here, we report a case of PSC in a 68-year-old woman.\n\n\nCase report\n\nA 68-year-old Nepali housewife with a 30-cigarette pack per year smoking history presented initially with dyspnea and dry cough of two months. The patient also had a history of long-term exposure to bio-mass fuel. Physical examination revealed clubbing in bilateral fingernails. A chest X-ray was done, which showed bilateral basal region haziness and fibrosis. Computed tomography (CT) scan showed features of interstitial lung disease with chronic obstructive airway disease changes (Figure 1). A pulmonary function test (PFT) was also performed, which was within normal limits. Hence, the patient was treated for interstitial lung disease (ILD). This led to an improvement in her symptoms and her condition was stable for 34 months.\n\nAfter 34 months, the patient again started having a dry cough with dyspnea. She also had a few episodes of hemoptysis. She was noted to be hypoxic with an oxygen saturation of 88%. Compared to the previous scan at initial assessment, a contrast-enhanced CT scan of the chest revealed an approximately 49 × 38 × 59 mm size heterogeneously enhancing lesion in the superior segment of the left lower lobe (Figure 2), extending into posterior basal segment with cavitation and surrounding septal thickening, with consolidation and ground-glass opacity. Minimal left pleural effusion and multiple enlarged mediastinal and hilar lymph nodes were seen. Also, minimal pericardial effusion was revealed. A core needle biopsy of the left lower lobe mass was performed. Histopathological examination revealed moderately pleomorphic epithelial cells with an oval hyperchromatic nucleus; stroma consisted of highly pleomorphic oval to spindle-shaped cells along with multinucleated malignant cells (Figure 3). A diagnosis of PSC was made.\n\nThe circular inlet shows irregular tubules and arrow shows sarcomatoid component (20×).\n\nEpidermal growth factor receptor gene (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement testing was negative. Eastern Cooperative Oncology Group (ECOG) performance status of the patient was noted to be 2. The patient was not a candidate for surgical resection as it was stage IIIb (T2N3M0) disease along with multiple other comorbidities. She was started on weekly nab-paclitaxel (80 mg/m2) and carboplatin (target area under the curve 2). The patient’s programmed death ligand-1 (PDL-1) expression status was not known with the initial diagnosis.\n\nThe patient’s condition quickly deteriorated within 72 hours of first cycle of chemotherapy with the development of massive pleural effusion. Therefore, the patient was started on immunotherapy with pembrolizumab 200mg intravenous, as her VENTANA PD-L1 (SP263) assay report revealed 85% tumor cells. A few days post-immunotherapy, the patient went into hypoxic respiratory failure requiring intubation. She died after two days of treatment in the intensive care unit.\n\n\nDiscussion\n\nSC of the lung is an extremely rare and poorly differentiated NSCLC, containing a sarcoma-like characteristic (malignant spindle or giant cells) or sarcomatous element (neoplastic bone, cartilage, or striated muscle) constituting of around 1% of all primary lung malignancies (Franks & Galvin, 2010; Rajdev et al., 2018).\n\nPSC is seen predominantly in male smokers with a higher male-to-female ratio. It is usually observed in the age group between 56 to 74 with the average age of diagnosis being 66 years (Fishback et al., 1994; Ishida et al., 1990). The patient in our case also had a long history of smoking cigarettes and was diagnosed at the age of 68. SC of the lungs can manifest as a central or peripheral lesion, most often in the upper lobes. It grows by invading the bronchial tree, pulmonary parenchyma, and adjacent anatomical structures (mediastinum and chest wall) in the form of necrotic and hemorrhagic large masses that are round to bosselated, soft to solid, and often rubbery to stiff (Pelosi et al., 2010). In our case, the patient had a mass in the superior segment of left lower lobe extending into the posterior basal segment with cavitation and hemoptysis as presentation.\n\nSymptoms like cough, hemoptysis, chest pain, shortness of breath, fever, and weight loss are common. Hemoptysis occurs in about half of all cases of proximal or central tumors, whereas peripheral tumors may be asymptomatic or may present with chest pain (Kim et al., 2002; Ouziane et al., 2014). Interstitial lung disease can co-exist, as in our case, where the patient was exposed to bio-mass fuel for a prolonged period. The tumor's morphology varies greatly on gross examination, ranging from soft to hard, or rubbery in consistency. The sliced surface ranges from whitish gray to tan-yellow, with patches of hemorrhage and necrosis (Rajdev et al., 2018). In our case, the tumor was hard in consistency, poorly circumscribed, light brown colored tissue with patches of hemorrhage.\n\nThere are no paraneoplastic syndromes identified in PSC, despite the fact that they exist in 15–20% of small cell lung cancers and 5–8% of NSCLCs (Jia et al., 2010). Metastasis to skin, stomach, pancreas, esophagus, jejunum, rectum, kidneys, bones and adrenal glands, brain, and mandibular gingiva have been reported with PSC (Fernandes De Oliveira et al., 2013; Park et al., 2006). The treatment of PSC is as difficult as the diagnosis. SC, though believed to evolve from the NSCLC, behaves aggressively and presents with locally advanced or metastatic disease (Rajdev et al., 2018). The mainstay of treatment, particularly for localized tumors, is still radical surgical resection. Radiotherapy and chemotherapy are being used as adjuvant treatment or in instances where the patient is a poor surgical candidate (Fernandes De Oliveira et al., 2013). Similarly, the patient in our case was a poor surgical candidate due to stage IIIb (T2N3M0) disease and compromised ECOG performance status with multiple co-morbidities like interstitial lung disease, hypertension, dyslipidemia, and type 2 diabetes mellitus. She was given chemotherapy with carboplatin and nab-paclitaxel. Studies have shown that EGFR mutations can be found in 8.8% (Li et al., 2020) and ALK rearrangement in 3.5% of SC of lung patients (Chen et al., 2017). Our patient reported negative for both. Previous studies have reported that patients with PSC have a high rate of PD-L1 expression (Maneenil et al., 2018). According to Velchet et al., 69.2% (9/13) of patients tested positive for PD-L1 (Velcheti et al., 2013). Likewise, our patient was tested for PD-L1 of 85%. PSC has a poorer outcome than conventional NSCLC. SC has a 5-year survival rate of around 20%, relative to NSCLCs, which has a 5-year survival rate of 50% and the median survival time is three months (Fernandes De Oliveira et al., 2013).\n\nThere are several limitations of our study; one of them being the lack of yearly CT scans after initial diagnosis of ILD, which hindered early diagnosis of the disease and curative treatment with local therapy like surgery. Comprehensive genomic analysis at diagnosis would have helped us to better deliver targeted therapy early on. This can be a problem in resource-limited countries like Nepal.\n\n\nConclusion\n\nPSC is an unusual biphasic malignant neoplasm of the lung that has a poor prognosis compared to other NSCLCs. A timely diagnosis is of vital importance to begin curative therapy, including surgery, chemotherapy, and radiotherapy. Our case highlights the aggressiveness of the disease and the importance of comprehensive investigations with a high index of suspicion. It might coexist sometimes with ILD like in our case, which should not be overlooked. Our case also highlights the timely resulting of comprehensive genomic profiling like PD-L1 which could have helped our patient. We believe that reporting this case with its overall clinical course can further add to the body of knowledge of this rare disease and aid in the development of successful treatment.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient’s sister.\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\nChen X, Zhang Y, Lu J, et al.: Pulmonary Sarcomatoid Carcinoma with ALK Rearrangement: Frequency, Clinical-Pathologic Characteristics, and Response to ALK Inhibitor. Transl Oncol. 2017; 10(2): 115–120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernandes De Oliveira M, Conde Watanabe S, Patrícia Guilhermina De Andrade M, et al.: Sarcomatoid carcinoma of the lung with brain metastases Carcinoma sarcomatoide de pulmão com metástases cerebrais. J Bras Pneumol. 2013; 39(6): 753–756. Publisher Full Text\n\nFishback NF, Travis WD, Moran CA, et al.: Pleomorphic (spindle/giant cell) carcinoma of the lung. A clinicopathologic correlation of 78 cases. Cancer. 1994; 73(12): 2936–2945. PubMed Abstract \| Publisher Full Text\n\nFranks TJ, Galvin JR: Sarcomatoid Carcinoma of the Lung: Histologic Criteria and Common Lesions in the Differential Diagnosis. Arch Pathol Lab Med. 2010; 134(1): 49–54. PubMed Abstract \| Publisher Full Text\n\nIshida T, Tateishi M, Kaneko S, et al.: Carcinosarcoma and spindle cell carcinoma of the lung. J Thorac Cardiovasc Surg. 1990; 100(6): 844–852. PubMed Abstract \| Publisher Full Text\n\nJia J, Ren J, Gu J, et al.: Predominant sarcomatoid carcinoma of the lung concurrent with jejunal metastasis and leukocytosis. Rare Tumors. 2010; 2(3): e44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim HM, Shin BS, Song YW, et al.: A case of pulmonary carcinosarcoma with persistent mild fever. Korean J Intern Med. 2002; 17(1): 78–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi X, Wu D, Liu H, et al.: Pulmonary sarcomatoid carcinoma: progress, treatment and expectations. Ther Adv Med Oncol. 2020; 12: 1758835920950207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManeenil K, Xue Z, Liu M, et al.: Sarcomatoid Carcinoma of the Lung: The Mayo Clinic Experience in 127 Patients. Clin Lung Cancer. 2018; 19(3): e323–e333. PubMed Abstract \| Publisher Full Text\n\nOuziane I, Boutayeb S, Mrabti H, et al.: Sarcomatoid carcinoma of the lung: A model of resistance of chemotherapy. N Am J Med Sci. 2014; 6(7): 342–345. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark J-Y, Kim H-S, Zo J-I, et al.: Initial Presentation of Lung Sarcomatoid Carcinoma as a Metastatic Lesion in the Mandibular Gingiva. J Periodontol. 2006; 77(4): 734–737. PubMed Abstract \| Publisher Full Text\n\nPelosi G, Sonzogni A, De Pas T, et al.: Pulmonary sarcomatoid carcinomas: A practical overview. Int J Surg Pathol. Los Angeles, CA: SAGE Publications Sage CA; 2010; 18(2): pp. 103–120. PubMed Abstract \| Publisher Full Text\n\nRajdev K, Siddiqui AH, Ibrahim U, et al.: Sarcomatoid Carcinoma of Lung Presenting as Localized Bronchiectasis: A Case Report and Review of Literature. Respir Med Case Rep. 2018; 24: 143–146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen XY, Lin ZF, Lin Q, et al.: Pulmonary sarcomatoid carcinoma: A case report. Wspolczesna Onkologia. 2013; 17(2): 210–213. Publisher Full Text\n\nSzkorupa M, Bohanes T, Neoral C, et al.: Sarcomatoid carcinoma of the lung a case report. Klin Onkol. 2015; 28(1): 57–60. PubMed Abstract \| Publisher Full Text\n\nTravis WD, Brambilla E, Burke AP, et al.: Introduction to the 2015 World Health Organization Classification of Tumors of the Lung, Pleura, Thymus, and Heart. J Thorac Oncol. Lippincott Williams and Wilkins; 2015; (Vol. 10, Issue 9, pp. 1240–1242). PubMed Abstract \| Publisher Full Text\n\nVelcheti V, Rimm DL, Schalper KA: Sarcomatoid lung carcinomas show high levels of programmed death ligand-1 (PD-L1). J Thorac Oncol. 2013; 8(6): 803–805. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "91016", "date": "27 Aug 2021", "name": "Alina Basnet", "expertise": [ "Reviewer Expertise lung cancer", "prostate cancer", "renal cell carcinoma", "bladder cancer" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case of an aggressive variant of NSCLC. This is unique due to the underlying history of ILD and the management in general in resource-limited countries like Nepal.\n\nThe clinical course has been summarised. If they could specify if the patient received radiation or not at any time during the course of the treatment? if not, why was the low dose chemotherapy for carboplatin and paclitaxel chosen?\nCan the authors elaborate on the staging scans that were performed? was any CT abdomen, bone scan, or PET scan done?\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": "7117", "date": "03 Sep 2021", "name": "Anirudra Devkota", "role": "Author Response", "response": "Radiotherapy was planned but couldn't be performed as the patient rapidly deteriorated. Low dose chemotherapy was preferred because our patient was elderly, and also the general condition was poor. We couldn't perform the Bone scan and PET Scan as it was not available in our hospital. Diagnostic centers were also closed due to COVID lockdown. CT chest together with upper abdomen was done, but CT abdomen separately was not performed." } ] }, { "id": "93529", "date": "15 Sep 2021", "name": "Jian-Hong Fang", "expertise": [ "Reviewer Expertise The molecular mechanism underlying the tumor metastasis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 report on an uncommon but aggressive variant of NSCLC. The authors have in detail described the clinical course of the patients, which will benefit patients with this uncommon disease. However, more information should be provided. Specifically, after receiving nab-paclitaxel and carboplatin, the patient’s condition quickly deteriorated within 72 hours. And then, the patient was started on immunotherapy with pembrolizumab for a few days before her death. During these treatments, was any physical examination or diagnostic test conducted for this patient. If so, what were the results? This information will be helpful to reveal the cause for the rapid progress of the disease.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "118657", "date": "14 Mar 2022", "name": "Ramila Shilpakar", "expertise": [ "Reviewer Expertise cost effective therapies" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a nice case report by Devkota et al. highlighting the rare case and difficult treating scenarios in resource-limited settings.\n\nThe patient had hemoptysis, so why was Radiotherapy not the first choice both for disease control as well as hemoptysis? If the authors could explain this it will be helpful for further reference.\nThe patient’s condition quickly deteriorated within 72 hours of the first cycle of chemotherapy with the development of massive pleural effusion: What was done to improve the dyspnea due to massive effusion, this should be written in brief to give an idea to the readers. And why was immunotherapy started rather than other supportive methods to decrease effusion related deterioration in patient condition. Please make these points clear for the readers.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-723
https://f1000research.com/articles/10-292/v1	16 Apr 21	{ "type": "Opinion Article", "title": "eLabFTW as an Open Science tool to improve the quality and translation of preclinical research", "authors": [ "Michael Hewera", "Daniel Hänggi", "Björn Gerlach", "Ulf Dietrich Kahlert", "Daniel Hänggi", "Björn Gerlach" ], "abstract": "Reports of non-replicable research demand new methods of research data management. Electronic laboratory notebooks (ELNs) are suggested as tools to improve the documentation of research data and make them universally accessible. In a self-guided approach, we introduced the open-source ELN eLabFTW into our lab group and, after using it for a while, think it is a useful tool to overcome hurdles in ELN introduction by providing a combination of properties making it suitable for small preclinical labs, like ours. We set up our instance of eLabFTW, without any further programming needed. Our efforts to embrace open data approach by introducing an ELN fits well with other institutional organized ELN initiatives in academic research.", "keywords": [ "Quality Management", "Electronic Lab Notebook", "ELN", "Open Science", "Reproducibility", "Transparency" ], "content": "Introduction\n\nIn the light of reports of non-replicable research results, new ways to strengthen an open science approach for better transparency, reproducibility and traceability are needed.1 Reproducibility meaning, that following the proper formatted documentation, any other lab group with the necessary equipment and personnel should be able to obtain the same results of any experiment performed.2 An easy way to increase the legibility, tidiness, and overall quality of documentation, can be the introduction of an electronic laboratory notebook (ELN) in a research unit. Publishing the ELN entries in an unaltered toegether with the manuscript enhances open science by creating transparency and will ultimately support reproducibilty.3 Here, ELNs will help in the context of research data management to make the entire life cycle of data transparent and better integrate it in the documentation process.4 The pros and cons of using ELNs have been discussed for a long time. The main fields of discussion are usability, accessibility, costs, time effort and data safety.5,6 In our experience, the use of an ELN has many benefits towards traditional paper-based notebooks, but it is often a challenge to convince researchers of the advantages of using an ELN. We believe that the use of eLabFTW, and open source software, is a particularly good approach to overcome this hurdle and ensure high research data quality in small, preclinical research groups like ours.\n\neLabFTW is a lightweight general-purpose ELN. It consists of an Experiment and a Database section, whereas the Experiment section is designed to create and edit experimental documentation and link it to entries of the Database section. These can be set by the administrator to be any type of object (Chemicals, cell lines, equipment, etc.). These two sections are complemented by a Teams section, in which equipment can be booked in a scheduler and experiment templates can be shared. Finally, eLabFTW provides an extensive search tool.7\n\n\nAdvantages of ELN on experimental and lab management level\n\nAlthough there are some disadvantages in using ELNs,6,8 we believe that the advantages outweigh those by far. Especially in aspects regarding open science and reproducibility, data safety and availability, as well as transparency and as a tool for thorough standardization. Additionally, to those most important factors, there are other advantages in using ELNs: digital recording of research data makes it much easier for researchers to find their older data as well as data of other users. Also, the sharing of data is simplified, which makes discussing, cooperation and troubleshooting of experimental procedures much easier. ELNs contain time saving mechanisms, such as automatic import of data, the possibility of linking experiments and resources, and the usage of templates for standard experiments, which can additionally simplify the planning of experiments. Furthermore, most ELNs can also contribute to the simplification of lab management aspects as inventory management, sample tracking, communication, and digitalization of paperwork. Finally, in our experience data recording and documentation is done more carefully, when researchers have in mind, that their records are visible to others.\n\n\nHurdles of introducing ELN\n\nNowadays, most data in life science are digital data. Thorough recording, documentation and annotation of this data is difficult in a traditional paper laboratory notebook. Using ELNs will not only ensure the data to be recorded in an efficient and comprehensive way but will also facilitate findability and reusability. Additionally, the possibility to create templates, standard operating procedures (SOPs) and predefined workflows in ELNs is time saving in routine lab work and sharing of knowledge. In combination with the search function, an ELN can become a pool of knowledge for the lab group and even for people outside of it.4\n\nHowever, as discussed in other sources, the overall adoption of ELNs is rather low in the academic field, as there are many perceived disadvantages in the use of ELN, as well as hurdles that must be overcome. These hurdles include the costs of obtaining the software and maintaining the necessary infrastructure, the time needed to implement the ELN and adapt the lab habits, as well as the fear of data being stored in a cloud on a foreign server.6 Another point that is important when working with primary samples of patient data is the protection of personal data (e.g., patient information) from unauthorized access.6 Other scientists fear that the software will be discontinued in the future, making it necessary to search for a new ELN, with the uncertainty whether the data from the previous system can still be accessed and imported to the new system.6\n\n\neLabFTW has a combination of features making it a powerful tool to overcome hurdles\n\nThe open-source ELN eLabFTW (https://www.elabftw.net/) was developed to provide lab groups with a free to use ELN. The developers of eLabFTW being researchers themselves, provided a general purpose ELN with limited features, but a powerful database and search function. Making it easy to learn and use, yet very suitable for all kinds of research fields. It consists of an Experiment section, used for documenting experiments with a simple built in text editor and a Database section, which can be used to create entries for all kinds of items like plasmids, enzymes, primers, etc. by using the same text editor. Furthermore, these items can be imported automatically via comma separated value (CSV) files by the admin. Subsequently, these items can be directly referenced from the experiment section. Experiments themselves also can be linked to other experiments, making it easy to keep a seamless documentation of projects and experiments. eLabFTW is built according to the open data principle, providing open-source code, as well as providing on board functions to easily share experiment and database entries with people in- and outside of eLabFTW via direct links, or export to PDF or ZIP files.\n\nIn our opinion, eLabFTW is well suited to overcome the aforementioned hurdles of ELN implementation in small preclinical labs, as well as in other environments and lead to an improvement of research data transparency and findability. Being an open-source software, there are no license or other costs to pay when acquiring eLabFTW. In addition, even if the development and support of the software is discontinued, the source code will always be available, preventing users to search for a new software solution. eLabFTW does not use any proprietary parts of code, so this property applies to the whole code of eLabFTW. Some other ELNs are open source but depend on pieces of proprietary third-party code. In addition, when needed, the code of eLabFTW can be adapted to individual needs, by trained IT-Staff in the organization. Providing the option to use Docker Containers, a kind of virtual machine, which contains all the necessary software to run eLabFTW. eLabFTW can be installed even by a person with only intermediate IT knowledge, when following the official documentation.9 The initial setup takes less than 30 minutes, on a Windows computer, as tested by us. The setup works on a server or even a simple computer running Linux, Windows or MacOS. That ability for local installation makes eLabFTW suitable for critical data and enables data storages independently from the availability of cloud services offered by other ELN companies. This ensures high data safety, as it prevents access to data from outsiders and protects from using services with uncompliant privacy policies. Backup of data can be set up using a simple script9 and the backup files can be stored on a second hard drive, or on other machines, like a Network Associated Storage. eLabFTW offers a full text search engine, meaning it searches for all elements of the database, as well as in the written text of entries, making it easy to find older research data from yourself, and others. Additionally, eLabFTW contains a powerful search tool, which allow users to search for entries with specific properties. All entries in the experiment section of eLabFTW can be timestamped after RFC 3161 standard, allowing for full audit trail documentation. In addition, all revisions in any entry are tracked, so that it can be proved that no data were altered. Every item in eLabFTW is identified by a unique identifier (eLabID), which is independent from the given title. Hence, all objects can be unambiguously identified. Finally, all entries in eLabFTW can be shared via a unique link with people inside or outside the lab group, if eLabFTW is running on a computer with internet connection.\n\nThe features mentioned above are the most outstanding differences between eLabFTW and a traditional paper-based lab notebook, or other ELNs. However, eLabFTW provides some more useful features, that can be beneficial for a lab. Firstly, it offers a simple interface with just a text editor and some additional features like file upload and the integration of image files into the text. Furthermore, items in eLabFTW are marked by tags, instead of being ordered in a folder structure. In our experience this works much better, because entries can be sorted with multiple tags, in contrast to just be in one single folder. Any item in the database can be exported to different formats (pdf, zip, json) to be shared or stored externally. The visibility for each item can be restricted to oneself, a specific group of people or set to public. This might be an important feature to secure rights of research. Lastly, eLabFTW provides an API that can be accessed with a specifically created python library. For example, this is useful for automatically upload raw data to eLabFTW.\n\n\nImplementation of eLabFTW in our lab\n\nThe implementation of an ELN was supposed as the first step of a bigger project of increasing the research data quality in our lab, as described in a prior publication.10 For this, we tested different trials of commercially and freely available ELNs. Our focus was on the usability rather than on the features, as we wanted to make transition to electronic documentation as easy as possible and many features offered by more expensive ELNs are not needed by our group. After initial testing, two commercial ELNs and eLabFTW came into closer selection. We got the notice that two ELNs in this selection are already hosted by our university, this being Labfolder and eLabFTW. After a two-week period during which both lab books were tested in a small group, we decided to introduce eLabFTW to the whole lab. Although Labfolder has some features that eLabFTW does not have (support of including Excel and Word files, adaptable page layout), we decided to implement eLabFTW because of the much quicker processing times, open-source format and in our opinion a much easier usability. After introducing eLabFTW to every lab member, we decided about the most useful setup for our lab environment, to make it suitable for our QM-Project.\n\nBefore the setup of eLabFTW, we already started to introduce SOPs for our basic lab procedures. Using the database of eLabFTW proved to be a good way to distribute new versions of SOPs to every lab member effectively. As a first step, all existing SOPs were transferred into eLabFTW. The first new SOP described how to use eLabFTW. This SOP describes the form of documenting and time stamping of experiments, as well as linking database items. Secondly, we imported our complete database of vectors, primers, antibodies, enzymes, chemicals, consumables, and equipment, as well as cell lines into eLabFTW, together with the matching documentation as there are vector maps, material safety datasheets, primer sequences and melting temperature, antibody datasheets, restriction sites of restriction enzymes, cell line properties, handbooks of machinery, and so on. We also use the database to create project items, that all experiments belonging to that project can be linked to, to establish a timeline of experiments performed in that project. Lastly, we use eLabFTW to upload meeting minutes and documentation of other events.\n\nThe visibility in our instance of eLabFTW can be set to the whole team, or subgroups working on specific projects, that we create as needed. In our lab we do not use the booking function of lab equipment, after we found it too inconvenient after a short test period.\n\nAs described in one of our previous publications,10 the ELN was also supposed to be used for one-on-one project communication in form of interactive weekly progress reports. This approach was ineffective, as it consumed too much time, lead to misunderstandings and was unsuitable for the discussion of some specific problems. Consequently, we switched back to personal, verbal meetings for project planning. In our experience, ELN-reporting is only suitable for one directional communication. Still this possibility of one-directional way of communication showed an unexpected advantage during the still ongoing COVID-19 pandemic. Due to electronic recording of research results and protocols, less face-to-face communication was necessary, which helped to keep the infection risk on a low level.\n\nOur effort embeds well in current initiatives from leading biomedical institutions, as the RE-PLACE Project at the Berlin Institute of Health (BIH),11 which is an institution-wide programme for adopting ELN software at Charité and BIH, by providing free licenses to the ELN Labfolder, as well as introductory courses and support. This whole project is combined with an evaluation study to see if ELN usage really is beneficial for fulfilling needs of researchers and to improve the quality and transparency of research documentation and data management.\n\nAnother project, that our lab is involved in, is the EQIPD quality system (QS).12 Our efforts on eLabFTW fit very well with the requirements of this first quality system for preclinical research. Implementing and maintaining an ELN supports the implementation and maintenance of several Core Requirements set forth by the EQIPD QS, i.e. those on data documentation and transparency. In fact, our efforts to make our science open and sharable are a major contribution towards certification of our internal processes. Hence, an ELN should be considered as one of the pillars towards quality processes in a small research lab, especially when it integrates seamless in the daily lab routine as eLabFTW does.\n\nThere are also the more general ELN guides from the Harvard Medical School Information Technology department13 and the BIH.14\n\nIn a forthcoming visionary standpoint, we think that stringent ELN implementation in more working units is facilitating the reduction of hurdles for interaction across disciplines, such as the interaction of basic scientists and patient-treating clinical faculty. We hypothesize this due to the fact that data can be rapidly and remotely available at any given time, not restricted to often incompatible time schedule of the participating stakeholders. Moreover, a growing body of large datasets will be summarized and presented in a more comprehensive and convenient way as compared to local storage solutions on core faculty servers or hidden folders in a given server infrastructure. Thereby, further exchanging interaction to data scientists and wet lab scientists is possible. Laboratory automation, as a standard for many work processes in industry and routine diagnostic labs, is embracing its way in many academic labs15 such as in our lab occurred for a liquid handling robotics.16 Direct linking of ELN to machine output, without human operator data handling step in between, saving the data file in a time stamped and logged version will minimize error introduction in parallel to increase transparency of the project. Moreover, linking lab automation to ELN with a time scheduling algorithm may improve economic effectivity of the working unit.17 Those aspects are only a few cornerstones that shall be a focus of operations developments of a state-of-the-art translational research lab, currently underway in the lab of neurosurgery at the University Clinic Düsseldorf.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nBaker M: Is there a reproducibility crisis? Nature. 2016; 533(7604): 452–454.\n\nDiggle PJ, Zeger SL: Editorial. Biostatistics. 2010; 11:3, p375. Publisher Full Text\n\nNielsen M: An informal definition of open science. The OpenScience Project. 2011. Reference Source visited on 22.02.2021\n\nAdam B, Lindstädt B: ELN-Wegweiser. 2nd edition2020. Publisher Full Text\n\nKihlén M: Electronic lab notebooks – do they work in reality? DDT. 2005; 10: 18. PubMed Abstract \| Publisher Full Text\n\nGerlach B, Untucht Ch, Stefan A: Electronic Lab Notebooks and Experimental Design Assistants. Handb Exp Pharmacol. 2020; 257: 257–275. PubMed Abstract \| Publisher Full Text\n\nCarpin MA, Piel M: eLabFTW: An open source laboratory notebook for research labs. JOSS 2017; 2(12): 146. Publisher Full Text\n\nWalsh E, Cho I: Using Evernote as an Electronic Lab Notebook in a Translational Science Laboratory. J Lab Autom. 2012; 18(3): 229–234. PubMed Abstract \| Publisher Full Text\n\nReference Sourcevisited on 22.02.2021\n\nHewera M, et al.: An inexpensive and easy-to-implement approach to a Quality Management System for an academic research lab. F1000Res. 2020; 30(9). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWetzel C, Pohlenz P, Schirmer D: Wissenschaft zwischen Konkurrenz und Kooperation - Zum Potenzial kooperationsfördernder Managementinstrumente. DUZ OPEN . Publisher Full Text\n\nBespalov A, et al.: Introduction to the EQIPD Quality System. OSF 2007. Publisher Full Text\n\nLoveluck J: Finding the Right Electronic Lab Notebook with the Corey Lab.Reference Sourcevisited on 22.02.2021\n\nDirnagl U, Przesdzing I: A pocket guide to electronic laboratory notebooks in the academic life sciences. F1000Res. 2016; 5: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLippi G, Da RG: Advantages and limitations of total laboratory automation: a personal overview. CCLM 2019; 75(6). PubMed Abstract \| Publisher Full Text\n\nVargas-Toscano A, et al.: Robot technology identifies a Parkinsonian therapeutics repurpose to target stem cells of glioblastoma. CNS Onkol. 2020; 9(2). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChung HJ, Song YK, Hwang SH, et al.: Experimental fusion of different versions of the total laboratory automation system and improvement of laboratory turnaround time. JCLA 2018; 32(5). PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "84047", "date": "06 May 2021", "name": "Sam Parsons", "expertise": [ "Reviewer Expertise Openness and reproducibility", "cognitive affective", "psychometrics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes the open-source electronic lab notebook eLabFTW and the authors experience with implementing this into their workflow. I see this as a potentially useful contribution to the literature, in part because I am broadly positive about the use of ELNs. The majority of my comments are based around improving the clarity of the manuscript – I have numbered suggestions for ease.\nThe main point (which ties in to many of the below suggestions for clarity) I am not clear on after reading the manuscript, is the framing or the take-home message I should be left with. The content included is informative, but moves between; support or advocacy for the eLabFTW program, reflections on the groups experiences implementing it, and reflections on the good work for open science the authors groups and institutions are doing. Throughout, it feels that these aims are merged together, which makes the flow of the paper difficult to follow. I have several general suggestions that could help improve this clarity:\nConsider more subheadings to distinguish the message of each subsection. At a minimum, I think it would be valuable to separate or clearly distinguish sections. e.g. descriptions of eLabFTW’s technical details and instructions from the authors reflections on implementing eLabFTW. Greater use of subheadings would help the flow of the manuscript and reader clarity. Given that the abstract describes the paper as providing a self-guided approach to using eLabFTW, this section could benefit from more detail. Reading the abstract, I thought (though this may be just my reading) that this would be the core element of the paper but this section was quickly moved into more general comments on other initiatives. Further detail, and examples such as the groups own ELN, would make this a much more useful contribution. Consider removing some references to work specific to the authors groups/institutions – or, revise so that it can be made relevant to the readers context in using ELNs. For example, as a reader I did not find the closing sentence to be useful to my understanding of the eLabFTW tool “Those aspects are only a few cornerstones that shall be a focus of operations developments of a state-of-the-art translational research lab, currently underway in the lab of neurosurgery at the University Clinic Düsseldorf”. The sentences describing the good work towards openness will be maximally useful to the reader if they are situated within how they can implement these tools themselves, otherwise they are not necessary to the manuscript and distract from the main content.\n\nThe manuscript mentions ‘As described in one of our previous publications’ several times. My original suggestion was that more description of what was discussed would be valuable in this manuscript, else the reader is missing potentially vital information to this context. Having now read the cited paper, I am less clear of the purpose of the “Implementation of eLabFTW in our lab” section of this manuscript, which in turn leaves me less clear of the purposes of this manuscript, which related to my first point also. Perhaps the authors can more clearly differentiate between the contribution both papers make and what this manuscript adds in particular?\nThe following comments relate to more specific sections of the manuscript.\nIn the opening paragraph, it may be useful to differentiate between the authors definition of reproducibility and replicability. Later in the paragraph focus is placed mainly on data management, so I thought that computational reproducibility was the focus (same data + same analyses = same results) whereas the definition used was what others might describe as replicability (new data with the same procedures = same results). Not that I have a problem with the definition used, but the clarity for the focus or benefits of ELNs could be improved with this.\n\nMinor typo: “unaltered toegether” I think that a word might also be missing here, perhaps ‘in an unaltered format together with…’ ?\n\n“The pros and cons of using ELNs have been discussed for a long time” – Perhaps tie this in to the following two sections, or provide citations here?\n\nI suggest moving around some of the text in the advantages and hurdles sections – it was slightly confusing to read as the advantages section first highlighted disadvantages and the hurdles also starts with an entire paragraph on the benefits. As mentioned above, citing examples that highlight the benefits would be valuable here too.\n\nPerhaps the authors could be clearer about the focus on life sciences. Given that this is the context the ELN was implemented in, it makes sense. But, it might be useful to highlight or discuss further the use of ELNs in other contexts, or state that this paper focuses on the life sciences solely.\n\nI suggest splitting the long paragraph discussing the open-source aspect of eLabFTW at least into a paragraph on open-source and a paragraph on the set up (further use of subheadings would assist here too).\n\nI suggest removing the SOP acronym as it is not needed.\n\nI was not sure of the purpose of the paragraph on EQIPD, it did not feel relevant to the ELN discussion.\n\nPerhaps the paragraph on other guides for ELNs could be integrated to somewhere else in the manuscript?\nIn the interest of openness I always sign my reviews, Sam Parsons\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6662", "date": "27 May 2021", "name": "Michael Hewera", "role": "Author Response", "response": "Dear Dr. Parsons, I (in the name of my co-authors, as well) thank you for reviewing our publication and for your valuable feedback. We tried to implemented most of your suggestions in the new version of our publication. These are (not only) - Condensing the part about pros and cons of ELNs and sortig it - Specifying references to \"prior publication\" - moving text, and creating new headings/sub-headings - Better explain the context of eLabFTW and EQIPD - Streamline the last chapter Best regards, Michael Hewera" } ] } ]	1	https://f1000research.com/articles/10-292
https://f1000research.com/articles/9-140/v1	24 Feb 20	{ "type": "Research Article", "title": "Eating versus skipping breakfast has no discernible effect on obesity-related anthropometric outcomes: a systematic review and meta-analysis", "authors": [ "Michelle M. Bohan Brown", "Jillian E. Milanes", "David B. Allison", "Andrew W. Brown", "Jillian E. Milanes", "David B. Allison" ], "abstract": "Background: Whether one should eat or skip breakfast for weight is of continued interest in both the scientific and lay communities. Our objective was to systematically review and meta-analyze causal effects of eating versus skipping breakfast on obesity-related anthropometric outcomes in humans. Methods: AltHealthWatch, CINAHL, Proquest Theses and Dissertations Global, PsycInfo, and Scopus were searched for obesity- and breakfast-related terms in humans (final search: 02 JAN 2020). Studies needed to isolate eating versus skipping breakfast in randomized controlled trials. Mean differences were synthesized using inverse variance random effects meta-analysis for each outcome measured in more than one study. Positive estimates indicate higher outcomes in breakfast conditions (e.g., weight gain). Leave-one-out analysis was used for sensitivity. Risk of bias was assessed using the Cochrane risk of bias tool. Results: Ten articles (12 comparisons) were included. Study lengths spanned 6 days to 16 weeks. Conditions included recommendations to eat versus skip breakfast, or provision of some or all meals. 95% confidence intervals of all main analyses included the null value of no difference for each outcome: body weight (0.17 kg [-0.40,0.74], k=12, n=486, I2=74.4), BMI (0.08 kg/m2 [-0.10,0.26, k=8, n=395, I2=53.9), body fat percentage (-0.27% [-1.01,0.47], k=6, n=179, I2=52.4), fat mass (0.24 kg [-0.21,0.69], k=6, n=205, I2=0.0), lean mass (0.18 kg [-0.08,0.44], k=6, n=205, I2=6.7), waist circumference (0.18 cm [-1.77,2.13], k=4, n=102, I2=78.7), waist:hip ratio (0.00 [-0.01,0.01], k=4, n=102, I2=8.0), sagittal abdominal diameter (0.19 cm [-2.35,2.73], k=2, n=56, I2=0.0), and fat mass index (0.00 kg/m2 [-0.22,0.23], k=2, n=56, I2=0.0). One study reported muscle mass and total body water percentage. Leave-one-out analysis did not indicate substantial influence of any one study. Conclusions: There was no discernible effect of eating or skipping breakfast on obesity-related anthropometric measures when pooling studies with substantial design heterogeneity and sometimes statistical heterogeneity. Registration: PROSPERO CRD42016033290.", "keywords": [ "Breakfast", "skipping", "obesity", "weight", "meta-analysis", "systematic review", "randomized controlled trials" ], "content": "Introduction\n\nWhether one should eat or skip breakfast for weight control or loss is a topic of continued interest in both the scientific and lay communities. In 20131, we documented how breakfast eating versus breakfast skipping served as an example of how beliefs about diet can go beyond the evidence within and beyond the scientific community. The evidence at the time was dominated by over 90 observational studies – most cross-sectional – leading us to conclude that eating versus skipping breakfast as a strategy for weight was a presumption: a belief “held to be true for which convincing evidence does not yet confirm or disprove their truth”2,3. The limited scientific evidence on the topic has been translated directly to the public. For instance, we noted in our prior paper that the website of the Dr. Oz Show included an article stating, “The fact is, when you’re trying to lose body fat, you can’t skip breakfast”4. More recently, Dr. Oz himself stated, \"I think for 2020, the first thing I’m going to do is ban breakfast”5, and using the social media hashtag of #TeamNoBreakfast. Meanwhile, continued scientific interest in the topic is evidenced by many more cross-sectional observational and other studies having been published; more recent narrative review articles summarizing existing literature on the topic6,7; a meta-analysis evaluating breakfast eating versus skipping on weight8 that confirmed our prior registered preliminary analyses9,10; and another group registering an analysis similar to ours after our registration (PROSPERO CRD42018110858).\n\nWith mixed messaging over time about the importance of eating or skipping breakfast for the ongoing obesity epidemic, and with continued interest in the topic both scientifically and generally, it is important to synthesize the causal evidence on the effect of breakfast eating versus skipping on obesity and related outcomes, rather than relying on weaker study designs or popular opinion.\n\nSince our earlier summaries, additional RCTs have been conducted and published (as reviewed herein). Herein, we extend our prior work to synthesize causal evidence from RCTs on eating versus skipping breakfast in humans on all reported obesity-related anthropometric outcomes we were able to extract from relevant literature.\n\n\nMethods\n\nOur study was registered with the PROSPERO international prospective register of systematic reviews (CRD42016033290) on 21 JAN 2016. The initial registration limited papers up to the registration date; however, because of the time between initial registration and this manuscript, the search was updated to 02 JAN 2020 (see Search and review strategy, below). Earlier versions of this work were published as abstracts for the American Society for Nutrition’s Annual Meeting and Scientific Sessions9,10.\n\nInclusion criteria were:\n\nthe study had at least one breakfast skipping condition and one breakfast eating condition regardless of modality (e.g., whether recommended or provisioned);\n\nthe study was a randomized, controlled trial (RCT);\n\nstudy length (i.e., time on intervention) was greater than 72 hr;\n\nparticipants were normal weight or greater, as defined by original study authors, who did not have diseases that influence weight; and\n\nthe study reported weight or other anthropometric outcomes.\n\nStudies were excluded if:\n\nparticipants had diseases or conditions that affected weight except for obesity, diabetes, and CVD;\n\nbreakfast eating versus breakfast skipping were confounded with other effects (could not isolate the effect of breakfast eating versus breakfast skipping from other intervention such as study design to maintain weight).\n\nOur first search was completed on 20 JAN 2016, the search refreshed on 26 JAN 2017, and the search finalized on 02 JAN 2020, with results from prior searches being deduplicated from subsequent searches.\n\nIn all search phases, searches were executed by using the application programming interfaces (APIs) of AltHealthWatch, CINAHL, Proquest Theses and Dissertations Global, PsycInfo, and Scopus using R (version 3.5.2). The following was used to search Scopus, with analogous search strategies adapted for the other databases:\n\nTITLE-ABS-KEY((Obesity OR obese OR adipose OR adiposity OR overweight* OR \"over weight\" OR \"weight gain\" OR \"weight reduc\" OR \"weight los\" OR \"weight maint\" OR \"weight decreas\" OR \"weight control\" OR \"weight restrict\" OR \"BMI\" OR \"FMI\" OR \"BMIz\" OR \"zBMI\" OR \"weight percentile\" OR \"gestational weight\" OR \"weight for height\" OR \"waist circumference\" OR \"skinfold thickness\" OR \"body composition\" OR \"body size\" OR \"fat mass\" OR \"body fat\" OR \"body mass\" OR \"body weight\" OR \"bodyweight\" OR \"waist hip ratio\") AND (breakfast OR \"break fast\" OR \"morning fasting\" OR \"morning meal\")) AND DOCTYPE(ar OR ip) AND SRCTYPE(j)\n\nSearch results across databases were compared for duplication, including by title, abstract, and PubMed ID. Studies with titles and abstracts addressing animals that did not also include words related to human subjects were excluded programmatically. Titles and abstracts were then coded independently by at least two authors for inclusion/exclusion criteria. If both authors excluded a study for violation of any inclusion or exclusion criterion, it was excluded; if at least one did not exclude it, the paper was passed on for full text review.\n\nAll data and code used to estimate effect sizes and meta-analyses are provided as Extended data at https://doi.org/10.5281/zenodo.366314811. Additional details are included as comments within the code, including exact approaches to estimating each effect size within a study.\n\nEffect sizes comparing breakfast eating versus skipping on each outcome were calculated for each study. Each effect size was calculated as a difference-in-difference in the native units of the outcome (e.g., kg for weight). Only outcomes for which there was more than one effect size were meta-analyzed: body weight, BMI, body fat percentage, fat mass, lean mass, fat free mass, adipose tissue mass, waist circumference, waist:hip ratio, fat mass index, sagittal abdominal diameter, and lean tissue mass. Lean mass, fat-free mass, and lean tissue mass were meta-analyzed together as ‘lean mass’; fat mass and adipose tissue mass were meta-analyzed together as ‘fat mass’. Total body water percentage and muscle mass are both reported only in Ogata et al.12; although muscle mass as an outcome was excluded, Ogata et al. also reported lean mass, which is captured in the pooled lean mass analysis.\n\nFarshchi et al.13 reported pre and post means and standard deviations separately for each treatment period in a two-arm cross-over design. Although the unbiased estimate of the difference-in-difference was calculable from the pre and post means in each condition, the lack of information on the correlation of change within or between conditions precluded us from directly calculating the variance of the effect. We requested summaries from the authors, but the authors informed us they no longer had the raw data given that the paper was published in 2005. Thus, within-condition and between-condition correlations had to be estimated. Sievert et al.8 used a correlation coefficient of 0.3 for post-only values. We chose to estimate within-period change scores based on the within-condition correlation coefficients we estimated from Geliebter et al.14 because Geliebter et al. had all values needed to estimate within-condition, pre-post correlation coefficients. All correlation coefficients from Geliebter were greater than 0.99. Effect sizes were estimated for each outcome. Because Farshchi et al. reported no statistically significant results for any outcome, any statistically significant estimates were recalculated using the largest within-condition correlation that resulted in non-significant effect sizes. This approach may underestimate the variance, which would provide the study more weight in the meta-analysis; however, the leave-one-out analysis described below gives Farshchi the lowest weight possible.\n\nGeliebter et al.14 reported three conditions: skipping, corn flakes, and oat porridge. We used the recommended method of the Cochrane Handbook, which is to “combine multiple groups that are eligible as the experimental or comparator intervention to create a single pair-wise comparison”15. Because we were interested in breakfast eating versus breakfast skipping, the two breakfast conditions were pooled together.\n\nLeidy et al.16 also reported three conditions: skipping, a normal protein breakfast, and a high protein breakfast. We requested summaries from Leidy et al., who graciously provided us with separate group means and standard deviations for the changes. We used the recommended method of the Cochrane Handbook to combine breakfast conditions as described above.\n\nNeumann et al.17 reported three conditions: skipping, high carbohydrate breakfast, and high protein breakfast. Again, we used the method recommended by the Cochrane Handbook to combine breakfast conditions. Neumann et al. reported individual-level data in their supplementary table. While reviewing the values in the supplement, we found some results to be implausible (e.g., multiple kg of weight or cm of height change in 8 days). We reached out to the authors, who clarified one subject’s data. For our analysis, we removed some implausible values as described in the code. We are in contact with the authors about additional data points of concern.\n\nSchlundt et al.18 reported follow-up data at 6 months, but the methods descriptions were unclear as to whether the interventions to eat or skip breakfast were continued past the 12-week intervention. Authors were contacted about this detail and for additional outcomes data at 12 weeks that were either not directly reported or reported as no significant strata (i.e., habitual breakfast eaters or skippers) or treatment effects; the authors informed us they no longer had the raw data given the study was published in 1992. We therefore chose to only use the change in body weight data from 12-weeks. Independent effect sizes were estimated for habitual breakfast eaters and habitual breakfast skippers.\n\nDhurandhar et al.19 reported body weight for the completers-only analysis in their paper. Because they registered their study as also measuring BMI, and because of the mention of an intention to treat analysis, we contacted the authors (one of whom, DBA, is a coauthor on the present meta-analysis), who provided us with summary data. Note that they also had a third group, in which participants received no specific breakfast eating or breakfast skipping recommendations; we limited our analysis to the intention to treat analyses of the breakfast eating and breakfast skipping groups. Independent effect sizes were estimated for habitual breakfast eaters and habitual breakfast skippers.\n\nLeCheminant et al.20 were contacted for estimates of change over time for data in their Table 3. The authors graciously provided estimates of change within each group for each outcome. The data used herein, as shared by the authors, differs slightly from their publication because of increased precision and because of a reporting error in which percent body fat did, in fact, have a small but non-significant increase in the no breakfast group. This error does not change the results of their study, but the corrected values are used herein.\n\nOgata et al.12, Betts et al.21, and Chowdhury et al.22 effect sizes were calculated with routine equations.\n\nMeta-analyses were calculated using the metafor package(version 2.1-0) in R. Each of 12 independent effects sizes (10 papers; 2 stratified by baseline habit) were included in each analysis as possible, depending on which outcomes were reported in which studies. Random effects analyses were calculated; no fixed effects analyses were calculated because design heterogeneity made the assumption of effect sizes being part of a homogenous distribution tenuous. The adjustment by Knapp and Hartung23 was used given the relatively small number of effect sizes. Two effect sizes were derived from separate papers of the Bath Breakfast Project (BBP; Betts et al. and Chowdhury et al.). Because these were independent samples (normal or with obesity) we treated them as independent even though they came from the same overarching study. Similarly, although there is plausibly some correlation amongst effect sizes calculated within the habit strata in Dhurandhar et al. and Schlundt et al. by nature of being part of the same overarching study, we treated the effect sizes as independent.\n\nLeave-one-out analysis was used as a sensitivity analysis to investigate the influence of any single study for each outcome, in which each study was omitted from the dataset at a time, and then the meta-analysis was recalculated.\n\nEffect estimates are displayed as mean differences with 95% confidence intervals in the native units of the outcome. I2 (%) and p-values for tests of heterogeneity are also reported. No multiple-comparison corrections are applied within or among outcomes. There are few effect sizes (k=12), there is substantial design heterogeneity (e.g., study length, types of breakfast, different populations), and there is statistical heterogeneity in several outcomes; therefore, funnel plot asymmetry is not presented because visual estimation of asymmetry is unreliable for small k24, the test is underpowered for small k25, and any association between effect size and variance may plausibly be explained by study design or other factors rather than just publication bias25.\n\nRisk of bias was assessed independently by two investigators (MMBB/JEM for all but Ogata 2019 and MMBB/AWB for Ogata 2019) using Cochrane’s Risk of Bias Tool26. Given that the interventions are obvious to participants (eating versus skipping breakfast), we only coded blinding of personnel, and readers should be aware of the risk of non-blinded interventions. We do not use the approach of assigning a binary risk of bias to an entire study (e.g., if one criterion is high risk in a study, the entire study is considered high risk); however, we provide the individual ratings for each article and readers can apply such an approach if they wish.\n\n\nResults\n\nThe search results are shown in the PRISMA diagram in Figure 1. The results of each of the three phases of the search are shown.\n\nThree searches were undertaken. For searches 2 and 3, the numbers in parentheses represent unique results to that search. Several ‘papers from other sources’ were identified in prior searches, but those papers were captured by the third search.\n\nTen papers were included with 12 effect sizes (see Table 1 for descriptions). Briefly, of the 10 studies included: six were conducted in the United States, three in the United Kingdom, and one in Japan; two were cross-over RCTs and eight were parallel arm RCTs; length ranged from 6 days to 16 weeks; five provisioned some or all foods and five were recommendations for dietary consumption; two stratified on baseline eating or skipping habits, two included only habitual breakfast eaters, three included only habitual breakfast skippers, two reported mixed baseline habits, and one did not specify baseline habits; four reported race/ethnicity of participants; four included females only, one included males only, and five included both females and males. For breakfast definitions, dietary compositions, and timing, see Table 1 and Figure 2. Breakfast definitions and timing of consumption varied amongst the studies included and ranged from highly controlled and prescribed to broad recommendations (Figure 2).\n\n1BF, Breakfast.\n\n2A, Asian; B, Black; BH, Black Hispanic; BNH, Black Non-Hispanic; C: Caucasian; H, Hispanic; I, Indian; O, Other; W, White; WH, White Hispanic; WNH, White Non-Hispanic.\n\n3Definitions paraphrased from each study paper.\n\n4ATM, adipose tissue mass; BF%, body fat percentage; BW, body weight; FFM, fat-free mass; FM, fat mass; FMI, fat mass index; LM, lean mass; LTM, lean tissue mass; MM, muscle mass; SAD, sagittal abdominal diameters; TBWP, total body water percentage; WC, waist circumference; WHR, waist:hip ratio. Some additional outcomes might have been mentioned in the paper, but quantitative results may not have been reported after the intervention.\n\nThe top section outlines the patterns for the included studies; the middle section shows a few examples of studies we did not classify as eating versus skipping breakfast that are explained further in the ‘Notable Exclusions’ section and in Table 3; and the bottom is a legend for the figure. ‘Inferred eating window’ represents the times we inferred that participants were permitted or recommended to consume food as reported in the papers; ‘specified eating window’, ‘breakfast eating window’, and ‘assigned eating times’ were reported by the authors in either absolute or relative times (e.g., number of hours since waking). For more details for the included studies, see Table 1.\n\nFigure 3 shows a composite forest plot that includes all meta-analyzable, obesity-related, anthropometric outcomes. In all cases, the 95% confidence intervals included the null of no differences between skipping and eating breakfast (frequently interpreted as “not statistically significant”). Table 2 shows the numerical estimates of the values displayed in the forest plots. Therefore, no discernible effects of breakfast eating or breakfast skipping on body weight (kg), BMI (kg/m2), body fat percentage (%), fat mass (kg), lean mass (kg), waist (cm), waist:hip ratio, sagittal abdominal diameter (cm) and fat mass index (kg/m2) were found in these primary analyses.\n\nSagittal abdominal diameter and fat mass index were only included in the two papers from the Bath Breakfast Project (Betts et al. and Chowdhury et al.), and are not plotted here; outcomes of muscle mass and total body water percent were only included in Ogata et al., and so no meta-analyzable estimate was possible. See Table 2 for the numerical values of these seven analyses, plus the sagittal abdominal diameter and fat mass index. Studies without point estimates and confidence intervals within an outcome indicates that the study did not report that outcome. 95% confidence intervals for individual studies and for the width of the diamond representing the summary estimate are presented. Horizontal dotted lines for the summary of the meta-analyses represents the 95% prediction interval. For the column ‘Habit’: e, habitual eaters; s, habitual skippers; u, unspecified or mixed.\n\nData are presented as mean [95% CI] for each study and the summary estimate, expressed as mean difference. Positive values are higher during breakfast conditions. n represents the total number of individuals within a study; k is the number of effect sizes in a meta-analytic estimate; MD is mean difference; I2 represents heterogeneity, with the associated p-value representing the statistical test for significant heterogeneity. Outcomes of muscle mass and total body water percent were only included in Ogata et al., and so no meta-analyzable estimate was possible.\n\n Studies were excluded for at least one reason; the reasons given in this column may not be the only reason for exclusion.\n\nRisk of bias varied by study (Figure 4). Two studies had low risk of bias across all categories: Dhurandhar 2014 and Ogata 201912. Two studies, Betts 201421 and Chowdhury 201622, were coded as high risk of bias for the criterion of blinding participants and personnel because the authors clearly indicated that personnel were not blinded. Given that the interventions are obvious to participants (eating versus skipping breakfast), we only focus on blinding of personnel, and readers should be aware of the risk of non-blinded interventions. On the other hand, many of the categories in the risk of bias in each study were unclear, and it is therefore uncertain whether the risk was high or low.\n\nEach included paper was assessed for risk of bias using the Cochrane Risk of Bias tool. Given that the interventions are obvious to participants (eating versus skipping breakfast), we only coded blinding of personnel, and readers should be aware of the risk of non-blinded interventions.\n\nThe leave-one-out analysis is shown in Figure 5. Little difference is noted among the analyses, with substantial overlap of confidence intervals in all cases. When considering statistical significance (i.e., confidence intervals that do not include 0), leaving Farshchi et al.13 out of the analysis results in significantly greater BMI in the breakfast conditions than the skipping conditions. When Leidy et al.16 is excluded, fat mass is greater in the breakfast than the skipping conditions. Waist:hip ratio is centered on zero with no estimable confidence interval when Chowdhury et al.22 is left out because the other three estimates are all 0.00. We reiterate that none of these summaries took multiple comparisons into account.\n\nWithin each column, the diamond represents the meta-analytic summary estimate when leaving out the study in a particular row. Row and column combinations without diamonds represent outcomes that are not reported for that particular study. *The waist:hip ratio had no estimable confidence interval because the three remaining estimates were all 0.00. Sagittal abdominal diameter and fat mass index were only included in the two papers from the Bath Breakfast Project (Betts et al. and Chowdhury et al.), and therefore a leave-one-out analysis would include only a single study; outcomes of muscle mass and total body water percent were only included in Ogata et al., and so a leave-one-out analysis is not possible.\n\nNotable exclusions are located in Table 3. Broad areas to note are the lack of a skipping group for comparison to breakfast groups, intervention periods that were less than 72 hr in duration, studies that had the comparison of interest but did not measure body weight, and studies whose primary purpose did not isolate breakfast eating versus breakfast skipping, such as time restricted feeding and shift in consumption periods. Two examples of the latter include Wehrens et al.,27 who shifted all meals by 5 hours (as well as not being in a randomized order), to extreme time restriction of Halberg et al.28 who assigned only breakfast or dinner (Figure 2).\n\nIn this meta-analysis, our included studies were all conducted in adults/adolescents, but, as noted in Table 3, there have been several related studies conducted in children; however, none of the studies in children had a true skipping group. For instance, Rosado et al.29 had a control group with no intervention, which is not equivalent to assigning children to skip breakfast. Similarly, Powell et al.30 did have a group that was assigned to consume a slice of orange as an attention placebo control, but again the children were not assigned to otherwise skip breakfast.\n\n\nDiscussion\n\nThe causal effect of eating versus skipping breakfast on obesity-related anthropometric outcomes was non-significantly different from zero across body weight, BMI, body fat percentage, fat mass, lean mass, waist circumference, waist:hip ratio, sagittal abdominal diameter, and fat mass index. Our results largely match our prior analyses9,10, as well as the analysis of body weight conducted by Sievert et al.8.\n\nThe choices of inclusion/exclusion criteria, adjustments, and assumptions to use when meta-analyzing data are often up for debate. While we cannot rule out that there may be some statistically significant combination of studies, subgroups, splitting-versus-pooling of different breakfasts, or different imputation strategies (e.g., using a different correlation coefficient to estimate Farshchi et al.), we note that the results are fairly consistently centered near zero. In the leave-one-out analyses, for instance, there were only two values that became statistically significantly different in favor of skipping breakfast: BMI when Farshchi et al. was excluded, and fat mass when Leidy et al. was excluded. We caution against over-interpretation of these statistically significant findings, however, because the 95% confidence intervals did not differ substantially from the other leave-one-out analyses and we did not adjust for multiple comparisons. Even if effects turned out to be non-zero, the 95% confidence and prediction intervals of the outcomes include effect sizes of low clinical significance.\n\nDespite this relative consistency in summary effect sizes, we note that there was substantial design heterogeneity. The length of studies, for instance, varied substantially. To be confident in effects of obesity-related interventions, longer term studies are desired. However, the need for longer-term studies is often to guard against concluding that early effects (weeks to months) will result in sustained weight loss over months to years. Given the overall null findings herein, suggesting a need for longer studies would serve to test whether these relatively acute null findings reflect long-term adaptations to establishing breakfast habits. In addition, some have argued that it is not merely eating versus skipping breakfast that is important, but rather that the type of breakfast matters (c.f., Leidy et al. 20167). Such an argument does not invalidate the question asked or the findings of this meta-analysis, however. If, for instance, a breakfast of a particular characteristic is what influences weight – be it fiber content, protein, energetic load, timing from waking, or others – then the question would not be whether eating versus skipping breakfast matters; rather, research would need to test the effects of that particular breakfast versus comparator groups, whether those comparator groups be different breakfasts or no breakfast at all.\n\nWe clarify that our results are limited to obesity-related anthropometric outcomes. As stated previously, “[j]ust because breakfast consumption may not have a statistically significant effect on weight does not make breakfast a bad recommendation”55, nor does it necessarily make it a good recommendation. Our results do not inform whether eating versus skipping breakfast is of value for blood glucose control, cardiometabolic risk, school performance, or other outcomes; nor do our results inform the effects of eating versus skipping breakfast as part of a broader intervention or time restriction paradigm (e.g., early vs late time-restricted feeding).\n\nThere was no discernible effect of eating or skipping breakfast on obesity-related anthropometric measures when pooling studies with substantial design heterogeneity and sometimes statistical heterogeneity.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo: Supplemental files for \"Eating versus skipping breakfast has no discernible effect on obesity-related anthropometric outcomes: a systematic review and meta-analysis.\". http://doi.org/10.5281/zenodo.366314811.\n\nThis project contains the following extended data:\n\ncalculations.R (calculates individual effect sizes for each study)\n\nmetaanalysis.R (reproduces the composite forest plot, leave-one-out plot, and the summary table)\n\nneumann2016.csv (contains the raw data from Neumann 2016 with authors’ correction)\n\nrho estimates for farshchi.R (uses data from Geliebter et al. to estimate within-condition pre-post correlations)\n\nZenodo: PRISMA checklist for \"Eating versus skipping breakfast has no discernible effect on obesity-related anthropometric outcomes: a systematic review and meta-analysis\". http://doi.org/10.5281/zenodo.366314811.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe authors would like to thank Xiwei Chen, MS, with the Indiana University School of Public Health-Bloomington Biostatistics Consulting Center for confirming the statistical approach used for the meta-analyses. We also thank the authors of the original studies who provided us with and permitted us to use additional data or information, as described in the methods.\n\n\nReferences\n\nBrown AW, Bohan Brown MM, Allison DB: Belief beyond the evidence: using the proposed effect of breakfast on obesity to show 2 practices that distort scientific evidence. Am J Clin Nutr. 2013; 98(5): 1298–1308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCasazza K, Brown A, Astrup A, et al.: Weighing the Evidence of Common Beliefs in Obesity Research. Crit Rev Food Sci Nutr. 2015; 55(14): 2014–2053. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCasazza K, Fontaine KR, Astrup A, et al.: Myths, presumptions, and facts about obesity. N Engl J Med. 2013; 368(5): 446–454. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLynn L: Breakfast Shakes: Drink Yourself Skinny. 2011; Accessed 17 January 2013. 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PubMed Abstract \| Publisher Full Text\n\nReeves S, Huber JW, Halsey LG, et al.: A cross-over experiment to investigate possible mechanisms for lower BMIs in people who habitually eat breakfast. Eur J Clin Nutr. 2015; 69(5): 632–7. PubMed Abstract \| Publisher Full Text\n\nRosi A, Martini D, Scazzina F, et al.: Nature and Cognitive Perception of 4 Different Breakfast Meals Influence Satiety-Related Sensations and Postprandial Metabolic Responses but Have Little Effect on Food Choices and Intake Later in the Day in a Randomized Crossover Trial in Healthy Men. J Nutr. 2018; 148(10): 1536–1546. PubMed Abstract \| Publisher Full Text\n\nYoshimura E, Hatamoto Y, Yonekura S, et al.: Skipping breakfast reduces energy intake and physical activity in healthy women who are habitual breakfast eaters: A randomized crossover trial. Physiol Behav. 2017; 174: 89–94. PubMed Abstract \| Publisher Full Text\n\nZakrzewski-Fruer JK, Plekhanova T, Mandila D, et al.: Effect of breakfast omission and consumption on energy intake and physical activity in adolescent girls: a randomised controlled trial. Br J Nutr. 2017; 118(5): 392–400. PubMed Abstract \| Publisher Full Text\n\nCarlson O, Martin B, Stote KS, et al.: Impact of reduced meal frequency without caloric restriction on glucose regulation in healthy, normal-weight middle-aged men and women. Metabolism. 2007; 56(12): 1729–1734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeim NL, Van Loan MD, Horn WF, et al.: Weight loss is greater with consumption of large morning meals and fat-free mass is preserved with large evening meals in women on a controlled weight reduction regimen. J Nutr. 1997; 127(1): 75–82. PubMed Abstract \| Publisher Full Text\n\nTinsley GM, Moore ML, Graybeal AJ, et al.: Time-restricted feeding plus resistance training in active females: a randomized trial. Am J Clin Nutr. 2019; 110(3): 628–640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsk AS, Hernes S, Aarek I, et al.: Changes in dietary pattern in 15 year old adolescents following a 4 month dietary intervention with school breakfast--a pilot study. Nutr J. 2006; 5: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrepinsek MK, Singh A, Bernstein LS, et al.: Dietary effects of universal-free school breakfast: findings from the evaluation of the school breakfast program pilot project. J Am Diet Assoc. 2006; 106(11): 1796–1803. PubMed Abstract \| Publisher Full Text\n\nDouglas SM, Byers AW, Leidy HJ: Habitual Breakfast Patterns Do Not Influence Appetite and Satiety Responses in Normal vs. High-Protein Breakfasts in Overweight Adolescent Girls. Nutrients. 2019; 11(6); pii: E1223 PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJakubowicz D, Froy O, Wainstein J, et al.: Meal timing and composition influence ghrelin levels, appetite scores and weight loss maintenance in overweight and obese adults. Steroids. 2012; 77(4): 323–331. PubMed Abstract \| Publisher Full Text\n\nSt-Onge MP, Buck C, Keller KL: Breakfast Cereal And Nutrition Education On Body Mass Index And Diet Quality In Elementary School Children: A Pilot Study. Int J Nutr. 2015; 1(1). Publisher Full Text\n\nVersteeg RI, Schrantee A, Adriaanse SM, et al.: Timing of caloric intake during weight loss differentially affects striatal dopamine transporter and thalamic serotonin transporter binding. FASEB J. 2017; 31(10): 4545–4554. PubMed Abstract \| Publisher Full Text\n\nZakrzewski-Fruer JK, Wells EK, Crawford NSG, et al.: Physical Activity Duration but Not Energy Expenditure Differs between Daily and Intermittent Breakfast Consumption in Adolescent Girls: A Randomized Crossover Trial. J Nutr. 2018; 148(2): 236–244. PubMed Abstract \| Publisher Full Text\n\nChowdhury EA, Richardson JD, Gonzalez JT, et al.: Six Weeks of Morning Fasting Causes Little Adaptation of Metabolic or Appetite Responses to Feeding in Adults with Obesity. Obesity (Silver Spring). 2019; 27(5): 813–821. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzalez JT, Richardson JD, Chowdhury EA, et al.: Molecular adaptations of adipose tissue to 6 weeks of morning fasting vs. daily breakfast consumption in lean and obese adults. J Physiol. 2018; 596(4): 609–622. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuttle W, Daum K, Larsen R: Relation of breakfast regimen to control of body weight. Research Quarterly American Association for Health, Physical Education and Recreation. 1954; 25(1): 100–108. Publisher Full Text\n\nBohan Brown MM: Digging into breakfast: serving up a better understanding of the effects on health of the \"most important meal of the day\". Am J Clin Nutr. 2019; 110(1): 4–5. PubMed Abstract \| Publisher Full Text" }	[ { "id": "62392", "date": "27 May 2020", "name": "Nerys Astbury", "expertise": [ "Reviewer Expertise Weight loss", "Obesity", "Appetite regulation", "body composition" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a systematic review of randomised controlled trials comparing the consumption of breakfast with skipping breakfast and follows other similar SR on breakfast eating/skipping.\nThere has been a long held debate regarding the potential effect of breakfast on energy intake, body weight and anthropometric measures, as the authors note this was driven by observational studies, but in recent years more RCT have emerged investigating effects.\nThe methods are meretriciously reported and follow standard or gold standard procedures. Justification for some inclusion criteria should be included - to allow the reader to make a judgement whether the methods of this paper match its intended objectives. Since outcome is bodyweight/composition what is the purpose of including studies of 72hrs duration where likelihood of effect is minimal?\n\nI feel that the discussion is weak and could be strengthened considerably. Limitations of this manuscript are not limited to the statistical methods and assumptions for dealing with missing data. Limitations of the current review should be discussed and critically analysed. Many of the studies included in this and other SR are small, and are often conducted in participants with a mixed usual breakfast habit who are not overweight or attempting to limit their energy intake or reduce their body weight. Further discussion on the clinical/research implications of this SR (and similar ones recently published) is warranted.\nThe absence of is a substantial clinical trial of breakfast eating/skipping within the context of weight loss attempt is notable, could this be related to issues surrounding potential conflicts of interest and industry involvement?, -would be worth discussion. Some mention of contextual setting is important- it would be difficult to determine if eating/skipping breakfast aids/impairs weight loss if the participants in are not attempting to limit daily energy intake or weight loss. However breakfast may affect appetite regulation, thus aiding/impairing adherence to energy restricted diets (or it may not)? Could usual skippers made to eat have different response than usual consumers made to skip? Could sub-group differences be affecting ability to detect effects of breakfast on body weight and composition?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5980", "date": "15 Oct 2020", "name": "Michelle M Brown", "role": "Author Response", "response": "We thank Dr. Astbury for the feedback. We address the critiques below: We have added to our discussion to outline more completely some of the limitations and choices we have made. We specifically address our choice of 72 hours, choices for defining breakfast and skipping, the fact that these effect estimates are pooled across weight loss backgrounds, weight categories, and breakfast compositions/timings. We have gone further to finish the subgroup analysis stratified by baseline breakfast habit. We initially presented a draft of the stratified analysis in our meeting abstracts. Completing it here addresses some of the reviewer’s concerns and allows us to uphold transparency by completely reporting analyses we have tried. This includes 5 new interaction-effect forest plots, a summary table, supplemental methods and results, and updated code. We now discuss Bonnet and Sievert’s meta-analyses in our discussion, and briefly discuss some methodological and analytical differences that may explain differences in conclusions. We also highlight that these minor differences in analyses result in qualitatively different conclusions (i.e., differences in statistical significance), which reinforces the overall uncertainty in conclusions that can be drawn from this body of evidence. We hope that these additions and clarifications, in addition to the limitations we initially discussed about length of studies, baseline breakfast habits, types of breakfast, and other considerations, more fully orient the reader to the limitations both of our analysis and the evidence base for these questions." } ] }, { "id": "62393", "date": "29 May 2020", "name": "Enhad A. Chowdhury", "expertise": [ "Reviewer Expertise Energy balance", "breakfast consumption", "appetite regulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview\nThe work of Bohan Brown et al examines the impact of breakfast skipping versus consumption on obesity related anthropometric measures using meta-analytical methods. The authors have selected a focused question and I found the description of the methods and decisions behind inclusion/exclusion of papers to be thorough. As a non-statistician, I do not feel best placed to provide a critique of the statistical approach, but at face value do not see anything unusual. Generally, the data presentation was clear, and the interpretation of the results was appropriate to the scope of the work. I have outlined some specific comments for the authors consideration below, relating to small clarifications and the inclusion of a very recently published piece of work as point of context.\nIntroduction\nI found the introduction appropriate to set the context for the work.\nAs an area of nutrition that has been historically dominated by cross-sectional studies, it is of clear interest to examine the causal data relating to breakfast and body composition.\nI am not completely clear on the timings of the relative publications, but at time of review, the work by Bonnet referenced in the Prospero registration has now been published in Obesity. https://onlinelibrary.wiley.com/doi/abs/10.1002/oby.227911 If possible, I would suggest this reference is added.\nMethods\nI generally found the description of the methods to be thorough and clear.\n“One breakfast skipping condition” and one “breakfast eating” condition. While the authors state regardless of modality – can I clarify whether this is based upon the original authors original definitions of these conditions (I assume this to be the case – but think it is useful to state here if so, in the same way that you have below in this section for weight status)?\nI found the description of included and excluded studies to be very thorough.\nAs a non-statistician I do not consider myself well placed to examine the meta-analytical methods used thoroughly, this would be more appropriate for a statistician to evaluate. At face value, there does not seem to be anything particularly unusual about the approach employed.\nResults\nI found the description of the results to be clear and thorough and the figures to be generally clear.\n\nFigure 2 – I think it is helpful to have a visual representation of the different implementations of the interventions. However, is it possible to make this figure colour and make the different elements more distinct? With these very similar grey shades and fill patterns this figure is a little difficult to interpret.\nFigure 3 – I think it would be helpful to write in simple terms what each side of the 0 line represents on this figure within the figure legend, as you do for the description of Table 2. I also appreciate this may be for practical reasons, but the figures with only one missing value on the axis look a little strange – I presume this might be because the labels do not fit, but if easily adjustable I would suggest it might be beneficial to add this single missing label.\nDiscussion\nI generally found the discussion clear and highlighted appropriately the limitations based on the scope of the work.\nAs outlined for the introduction – the work of Bonnet is now published, and is potentially of interest, as they suggest based on their analysis a small but significantly reduced body weight (-0.54kg (CI: -1.05 to -0.03), which is also broadly in line with Sievert, suggesting a very small reduction in body mass with breakfast skipping. If possible, I would suggest this study is cited.\nI understand that clearly any effects in favour of breakfast skipping for body mass are small, but I think as the two other meta-analyses conducted conclude this, this is worth incorporating into your second paragraph about the potential for non-zero effects as a point of context. I don’t think this impacts upon your general point that any potential for effects are likely of low clinical significance.\nI think that with such a small set of studies with a very large variation between them, narrative reviews can often be valuable in this context for examining and considering the nuance of the results from different designs. I think it is good that you have clearly highlighted the limits of the meta-analytical approach used.\nI appreciate your point that longer term studies are often to determine the longer-term trajectory of interventions that may show short term impacts on body composition. Given that two other meta-analyses conclude that there may be a significant reduction overall (albeit small) with breakfast skipping is there also added value in determining if breakfast skipping over long durations causes more substantial weight loss?\nI am unclear as to the relevance of the “[j]ust because”? If this is a typo – it should be amended.\nWhile I think it is helpful that you have outlined the limitations of your analysis for informing the overall benefit of breakfast, I have two suggestions:\nI think it is relevant to acknowledge that breakfast consumption/omission has previously been shown to impact upon specific components of energy balance that may have independent effects upon health.\n\nI would suggest providing some references for studies/reviews examining the different factors that you highlight as not being examined in the present work (i.e. blood glucose control, cardiometabolic risk etc), should the reader be interested in those aspects.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5981", "date": "15 Oct 2020", "name": "Michelle M Brown", "role": "Author Response", "response": "We thank Dr. Chowdhury for the feedback. We address the critiques below: Regarding Bonnet et al, we have now added the reference. It was published between our first submission and our response to reviewers. Regarding ‘one breakfast skipping condition’, we depend on the original authors’ definition where possible. We have expanded our exclusion criteria to give examples of situations where we would have excluded a breakfast/skipping group. We have updated Figure 2. We prefer to avoid color to make figures color-blind friendly, and amenable to gray scale printing. We therefore have increased the contrast among three of the bars (light gray, dark gray, black), and increased the contrast and pattern of the one that previously had a dotted fill pattern. We hope this makes the figure more distinct. We have updated the legend to Figure 3 to describe what left and right of zero means. We reprogrammed the code to generate the figures to make the tick marks consistent, rather than depending on the software defaults. We now discuss Bonnet and Sievert’s meta-analyses in our discussion. It would seem that the instability of findings, including across meta-analyses that included only studies of longer duration, may warrant longer studies to confirm the effects of eating versus skipping on weight. We changed the “[j]ust”, which was a stylistic artifact that was no longer needed. We now cite papers that talk about outcomes beyond obesity, and we try to better emphasize that our analysis is limited to the binary eat versus skip breakfast on anthropometric measurements." } ] } ]	1	https://f1000research.com/articles/9-140
https://f1000research.com/articles/10-703/v1	30 Jul 21	{ "type": "Data Note", "title": "Organizing knowledge to enable faster data interpretation in COVID-19 research", "authors": [ "Joseph Hearnshaw", "Marco Brandizi", "Ajit Singh", "Chris Rawlings", "Keywan Hassani-Pak", "Joseph Hearnshaw", "Marco Brandizi", "Ajit Singh", "Chris Rawlings" ], "abstract": "Enormous volumes of COVID-19 research data have been published and this continues to increase daily. This creates challenges for researchers to interpret, prioritize and summarize their own findings in the context of published literature, clinical trials, and a multitude of databases. Overcoming the data interpretation bottleneck is vital to help researchers to be more efficient in their quest to identify COVID-19 risk factors, potential treatments, drug side-effects, and much more. As a proof of concept, we have organized and integrated a range of COVID-19 and human biomedical data and literature into a knowledge graph (KG). Here we present the datasets we have integrated so far and the content of the KG which consists of 674,969 biological concepts and over 1.6 million relationships between them. The COVID-19 KG is available via KnetMiner, an interactive online platform for gene discovery and knowledge mining, or via RDF and Neo4j graph formats which can be searched programmatically through SPARQL and Cypher endpoints. KnetMiner is a road mapped ELIXIR UK service. We hope this integrated resource will enable faster data interpretation and discovery of linkages between genes, drugs, diseases and many more types of information relating to COVID-19.", "keywords": [ "Coronavirus", "COVID-19", "SARS-CoV-2", "knowledge graph", "knowledge base", "target discovery", "knowledge mining", "bioinformatics" ], "content": "Introduction\n\nThe global COVID-19 pandemic, caused by the SARS-CoV-2 virus, has infected millions worldwide, causing many more infections and deaths than the previous severe acute respiratory syndrome (SARS) outbreak that occurred in 2002 and 2003 (WHO, 2020). The race to find plausible treatment strategies and management plans for the coronavirus pandemic, concurrent with investigating the biological underpinnings of how SARS-CoV-2 operates has driven a very rapid rise in the number of SARS-CoV-2-related publication, pre-prints and biological data. The White House, alongside other groups, made a global call to action to find a way to rapidly sift through this mountain of COVID-19 literature. They released the COVID-19 Open Research Dataset (CORD-19), which has over 150,000 COVID-19-related full text articles (Kaggle, 2020). Additionally, the COVID-19 data portal contains over 360,000 publications and many other datasets related to the disease. The rate of growth and sheer volume of data related to the pandemic and the virus has become so large that it has led to something of a new phenomenon for many researchers: ‘too much information’. As such, there lies a challenge to organize the growing mountain of biomedical knowledge in order to overcome the data interpretation bottleneck (Good et al., 2014).\n\nOur goal was to repurpose the KnetMiner software, which we originally developed for crop scientists to help identify the most important genes involved in complex plant traits (Hassani‐Pak et al., 2021), to provide medical researchers with quick and intuitive access to all documented linkages between genes, potential therapeutic compounds, and the virus. KnetMiner contains fast algorithms for scanning millions of relationships from across a range of datasets and literature, scoring all evidence and displaying the links in easy to understand knowledge graphs. KnetMiner for COVID-19 would enable users to search for genes and keywords related to COVID-19 and explore the surrounding connected gene networks and pathways, for example, for negative downstream effects of drugs, or genetic associations with known diseases, or human-pathogen interactions.\n\nKnetMiner requires as input a knowledge graph (KG) which is a flexible semantic data model to represent heterogeneous, interconnected data where graph nodes represent biological concepts and graph edges the relationships between them. Such biological concepts can include, but are not limited to, genes, drugs, diseases, and publications. There have been a few parallel efforts to construct KGs from COVID-19 data (Reese et al., 2020). These mostly differ in data coverage and modelling approaches and would require further work to be made usable by KnetMiner. Hence, we decided to use the toolkit provided by KnetMiner to build a unified knowledge graph from public datasets and the scientific literature.\n\nHere, we describe how we organized the biomedical knowledge in a format that is compatible with KnetMiner and present a use case.\n\n\nMethods\n\nFirstly, we identified the datasets that we considered as important for the first iteration of the COVID-19 KG, based on discussions held with participants of the COVID-19 Biohackathon (5–11 April 2020). We downloaded over 20 datasets including COVID-19 related publications and pre-prints, human genetics studies, virus-host protein interactions, drug-target interactions, pathway information, ontology annotations and mouse knock-out mutant data with links to diseases and phenotypes (see Table 1). The data download and processing steps were automated using custom Python scripts (see Extended data). Various data formats are supported by the down-stream integration pipeline, including PubMed XML, UniProt XML and OWL, and most importantly tabular format, which required the development of an XML-based configuration to map the tabular content to a labelled property graph representation.\n\nThe KnetBuilder (https://github.com/Rothamsted/knetbuilder) software was used to integrate the various data sources into a KG with unified semantics and augment it with additional text mining-based relationships (Hassani-Pak et al., 2010). The data loading and integration steps are specified using an XML-based configuration. KnetBuilder uses an in-memory graph implementation (Köhler et al., 2006) and provides exports to RDF and tooling to convert RDF to Neo4j (Brandizi et al., 2018b).\n\nKnetMiner is an open-source software allowing to index and serve the contents of a knowledge graph in order to accelerate gene discovery. It requires as input a dataset folder with configuration files and a compatible knowledge graph in OXL format. We used the version 4.0 of KnetMiner and deployed it with our COVID-19 KG (see Underlying data). The COVID-19 KG contains all human genes; however, KnetMiner was configured to index a subset of 9,141 COVID-19-related genes, derived from SciBite gene annotations of CORD-19 version 54 (Giles et al., 2020). In the presented use case, KnetMiner was searched with a list of differentially expressed genes (Table 3) and the following keywords:\n\n\"Defense Response To Virus\" Remdesivir \"Killing Of Cells Of Other Organism\" \"Antimicrobial Humoral Immune Response Mediated By Antimicrobial Peptide\" \"Response To Virus\" \"Cellular Response To Lipopolysaccharide\" \"Positive Regulation Of Transcription By RNA Polymerase II\" \"White blood cell count\" \"Blood protein levels\"\n\nConceptID:500458 ConceptID:518058 ConceptID:511770 ConceptID:513484 ConceptID:503458 ConceptID:512650 ConceptID:471625 ConceptID:379514 ConceptID:370288\n\n\nCOVID-19 Knowledge Graph\n\nThe combined COVID-19 KG has 674,969 biological concepts and over 1.6 million relationships between them, and is available in OXL, RDF and Neo4j formats. It contains data on 27,599 human genes that are present in the KG as “type gene” concepts that are linked to a range of other concepts through different relation types and evidences. The content of the KG with a breakdown by concept type is shown in Table 2.\n\nThe most up-to-date statistics can be accessed at: https://knetminer.com/COVID-19/html/release.html\n\nProgrammatic access is available via public SPARQL and Cypher endpoints (Brandizi et al., 2018a) (see Underlying data). We have developed sample Jupyter notebooks to illustrate how the KG can be queried (Brandizi, 2020). This maximizes the adhesion to FAIR data principles, ensuring the data is reusable for a variety of applications. Availability, including pointers to data repositories on the Open Science Framework (OSF), is listed in the README file (see data availability section). Using Neo4j and Cypher, we can query the data and walk the knowledge graph in multiple directions. For example, we can look at the most common pathways related to proteins which are targeted by literature cited drugs using the following Cypher query:\n\n\n\n\nCOVID-19 KnetMiner\n\nCOVID-19 KnetMiner (https://knetminer.com/COVID-19/) provides gene-centric and interactive access to the integrated knowledge base created by KnetBuilder. KnetMiner can be used to search the KG with keywords, gene lists, genomic regions of interest, and/or any combinations of these user inputs. We have created several example queries for different use cases and search strategies. These are available on the COVID-19 KnetMiner website for easy use.\n\nIn June 2020, a gene expression study was published which investigated the transcriptional response of human lung epithelial cells to SARS-CoV-2 infection (Enes & Pir, 2020). The data and the analysis between infected vs mock cells are available at the EBI Gene Expression Atlas (Table 3; https://www.ebi.ac.uk/gxa/experiments/E-GEOD-147507).\n\nWe chose this dataset because of its relevance and tractable size to showcase the use of KnetMiner for the interpretation of differentially expressed genes (DEGs). The approach can be equally applied to any data driven analysis and derived gene lists. All DEGs from the above study were used in KnetMiner to investigate linkages to drugs, diseases, biological processes, and the CORD-19 literature. As such the DEG IDs were entered into the Gene List search interface of KnetMiner and analyzed in several iterations (Figure 1): (i) without any keywords to get an unbiased view of the linked and enriched knowledge, (ii) with the keyword SARS-CoV-2 to review recent CORD-19 articles in relation to these genes, and (iii) with specific drugs, diseases and GO terms to visualize and share the organized knowledge with other scientists (Figure 2).\n\nA) Search result for gene list and search terms. B) Gene View: Ranked list of genes and evidence summaries. C) Map View: Interactive genomic map with related GWAS studies and top scoring genes. D) Evidence View: Enriched linked knowledge.\n\nInformation from various sources are visualized in a single view. Users can add or hide information using the legend. In total, the network contains 1216 nodes and 1808 edges, of which 101 and 143 are shown respectively. For example, 2 out of 72 Trait concepts are visible and other linked Traits (GWAS studies) can simply be added by double-clicking the Trait symbol in the legend.\n\nKnetMiner user stories usually follow a common pattern: search, prioritize, explore, and share knowledge. All views shown in Figure 1 can generate knowledge networks for selected genes and/or evidence terms. As an example, an interactive network was generated for the top 20 scoring DEGs and keywords related to white blood cell count (GWAS study), Remdesivir (Drug), Response to Virus (GO).\n\n\nSummary\n\nKnetMiner provides open-source tools to build, search, visualize and share knowledge graphs, and its species agnostic architecture has provided a cost-efficient toolkit for building the first COVID-19 KnetMiner resource. By organizing COVID-19 related data in one place, integrated through a clear semantic data model, the hope is that this will enable data interpretation using more reproducible and objective approaches and support the international search for useful drugs, stop researchers repeating work done elsewhere, avoid harmful interventions, and ultimately, help pave the way to effective treatments. In an effort to maximize our data usefulness and grow the knowledge graph faster, we plan to offer mappings and conversions between our data and the KGX/Biolink ecosystem (Biolink, 2020). For instance, we are investigating methods to ingest the covid-19-kg produced by the Berkely Lab (Reese et al., 2020) and integrate it into the KnetMiner platform. This is work that will be conducted within the COVID-19 international research team (COV-IRT).\n\n\nData availability\n\nCOVID-19 KG: https://github.com/Rothamsted/covid19-kg/\n\nRDF Endpoint: http://knetminer-data.cyverseuk.org/lodestar/sparql\n\nNeo4j Endpoint: http://knetminer-covid19.cyverseuk.org:7476/browser/\n\nDocumentation and custom scripts are available from https://github.com/Rothamsted/covid19-kg/ (see README)\n\nArchived scripts as at time of publication: https://doi.org/10.5281/zenodo.5094695\n\nLicense: AGPL-3.0\n\n\nSoftware availability\n\nKnetMiner web app: https://knetminer.com/COVID-19\n\nELIXIR bio.tools: https://bio.tools/covid-19_knetminer\n\nSoftware available from: https://hub.docker.com/u/knetminer\n\nSource code available from: https://github.com/Rothamsted/knetminer\n\nArchived source code at time of publication (usually Zenodo): https://doi.org/10.5281/zenodo.3891097\n\nLicense: AGPL-3.0", "appendix": "Acknowledgements\n\nWe are grateful to the organizers of COVID-19 Biohackathon and the participants of the Knowledge Graph topic, especially Justin Reese, Deepak Unni and the SciBite lab for their support with our queries. We would also like to thank the COVID-19 International Research Team (https://www.cov-irt.org/) for allowing us to present KnetMiner and for providing an excellent networking channel. We additionally thank CyVerse UK and acknowledge UKRI BBSRC support (BB/R000662/1) for providing us with resources to host our Neo4j and RDF COVID-19 knowledge graphs.\n\n\nReferences\n\nBiolink: Biolink Model. 2020. Reference Source\n\nBrandizi M: The Power of Standardised and FAIR Knowledge Graphs. 2020. Reference Source\n\nBrandizi M, Singh A, Rawlings C, et al.: Towards FAIRer Biological Knowledge Networks Using a Hybrid Linked Data and Graph Database Approach. J Integr Bioinform. 2018a; 15(3): 20180023. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrandizi M, Singh A, Rawlings C, et al.: Getting the Best of Linked Data and Property Graphs: rdf2neo and the KnetMiner Use Case. 2018b. Publisher Full Text\n\nEnes A, Pir P: Transcriptional response of signalling pathways to SARS-CoV-2 infection in normal human bronchial epithelial cells. bioRxiv. 2020; 2020.06.20.163006. Publisher Full Text\n\nGiles O, Huntley R, Karlsson A, et al.: Reference ontology and database annotation of the COVID-19 Open Research Dataset (CORD-19). bioRxiv. 2020; 2020.10.04.325266. Publisher Full Text\n\nGood BM, Ainscough BJ, McMichael JF, et al.: Organizing knowledge to enable personalization of medicine in cancer. Genome Biol. 2014; 15(8): 438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHassani-Pak K, Legaie R, Canevet C, et al.: Enhancing Data Integration with Text Analysis to Find Proteins Implicated in Plant Stress Response. J Integr Bioinform. 2010; 7(3). PubMed Abstract \| Publisher Full Text\n\nHassani‐Pak K, Singh A, Brandizi M, et al.: KnetMiner: A Comprehensive Approach for Supporting Evidence‐based Gene Discovery and Complex Trait Analysis across Species. Plant Biotechnol J. 2021. PubMed Abstract \| Publisher Full Text\n\nKaggle: CORD-19. 2020. Reference Source\n\nKöhler J, Baumbach J, Taubert J, et al.: Graph-based analysis and visualization of experimental results with ONDEX. Bioinformatics. 2006; 22(11): 1383–90. PubMed Abstract \| Publisher Full Text\n\nReese J, Unni D, Callahan TJ, et al.: KG-COVID-19: A Framework to Produce Customized Knowledge Graphs for COVID-19 Response. bioRxiv. 2020; 2020.08.17.254839. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: COVID-19 Situation Reports. 2020. Reference Source" }	[ { "id": "91031", "date": "27 Aug 2021", "name": "Justin Reese", "expertise": [ "Reviewer Expertise Knowledge graphs", "machine learning", "data science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author describe a knowledge graph that integrates public datasets about COVID-19, as well as existing tooling to query and extract knowledge from the KG, i.e. Knetminer.\nTransform code is publicly available on GitHub, and source data are well explained.\nDatabase dumps are available on request, but I think these should be made available in some public place.\nKnetminer code is also available on GitHub, and is usable under an MIT license that is permissive and favorable for academic use.\nAuthors do an excellent job of conforming to existing data standards (OXL, SPARQL/Neo4J endpoints, code to convert to Neo4J, etc) and demonstrating how to use their tools. Again though, I don't see why these RDF or other dumps should not be public at some stable URL.\nThe DEG use case the authors use to demonstrate their KG and tooling is compelling and interesting. The display components of this tool are particularly strong. For example, network output for results is a very expressive way of displaying results. Could this not be linked to some public instance of Knetminer, so readers can see this result for themselves, and interact with the network?\nThe authors should probably also compare their tools/KG with existing tools. They compare with Reese et al fairly well, but there are several other COVID-19 KG efforts that probably should be included in the comparison.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "90852", "date": "01 Sep 2021", "name": "Keith Hall", "expertise": [ "Reviewer Expertise Natural Language Processing", "Machine Learning" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents an automatically constructed and normalized Knowledge Graph of Covid-19 related literature and structured data sources. An existing tool KnetMiner is used to perform the ingestion and normalization of the data.\n\nIt is not clear from the paper what the actual normalization algorithms are. When combining entities and relations from different sources, what is the algorithm which determines that two entities are actually the same entity? The same question applies to relations. While there are pointers to the available tool, without knowing the details, it's hard to determine the quality is of the created resource.\n\nAn evaluation of the quality of the data returned from this resource would have been very helpful. This would fill in the gaps where we do not know the actual algorithm/models being used.\n\nI suspect the tool may be very useful, but without some notion of the quality of the data, it is hard to determine its utility. Without knowing the precision of the data, it would be difficult to rely on this resource for active research.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-703
https://f1000research.com/articles/10-701/v1	30 Jul 21	{ "type": "Research Article", "title": "Predicting coronary artery disease risk in firefighters – a cross-sectional study", "authors": [ "Jaron Ras", "Lloyd Leach", "Lloyd Leach" ], "abstract": "Background: Firefighters are placed under severe cardiovascular load in performing active duty and, when carrying various coronary artery disease (CAD) risk factors, firefighters are predisposed to significant morbidity and mortality. Reducing the incidence of these risk factors is paramount. The purpose of this study is to determine the predictors of CAD risk.\n\nMethods: This study used a quantitative, cross-sectional and correlational design. The researchers conveniently sampled 124 full-time firefighters from the City of Cape Town Fire and Rescue Service. A researcher-generated questionnaire was used to collect sociodemographic and CAD risk factors information, such as age, gender, ethnicity, family history of CAD, cigarette smoking and physical activity levels, and all research procedures were conducted according to the American College of Sports Medicine guidelines. Data collection took place between September and November 2019. Linear and logistic regression were used to determine the relationship between the various CAD risk factors and the predictors of CAD risk.\n\nResults: Age was a significant predictor of hypertension (p <0.01), dyslipidemia (p <0.01), diabetes (p <0.01), obesity (p <0.01) and central obesity (p <0.01). Gender was a significant predictor of obesity, central obesity and cigarette smoking (p <0.05). Waist circumference was a significant predictor of hypertension (p <0.01), dyslipidemia (p <0.01) and diabetes (p <0.05).\n\nConclusion: Age was a significant predictor of various modifiable CAD risk factors, including obesity, in both genders and all ethnicities. Attentive monitoring should be in place as firefighters age, along with behavioural modifications designed to reduce age-related increases in CAD risk factors.", "keywords": [ "cardiovascular", "CAD risk factors", "age", "obesity", "firefighters" ], "content": "Introduction\n\nFirefighting is a hazardous occupation, where firefighters are constantly exposed to harmful chemicals, fumes, and severe temperatures. They routinely function in oxygen-deprived environments, which require the use of breathing apparatus and heavy insulated personal protective equipment (Smith et al., 2013; Smith et al., 2016; Smith et al., 2020). These stressful situations and while wearing their protective equipment place a tremendous load on the cardiovascular system, and, consequently, nearly 50% of firefighter mortalities are related to sudden cardiac death (SCD) (Smith et al., 2013; Smith et al., 2016; Yang et al., 2013). The prevalence of multiple coronary artery disease (CAD) risk factors, particularly obesity, diabetes, hypertension and age, significantly increase the risk of SCD (Smith et al., 2013; von Koenig Soares et al., 2020; Yang et al., 2013). Obesity and age are well-known risk factors of CAD, which augment the development of other modifiable CAD risk factors (Smith et al., 2013; von Koenig Soares et al., 2020).\n\nHealthy dietary practices, behavioural modification and regular exercise have been recommended, by previous researchers, to reduce the incidence of obesity in firefighters, as well as the onset and development of other CAD risk factors (Farioli et al., 2014; Smith et al., 2012a; Smith et al., 2019; Soteriades et al., 2011). However, age is a non-modifiable risk factor and has been associated with the development of obesity, hypertension, dyslipidemia and diabetes. The age-related increases in risk are due to hormonal changes, elasticity of arteries, disrupted cholesterol synthesis and increasing insulin resistance (Choi et al., 2016a; Choi et al., 2016b; Martin et al., 2019; Soteriades et al., 2003; Soteriades et al., 2008). The development and progression of CAD are compounded by the duties related to firefighting, particularly the erratic work schedules, irregular sleep-wake cycles and exposure to hazardous chemicals and fumes (Navarro et al., 2019; Reinberg et al., 2017; Riedel et al., 2019). Furthermore, males and specific ethnic groups have been known to be particularly predisposed to developing certain risk factors, such as hypertension, diabetes and dyslipidemia in firefighters, globally and also in South Africa (Choi et al., 2016a; Choi et al., 2016b; Choi et al., 2016c; Ras & Leach, 2021; van Zyl et al., 2012). Our previous study, conducted on the same population, indicated that increasing CAD risk factor prevalence was significantly related to age, obesity and gender in firefighters (Ras & Leach, 2021). Therefore, this study aimed to predict CAD risk in this population of firefighters, particularly in relation to gender and ethnicity.\n\n\nMethods\n\nThe current study used a quantitative, cross-sectional and correlational research design. The researchers approached each fire station individually to inform firefighters of the purpose of the study and then recruit those that were interested, using convenient sampling. In total, 124 full-time firefighters from the City of Cape Town Fire and Rescue Service were recruited to participate in the study. To address the potential bias, 10 fire stations (30 platoons) were randomly selected from the 33 fire stations in the City of Cape Town, which dispersed among the four major firefighting districts. The researchers were limited to 124 participants, as part of the agreement with the City of Cape Town. Demographic characteristics were collected, which included age, gender and ethnicity. A researcher-generated questionnaire, that included an open-ended question related to a participant information section, a medical information and lifestyle information section, which included both open-ended and closed-ended questions, and the last section included physical measures performed by the researcher. This questionnaire was used to collect subjective CAD risk factor information, such as family history of CAD, cigarette smoking and physical activity levels. The physical activity section of the questionnaire was based on the International Physical Activity Questionnaire (IPAQ) (Bohlmann et al., 2001), which is considered an accurate tool for collecting physical activity data in a South African context. Physical measures were objectively collected by the researcher (TEST DATA section). Data collection took place between September and November 2019. A copy of the questionnaire used can be found here: https://doi.org/10.6084/m9.figshare.14991447\n\nThe research procedures in the current study has been repeated from a previous published article (Ras & Leach, 2021). The principle researcher (Jaron Ras) performed all the physical measures and was responsible for administering the data recording sheet (questionnaire). For more information on the testing procedures followed to determine firefighter stature, body mass, blood pressure, blood glucose and cholesterol, please refer to the article that was previously published (Ras & Leach, 2021). The procedures used for all measurements were based on the recommendation by the American College of Sports Medicine (American College of Sports Medicine, 2018). A portable stadiometer was used to measure stature, body mass was measured using a precision electronic scale and blood pressure was measured using a standard blood pressure sphygmomanometer and stethoscope (American College of Sports Medicine, 2018). Total cholesterol and non-fasting blood glucose (NFBG) were measured using an AcuTrend® Plus GC meter. The recommended finger-prick method was used to collect blood samples. The cross-hand technique was used to measure waist circumference (WC) and Hip circumference (HC), using a steel tape measure. Both circumferences were measured at the end of normal expiration and to the nearest 0.1 cm (Geeta et al., 2009). The research instruments used for data collection were calibrated using a criterion, supplied by manufacturers, prior to testing. Calibration involved determining the test-retest reliability of the instruments, using the manufacturer’s specifications, and against a calibrated instrument. Only one tester was used in the study to ensure inter-tester reliability and a minimum test-retest reliability coefficient of 0.8 was required prior to the commencement of the study (Geeta et al., 2009).\n\nThe double-entry method was used to capture data in a Microsoft Office Excel spreadsheet, and then cleaned of errors, which involved removal of extra spaces, case and spell checking, and error removal. Thereafter, it was exported to the Statistical Package for the Social Sciences (SPSS) version 27 for descriptive and inferential data analysis. Linear and logistic regression statistics were generated to predict CAD risk in firefighters. A p-value of less than 0.05 was used to indicate statistical significance. Coefficient of determination (R2), Nagelkerke R square value and odds ratios were used to predict CAD risk. All assumptions prior to performing the regression analysis were met. For linear regression, the assumptions met included to following: (1) the data was continuous, (2) the data had a linear relationship, (3) there were no significant outliers, (3) there were independence of observations, (4) there was homoscedasticity and (5) the residuals of the regression line were approximately normally distributed. For binary logistic regression, the following assumptions were met: (1) the dependent variable was dichotomous, (2) the independent variables were continuous, (3) there were independence of observations and (4) there was a linear relationship between the logit transformation of the dependent variable and the continuous variable.\n\n\nEthics statement\n\nThe study protocol was approved by the Biomedical Research Ethics Committee (BMREC) at the University of the Western Cape (Ethics reference number: BM19/4/3). The study was also approved by the City of Cape Town. The researcher provided firefighters with an information sheet on the day of testing which explained that data would only be disclosed to the principle researcher and supervisor involved (Jaron Ras and Lloyd Leach). All participants gave their written informed consent to participate in the study and for the publication of their data. Participants were given alpha-numeric codes when capturing the data to ensure confidentiality and anonymity.\n\n\nResults\n\nThe mean age, body mass and stature of the firefighters were 37.53±9.05 years, 87.4±17.9 kg and 172.6±7.3 cm, respectively. Male firefighters represented 79.1% of the participants, and had a mean age of 37.8±9.8 years, a mean body mass of 87.8±18.5 kg and a mean stature of 174.7±6.5 cm. In female firefighters, the mean age was 36.4±5.4 years, the mean body mass was 85.9±16.2 kg and the mean statue was 164.8±4.5 cm. After firefighters were separated into age groups, the 20-29, 30-39, 40-49- and 50-65-years age-groups represented 19.4%, 44.4%, 24.2% and 12.1% of firefighters, respectively. Regarding ethnicity, 56.5% were of mixed ethnicity, 25.8% were of Black ethnicity and 16.9% were of White ethnicity. The prevalence of CAD risk factors in firefighters was diabetes in 8.9%, physical inactivity in 13.7%, a family history of CAD in 20.9%, age in 23.4%, hypertension in 33.1%, obesity in 37.1%, cigarette smoking in 39.5%, and dyslipidemia in 40.3% (Ras & Leach, 2021). In addition, 10.5% of firefighters were on anti-hypertension medication, 6.4% were on diabetes medication, and 4.8% were on lipid-lowering medication. For more information on the CAD risk factor prevalence’s or mean values for each risk factor, please refer to the previously published article (Ras & Leach, 2021).\n\nIn Table 1, age was a significant predictor of body mass index (BMI) (β = 0.25, F = 23.1, R2 = 0.16, p <0.001), WC (β = 0.79, F = 42.4, R2 = 0.26, p <0.001), systolic blood pressure (SBP) (β = 0.49, F = 11.3, R2 = 0.17, p <0.001) and diastolic blood pressure (DBP) (β = 0.24, F = 13.2, R2 = 0.09, p <0.001). The model found that 16%, 26%, 17% and 9% of the variation in BMI, WC, SBP and DBP, respectively, could be explained by an increase in age. BMI was a significant predictor of SBP (β = 0.11, F = 12.1, R2 = 0.09, p = 0.001) and DBP (β = 0.20, F = 25.9, R2 = 0.18, p <0.001). The model found that 9% and 18% of the variance in SBP and DBP, respectively, could be explained by an increase in BMI. WC was found to be a significant predictor of SBP (β = 0.29, F = 13.5, R2 = 0.10, p <0.001) and DBP (β = 0.56, F = 32.1, R2 = 0.21, p <0.001). The model found that 10% and 21% of the variance in SBP and DBP, respectively, could be explained by an increase in WC.\n\nNote: indicates statistical significance <0.05, indicates statistical significance <0.01; F – ANOVA; R2 – coefficient of determination.\n\nCAD, coronary artery disease; BMI, body mass index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; NFBG, non-fasting blood glucose; TC, total cholesterol.\n\nIn male firefighters, age was a significant predictor of BMI (β = 0.228, F = 22.9, R2 = 0.19, p <0.001), WC (β = 0.364, F = 37.1, R2 = 0.28, p <0.001), SBP (β = 0.434, F = 8.0, R2 = 0.08, p = 0.006) and DBP (β = 0.351, F = 9.5, R2 = 0.09, p = 0.003) (Table 2). The model (age) explained 19%, 28%, 8% and 9% of the variation in BMI, WC, SBP and DBP, respectively. Increasing age resulted in an increase in BMI, WC, SBP and DBP. BMI was a significant predictor of SBP (β = 1.009, F = 13.4, R2 = 0.12, p <0.001) and DBP (β = 0.899, F = 20.1, R2 = 0.17, p <0.001), explaining 12% and 17% of the variation in SBP and DBP, respectively. WC was a significant predictor of SBP (β = 0.351, F = 11.5, R2 = 0.11, p <0.001) and DBP (β = 0.329, F = 19.2, R2 = 0.17, p <0.001), with the model explaining 11% and 17% of the variation in SBP and DBP, respectively. In female firefighters, age was a significant predictor of BMI (β = 0.2541 F = 6.3, R2 = 0.21, p = 0.019), WC (β = 1.312, F = 7.6, R2 = 0.21, p = 0.011), and DBP (β = 0.909, F = 4.8, R2 = 0.17, p = 0.039). The model explained 21%, 21% and 17% of the variation in BMI, WC, and DBP, respectively. BMI was a significant predictor of DBP (β = 1.239, F = 18.3, R2 = 0.43, p <0.001), and the model explained 43% of the variation in DBP. WC was a significant predictor of DBP (β = 0.582, F = 23.1, R2 = 0.49, p <0.001), and explained 43% of the variation in DBP.\n\nNote: indicates statistical significance <0.05; ** indicates statistical significance <0.01; β – Beta; F – ANOVA; R2 – coefficient of determination.\n\nCAD, coronary artery disease; BMI, body mass index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; NFBG, non-fasting blood glucose; TC, total cholesterol.\n\nIn firefighters of mixed-ethnicity, age was a significant predictor of BMI (β = 0.209, F = 7.7, R2 = 0.10, p = 0.007), WC (β = 0.648, F = 12.9, R2 = 0.16, p = 0.001), SBP (β = 0.592 F = 9.5, R2 = 0.12, p = 0.003), DBP (β = 0.362, F = 5.3, R2 = 0.07, p = 0.024), and NFBG (β = 0.0.057, F =9.6, R2 = 0.12, p = 0.032) (Table 3). The model explained 10%, 16%, 12%, 7% and 12% of the variation in BMI, WC, SBP, DBP and NFBG, respectively. BMI was a significant predictor of SBP (β = 0.687, F = 5.1, R2 = 0.07, p = 0.027), DBP (β = 0.058, F = 14.2, R2 = 0.17, p = <0.005), and NFBG (β = 0.069, F = 5.8, R2 = 0.08, p = 0.018). The model explained 7%, 17% and 8% of the variation in SBP, DBP and NFBG, respectively. WC was a significant predictor of SBP (β = 0.273, F = 4.9, R2 = 0.07, p = 0.029), DBP (β = 0.347, F = 14.3, R2 = 0.17, p <0.001), and NFBG (β = 0.028, F = 5.9, R2 = 0.08, p = 0.018). The model explained 7%, 17% and 8% of the variation in SBP, DBP and NFBG, respectively.\n\nIn Black firefighters, age was a significant predictor of BMI (β = 0.331, F = 9.9, R2 = 0.22, p = 0.004), WC (β = 1.189, F = 25.5, R2 = 0.46, p <0.001), SBP (β = 0.824, F = 5.3, R2 = 0.15, p = 0.028), and DBP (β = 0.854, F =13.3, R2 = 0.31, p = 0.001). The model explained 22%, 46%, 15% and 31% of the variation in BMI, WC, SBP, and DBP, respectively. BMI was a significant predictor of DBP (β = 1.104, F = 8.8, R2 = 0.20, p = 0.006), with the model explaining 20% of the variation in DBP. WC was a significant predictor of SBP (β = 0.501, F = 6.2, R2 = 0.14, p = 0.018), and DBP (β = 0.488, F = 13.3 R2 = 0.28, p = 0.001), with the model explaining 14% and 28% of the variation in SBP and DBP, respectively.\n\nIn White firefighters, age was a significant predictor of BMI (β = 0.176, F = 6.2, R2 = 0.14, p = 0.018), and WC (β = 0.698, F = 9.9, R2 = 0.34, p = 0.005), where the model explained 14% and 34% of the variation in BMI and WC, respectively. BMI was a significant predictor of SBP (β = 1.158, F = 6.1, R2 = 0.20, p = 0.023), with the model explaining 20% of the variation in SBP.\n\nNote: * indicates statistical significance <0.05; ** indicates statistical significance <0.01; B – Beta; F- ANOVA; R2 – coefficient of determination.\n\nCAD, coronary artery disease; BMI, body mass index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; NFBG, non-fasting blood glucose; TC, total cholesterol.\n\nIn Table 4, gender was a significant predictor of obesity [β = 0.878, χ2 = 23.1, R2 = 0.04, p = 0.050, OR (95% CI): 2.4 (0.9, 5.8)], central obesity [β = 1.503, χ2 = 10.9, R2 = 0.12, p = 0.001, OR (95% CI): 4.5 (1.8, 11.2)], and cigarette smoking [β = 1.230, χ2 = 6.1, R2 = 0.07, p = 0.022, OR (95% CI): 3.4 (1.2, 9.8)]. The model explained 4%, 12% and 7% of the variation in obesity, central obesity and cigarette smoking, respectively. Furthermore, females were 2.4 times more likely to be obese, and 4.5 times more likely to have central obesity, whereas males were 3.4 time more likely to be cigarette smokers. Physical inactivity was a significant predictor of central obesity [β = 0.035, χ2 = 8.3, R2 = 0.06, p = 0.046, OR (95% CI): 2.4 (0.9, 5.8)]. The model explained 6% of the variation in central obesity. Age was a significant predictor of a family history of CAD [β = 0.052, χ2 = 9.1, R2 = 0.06, p = 0.035, OR (95% CI): 1.1 (1.0, 1.1)]. The model explained 6% of the variation in family history of CAD, and aged firefighters were 1.1 times more likely to have a family history of CAD. Age was a significant predictor of hypertension [β = 0.093, χ2 = 4.3, R2 = 0.18, p <0.001, OR (95% CI): 1.1 (1.0, 1.2)], dyslipidemia [β = 0.058, χ2 = 10.3, R2 = 0.08, p = 0.007, OR (95% CI): 1.1 (1.0, 1.1)], diabetes [β = 0.138, χ2 = 8.5, R2 = 0.24, p = 0.001, OR (95% CI): 1.1 (1.1, 1.2)], obesity [β = 0.066, χ2 = 12.3, R2 = 0.10, p = 0.003, OR (95% CI): 1.1 (1.0, 1.1)], and central obesity [β = 0.079, χ2 = 8.5, R2 = 0.14, p = 0.001, OR (95% CI): 1.1 (1.0, 1.1)]. The model explained 18%, 8%, 24%, 10% and 14% of the variation in hypertension, dyslipidemia, diabetes, obesity and central obesity, respectively. Aging increased the likelihood of firefighters having hypertension, dyslipidemia, diabetes, obesity, and central obesity by 1.1 times. BMI was a significant predictor of hypertension [β = 0.091, χ2 = 6.2, R2 = 0.08, p = 0.010, OR (95% CI): 1.1 (1.0, 1.2)], and dyslipidemia [β = 0.077, χ2 = 10.9, R2 = 0.06, p = 0.025, OR (95% CI): 1.1 (1.0, 1.1)] in firefighters. The model explained 8% and 6% of the variation in hypertension and dyslipidemia, respectively, where increasing BMI increased the likelihood of hypertension and dyslipdemia by 1.1 times. WC was a significant predictor of hypertension [β = 0.052, χ2 = 12.1, R2 = 0.15, p = 0.001, OR (95% CI): 1.1 (1.0, 1.1)], dyslipidemia [β = 0.039, χ2 = 18.9, R2 = 0.09, p = 0.006, OR (95% CI): 1.1 (1.0, 1.1)], and diabetes [β = 0.046, χ2 = 8.9, R2 = 0.06, p = 0.026, OR (95% CI): 1.1 (1.0, 1.1)]. The model explained 15%, 9% and 6% of the variation in hypertension, dyslipidemia and diabetes, respectively. Additionally, when WC increased, the firefighters were 1.1 times more likely to have hypertension, dyslipidemia and diabetes.\n\nNote: * indicates statistical significance <0.05; ** indicates statistical significance <0.01; B – Beta; X2 - Chi-square; df - degree of freedom; OR (95% CI) = odds ratio (95% confidence interval; R2 - Nagelkerke R square value.\n\nCAD, coronary artery disease; BMI, body mass index; WC, waist circumference.\n\n\nDiscussion\n\nAge was a significant predictor of an increase in BMI and WC. Furthermore, age, BMI and WC were significant predictors of SBP and DBP. This is consistent with previous literature which indicated that age, BMI and WC were significant catalysts for the development of major CAD risk factors, particularly hypertension (Choi et al., 2016a; Choi et al., 2016b; Damacena et al., 2020; Jang et al., 2020; Soteriades et al., 1997; Soteriades et al., 2003). Interestingly, age was the highest predictor of CAD risk in the current study, followed by WC and BMI. This is supported by previous literature, which consistently reported that increasing age was the most important determinant in the development of CAD, followed by BMI and WC (Choi et al., 2016a; Choi et al., 2016b; Choi et al., 2016c; Damacena et al., 2020; Lee & Kim, 2017; Smith et al., 2013; Soteriades et al., 2003). Dyslipidemia was the most prevalent CAD risk factor in firefighters, yet, was not predicted by age, BMI or WC. Firefighters’ diets appear to be the most significant cause of dyslipidemia (de Ridder et al., 2017; Liska et al., 2016; Sanders et al., 2016).\n\nIn male firefighters, age was a significant predictor of BMI and WC, and age, BMI and WC were significant predictors of SBP and DBP. In female firefighters, age was a significant predictor of BMI and WC, and age, BMI and WC were significant predictors of DBP. Philippe Gendron et al. (2018a) reported that, in male firefighters, BMI was significantly different between the group that presented with no CAD risk factors compared to the group with one or more risk factors. Choi et al. (2016c) reported that in both male and female firefighters, age was significantly correlated with BMI and WC in firefighters. Li et al. (2017) reported that, among male and female firefighters, age and obesity were significantly associated with metabolic syndrome. Smith et al. (2020) reported a similar result, where BMI significantly increased as firefighters aged.\n\nIn firefighters of mixed ethnicity, age, BMI and WC were significant predictors of SBP, DBP and NFBG, and age and WC were significant predictors of SBP in Black firefighters. In White firefighters, BMI was a significant predictor of SBP. In all ethnic groups, age was a significant predictor of BMI and WC. Previous literature indicated that all ethnicities were prone to developing CAD with increasing age, BMI and WC. Age was significantly related to obesity across all ethnic groups of firefighters (Choi et al., 2016a; Choi et al., 2016c; Damacena et al., 2020; Poston et al., 2015), which corresponds to the results in the present study.\n\nThe logistic regression model indicated that females were 2.4 and 4.5 times more likely to be obese and have central obesity, compared to males. However, males were significantly more likely (3.4 times) to be cigarette smokers. This is contrary to previous studies, which indicated that male firefighters were more likely to be obese than female firefighters (Crespo-Ruiz et al., 2020; Gendron et al., 2018b; Gendron et al., 2018a; Jahnke et al., 2012a; Jahnke et al., 2012b; Li et al., 2017). In addition, previous studies indicated that female firefighters were more likely to be cigarette smokers compared to males (Gendron et al., 2018b; Gendron et al., 2018a; Jitnarin et al., 2013; Jitnarin et al., 2019; Li et al., 2017). Central obesity was a significant predictor of physical inactivity, which was similar to Damacena et al. (2020) who indicated that central obesity was a significant predictor of physical inactivity, where firefighters who had central obesity were 3.43 times more likely to be physically inactive.\n\nAge was a significant predictor for hypertension, dyslipidemia, diabetes, obesity and central obesity. Damacena et al. (2020) reported that central obesity was significantly associated with increased age in firefighters, with the 40-49 years age-group being 4.9 times more likely to have central obesity, and the 50-59 years age-group being 5.41 times more likely to have central obesity. This is consistent with previous literature which indicated a linear relationship between increased age and the incidence of hypertension, dyslipidemia, diabetes and obesity (Choi et al., 2016a; Choi et al., 2016b; Choi et al., 2016c; Damacena et al., 2020; Smith et al., 2012b; Smith et al., 2020; Soteriades et al., 1997; Soteriades et al., 2002). Eastlake et al. (2015) reported that age in firefighters had a significant association with high blood cholesterol and high blood pressure, with aged firefighters being 1.08 times and 1.06 times more likely to have elevated blood cholesterol and high blood pressure, respectively. Burgess et al. (2012) reported that age was significantly associated with dyslipidemia, and that aged firefighters were 3.3 times more likely to be dyslipidemic. The role of age in the development and progression of major CAD risk factors may be attributed to the cascade of age-related alterations in normal homeostatic functioning (Costantino et al., 2016; Ferrucci & Fabbri, 2018; Morgan et al., 2016). Aging was reported to reduce the growth hormones essential for angiogenesis and vascular maintenance, contributing to the increase in blood pressure (Costantino et al., 2016; Lakatta, 2002). The increased inflammatory response due to aging, the increased catabolic metabolism due to a decrease in anabolic hormones, specifically, testosterone, oestrogen, and growth hormone, and the reduced insulin sensitivity and cholesterol regulation, all collectively result in an increase in adipose tissue accumulation, particularly around the abdomen (Gadde et al., 2018; Pandey et al., 2018). All these age-related changes were associated with the increased incidence of CAD risk in firefighters, specifically obesity, hypertension and dyslipidemia (Costantino et al., 2016; de Schutter et al., 2014; Ferrucci & Fabbri, 2018; Gadde et al., 2018). However, the stressful nature of firefighting, the constant inhalation of toxic chemicals and fumes, irregular sleep-wake cycles, and poor dietary practices, further compounded the negative effects of aging in this population, and augment the CAD risk (Adetona et al., 2016; Bonnell et al., 2017; Costantino et al., 2016; Ferrucci & Fabbri, 2018; Lakatta, 2002; Navarro et al., 2019; Reinberg et al., 2017; Riedel et al., 2019; Sanders et al., 2016; Yang et al., 2013). The longer firefighters are in service, the more this effect is compounded (Adetona et al., 2016; Bonnell et al., 2017; Costantino et al., 2016; Ferrucci & Fabbri, 2018; Lakatta, 2002; Navarro et al., 2019; Reinberg et al., 2017; Riedel et al., 2019; Sanders et al., 2016; Yang et al., 2013).\n\nIn the present study, BMI was a significant predictor of hypertension and dyslipidemia. Eastlake et al. (2015) reported that BMI was a significant predictor of high cholesterol, where firefighters were 1.09 times more likely to have high cholesterol as BMI increased. Previous research indicated a linear relationship between increased BMI and the incidence of hypertension and dsylipidemia (Choi et al., 2016a; Choi et al., 2016b; Choi et al., 2016c; Soteriades et al., 1997; Soteriades et al., 2002; Soteriades et al., 2003; Soteriades et al., 2008). The strong predictive value of BMI, particularly to blood pressure and dyslipidaemia, can be explained by the increase in peripheral vascular resistance associated with an increase in total body mass related to adipose tissue accumulation, and the resultant cholesterol synthesis dysregulation associated with increased adiposity (Alpert et al., 2014; Ariyanti & Besral, 2019; de Schutter et al., 2014; Shulman, 2014).\n\nIn the present study, WC was a significant predictor of hypertension, dyslipidemia and diabetes. This is similar to the results reported by Damacena et al. (2020), where increased WC was a significant predictor of total cholesterol, blood glucose and blood pressure, with firefighters being 1.71 and 2.94 more likely to have elevated total cholesterol and blood glucose concentrations, respectively. In the literature, WC has a linear relationship with blood pressure, blood cholesterol and glucose concentration (Choi et al., 2016c; Soteriades et al., 1997; Soteriades et al., 2002). The strong predictive value of WC related to CAD risk can be attributed to a similar mechanism implicated in increased BMI, in which an increase in adiposity also increases peripheral vascular resistance, resulting in hypertension and cholesterol synthesis dysregulation (Alpert et al., 2014; Ariyanti & Besral, 2019; de Schutter et al., 2014; Shulman, 2014). Abdominal fat, especially when central obesity is present, is associated with an increased risk of diabetes and, presumably, due to abdominal adipose tissue being more insulin resistant (Emdin et al., 2017; Shulman, 2014).\n\nThis was the first study in South Africa to predict CAD risk in firefighters according to age, gender and ethnicity. This study provides valuable information for the City of Cape Town to consider how to increase the longevity of firefighter careers.\n\nA limitation was that the study used convenient sampling that negatively impacted the external validity as selection bias may have occurred, due to firefighters opting not to participate that have known CAD risk factors. Also, the relatively small sample size of 124 firefighters negatively impacted the power of the study, which could be seen where variables trended towards significance, but required a larger sample size to be realized. The study was also under-represented by female participants.\n\nIt is recommended that future studies use random sampling and a larger sample, which is sufficiently powered in order to ensure external validity. In addition, a more representative sample of female firefighters is recommended, as females are notably underrepresented, both in the present study and in global firefighter research.\n\n\nConclusion\n\nIn conclusion, age was a significant predictor of CAD risk, including obesity, and this was consistent across both genders and all ethnicities. WC was a significant predictor of blood pressure and cardiometabolic abnormalities, particularly in relation to firefighters of mixed ethnicity. The City of Cape Town Fire and Rescue service should emphasise behavioural modification, such as a healthier diet and an exercise routine designed for firefighters, to reduce the likelihood of obesity and, in particular, central obesity. As firefighters age, attentive monitoring, such as annual or biannual medical, cardiovascular and fitness screenings should be in place to reduce age-related obesity, and the subsequent development of lifestyle-related CAD risk factors, specifically hypertension, dyslipidemia and diabetes.\n\n\nData availability\n\nThe captured data contains confidential information on firefighters that cannot be made publicly available as part of the agreement with the City of Cape Town Fire and Rescue Service. Only the researchers directly involved in the study, i.e., Jaron Ras and Lloyd Leach, have access to this data. If researchers require the data, requests should be submitted to the corresponding author (Jaron Ras: jaronras@gmail.com), where permission will then be requested from the City of Cape Town Fire and Rescue Service and upon signing a data access agreement in compliance with the City of Cape Town data regulations.\n\nFigshare: Prevalence of Coronary Artery Disease Risk Factors in the City of Cape Town Fire and Rescue Service. CC0 License (https://doi.org/10.6084/m9.figshare.14991447) and (https://doi.org/10.6084/m9.figshare.14991576)\n\nThis project contains the following extended data:\n\nData recording sheet (Questionnaire)\n\nStudy protocol", "appendix": "Acknowledgments\n\nWe thank the City of Cape Town for granting permission to conduct the study, Mr. Ian Bell from the City of Cape Town Fire and Rescue Service and the firefighters who participated in the study.\n\n\nReferences\n\nAdetona O, Reinhardt TE, Domitrovich J, et al.: Review of the health effects of wildland fire smoke on wildland firefighters and the public. Inhal Toxicol. 2016; 28(3): 95–139. PubMed Abstract \| Publisher Full Text\n\nAlpert MA, Lavie CJ, Agrawal H, et al.: Obesity and heart failure: Epidemiology, pathophysiology, clinical manifestations, and management. Transl Res. 2014; 164(4): 345–356. 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PubMed Abstract \| Publisher Full Text\n\nRiedel M, Smolensky MH, Reinberg A, et al.: Twenty-four-hour pattern of operations-related injury occurrence and severity of off-site/on-call volunteer French firefighters. Chronobiol Int. 2019; 36(7): 979–992. PubMed Abstract \| Publisher Full Text\n\nSanders D, Puoane T, Hoelzel P, et al.: Diet-related non-communicable diseases in South Africa: determinants and policy responses. S Afr Health Rev. 2016; 2016(1): 35–42. Reference Source\n\nShulman GI: Ectopic fat in insulin resistance, dyslipidemia, and cardiometabolic disease. N Engl J Med. 2014; 371(12): 1131–1141. PubMed Abstract \| Publisher Full Text\n\nSmith DL, Barr DA, Kales SN: Extreme sacrifice: Sudden cardiac death in the US Fire Service. Extrem Physiol Med. 2013; 2(1): 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith DL, DeBlois JP, Kales SN, et al.: Cardiovascular strain of firefighting and the risk of sudden cardiac events. Exerc Sport Sci Rev. 2016; 44(3): 90–97. PubMed Abstract \| Publisher Full Text\n\nSmith DL, Fehling PC, Frisch A, et al.: The prevalence of cardiovascular disease risk factors and obesity in firefighters. J Obes. 2012a; 2012: 908267. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith DL, Fehling PC, Frisch A, et al.: The prevalence of cardiovascular disease risk factors and obesity in firefighters. J Obes. 2012b; 2012: 908267. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith DL, Graham E, Stewart D, et al.: Cardiovascular Disease Risk Factor Changes Over 5 Years Among Male and Female US Firefighters. J Occup Environ Med. 2020; 62(6): 398–402. PubMed Abstract \| Publisher Full Text\n\nSmith DL, Haller JM, Korre M, et al.: The Relation of Emergency Duties to Cardiac Death Among US Firefighters. Am J Cardiol. 2019; 123(5): 736–741. PubMed Abstract \| Publisher Full Text\n\nSoteriades ES, Hauser R, Kawachi I, et al.: Obesity and risk of job disability in male firefighters. Occup Med (Lond). 2008; 58(4): 245–250. PubMed Abstract \| Publisher Full Text\n\nSoteriades ES, Kales SN, Liarokapis D, et al.: Prospective surveillance of hypertension in firefighters. J Clin Hypertens (Greenwich). 2003; 5(5): 315–320. PubMed Abstract \| Publisher Full Text\n\nSoteriades ES, Kales SN, Liarokapis D, et al.: Lipid Profile of Firefighters Over Time: Opportunities for Prevention. J Occup Environ Med. 2002; 44(9): 840–6. PubMed Abstract \| Publisher Full Text\n\nSoteriades ES, Liarokapis D, Christoudias SG, et al.: Lipid Profile of Firefighters Over Time. 1997; 840–846.\n\nSoteriades ES, Smith DL, Tsismenakis AJ, et al.: Cardiovascular disease in US firefighters: A systematic review. Cardiol Rev. 2011; 19(4): 202–215. PubMed Abstract \| Publisher Full Text\n\nvan Zyl S, van der Merwe LJ, Walsh CM, et al.: Risk-factor profiles for chronic diseases of lifestyle and metabolic syndrome in an urban and rural setting in South Africa. Afr J Prim Health Care Fam Med. 2012; 4(1): 346. Publisher Full Text \| Free Full Text\n\nvon Koenig Soares EMK, Smith D, Grossi Porto LG: Worldwide prevalence of obesity among firefighters: A systematic review protocol. BMJ Open. 2020; 10(1): e031282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang J, Teehan D, Farioli A, et al.: Sudden cardiac death among firefighters ≤45 years of age in the United States. Am J Cardiol. 2013; 112(12): 1962–1967. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "91057", "date": "31 Aug 2021", "name": "Francois Trudeau", "expertise": [ "Reviewer Expertise work physiology", "cardiac 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\nVery interesting manuscript. I present my comments in the form provided, but also a copy of your manuscript (converted to Word) with annotations/suggestions.\nA major weakness of the study is the lack of comparison with the corresponding population. Is the risk factors magnitude comparable to a paired group from the population? This should at least be mentioned in the Strengths and limitations section.\nAbstract:\nI doubt it is necessary to mention: \"according to the American College of Sports Medicine guidelines’’.\nKeywords: You include ‘’ cardiovascular’’ as a keyword, I suggest that you include a name with cardiovascular. Cardiovascular health?\nIn your text, you refer to ‘’gender’’ while you should refer to ‘’sex’’ since in your questionnaire you ask for female or male.\nMethodology: What is the statistical power of your sample? It is important since in the recommendations you indicate that:’’ It is recommended that future studies use random sampling and a larger sample, which is sufficiently powered’’. It is important since you perform regression analysis that needs a minimum of participants per predictors.\nIn the methodology, you mentioned ‘’ The recommended finger-prick method was used to collect blood samples.’’ Which method is it ? The one provided by the maker of the AccuTrend® AccuTrend® Plus GC meter?\nAcuTrend is AccuTrend.\nResults: In tables, you often use different fonts (Arial and Sans Serif vs. Times New Roman).\nPage 4: you mentioned : ‘’ For more information on the CAD risk factor prevalence’s or mean values for each risk factor, please refer to the previously published article (Ras & Leach, 2021).’’ PLease explain the relationship between the manuscript and the cited paper. Is it the same data base?\nDiscussion: General comment: With your correlational design is it appropriate to use the term predictors instead of correlates? Why not physical inactivity a predictor of central obesity?\nPage 8 : You present an interesting but speculative hypothesis: ‘’This suggests that firefighters’ diet appear to be the most probable cause of dyslipidemia’’ Please develop how diet impact risk factors.\nThe paragraph starting by : In male firefighters,…is an enumeration of facts, and a sentence should wrap-up or synthesize the paragraph.\nI have some difficulties with this paragraph: ‘’Central obesity was a significant predictor of physical inactivity, which was similar to Damacena et al. (2020) who indicated that central obesity was a significant predictor of physical inactivity, where firefighters who had central obesity were 3.43 times more likely to be physically inactive.’’ What would be the mechanism of this relation (causality of central obesity leading to inactivity)? The other way around is probable biologically.\nPage 9: 2 successive paragraphs start with ‘’In the present study,…’’ Change one of them to avoid repetition.\nReferences: Please standardize the style of references. 1) Some journal titles are abbreviated, 2) Sometimes the first letters of words from the article title are capitalized sometimes not, etc.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "301518", "date": "16 Jul 2024", "name": "Koulla Parpa", "expertise": [ "Reviewer Expertise Exercise Physiology", "Sports Medicine", "Exercise Performance", "Disease Prevention" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview: Predicting coronary artery disease risk in firefighters- a cross-sectional study\nFirst, I would like to recognize the authors for preparing this manuscript which aimed to determine the predictors of CAD risk in firefighters.\nThe Abstract presents the rationale of the study. In addition, it provides an objective representation of the article and presents the study's results.\nKeywords: I would say cardiovascular health instead of cardiovascular.\nThe Introduction is well-structured, includes the relevant literature on the topic and presents the study's rationale. The purpose of this study is clearly presented.\nMethodology: Including only those who were interested in participating could be a limitation which is already mentioned by the authors in the limitations section.\n\nThe recommended finger-prick method was used to collect blood samples. This statement is fine by me as I use this method frequently. You can include a previously published study (as a citation) which explains the procedure in detail so that you don’t have to provide information as to the amount of blood (drop) that you got, the time you waited for the reading to appear, the finger used, the cleaning of fingers before etc.\nThe statistical analysis is fine by me. I would have added a power analysis in case anyone is interested in replicating this study.\nThe only question I have on the results section is relevant to the following study [Ras J et al. (2021 [Ref-2]). Is that the same study as this one or a different one? It is a little confusing as you start your procedures section by presenting that study which was published in 2021 (while the data collection for this study was done in 2019). Thereafter in the results section, you also state that we should refer to the 2021 study for the mean scores and prevalence etc. You need to clarify the above.\nDiscussion Did you control for dietary habits, or do you assume that dietary habits are the most significant cause of dyslipidemia? I saw your questionnaire which did not include any questions on dietary habits. Could this be related to genetic predisposition? We do not really know if they have familiar dyslipidemia. You could assume based on previous studies but I believe this statement needs to be re-written.\nParagraph 4: I would have assumed that physical inactivity is a predictor of obesity based on the results presented in a previously published study [Firefighters who were physically inactive were 6.0, 2.9 and 3.7 times more likely to be aged, obese and to have central obesity, respectively- Ras and Leach, 2022]. To be honest I am not sure which one comes first. Are they inactive because they are obese or are they obese because they are inactive? I think you could explain this prediction that you present in paragraph 4 a little further.\nI know this is beyond the purpose of this study but in the discussion section, I was expecting to see some comparison with the general population. Are those predictors different from those presented for the general population? This is a minor issue but it may be nice to make a short comparison (within the existing paragraphs maybe) as some researchers state that firefighters are at a higher risk for CAD compared to the general population.\nLimitations are accurately presented.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-701
https://f1000research.com/articles/9-179/v1	11 Mar 20	{ "type": "Research Article", "title": "The Norwegian public’s ability to assess treatment claims: results of a cross-sectional study of critical health literacy", "authors": [ "Astrid Dahlgren", "Kjetil Furuseth-Olsen", "Christopher James Rose", "Andrew David Oxman", "Kjetil Furuseth-Olsen", "Christopher James Rose", "Andrew David Oxman" ], "abstract": "Background: Few studies have evaluated the ability of the general public to assess the trustworthiness of claims about the effects of healthcare. For the most part, those studies have used self-reported measures of critical health literacy. Methods: We mailed 4500 invitations to Norwegian adults. Respondents were randomly assigned to one of four online tests that included multiple-choice questions that test understanding of Key Concepts people need to understand to assess healthcare claims. They also included questions about intended behaviours and self-efficacy. One of the four tests was identical to one previously used in two randomised trials of educational interventions in Uganda, facilitating comparisons to Ugandan children, parents, and teachers. We adjusted the results using demographic data to reflect the population. Results: A total of 771 people responded. We estimate that at least half of Norwegian adults understand 18 of the 30 Key Concepts (i.e. the adjusted proportion of correct answers was > 50%). On the other hand, less than half understood 13 of the concepts. The results for Norwegian adults were better than the results for Ugandan children in the intervention arm of the trial and parents, and similar to those of Ugandan teachers in the intervention arm of the trial. Based on self-report, most Norwegians are likely to find out the basis of treatment claims, but few consider it easy to assess whether claims are based on research and to assess the trustworthiness of research. Conclusions: Norwegian adults do not understand many concepts that are essential for assessing healthcare claims and making informed choices. This can result in poorly informed decisions, underuse of effective interventions, and overuse of ineffective or harmful interventions.", "keywords": [ "health literacy", "critical health literacy", "evidence-informed decision-making", "evidence-based practice", "critical thinking", "public health", "shared decision making", "public understanding of science" ], "content": "Introduction\n\nEnabling people to make informed decisions about healthcare by improving their ability to think critically about such claims is an important public health initiative1. Health literacy has been defined in many ways. Commonly this includes functional, interactional, and critical health literacy skills2. Health literacy research efforts in Norway and elsewhere have mainly been directed at providing reliable health information and education in functional literacy, focusing on improving understanding of medical terminology and self-management of health conditions3. Very little research has targeted critical health literacy, focused on enabling members of the public to make informed healthcare choices4–6.\n\nTraining in critical thinking about treatment decisions includes courses in evidence-based practice and education in basic research methodology. Such training is usually directed at health professionals. Despite the importance of enabling patients and the public to make informed decisions about treatment choices, such training has rarely been offered4–8.\n\nOver the past decade, there has been increasing interest in enabling patients and the public to think critically about healthcare4,5,9–14. For example, in Norway three websites have been developed that aim to empower patients and the public to assess the trustworthiness of health claims15–17. Critical thinking has also been the focus of popular television shows that target common claims about treatment effects and illustrate how such claims can be tested using rigorous study designs. In collaboration with the Norwegian national television network (NRK), we carried out randomised trials with the aim of educating the public about the need for fair comparison of treatments18–22. More recently, we have sought to improve critical thinking in school children through the Informed Health Choices (IHC) project, an international collaboration of researchers in Uganda, Rwanda, Kenya, the UK, and Norway. We developed an educational intervention to teach primary school children to assess healthcare claims and an educational podcast for their parents. Both interventions were shown to be effective in randomized trials in Uganda23,24.\n\nFew studies have been done to evaluate this ability in the general public to assess the trustworthiness of claims about the effects of healthcare and to make informed health choices6. Most such studies have relied on self-assessment of critical health literacy skills. One such project is the HLS-EU survey, mapping health literacy skills in several European countries25.\n\nThe Claim Evaluation Tools item bank was originally developed in English for use in the two randomised trials described above23,24. Since the item bank was first created items have been translated and validated in several settings and languages, including Uganda (in Luganda and English), Mexico, and China26–30.\n\nOur starting point for developing the educational interventions and the Claim Evaluation Tools item bank was a list of Key Concepts that people need to understand and apply to assess claims about treatment effects and make informed health choices (hereafter referred to as Key Concepts)31 (Table 1). We define “treatment” broadly to include any intervention (action) intended to improve health, including preventive, therapeutic and rehabilitative interventions, and public health or health system interventions. The list of concepts provides a framework for researchers, teachers and others to develop interventions and measure people’s ability to assess treatment claims and make informed choices. We review and amend the list yearly9.\n\nThe item bank is an open-access resource. When we conducted this study, it included at least four multiple-choice questions (MCQs) for 32 of the Key Concepts shown in Table 1. The item bank also includes questions that assess intended behaviours and self-efficacy. Teachers and researchers can select items for tests tailored to their specific needs.\n\nTo our knowledge only one survey has attempted to measure the ability to understand and apply any of the IHC Key Concepts in a representative sample of Norwegian adults32. That study only addressed four of the Key Concepts. The purpose of this study was to map the ability of Norwegian adults to assess treatment claims and make informed health choices, using MCQs from the Claim Evaluation Tools item bank. The findings can be used to inform the development of learning resources and communication of information to patients and the public, and for international comparisons.\n\n\nObjective\n\nTo map the ability of Norwegian adults to assess treatment claims and make informed choices, and to compare these results to the findings of two studies in Uganda23,24.\n\n\nMethods\n\nThis study was considered for ethical approval by the Norwegian Institute of Public Health. This project was considered to not to require a full ethical review (reference 18/11854), because no sensitive data was included. All information was collected through Nettskjema (a web-based survey system), ascertaining a high level of data security and safety.\n\nThe population was drawn from the National registry who provided us with a CD-ROM with respondents’ addresses. This CD was destroyed once data collection was terminated.\n\nAll respondents were given information about the purpose of the study and how the results would be managed and presented. To gain access to the questionnaire, each participant had to provide written consent electronically. The questionnaire was anonymous and once submitted, the information could not be traced back to the respondent.\n\nThe Claim Evaluation Tools item bank was developed in English. For this study, a language specialist (KFO) translated all items to Norwegian. Three researchers trained in evidence-based medicine and epidemiology reviewed this translation (AD, ADO, and Atle Fretheim). The questions were not modified other than changing some of the names of people in the scenarios to more familiar names in Norwegian. For example, “Dr. Acheng” was changed to “Dr. Anker” (a more familiar name in Norwegian). MCQs for 32 Key Concepts were administrated as part of this study. However, as previously mentioned, the Key Concepts are reviewed and revised annually. Over the duration of this study, two Key Concepts were revised into the new Key Concept 1.1a. Considering that the MCQs were no longer appropriately covering this concept, these MCQS were therefore taken out of the analysis.\n\nAll MCQs in the item bank include a scenario with a treatment claim, and response options, with one answer being the “best” (correct) and the remaining options considered “worse” (incorrect). An example of a multiple-choice question is shown in Box 1. We planned on including questions about intended behaviours and self-efficacy, in addition to two MCQs for each Key Concept. Anticipating that few respondents would be prepared to answer this number of MCQs, we split the questions across four tests (see Additional file 1 and Additional file 5, Extended data)33,34.\n\nJudith wants smoother skin. The younger girls in her school have smoother skin than the older girls. Judith thinks this is because the younger girls use cream on their skin to make the skin smoother.\n\nQuestion: Based on this link between using cream and smooth skin, is Judith correct?\n\nOptions:\n\nA) It is not possible to say. It depends on how many younger and older girls there are\n\nB) It is not possible to say. There might be other differences between the younger and older girls\n\nC) Yes, because the younger girls use cream on their skin and they have smoother skin\n\nD) No, Judith should try using the cream herself to see if it works for her\n\nWe included a common set of eight MCQs in all four tests, two for each of four Key Concepts:\n\nAn outcome may be associated with a treatment but not caused by it.\n\nThe results of one study considered in isolation can be misleading.\n\nWidely used treatments or those that have been used for decades are not necessarily beneficial or safe.\n\nComparison groups should be as similar as possible.\n\nWe considered these four Key Concepts to be amongst the most important Key Concepts that can be understood and used by children, as well as adults. These MCQs were also included in the test used in the randomised trials in Uganda35.\n\nThe first test was identical to the test used for the randomised trials in Uganda23,24, facilitating comparison between Norwegian adults and Ugandan children, parents, and teachers. The second test included the remaining items that address concepts in the first (Claims) category as well as items from the second (Comparisons) category (Table 1). The third test included items from the third (Choices) category. This test also included three items evaluating intended behaviours regarding assessing treatment claims and agreeing to participate in a study evaluating treatments, and four items regarding self-efficacy. The fourth test included MCQs addressing Key Concepts that we judged to be more difficult, mostly belonging to the Comparisons category.\n\nAll four tests included the same set of questions regarding the participant’s sex, level of education, health professional background, training in research methods, and prior participation in research.\n\nBased on evidence from two systematic reviews that evaluated strategies to improve participation in research36,37, we developed attractive postcards personally addressed to each potential participant inviting people to take part in the study. The postcard is shown in Figure 1. The postcard included a short description of the purpose of the study as well as a URL (“link”) to one of the four online tests. Participants received full information about the study when they accessed the website and were asked to provide informed consent. Evidence also suggests greater participation rates can be expected if people are informed how the research may benefit them. This was stated on the postcard and the website provided information about how the study results can be accessed.\n\nIn January and February 2019, we mailed postcards to a representative sample of 4500 adults (≥18 years) living in Norway. The sample was provided to us by the Norwegian National Registry, considering level of education, sex and geographical spread38. The questionnaires were administrated electronically using Nettskjema, a service provided by University of Oslo39. In addition to the first postcard, one reminder postcard was sent out to each person.\n\nThe tests were evaluated for their psychometric properties as part of this study. Although some MCQs were tested in Norway as part of a previous pilot, this is the first full-scale psychometric evaluation of the complete available battery of the Claim Evaluation Tools Item bank26,27.\n\nRasch analysis is a dynamic and practical approach to address important measurement issues required for validating an outcome measure and can be used for both dichotomous and polytomous data40–42. In this study we followed the fundamental steps of Rasch analysis including testing for internal construct validity (multidimensionality), invariance of the items (Item-Person Interaction), and item bias (differential item functioning), as well as testing for reliability41,42. Rasch analysis can be used for both dichotomous and polytomous data41,43,44. Raw data were exported from the electronic data collection service (Nettskjema) as Excel-files and entered into RUMM2030 for Rasch analysis. Results can be replicated using the open-access software WINSTEPS45. Each test set was evaluated separately. Test 3 included also polytomous questions assessing intended behaviour and self-efficacy questions. Therefore, for test 3 specifically, additional analysis was done for each item block separately (MCQ- item block and intended behaviour and self-efficacy questions-block respectively).\n\nEach MCQ was scored as correct or incorrect. The Key Concepts for which there were two MCQs were scored as “understood” if a participant answered both MCQs correctly. For questions about intended behaviours and self-efficacy, we dichotomised the responses in the analysis, for example as likely (very likely or likely) vs not likely (very unlikely, unlikely, or don’t know).\n\nBased on a previous survey we conducted32, we anticipated that women and respondents with higher education levels would be more likely to respond. To address such non-random non-response, we used iterative post-stratification to match marginal distributions of sex and educational attainment level of the sample to the Norwegian population46. We also adjusted for region of residence. Due to an error, we did not collect data on participants’ age and could not use it for post-stratification, as planned.\n\nWe obtained Eurostat data on the marginal distributions of sex and region of residence for all Norwegians, and educational attainment level for Norwegians aged 15 to 64 years47,48. Participants reported their county of residence. We mapped counties to the corresponding Nomenclature of Territorial Units for Statistics (NUTS 2) regions of Norway used by Eurostat49. Participants reported the highest level of education they attained. We mapped these to the corresponding International Standard Classification of Education (ISCED) 2011 categories used by Eurostat (levels 0–2, 3–4, and 5–8). We used multiple imputation with chained equations to account for the uncertainty introduced by missing values of the post-stratification variables sex, region of residence, and educational level50, and iteratively post-stratified each imputed data set. Based on these adjustments, we estimated the percentage of the Norwegian population that understands each key concept, and that responded positively for each question about intended behaviours and self-efficacy. We used the R packages tidyverse, mice, mitools, and survey.\n\nWe present summaries of the results for participants and the post-stratified population estimates for understanding of the 30 Key Concepts, and the intended behaviours and self-efficacy questions. We quantified the uncertainty of our estimates using 95% confidence intervals and protected the family-wise coverage probability of the confidence intervals for the four Key Concepts included in all questionnaires via Bonferroni-correction (i.e., we report a 98.75% CI for each of those concepts). For each Key Concept, we calculated the likelihood of answering both questions (or the one question for two Key Concepts) correctly if participants randomly guessed the answer. These probabilities vary between six and 25%, depending on the number of MCQs and the number of response options (between two and four) for each MCQ.\n\nWe compared Norwegian and Ugandan adults’ understanding of the Key Concepts to that of Ugandan children in the intervention arm of our randomized trial. We compared mean test scores, and probabilities of achieving passing and mastery scores, using estimates from one year after the IHC primary school intervention for the Ugandan children and their teachers51,52 and estimates from the control group for parents in the IHC podcast trial, one year after parents in the intervention group listened to the podcast52,53. We used predetermined thresholds of at least 13 of 24 questions answered correctly for a passing score and at least 20 of 24 for mastery.\n\nWe estimated mean scores and odds ratios using generalized linear mixed-effects models (GLMMs; normal errors and identity link for mean scores, binomial errors and logit link for passing and mastery) using the lme4 R package. In the trials, children and teachers were randomized in clusters (schools), while parents were individually randomized. We modelled this clustering structure as a random intercept for each randomized unit. We did not adjust for covariates such as those used for stratified sampling in the Ugandan trials because those variables are not defined for all samples (e.g., school ownership was used in the trial on Ugandan children, but there is no analogous concept for Norwegian adults). No data were missing. It is not possible to use the lme4 package to apply post-stratification weights for the Norwegians.\n\nWe conducted exploratory analyses to investigate associations between understanding each Key Concept and the demographic covariates sex, research training, research participation, education level, and health professional background. Based on findings from our previous survey32, we hypothesised that better understanding of the Key Concepts would be associated with having a research background or a higher education level; that there would be little difference between health professionals and others; and that there would be little difference between women and men. We used generalized linear models (GLMs; quasibinomial errors and logit link) as before, modelled the covariates as categorical variables, and used multiple imputation and post-stratification as in the main analysis.\n\nWe used data from the first questionnaire to perform exploratory analyses to investigate how Norwegians’ mean scores and achievement of passing and mastery scores are associated with the demographic covariates sex, research training, research participation, education, and health professional background. We used GLMs (normal errors and identity link for mean score; quasibinomial errors and logit link for passing and mastery) to model the outcomes in terms of the covariates, which were modelled as before. Multiple imputation and post-stratification were used as in the main analysis. The variables included in imputation were the post-stratification variables (sex, region of residence, and educational attainment); demographic variables that coded for whether participants had research training or a health professional background, and whether they had been a research participant; and mean score. We did not include passing and mastery in imputation, as they can be calculated from the mean score.\n\nWe performed three power analyses. The first two analysed power to estimate differences, of at least 5% from random guessing, in the proportion of the Norwegian population that understands the concepts. The first analysis focused on the four Key Concepts that would be included on all four tests while protecting the 95% coverage of the four confidence intervals as a family, and the second analysis focused on a single prototypical Key Concept that would be included on only one test. We assumed that each concept would be probed by two MCQs, each having four options, such that we would expect that 6.25% of Norwegians would appear to “understand” a concept if all participants guessed at random. We further calculated confidence interval widths, aiming to estimate proportions with precision no worse than ±0.05 (i.e., ±5%) for those four Key Concepts and no worse than ±0.08 (i.e., ±8%) for the other Key Concepts. The third analysis estimated power to detect a difference of at least 10% in the mean score (proportion of correct answers) between Norwegians and Ugandan children, parents, and teachers. Finally, drawing on experience with our previous survey, we explored a range of response rate scenarios.\n\nThese analyses suggested that sending a total of 4500 postcards would be sufficient to achieve a margin of error no greater than ±5% and ±8% for the first two analyses, and approximately 97% power to detect differences greater than 10% in the mean score compared to results from the Ugandan trials.\n\n\nResults\n\nTable 2 shows participant characteristics across the four questionnaires54. Of the 771 respondents, 21 (2.7%) did not provide data on at least one of the covariates sex, research training, research participation, education, health professional background, and county of residence.\n\nThe log describing the Rasch analysis is described in more detail in Additional file 2 (Extended data)55. Overall, the four tests showed acceptable fit to the Rasch model. Three out of four tests were found to be unidimensional. The test including intended behaviour and self-efficacy items suggested that this sub-scale may include more than one dimension. Separate analyses of the two item-sets in Test 3 (Key Concept MCQs and intended behaviour and self-efficacy items) suggest that the MCQ sub-test works very well, with no apparent validity issues. Overall, the analysis of the sub-test consisting of items evaluating intended behaviour and self-efficacy items also shows satisfactory fit. However, two of the intended behaviour items show disordered thresholds. We did not observe any important local dependency in Test 2, 3 and 4. However, in Test 1, twelve correlations was observed. Two tests were underpowered for this analysis (Test 1 and 4). However, based on the available data, most MCQs in all tests showed acceptable fit to the ICC-curve, and no important differential item functioning by gender was identified in any of the tests. Five MCQs showed evidence of under discriminating with one ability class deviating: two MCQs in Test 1 (q11 and q19.1), two MCQs in Test 2 (q3 and q18.3), and one MCQ in Test 4. In the two tests that were adequately powered, Cronbach’s alpha was 0.6 and 0.7, respectively.\n\nThe mean width of the confidence intervals for the four concepts common to all questionnaires was ±4.6% (range ±3.6% to ±5.9%). One concept (An outcome may be associated with a treatment but not caused by it) was not estimated to the desired precision. The mean width of the other confidence intervals was ±7.92% (range ±3.37% to ±11.9%; 15 of the 30 concepts had confidence intervals wider than ±8%).\n\nFigure 2 shows estimates of the percentage of Norwegian adults who understand each Key Concept. We estimate that more than 80% of Norwegian adults understand five of the Key Concepts and more than 50% understand 18 concepts. On the other hand, less than half of Norwegian adults understand 13 of the Key Concepts, and for seven of those concepts, the proportions of correct answers were no better than if people had randomly guessed.\n\nIntended behaviours and self-efficacy are presented in Figure 3. The results suggest that most Norwegian adults are likely to question the basis of treatment claims (75%; 95% CI 66–85%), find out if treatment claims are based on research evidence (70%; 95% CI 59–81%), and say “yes” if asked to participate in a study comparing treatments (67%; 95% CI 63–72%). However, only about one out of five Norwegian adults find it easy to assess the relevance of studies that compare treatments (21%; 95% CI 9–33%), assess whether a treatment claim is based on a study comparing treatments (18%; 95% CI 12–25%), find studies that compare treatments (18%; 95% CI 9–28%), or assess the trustworthiness of the results of studies that compare treatments (16%; 95% CI 6–26%).\n\nThe results for Norwegian and Ugandan adults are compared to results for Ugandan children in Table 3 to Table 5. The mean score for Norwegian adults who participated in the survey (Test 1) was 17% higher (95% CI 14–20%) than the mean score for Ugandan children in the intervention arm of the randomised trial of the IHC primary school resources one year after the intervention and similar to the mean score for Ugandan teachers in the intervention arm of the trial (Table 3)23. The scores for Ugandan teachers in the control group were similar to those of the children in the intervention group, and the scores of Ugandan parents in the control group of the IHC podcast trial24 were 16% lower (95% CI -19 to -13%).\n\nNearly all the Norwegian adults (209 out of 210) who participated in the survey and Ugandan teachers in the intervention group (77 out of 78) had a passing score (Table 4). This was 15% more than the Ugandan children in the intervention group. However, because only one Norwegian and one Ugandan teacher in the intervention group did not have a passing score, the estimated differences may be unreliable for those comparisons. The proportion of Ugandan teachers in the control group with a passing score (88%) was similar to that of the children (84%), and 47% fewer (95% CI -56% to -37%).\n\nThree quarters (66%; 95% CI 56–74%) of the Norwegian adults who participated in the survey and 70% (95% CI 59–80%) of the Ugandan teachers in the intervention group had a mastery score (Table 5). Compared to Ugandan children in the intervention group, the Odds for achieving a mastery score by the Norwegian participants was 5.3 (95% CI 3.5–7.9%), whereas the Odds for achieving a mastery score was 0.24 (95% CI 0.15–0.4) for the Ugandan parents compared to the children.\n\nWe did not find strong associations between gender, health professional background, or having participated in research and how well participants did (Table 6). Having 1–2 years of tertiary school education (ISCED levels 5–8) was associated with a mean score that was 11% higher (95% CI 4.0–19%), and a higher likelihood of having a mastery score (OR 3.5; 95% CI 1 to 12). Norwegian men were more likely than women to state that they are likely to find out the basis of treatment claims (OR 3.0; 95% CI 1.1 to 8.2), and more likely to find it easy to assess the trustworthiness of the results of studies that compare treatments (OR 3.8; 95% CI 1 to 14). People who have participated in research may be more likely to find it easy to find research based on studies that compare treatments (OR 5.7; 95% CI 1.9 to 17). People with secondary school education (ISCED levels 3–4) may be less likely than people with no more than primary school education (ISCED levels 0-2) to find it easy to assess the trustworthiness of results of studies that compare treatments (OR 0.057; 95% CI 0.0049 to 0.68). Besides these associations, we did not find evidence suggesting that gender, education level, health professional background, or prior participation in research were associated with intended behaviours or self-efficacy. Associations between participant characteristics and their understanding of specific Key Concepts, intended behaviour and self-efficacy are reported in Additional file 3 (Extended data)56.\n\nISCED, International Standard Classification of Education.\n\n\nDiscussion\n\nAccording to Statistics Norway, 24.7% of Norwegian adults have a primary school education, 41.7% secondary school education, 22.4% tertiary school education, 7.3% have a master’s degree, and 0.7% of the population have a PhD. As we anticipated, participants in our study had a somewhat higher educational level than the general population57. In Norway, approximately 18% of the population (15–74) have an educational background in health or welfare58. This is comparable to participants in this study.\n\nParticipants in our survey had a good understanding of the 12 Key Concepts that were addressed in our randomised trial of the IHC primary school intervention23. Their understanding was comparable to Ugandan teachers in the intervention arm of our randomized trial of a primary school intervention, and better than that of Ugandan children in the intervention arm of that trial, and teachers in the control group. It was also better than parents in the control group of our randomised trial of an educational podcast for parents of primary school children in Uganda24.\n\nWe estimate that more than 80% of Norwegian adults understand these five concepts:\n\nIncreasing the amount of a treatment does not necessarily increase its benefits and may cause harm.\n\nCompeting interests may result in misleading claims.\n\nPersonal experiences or anecdotes alone are an unreliable basis for most claims.\n\nThe people being compared should be cared for similarly apart from the treatments being studied.\n\nWeigh the benefits and savings against the harms and costs of acting or not.\n\nOn the other hand, Norwegian adults appear to do no better than if they were to randomly guess the answers to questions about these seven Key Concepts:\n\nBeliefs alone about how treatments work are not reliable predictors of the presence or size of effects.\n\nWidely used treatments or those that have been used for decades are not necessarily beneficial or safe.\n\nComparison groups should be as similar as possible.\n\nPeople’s outcomes should be counted in the group to which they were allocated.\n\nResults for a selected group of people within a study can be misleading.\n\nDeeming results to be “statistically significant” or “nonsignificant” can be misleading.\n\nConsidering that people who responded to our survey had a somewhat higher educational level than the general population, the ability of our respondents may be higher than in the general population57.\n\nBased on self-report, most Norwegians are likely to find out what the basis of a treatment claim is and to find out if a treatment claim is based on research. However, they do not consider it easy to assess the relevance of a claim, whether it is based on research, or its trustworthiness. They also do not consider it is easy to find relevant studies.\n\nIn the previous Norwegian study, four Key Concepts were evaluated32. In both studies the majority responded correctly to the question “Weighing the benefits and harms”. However, in the present study, the respondents were less likely to respond correctly to “Relative effects of treatments can be misleading” (29% vs 64%) and “The use of p-values may be misleading” (19% vs 51.5%). In contrast, more responded correctly to “An outcome may be associate with a treatment but not caused by it” (64% vs 30%). It is difficult to interpret these differences, other than that these evaluations were done using different question sets. Consequently, one explanation may be difference in the questions’ difficulty level. The gender distribution in the studies were identical, however a higher percentage in the present study had at least one year of education beyond secondary school (68% vs 52%). We did not find gender or having a health professional background to be a good predictor of participants’ understanding of the Key Concepts in any of studies. This is consistent with other studies and findings that both health professionals and patients feel challenged finding, appraising, and applying relevant evidence for use in health decisions59–64.\n\nThere are few other studies to which we can compare these results6. The European health literacy survey included all domains of health literacy, responses were given as self-assessments, and results are reported as overall scores25. Consequently, comparison with our study is difficult. They concluded that a little more than one tenth (12%) of those surveyed had insufficient health literacy and almost one half (47%) had limited (insufficient or problematic) health literacy. Both our survey and the European health literacy survey examined associations with education and, unsurprisingly, found that people with higher education have higher health literacy skills. However, the European health literacy survey reported that this result varies by country.\n\nThe strength of this study is that we provide new evidence on people’s ability to assess treatment claims using multiple-choice questions. To our knowledge, few such objective surveys have been conducted in Europe. Our work complements studies in which participants assessed their own abilities.\n\nMany conceptualisations of critical thinking exist. Not all are based on explicit criteria, and unlike the Key Concepts used in this survey, few if any are subject to annual revision65. The questionnaires we administrated were based on a framework that has been developed from methodological literature and input from multiple disciplines and people with methodological expertise66.\n\nWe validated the questionnaires using robust methods. Although the results of the Rasch analysis are promising (Additional file 2, Extended data)55, it suggests the potential for improvements. Across all four tests, we found that only five MCQs warranted improvement. However, Rasch analysis of two of the tests was underpowered, so the validity and reliability of these should be assessed again in future studies.\n\nOur analysis of the test including intended behaviours and self-efficacy suggests that intended behaviour and self-efficacy items measure two different dimensions. This might be because intended behaviour is more complex and dependent on self-efficacy, values, knowledge, and other factors. Except for Test 1, we did not identify any important dependencies between items.\n\nWe did not collect information on age for this study, and thus the association between age and ability to assess treatment claims should be explored in future studies. In our previous study conducted in Norway, younger respondents had a higher proportion of correct responses and higher total scores. This may suggest that there may have been improvements over time in the ability of both health professionals and patients to assess treatment claims32.\n\nAnother possible limitation of our study is the low response rate, which we anticipated. Our sample was similar to the general population in terms of the percentage of health professionals, but people with a higher education were over-represented among respondents. We addressed this issue using prespecified post-stratification in the survey analyses but did not address this in the comparisons to Ugandans. The results of those analyses therefore cannot be assumed to apply to the general population of Norwegian adults.\n\nThe results of this study can inform the development and evaluation of educational interventions that address Key Concepts that Norwegians appear to poorly understand. Up to now, few such interventions have been evaluated. There is a need to evaluate interventions for health professionals as well as for the general public to help ensure that they can think critically about treatment claims and choices. The results also can inform the development and evaluation or strategies for improving communication of information about the effects of treatments by researchers, health professionals, and others.\n\nStudies like this one in other countries would help to map similarities and differences in people’s abilities across different countries and settings. Such information could help to determine the extent to which interventions should be tailored to address different Key Concepts for different populations.\n\nHealth professionals and others who communicate health information should be aware that patients may not be able to think critically about treatment claims and may therefore struggle to process information necessary to making informed decisions.\n\nOur Rasch analysis suggests that the MCQs can be used in Norway. Most MCQs performed well, and this evaluation is the first step in developing a calibrated item bank that can be used for Computer Assisted Testing. The results of our Rasch analysis can be used to improve the multiple-choice questions that did not perform well.\n\n\nConclusion\n\nNorwegian adults’ understanding of Key Concepts for assessing treatment claims and making choices varies from over 80% who understand five Key Concepts to 20% or less who understand seven Key Concepts. We did not find strong evidence that gender, being a health professional, or having participated in research are associated with the ability to assess treatment claims, intended behaviours, or self-efficacy. We did find evidence that people with higher education have a better understanding of Key Concepts.\n\nUnderstanding the need for systematic reviews of fair comparisons as the basis for trustworthy treatment claims has the potential to reduce waste and harm from trusting and acting on misleading claims, and from not trusting and acting on reliable claims. Future interventions should be tailored to address these essential Key Concepts as well as other Key Concepts for which there appears to be a lack of understanding.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Raw data Norwegian Claim Study_2019. https://doi.org/10.7910/DVN/R3DHA554\n\nThis project contains the following underlying data:\n\n- Quiz-1-data.xlsx\n\n- Quiz-2-data.xlsx\n\n- Quiz-3-data.xlsx\n\n- Quiz-4-data.xlsx\n\nWith respect to the potential ability to triangulate identities, access to data is restricted. Ethical approval was granted with an informed consent including a statement were people were guaranteed to be anonymous. Prospective data users can contact the authors to access the data, which can be rejected or approved by the data owner based on certain conditions (must be for legitimate research purposes and requestors are required to sign a data use agreement).\n\nZenodo: multinormal/fhi.informed-health-choices-norway.2019: Version 1.0. https://doi.org/10.5281/zenodo.366996452\n\nThis project contains the following underlying data:\n\nUganda-children-one-year-follow-up.csv\n\nUganda-parents-immediate.csv\n\nUganda-parents-one-year-follow-up.csv\n\nUganda-teachers-one-year-follow-up.csv\n\nFigshare: Additional file 1. Claim Evaluation Tools_tests administrated in Norway_2019. https://doi.org/10.6084/m9.figshare.11439651.v133\n\nFigshare: Additional file 2 Log Rasch analysis Norwegian Claim Study_2019.docx. https://doi.org/10.6084/m9.figshare.10109840.v155\n\nFigshare: Additional file 3. Supplementary tables and figures_Norwegian Claim Study_2019. https://doi.org/10.6084/m9.figshare.11439915.v156\n\nFigshare: Additional file 5. English versions of Claim Evaluation Tools_tests administrated in Norway_2019. https://doi.org/10.6084/m9.figshare.11652342.v134\n\nFigshare: Additional file 4. STROBE_checklist_cross-sectional_Norwegian Claim Study_2019. https://doi.org/10.6084/m9.figshare.11439753.v267\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSource code available from: https://github.com/multinormal/fhi.ihc.norway.2019\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.360625952\n\nLicense: MIT", "appendix": "Acknowledgments\n\nWe would like to thank Atle Fretheim for his feedback on the Norwegian translation of the Claim Evaluation Tools Item bank assuring face validity and applicability to the Norwegian context of the translated content. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOxman AD, Martínez GL: Comparison of the Informed Health Choices Key Concepts to other frameworks that are relevant to learning how to think critically about treatment claims, comparisons, and choices: protocol for a mapping review. IHC Working Paper. 2018. Reference Source\n\nAronson JK, Barends E, Boruch R, et al.: Key concepts for making informed choices. Teach people to think critically about claims and comparisons using these concepts, urge Andrew D. Oxman and an alliance of 24 researchers — they will make better decisions. Nature. 2019; 572. Reference Source\n\nDahlgren A: Additional file 4. STROBE_checklist_cross-sectional_Norwegian Claim Study_2019. figshare. Journal contribution. 2019. http://www.doi.org/10.6084/m9.figshare.11439753.v2" }	[ { "id": "80622", "date": "17 Mar 2021", "name": "Julia Lühnen", "expertise": [ "Reviewer Expertise evidence-based health information", "informed decision-making", "trainings in evidence-based 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 provided a very interesting and highly relevant paper. Critical health literacy and the ability to assess the trustworthiness of claims about healthcare are prerequisites for informed choices. For the assessment of public health literacy, only self-reported measures are used in most of the studies.\nThe authors performed a cross-sectional study to assess the Norwegian public’s ability to assess treatment claims. The questionnaires, assessing understanding, are based on the key concepts people need to understand to assess healthcare claims, developed in the IHC project. The key concepts and the item bank are regularly up-dated, open access resources. The item bank included at least four multiple-choice questions for 32 Key Concepts. From this item pool four questionnaires were designed and randomly assigned to the participants.\n771 out of 4500 invited Norwegian adults responded. Rasch analysis were performed to evaluate the psychometric properties, the percentage of the Norwegian population that understands each key concept was calculated, the results were compared to the results of a previous study in Uganda and analyses were conducted to explore associations between understanding of the key concepts and demographic characteristics. The results are reported in detail. The authors’ discussed strength and limitations of their work and provided implications for practice and future research.\nI have only few comments to optimize the manuscript:\nThe description of the results in the abstract and on page 7 last paragraph is confusing. Maybe there better way providing the results than using “…more than half understood x...” and “…less than half…”?\n\nPage 6 in the paragraph “Rasch analysis”: There is a repetition of the sentence “Rasch analysis can be used for both dichotomous and polytomous data.”\n\nThe authors used the STROBE checklist. Looking at reporting guidelines for online surveys (e.g. CHERRIES, Eysenbach 2004), some aspect are not entirely addressed such as the number of items in each questionnaire, number of screens / pages, completion rate (besides covariates) or the handling of incomplete questionnaires.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6916", "date": "30 Jul 2021", "name": "Astrid Dahlgren", "role": "Author Response", "response": "Thank you for considering our paper and for this useful feedback. We have revised the manuscript and considered all suggested revisions carefully. Our revisions and comments are provided below: The description of the results in the abstract and on page 7 last paragraph is confusing. Maybe there better way providing the results than using “…more than half understood x...” and “…less than half…”? Our response: these sections have now been revised accordingly to improve clarity. Page 6 in the paragraph “Rasch analysis”: There is a repetition of the sentence “Rasch analysis can be used for both dichotomous and polytomous data.” Our response: typo is corrected. The authors used the STROBE checklist. Looking at reporting guidelines for online surveys (e.g. CHERRIES, Eysenbach 2004), some aspect are not entirely addressed such as the number of items in each questionnaire, number of screens / pages, completion rate (besides covariates) or the handling of incomplete questionnaires. Our response: a description of the number of items per questionnaire has now been added to the description of the questionnaires in the methods section. Responses were mandatory in the software we used and thus all questionnaires were complete (thus it was not necessary to handle any incomplete questionnaires) For missing values on specific questions, we used multiple imputation with chained equations to account for the uncertainty introduced by missing values of the post-stratification variables sex, region of residence, and educational level. This is stated under the subheading “Survey analysis”" } ] }, { "id": "84745", "date": "07 Jun 2021", "name": "Gerardine Doyle", "expertise": [ "Reviewer Expertise Health Literacy", "Value measurement in health care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for a very innovative research study around critical health literacy competencies and a comparative study across two countries. The educational intervention at primary school level and the podcast for parents are novel. Below are some suggestions that may help to improve your manuscript:\nTitle:\nThe title of your paper could be amended to more accurately reflect your study. The use of the word ‘claims’ in a health care setting can very easily be confused with health insurance claims! This will then need to follow through your entire manuscript. Perhaps consider using the word ‘statement’ rather than ‘claims‘ so your title might be something like the following:\nThe Norwegian public’s ability to assess statements about health treatments: …\n\nAbstract:\nFor the above reason the Background section of the Abstract needs to be rephrased.\n\nResearch Design:\nSurrounding the research design, the rationale for comparing Uganda with Norway is not clearly explained to the reader. Why are Uganda and Norway comparable? A justification is necessary.\n\nMethods and Analysis:\nWhile a rationale can be understood for a comparison of adults in one country with parents and teachers in another country (all are adults), the rationale for comparing adults in one country with primary school children in another country remains a puzzle. This is so especially because the quizzes collect data on education level attained and so can be controlled for. Again this needs to be explained more clearly to the reader.\n\nThe structure of the Methods section is difficult for the reader to follow. For example the methods section begins with explaining through Table 1 the three ‘Health Choices Key Concepts’ of ‘Claims, Comparisons and Choices’ and then moves on to the four ‘concepts’ immediately following Box 1. The use of the identical term ‘concept’ to describe both of the above is very confusing to the reader. It is not clear how the three ‘Health Choices Key Concepts’ link or map to these four ‘concepts’. Furthermore the label of ‘concepts’ does not seem to be accurate for the four ‘concepts’. Are these concepts? Perhaps ‘contentions’ is a more accurate label. This entire section needs to be redrafted.\n\nIt is not clear from the manuscript if the quizzes went through a plain English audit process. This should be stated.\n\nFor an English journal publication, it might be best to move the English version of the quizzes to be Additional File 1, rather than the Norwegian version.\n\nConclusions:\nThe conclusion stated in the Abstract ‘This can result in poorly informed decisions, underuse of effective interventions, and overuse of ineffective or harmful interventions’ does not align with the conclusions at the end of the manuscript. Indeed it is difficult to see how these conclusions set out in the Abstract can be made from this particular study.\n\nTwo typing errors were noted:\nEthical Statement, second sentence Page 13 should read ‘including a statement where people were guaranteed…’\n\nThank you for a very interesting piece of research and I wish you every success enhancing your paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6917", "date": "30 Jul 2021", "name": "Astrid Dahlgren", "role": "Author Response", "response": "Thank you for considering our paper and for this useful feedback. We have revised the manuscript and considered all suggested revisions carefully. Our revisions and comments are provided below: 1. Suggestion to change the name “Claim”. Our response: we understand that this term may be understood differently based on context. However, this term has been evaluated for relevance in several country contexts and be considered as the most precise term to be used. The Claim Evaluation Tools is also the name of the item bank this study was based on and thus cannot be changed. 2. Suggestion to add rationale for comparing Uganda and Norway Our response: the rationale for this comparison has now been added. 3. Suggestion to add rationale for comparing children and adults. Our response: the rationale for this comparison has now been added. 4. Suggestion to restructure the methods section describing the Key Concepts list and the claim Evaluation Tools item bank. Our response: these sections have now been revised accordingly to improve clarity. 5. Suggestion to clarify whether the tests went through a plain English audit process. The test was based on a set of MCQS translated from the English to Norwegian. Our response: statement now added to the manuscript. 6. Suggestion to replace the Norwegian version of the tests with the English version in the additional file. Our response: F1000 publishes international studies reporting evidence from a range of contexts. The evaluation instruments used in this study was designed and adapted to a Norwegian setting. Consequently, our study describes the validation and results of the Norwegian tests. Responses and psychometric properties of the tests may be different for the English versions of these test. Thus, we believe it to be most appropriate to include the tests that was used in the study. However, examples of the questions in English has been provided in the manuscript. Other studies have used the English versions of the MCQs in other contexts (such as in Uganda). 7. Suggestion to revise the conclusion taking into account: the conclusion stated in the Abstract ‘This can result in poorly informed decisions, underuse of effective interventions, and overuse of ineffective or harmful interventions’ does not align with the conclusions at the end of the manuscript. Indeed, it is difficult to see how these conclusions set out in the Abstract can be made from this particular study. Our response: the sentence has been deleted. 8. Typos were corrected were noted" } ] } ]	1	https://f1000research.com/articles/9-179
https://f1000research.com/articles/9-1208/v1	08 Oct 20	{ "type": "Brief Report", "title": "Interprofessional grant writing seminar for early career faculty in a small, isolated teaching center", "authors": [ "India King", "Andrea Christopher", "Ann Hansen", "Ami Student", "Jeff Sordahl", "Sarah Naidoo", "Elaine Nguyen", "Amber Fisher", "Rick Tivis", "C. Scott Smith", "India King", "Andrea Christopher", "Ann Hansen", "Ami Student", "Jeff Sordahl", "Sarah Naidoo", "Elaine Nguyen", "Amber Fisher", "Rick Tivis" ], "abstract": "Small, isolated teaching centers have difficulty mentoring interprofessional junior faculty in research methods and grant writing. Peer mentoring programs for grant writing at larger institutions have been successful. In this short report, we describe our program that leveraged mentor experience using four framing seminars followed by project refinement in three-person peer groups and monthly mentored works in progress meetings. In its first year, ten faculty from medicine, psychology, and pharmacy completed the program and successfully obtained six funded grants. Five of the projects transitioned from single profession applications to interprofessional applications as participants connected and profession-specific expertise was identified. Refinements for future cohorts are discussed.", "keywords": [ "Grants", "Organized Financing", "Mentoring", "Peer", "Interprofessional" ], "content": "Introduction\n\nOver the past three decades there has been a decline in research funding (Alberts et al., 2014). The funding that is distributed in many disciplines goes to better established scientists and large, highly networked institutions (Szell & Sinatra, 2015; Traynor & Rafferty, 1999). Yet, research is an important element for early career faculty promotion in healthcare disciplines (Yeh et al., 2015). One solution that has been successful at larger institutions is self-organized peer mentoring (Johnson et al., 2011). This short report describes a year-long seminar at a small, isolated teaching center designed to expose early-career interprofessional faculty to basic research principles and then to facilitate small group peer mentoring in order that they might obtain their first research awards.\n\nThe Boise Veterans Affairs Medical Center (VAMC) provides primary, secondary, and specialty care to over 28,000 veterans each year in an outpatient system that includes five community-based outreach clinics around the state, a 46-bed hospital, an 11-bed inpatient substance abuse treatment center, and a collocated 28-bed nursing home. Boise VAMC has affiliations with the University of Washington School of Medicine (600 miles away), Gonzaga University Nurse Practitioner program (400 miles away), Idaho State University School of Pharmacy (250 miles away) and Boise State University School of Nursing. The facility maintains multiple training programs, including a nurse practitioner residency, pharmacy PGY 1 and PGY 2 residencies, psychology internship and postdoctoral residency, and an internal medicine residency.\n\n\nMethods\n\nOur local research office, The Boise VA Office of Research and Development, determined that our outcome data collection would constitute Quality Improvement work and would be considered exempt from IRB approval. The data collected was provided voluntarily by the group participants who gave permission to publish the results in a de-identified format.\n\nBeing a small academic facility, far from our major affiliates, and without a deep and rich network of research mentors, we decided to create a grant writing program with the following objectives:\n\n1) Help interprofessional junior clinician educators obtain their first research or program development/evaluation funding.\n\n2) Create a small group of peer-mentors with the experience to recapitulate this effort with future cohorts.\n\nWe developed a year-long program that consisted of four kick-off seminars delivered weekly followed by dividing into small project groups of three that collaborated to refine their questions and methods and share the work of information gathering such as grant opportunities, where to find online human subjects forms, etc. Each group reported back to the larger group approximately monthly during ‘works in progress’ (WIP) meetings.\n\nThe kick-off seminars were developed by a biostatistician (RT) and a local mentor (CSS) with previous success in obtaining National Institutes of Health, Veterans Affairs (VA), and foundation research funding as well as large program grants. They used a “flipped classroom” format with a research article covering the topic for the session handed out ahead of time to focus and stimulate class discussion and small group interaction. The topics and structure of these seminars are provided in Table 1.\n\n(1) Wong G, et al. (2012). Realist methods in medical education research: What are they and how can they contribute. Medical Education;46:89–96.\n\n(2) Sanders, J., Brown, J., & Walsh, K. (2017). Producing useful evaluations in medical education. Education for Primary Care, 28, 137–140.\n\n(3) Squire 2.0 guideliens http://squirestatement.org/index.cfm?fuseaction=Page.ViewPage&PageID=471\n\n(4) VanderWeele, T.J., Ding, P. (2017). Sensitivity analysis in observational research: Introducing the E-value. Annals of Internal Medicine, 167, 268–274.\n\n(5) Strobe guidelines https://www.strobe-statement.org/fileadmin/Strobe/uploads/checklists/ STROBE_checklist_v4_combined.pdf\n\n(6) Inui TS (1996) The virtue of qualitative and quantitative research. Ann Intern Med;125:770–771.\n\nFollowing these seminars, the participants were divided into groups of three based on rough similarity of research questions and/or proposed methods. These groups decided how often to meet and how to support each other with moving forward on their projects.\n\nApproximately monthly the large group had a WIP update. There was a report on the current state of each three-person group’s projects and any questions or barriers would be outlined. These sessions tended to distribute early expertise. For example, when one member discovered a new funding opportunity it was shared with the entire group. Or, if someone wondered how to navigate the IRB process, someone else who had navigated it would explain where the electronic forms were and offer to help. In addition, an early career faculty member from our affiliate, who had been a successful career development awardee, spoke to the group about the career development program and offered her contact information and assistance.\n\n\nResults\n\nTwelve (12) participants started the grant writing program. This included five (5) physicians, four (4) psychologists, and three (3) pharmacists. All but two completed the series (both withdrew from the program due to competing demands). Ten (10) were within three years of being hired to the Boise VA, and ten were academically early career, having entry level faculty appointments. Most had completed programs with less emphasis on research (e.g., PsyD or PharmD versus PhD, fellowships with clinical versus research emphasis).\n\nAfter completing the grant writing program, six of the remaining ten participants submitted at least one research grant or program evaluation proposal for a total of 10 proposals. Six of these proposals were funded (see Table 2).\n\nInvest. = investigator; PI = principal investigator; NCI = National Cancer Institute; VA = Veterans Affairs; HSR&D = Health Services Research & Development; NIH = National Institutes of Health.\n\n\nDiscussion\n\nThis grant writing seminar and peer support group met the initial objectives of the program and had some unintended positive consequences. The majority of the completing group members submitted a grant application. For most this was their first attempt. Five of the funding applications transitioned from single profession applications to interprofessional applications as connections were made between attendees in their small groups and profession-specific expertise was identified. Because funding sources look for team-based applications from multiple professions (Wuchty et al., 2007), this consequence may have a positive impact on funding success as interprofessional teams at this facility create connections and expand expertise and project success.\n\nLessons learned include starting the seminar series earlier in the academic year. We started the seminars in mid-summer and this created unnecessary time pressure for proposal submissions, which are usually due in the fall. Also, we would now consider prior experience as a factor as we create the small groups of three.\n\nOur next step involves further partnership with the Boise VAMC’s research department. The research department is interested in expanding the scope and number of research projects and our modest success has demonstrated value. Our research department has agreed to partner in the ongoing development and expansion of this group. This partnership will increase access to instructors from the IRB, VA Health Services Research and Development (HSR&D) office and other major funders of VA research, and the Office of Research Oversight, to discuss distinctions between program evaluation/quality improvement research. These opportunities for direct discussion and questions will be invaluable to the next round of beginning grant writers.\n\nThis project had some weaknesses. Because it was not designed as research, no systematic survey of participants (satisfaction, skill improvements, etc.) was obtained. Several topics were identified that might have made the project more effective including types of grants available, navigating the IRB, writing your letter of intent, and budgets. These should be incorporated into future versions.\n\n\nConclusions\n\nSmall, isolated teaching centers struggle to provide support for designing and submitting research grants for their early career faculty. Any experienced potential mentors are quickly overwhelmed with mentees. Yet, research is an important element for these same early career faculty’s success. This program of four orienting seminars, three-person project groups, and monthly works in progress meetings was successful in obtaining initial grants for individuals and group expertise that could guide future cohorts.\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\nAlberts B, Kirschner MW, Tilghman S, et al.: Rescuing US biomedical research from its systemic flaws. Proc Natl Acad Sci U S A. 2014; 111(16): 5773–5777. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson KS, Hastings SN, Purser JL, et al.: The junior faculty laboratory: an innovative model of peer mentoring. Acad Med. 2011; 86(12): 1577–1582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSzell M, Sinatra R: Research funding goes to rich clubs. Proc Natl Acad Sci U S A. 2015; 112(48): 14749–14750. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTraynor M, Rafferty AM: Nursing and the research assessment exercise: past, present and future. J Adv Nurs. 1999; 30(1): 186–92. PubMed Abstract \| Publisher Full Text\n\nWuchty S, Jones BF, Uzzi B: The increasing dominance of teams in production of knowledge. Science. 2007; 316(5827): 1036–1039. PubMed Abstract \| Publisher Full Text\n\nYeh HC, Bertram A, Brancati FL, et al.: Perceptions of division directors in general internal medicine about the importance of and support for scholarly work done by clinician-educators. Acad Med. 2015; 90(2): 203–208. PubMed Abstract \| Publisher Full Text" }	[ { "id": "72758", "date": "06 Nov 2020", "name": "Mamta Singh", "expertise": [ "Reviewer Expertise QI", "Patient Safety" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a brief report describing a grant writing seminar for early career interprofessional faculty in a small teaching center. The article presents the description of the 4 seminars that were included in this larger grant writing program and the authors give clear descriptions of the participants and their demographics. The article only covers one year and is limited to 10 persons (started with 12- but 2 faculty left the program due to completing demands). Below are some areas that need to be addressed:\nThis is a brief report on a self select group of faculty attending a series of seminars over one year. This narrows the impact of such a program and also speaks to feasibility. Moreover, this poses questions of whether this program could be reproduced at other sites. The authors point out that small isolated teaching centers struggle to provide support for early career faculty and suggest that such a seminar format may be a method to provide support. However, without evidence that this is sustainable, the report does not help the feasibility argument. If the program is still going on, then comments on how it is being sustained would be helpful.\n\nThe authors may want to interview the faculty and present some data as to what the faculty thought and how this was helpful. This will provide a \"lessons learned\" part which again can make the findings helpful to other academic centers in similar situations.\n\nIt is commendable that the 6/10 programs were funded and is a key outcome for the program. It would be helpful to know if the faculty participants went on to get other funding or seek other funding after completion of the program.\n\nIn summary, this is a descriptive report on a grant writing seminar and it is well described and I commend the authors for their work on this. However, in response to their own concern that faculty in isolated centers do not get mentoring in such areas, it would be helpful if they can show evidence of the sustainability of the program, or demonstrate if faculty continue to write grants or bring in funding. As the faculty are self select, if the authors could comment on what worked so other places could reproduce this at their home institutions- it would further the experience for others.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6128", "date": "30 Jul 2021", "name": "C. Scott Smith", "role": "Author Response", "response": "Thank you for your time and your comments. We agree that components of the seminar are site-specific, but still believe that it is feasible for other sites to adopt and adapt it to meet their individual needs. Regarding sustainability, unfortunately this seminar is not still happening due to loss of key organizers (no longer working at the site) and a restructuring of the research department. Satisfaction: We devised a brief impact questionnaire consisting of 5-point Likert-style questions (strongly disagree-1 to strongly agree-5) and a free text area. This was administered two years after the program. Response rate was 60%. The results were as follows: Knowledge and skills for design/grant writing 4.4 +/- 0.55 Developed collaborators 4 +/- 0 Learned about new funding opportunities 4 +/- 0.71 Gained confidence 4.2 +/- 0.45 Improved academic career 4.2 +/- 0.45 Comments identified networking as the most important element (67% mentioned it) followed by increased confidence, new grant opportunities, and new skills mentioned by one responder each. Other Funding: This cohort has gone on to submit at least 17 new grants, 6 (35%) of which were funded for nearly $800,000. Funders included HRSA, VA Office of Academic Affairs, Idaho Dept. of Health and Welfare, and Alaska Dept. of Health & Social Services. As you can see, sustainability of the program was poor, while sustainability of the initial cohorts' grant writing was excellent, largely due to networking, skills, and confidence. We will add these data in a post script." } ] }, { "id": "72757", "date": "17 Nov 2020", "name": "Dawn DeWitt", "expertise": [ "Reviewer Expertise Medical education", "career choice", "diabetes", "inter professional education." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written brief, descriptive report of a capacity-building program at a relatively isolated small academic center. The detail is enough to replicate the program and although some of the references are relatively old (Inui), they are sound and useful. I would refer others to this article if they were interested in building a similar program.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": [ { "c_id": "6129", "date": "30 Jul 2021", "name": "C. Scott Smith", "role": "Author Response", "response": "Thank you for your time, effort, and suggestions. We will be adding a post-script in response to another reviewer's suggestions you may want to see." } ] } ]	1	https://f1000research.com/articles/9-1208
https://f1000research.com/articles/9-1045/v1	26 Aug 20	{ "type": "Case Report", "title": "Case Report: Sudden cardiac death due to ventricular myocardial non-compaction", "authors": [ "Syrine Mannoubi", "Med Amin Mesrati", "Ibn Hadj Amor Hassen", "Taha Hasnaoui", "Hiba Limem", "Marwa Boussaid", "Nouha Ben Abdejlil", "Abir Aissaoui", "Syrine Mannoubi", "Med Amin Mesrati", "Ibn Hadj Amor Hassen", "Hiba Limem", "Marwa Boussaid", "Nouha Ben Abdejlil", "Abir Aissaoui" ], "abstract": "Ventricular non-compaction (VNC) is a rare myocardium disorder, which can be both genetic and sporadic. A poor wall compaction process or an excessive trabeculae formation may be at the genesis of myocardial hypertrabeculation with multiple recesses. It is often complicated by ventricular dysfunction, arrhythmias and cardiac embolism. Herein we report a case of a 20-year-old male patient with no particular past medical history who was followed up at the cardiology department for dyspnea. Echocardiography showed reduced ejection fraction of the left ventricle with potential hypertrabeculation in the right ventricle, confirmed by cardiac MRI. The patient was not put under medication and was later lost to follow-up. He died few months later without a clear cause explaining death. A forensic autopsy was performed that attributed death to acute ventricle arrhythmia secondary to VNC, emphasizing the major role of an early and specific treatment to avoid such a fatal outcome.", "keywords": [ "Isolated Non compaction of the Ventricular Myocardium", "Cardiomyopathy", "Histology", "Sudden Cardiac Death", "Autopsy." ], "content": "Introduction\n\nVentricular non-compaction (VNC) is a complex and heterogeneous cardiomyopathy first described in 1926. It is a rare disease with a reported prevalence of 0.014–0.17%1. Several terms are used to describe this disease namely: “cardiac hyper and excessive trabecularization”, “spongy myocardium”, “honeycomb myocardium”, or “persisting myocardial sinusoids”, and even “isolated ventricular abnormal trabeculation”. It is characterized by hypertrabeculation with an excessive lace-like network of trabeculae and deep trabecular pockets in the ventricle, creating a perfect environment for thrombi formation. VNC can be detected in all age groups, ranging from the fetal period to adulthood2. It can occur sporadically or is hereditary secondary to chromosomal abnormalities. It can also be associated with other cardiac diseases, which may be congenital. Additionally, VNC is represented by a large spectrum of symptoms and clinical features ranging from normal variants to pathological phenotypes. Indeed, VNC may remain asymptomatic until a complication occurs. Cardiologists must pay more attention to various clinical manifestations, including heart failure, arrhythmias and cardio embolic events, which can be related to VNC, to initiate an early treatment and avoid potentially fatal complications3.\n\nHerein, we report a fatal case of VNC in a 20-year-old male, attested by the autopsy, and we discuss different mechanisms involved in the occurrence of death.\n\n\nCase report\n\nA 20- year -old male, who presented with dyspnea in March 2019, was followed up by the cardiology department and classified as stage 2 on the New York Heart Association (NYHA) Functional Classification. The patient had no other relevant personal or family past medical history. As part of an etiological assessment of dyspnea, a transthoracic echocardiography was requested showing a dilated left ventricle, reduced left ventricular ejection fraction at 40%, septo-apical myocardial hypokinesia and left ventricular hypertrabeculation (Figure 1).\n\nThe diagnosis made was a heart failure, and patient was put under treatment. A cardiac magnetic resonance imaging (CMRI) was requested and revealed reduced left ventricular ejection fraction (LVEF) at 30%, globular shape of the left ventricle with an overall wall hypokinesia, hypertrabeculation located at the left ventricle, a thickness of 2.8cm of the trabeculated myocardium on the compact myocardium, less contrast enhancement of the trabeculated myocardium compared to normal, and no thrombus in the left ventricle (Figure 2). It was concluded that the appearance was most likely left VNC cardiomyopathy with a LVEF at 30% without intra-cardiac thrombus or mitral insufficiency Figure 2.\n\nThe patient was later lost to follow-up. A few months later, he died without a clear cause explaining the death. A forensic autopsy was performed.\n\nThe body was that of a man of average build, with an approximate weight of 70kg. There was no specific sign on the external examination particularly, no asymmetry in comparing the circumference of the two calves.\n\nThe heart was globose weighing 325g. The coronary arteries were in a normal position without any significant lesion. The axial dissection of the heart found left ventricle hypertrabeculation with parietal thinning measured at 0.5cm and some intra-cavitary adhering thrombi Figure 3. Left ventricle examination found wall thickening at 1.2cm. Valvular examination was normal. No systemic thrombi was found. The lungs were the site of profuse oedema. The rest of the organs were congestive without any abnormality. Toxicology was negative. Based on clinical history and the necropsy findings, death was attributed to acute ventricle arrhythmia secondary to a myocardium non-compaction.\n\n\nDiscussion\n\nThe European Society of Cardiology (ESC) categorize VNC as “unclassified cardiomyopathy” with a structural and functional abnormal heart muscle without any other diseases sufficient to cause the observed myocardial abnormality”. However, The American Heart Association (AHA) categorize it as “genetic cardiomyopathy”4. The etiology and embryogenetic mechanisms leading to VNC are still unknown and several hypothesis are suggested. The most frequent on is that hypertrabeculation may result from excessive trabeculae formation and/or a defect in the later compaction processes1,5.\n\nVNC, and specifically on the left side, has been found in association with more than 40 mutated genes, which encode for several cell structures. The most common are MYH7, MYBPC3, TTN, at a rate of 71%6. Some cases of VNC associated with congenital hemoglobinopathies have been described in the literature.\n\nAlthough the usual site of hypertrabeculation involvement is the left ventricle, the right ventricle is rarely affected. Right VNC can lead to ventricular tachycardia or right heart failure. In addition, patients with right VNC can be perfectly asymptomatic with only electrocardiographic disorder. In addition, concomitant damage of right VNC is not rare and it can be difficult to distinguish between non-compaction and arrhythmogenic right ventricular cardiomyopathy (ARVD). Diagnosis criteria for ARVD, even if it coexists with typical VNC, may lead to a diagnosis of ARVD rather than VNC. Less frequently, both ventricles can be affected leading to entirely non-compaction cardiomyopathy7.\n\nDifferent imaging-based classification systems have been used to make VNC diagnosis. Cardiac Magnetic Resonance Imaging (CMRI)-based criteria (Petersen criteria) is considered as the gold standard8. Not all definitions are anatomically controlled and these criteria are nonspecific. Autopsy performed on individuals with known VNC can be a good way to compare radiological criteria to anatomical findings. Collaboration between forensic medicine and cardiology should be take into consideration with the aim to standardize diagnostic criteria and to avoid over diagnosis in healthy people with a benign prognosis.\n\nNonspecific histopathological findings have been described, including hypertrophy of the cardiomyocytes, ischemic necrosis with fibrosis due to insufficient vascular supply of the trabeculations, and disorganization of cardiomyocytes9. VNC leads to variable complications that can be misdiagnosed and at the origin of sudden cardiac death. The most common is conduction defects in approximately 90% of patients followed by myocardium arrhythmia. Thromboembolic events are not very frequent and occur in only 10% of cases, mostly in adults. Three main factors are involved in the occurrence of thromboembolic events: the presence of thrombi into ventricular trabeculations, left ventricular systolic dysfunction with reduced ejection fraction, and/or atrial fibrillation.\n\nCurrently, there are no guidelines for the management of patients with VNC. Recommendations for treatment include prophylactic anticoagulation therapy and the implantation of a cardiac defibrillator. Treatment for VNC is therefore that of any cardiomyopathy with heart failure10. A periodic check with a 24-hour ECG holter is indicated in order to assess the risk of a possible asymptomatic arrhythmia. Finally, first-degree family members of all patients diagnosed with VNC should undergo an echocardiographic screening examination and genetic exploration11.\n\nIn summary, there are multiple controversies related with VNC comprising etiology and pathogenesis, genetic findings, relation with extra-cardiac diseases, diagnostic criteria, treatment, and prognosis. Cardiologists have to pay attention to various clinical manifestations, including heart failure, arrhythmias and cardio embolic events, which can be related to VNC in order to initiate an early treatment and avoiding potentially fatal complications.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication was obtained from the legally authorized representative of the decedent.", "appendix": "References\n\nChoquet C, Kelly RG, Miquerol L: Defects in Trabecular Development Contribute to Left Ventricular Noncompaction. Pediatr Cardiol. 2019; 40(7): 1331–8. PubMed Abstract \| Publisher Full Text\n\nKido K, Guglin M: Anticoagulation Therapy in Specific Cardiomyopathies: Isolated Left Ventricular Noncompaction and Peripartum Cardiomyopathy. J Cardiovasc Pharmacol Ther. 2019; 24(1): 31–6. PubMed Abstract \| Publisher Full Text\n\nStöllberger C, Finsterer J: Understanding left ventricular hypertrabeculation/noncompaction: pathomorphologic findings and prognostic impact of neuromuscular comorbidities. Expert Rev Cardiovasc Ther. 2019; 17(2): 95–109. PubMed Abstract \| Publisher Full Text\n\nLoria V, Colizzi C, Vaccarella M, et al.: Left Ventricular Noncompaction: Cause or Consequence of Myocardial Disease? A Case Report and Literature Review. Cardiology. 2019; 143(3–4): 100–4. PubMed Abstract \| Publisher Full Text\n\nKayvanpour E, Sedaghat-Hamedani F, Gi WT, et al.: Clinical and genetic insights into non-compaction: a meta-analysis and systematic review on 7598 individuals. Clin Res Cardiol. 2019; 108(11): 1297–308. PubMed Abstract \| Publisher Full Text\n\nBermudez-Jiménez FJ, Jiménez-Jáimez J: Genotype, Family History, and Outcomes in Noncompaction Cardiomyopathy. J Am Coll Cardiol. 2018; 71(24): 2864. PubMed Abstract \| Publisher Full Text\n\nLuckie M, Irwin B, Nair S, et al.: Left ventricular non-compaction in identical twins with thalassaemia and cardiac iron overload. Eur J Echocardiogr. 2009; 10(4): 509–12. PubMed Abstract \| Publisher Full Text\n\nStöllberger C, Finsterer J: Pitfalls in the diagnosis of left ventricular hypertrabeculation/non- compaction. Postgrad Med J. 2006; 82(972): 679–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRambhatla T, Mountantonakis S, Bhasin K, et al.: Ventricular tachycardia due to isolated non-compaction of the right ventricle. Eur Heart J Cardiovasc Imaging. 2018; 19(8): 878. PubMed Abstract \| Publisher Full Text\n\nGomathi SB, Makadia N, Ajit SM: An unusual case of isolated non-compacted right ventricular myocardium. Eur J Echocardiogr. 2008; 9(3): 424–5. PubMed Abstract \| Publisher Full Text\n\nAggarwal S, Kalavakunta J, Gupta V: A case of isolated right ventricle noncompaction with ST-Elevation chest leads. Hear Views. 2016; 17(1): 30. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70295", "date": "09 Sep 2020", "name": "Mokhles lajmi", "expertise": [ "Reviewer Expertise cardiovascular surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a case report about a young male deceased from ventricular arrhythmia due to left ventricular non compaction (VNC) syndrome, a rare but quite controversial pathology. In fact, this condition can express itself in various ways (ranging from completely asymptomatic patients to severe heart failure or arrhythmias) misleading the diagnosis, and imagery criteria are lacking specificity.\nWe thank the authors for this quality case report.\nQ1:\nThe case description had all the necessary information to fully understand the case.\n\nQ2:\nClinical findings (autopsy findings in this case) are accurate and concise.\n\nQ3:\nI congratulate the author's for their interesting and insightful point of view regarding the potential importance of collaboration between forensics and cardiology to further improve the diagnosis accuracy. In fact diagnosis of VNC is very controversial and misleading, and imaging criteria are perplexing, thus dealing with VNC from a cardiology-only point of view is narrow minded, forensics could be a huge asset to correlate imagery to anatomy.\n\nWe know that some degree of non compaction of the left ventricle is not uncommon in the anterior apical region and trabeculations of the left ventricle can be increased in physiological conditions such as pregnancy and exercise training, would this be a diagnostical challenge for forensics? Can this mislead the autopsy findings? Is there any differential diagnosis in VNC?\n\nQ4:\nAuthor's succeeded in explaining a relatively complex pathology, in a way that it's understandable for non cardiologists/forensics.\n\n5: The author's should consider the following points:\nIn accordance with CARE guidelines for case reports, \"case report\" should appear in keywords, with no more than 5 keywords\n\nAs mentioned in the introduction, ventricular non compaction was first described in 1926. I think it would be interesting to add that it was done by Mr Grant and cite the reference: Grant RT. An unusual anomaly of the coronary vessels in the malformed heart of a child. Heart 1926; 13: 273–83.1\n\nRegarding the prevalence of this disease, although it was deemed rare by many investigators, the distribution of this disease in the global population is very difficult to evaluate, and numbers diverge hugely in the literature. One referenced source in a Lancet paper even stated that it “seems to be the third most commonly diagnosed cardiomyopathy”.2 Thus I advise to not mention a particular prevalence and just state that it is rare and that its prevalence is uncertain.\n\nFigure 2: it would be more accurate to mention: non compacted left trabeculations.\n\nDiscussion section: Some cases of VNC associated with congenital hemoglobinopathies have been described in the literature. Author's should add a reference relating to this fact.3,4\n\nVNC abbreviation is used in abstract. Abstracts should not contain abbreviations unless it is necessary.\n\n6/ We noted some spelling errors:\n\nAutopsy findings, line 5; change \"found wall thickening at 1.2cm\" to \" found a wall thickening of 1.2cm\".\n\nAutopsy findings, line 7; \"abnormalities\" instead of \"\"abnormality\".\n\nDiscussion, line 12; \"disorders\" instead of \"disorder\".\n\nDiscussion, line 12: \"complications of right VNC are not rare \" instead of \"concomitant damage\".\n\nDiscussion, line 19: \"taken\" instead of \"take\".\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": "5930", "date": "11 Sep 2020", "name": "med amin mesrati", "role": "Author Response", "response": "Q1: The case description had all the necessary information to fully understand the case. No comments Q2: Clinical findings (autopsy findings in this case) are accurate and concise. No comments Q3: I congratulate the author's for their interesting and insightful point of view regarding the potential importance of collaboration between forensics and cardiology to further improve the diagnosis accuracy. In fact diagnosis of VNC is very controversial and misleading, and imaging criteria are perplexing, thus dealing with VNC from a cardiology-only point of view is narrow minded, forensics could be a huge asset to correlate imagery to anatomy. We know that some degree of non compaction of the left ventricle is not uncommon in the anterior apical region and trabeculations of the left ventricle can be increased in physiological conditions such as pregnancy and exercise training, would this be a diagnostical challenge for forensics? Can this mislead the autopsy findings? Is there any differential diagnosis in VNC? R: After excluding dilated and hypertrophic cardiomyopathy, forensic doctor should consider other diagnoses that can present with similar features. Thus, it is essential to distinguish left ventricular non-compaction from acquired changes seen in pulmonary atresia, mycotic invasion of the heart… and literature disclosed many other differential diagnoses. In our context, the MRI helped us to retain VNC as final diagnosis. Q4: Author's succeeded in explaining a relatively complex pathology, in a way that it's understandable for non cardiologists/forensics. No comments 5: The author's should consider the following points: In accordance with CARE guidelines for case reports, \"case report\" should appear in keywords, with no more than 5 keywords R: it is considered As mentioned in the introduction, ventricular non compaction was first described in 1926. I think it would be interesting to add that it was done by Mr Grant and cite the reference: Grant RT. An unusual anomaly of the coronary vessels in the malformed heart of a child. Heart 1926; 13: 273–83.1 R: it is considered Regarding the prevalence of this disease, although it was deemed rare by many investigators, the distribution of this disease in the global population is very difficult to evaluate, and numbers diverge hugely in the literature. One referenced source in a Lancet paper even stated that it “seems to be the third most commonly diagnosed cardiomyopathy”.2 Thus I advise to not mention a particular prevalence and just state that it is rare and that its prevalence is uncertain. R: it is considered Figure 2: it would be more accurate to mention: non compacted left trabeculations. R: It is considered Discussion section: Some cases of VNC associated with congenital hemoglobinopathies have been described in the literature. Author's should add a reference relating to this fact. VNC abbreviation is used in abstract. Abstracts should not contain abbreviations unless it is necessary. R: it is considered 6/ We noted some spelling errors: Autopsy findings, line 5; change \"found wall thickening at 1.2cm\" to \" found a wall thickening of 1.2cm\". Autopsy findings, line 7; \"abnormalities\" instead of \"\"abnormality\". Discussion, line 12; \"disorders\" instead of \"disorder\". Discussion, line 12: \"complications of right VNC are not rare \" instead of \"concomitant damage\". Discussion, line 19: \"taken\" instead of \"take\". R: considered" } ] }, { "id": "88980", "date": "05 Jul 2021", "name": "Citrawati Dyah Kencono Wungu", "expertise": [ "Reviewer Expertise Molecular cardiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report, Dr. Mannoubi and colleagues reported a case of a man with ventricular non-compaction and sudden cardiac death. This case shows a good collaboration between cardiologists and forensics. However, I have some major and minor comments to improve the quality of the article, especially regarding the completeness of the data:\nSeveral data are lacking about the patient, such as: the patient’s race (this is important due to the influence of genetic factors); how long the symptoms have occurred; symptoms other than dyspnea; the patient’s occupation; medications that have been taken; physical examination; laboratory findings; and how the patient was diagnosed with heart failure. Is there any cardiomegaly? Was the patient in shock at his admission to the hospital? ECG data is also important because ventricular non-compaction is closely related to arrhythmia.\n\nHistology is present in the Abstract but no histopathological findings mentioned. The histopathological findings should be added.\n\nData regarding therapy administered to the patient is also not mentioned.\n\nHow much is the ratio of non-compacted myocardium to compact myocardium at the end of systole/diastole (Petersen criteria)?\n\nCompleting the patient’s data is important for the diagnosis of LVNC, as hypertrabeculation can also be observed in other comorbidities or even athletes. Will these conditions give different results in the autopsy findings? This should also be mentioned in the Discussion.\n\nAs the death of the patient was attributed to acute ventricle arrhythmia secondary to a myocardium non-compaction, the prognosis and mortality of the patients with LVNC should also be explained, like the mortality/survival rate.\n\n“Some cases of VNC associated with congenital hemoglobinopathies have been described in the literature.” Please put citations and more explanations regarding this.\n\nIn the Discussion, please explain ECG findings that should be found in VNC.\n\n“VNC leads to variable complications that can be misdiagnosed and at the origin of sudden cardiac death. The most common is conduction defects in approximately 90% of patients followed by myocardium arrhythmia. Thromboembolic events are not very frequent and occur in only 10% of cases, mostly in adults. Three main factors are involved in the occurrence of thromboembolic events: the presence of thrombi into ventricular trabeculations, left ventricular systolic dysfunction with reduced ejection fraction, and/or atrial fibrillation.”: Please put references to these statements.\n\nThe role of autopsy and collaboration between cardiologists and forensics should be added in the summary.\n\nThere are many grammatical errors, for example:\n“Ventricular non-compaction (VNC) is a rare myocardium disorder…” should be “myocardial disorder”.\n\n“...a transthoracic echocardiography was requested showing”. The word \"requested\" is unneeded.\n\n“...and patient was put under treatment” should be “and the patient”.\n\n“A cardiac magnetic resonance imaging (CMRI) was requested”, \"requested\" should be changed to \"performed\".\n\n“Left ventricle examination” should be “left ventricular”.\n\n“Toxicology was negative” should be \"toxicology examination\" or \"toxicology test\".\n\n“The European Society of Cardiology (ESC) categorize VNC” should be \"categorizes\".\n\n“However, The American Heart Association (AHA) categorize” should be \"categorizes\".\n\n“The most frequent on” should be “the most frequent hypothesis”.\n\n“...at the origin of sudden cardiac death” should be “lead to sudden cardiac death”.\n\n“...in order to initiate an early treatment and avoiding” should be \"avoid\".\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6885", "date": "29 Jul 2021", "name": "med amin mesrati", "role": "Author Response", "response": "Dear Reviewer, First, we thank you for the interest and the time that you have gave to the article. This manuscript was revised according to your relevant comments. We realize that the quality of the article is now improved. We hope that these changes make our work acceptable for indexing. Best regards," } ] } ]	1	https://f1000research.com/articles/9-1045
https://f1000research.com/articles/10-693/v1	29 Jul 21	{ "type": "Research Article", "title": "Association between depression, anxiety, stress and perceived quality of life in a Malaysian B40 urban community during the COVID-19 lockdown: A cross-sectional study", "authors": [ "Gan Sing Joo", "Daniel Mahalingam Owen Devan", "Chuah Shao Qi", "Sapna Shridhar Patil", "Gan Sing Joo", "Daniel Mahalingam Owen Devan", "Chuah Shao Qi" ], "abstract": "Background: In Malaysia, B40 communities are those with a median monthly household earning of under RM 3166. With the prolonged COVID-19 pandemic and the resulting lockdown, the livelihoods of those in these areas has been severely impacted. This has increased their vulnerability to psychological afflictions and has led to a poorer perception of their quality of life (QoL) in comparison to the rest of the population. We investigated the association between perceived levels of depression, anxiety and stress and their impact on quality of life (QoL) among B40 residents in a low-cost urban housing area in Kuala Lumpur, Malaysia. Methods: A cross-sectional study was conducted between July 2020 and February 2021 in the Seri Pantai housing settlement. The validated Malay versions of the depression, anxiety, and stress scale-21 (DASS-21) and the World Health Organization Quality of Life, brief (WHOQOL-BREF) were distributed to the participants using Google forms. The statistical significance of the association between subscales of depression, anxiety, stress and QoL domains were assessed using the Pearson’s correlation test. Results: Of the 180 participants, the majority were Malays (87.2%) and females (82.2%). The average scores were the highest for stress (5.66 ± 4.59) and the score in the environment domain of QoL (59.27 ± 17.23) was the lowest. A statistically significant negative correlation was found between the subscales of DASS-21 and the four domains of the QoL, with the social relationships and psychological domains showing a highly significant association (p < 0.001). The strongest correlation was observed between the psychological domain and depression (r= -.520) followed by psychological domain and stress (r= -.496). Conclusion: The strongest correlation was observed between psychological domain and depression. This suggests a need to address potential devastating mental health consequences of the COVID-19 pandemic and its effect on the QoL of residents in B40 communities.", "keywords": [ "Malaysian B40 community", "Quality of Life", "DASS-21", "WHOQOL-BREF", "Association", "COVID-19 pandemic", "lockdown" ], "content": "Introduction\n\nQuality of life (QoL) is defined as an individual's perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns (World Health Organization Quality of Life [WHOQOL], 2012). This humanistic element was introduced to expand the focus of measuring health holistically beyond the norm of mortality and morbidity (WHOQOL, 1994). Globally, there are more than 150 measures available which are used to assess perceived quality of life (Gill, 1994). This includes the Short Form-36 Health Survey (Rand, 1980), The Quality-of-Life Scale (Flanagan, 1978) and EuroQoL-5D or EQ-5D (Devlin & Brooks, 2017). However, the tool used most worldwide is the WHOQOL questionnaire - the WHOQOL-100 being the original complete version and the WHOQOL-BREF assessment being the abbreviated version, which are applicable cross-culturally and translated accordingly, and include translation to the Malay language (Cheung et al., 2019; Hasanah et al., 2003; Iqbal et al., 2020).\n\nThe Department of Statistics Malaysia (DOSM, 2020) categorized the Malaysian population into T20 (top 20), M40 (middle 40) and B40 (bottom 40) according to monthly household income. B40 communities are those with monthly household earnings of under RM 4850 and a mean monthly income of RM 3166.\n\nPerceived QoL has been shown to differ between various communities and is affected by several factors such as income stability, health status, and socioeconomic status (Bielderman et al., 2015; Cruz et al., 2011; Khan & Tahir, 2014; Shahar et al., 2019; Wan Puteh et al., 2019). Locally, a study done by Wan Puteh et al. in 2019 found that statistically significant low QoL was observed in single, male, low-income households, and in households with chronic illness. These results concurred with those reported by Cruz et al. (2011) and Skevington and McCrate (2012).\n\nPoor mental health is associated with low perceived QoL (Bujang et al., 2015; Clavecillas & Perez, 2020; Farris et al., 2020; Gan & Hue, 2019; Tan and Yadav, 2013). Lower socioeconomic groups are two to three times more likely to suffer from mental health issues than those in higher socioeconomic groups (Kim & Cho, 2020). Meta-analysis by Lorant et al. (2003) also revealed significant evidence of the correlation between socioeconomic inequality and depression. Income inequality during rapid urbanization in Malaysia has led to the urban poor being more susceptible to mental health disorders than the others. The DASS-21 (Depression, Anxiety and Stress Scale - 21 items) questionnaire has been proven to be reliable and valid in the context of Malaysians, and was used to assess the mental health of the participants in this study (Musa et al., 2007; Oei et al., 2013; Ramli et al., 2009).\n\nThe current COVID-19 lockdown has led to a substantial increase in symptoms of depression, anxiety, and stress among the general population (Farris et al., 2020; Patel et al., 2020). Income loss, social isolation, and poor physical health were shown to be factors which exacerbated mental ill-health (Pfefferbaum & North, 2020). In a study conducted mid-pandemic, Ali et al. (2020) assessed the QoL and levels of DAS (Depression, Anxiety, Stress) in the general population of India. Out of 847 participants, 61.9% were found to have psychological symptoms of DAS, and 36.1% were reported having poor QoL after 45 days of lockdown.\n\nIn Malaysia, Gan and Hue (2019) investigated the levels of anxiety, depression and QoL among medical students using the Hospital Anxiety and Depression Scale (HADS) and WHOQOL-BREF. Among 149 medical students, the prevalence rates of anxiety and depression were 33% and 11% respectively. Anxiety was significantly associated with lower psychological, social, and environmental scores, while depression was significantly associated with lower physical, psychological, and environmental scores.\n\nRecently, a sample of 326 participants was recruited from several urban communities in Malaysia and participants were assigned the DASS-21 and WHOQOL-BREF (Farris et al., 2020). Similar to previous studies by Shamsuddin et al. (2013) and Aziella et al. (2017), it was shown that anxiety had the highest prevalence (41.7%) among the participants. The sample was only drawn from urban communities because the authors presumed a higher prevalence of mental issues in urban settings (Farris et al., 2020). This supports Gruebner et al. (2017) who proved that in the urban population, there are higher odds of developing mood disorders (1.4-fold) and anxiety disorders (1.2 fold) than in the rural population.\n\nThe academic community has extensively explored the relationship between perceived QoL and DASS-21 (Ali et al., 2020; Clavecillas & Perez, 2020; Farris et al., 2020; Patil, 2021). However, studies on the association between DAS and QoL are still scarce in the international literature and few studies have been conducted among the B40 community in Malaysia (Bujang et al., 2015; Gan & Hue, 2019). Moreover, the study of these associations is of particular importance during the ongoing COVID-19 pandemic. Understanding the association between DAS and QoL may shed light on the need for future policies relating to the welfare of the urban poor. Thus, the aim of this research is to investigate the association between DAS and QoL in a low socioeconomic community in a developing country during the pandemic lockdown.\n\n\nMethods\n\nThis cross-sectional study was conducted among the inhabitants of the Seri Pantai Community Housing Program, also known as Program Perumahan Rakyat (PPR) in Kuala Lumpur, Malaysia. The PPR is a community housing project which was developed by the National Housing Department (Jabatan Perumahan Negara) in 1998 in an effort to improve the housing environment of the low income (B40) urban dwellers (Hami Erina, 2013). The low-cost, high-rise apartments of Seri Pantai PPR comprises two, 21-storey blocks with approximately 20 households per floor.\n\nThe recruitment of participants and data collection took place from July 2020 to February 2021 with the help of community leaders. These community leaders are residents of PPR Seri Pantai who are a link between the local government bodies and the PPR residents. Due to the current COVID-19 lockdown, face-to-face interviews were not possible. Hence, an online questionnaire using Google forms was designed, which was disseminated by the community leaders to all eligible participants. Eligible participants were all residents of PPR Seri Pantai aged ≥ 18 years of age, and we included all of those who were willing to participate. We employed a convenience sampling technique to include as many eligible residents as possible to participate in the survey. This was essential due to the ongoing pandemic as access to the community was not possible due to the imposed lockdown. To improve participation, the community leaders informed the community about the purpose of the survey and a convenient time for completing the data collection form was ascertained. The community leaders informed the residents about the need to read through and understand the consent form provided alongside the questionnaire. After the participant recorded their consent using a tick box, the survey form became available for them to complete. A total of 180 participants completed all elements of the survey.\n\nWe used the Malay version of the WHOQOL-BREF questionnaire to assess the QoL of the respondents and the DASS-21 to study the perceived depression, anxiety, and stress of the respondents. Sociodemographic data, including age, gender, race, educational level, marital status, and occupation were also recorded.\n\nThe WHOQOL-BREF questionnaire\n\nHarper et al. (1998) has validated the 26-item WHOQOL-BREF as a reliable alternative to WHOQOL-100. The WHOQOL-BREF has been demonstrated to be suitable for assessing the perceived QoL of community-dwelling populations. It comprises 26 items, categorised into a physical domain (seven items), a psychological domain (six items), a social relations domain (three items), and an environment domain (eight items). Each item is ranked using a 5-point Likert scale. The 5-point Likert scale measures the level of agreement to a given statement. The points 1, 2, 3, 4 and 5 each corresponds to ‘strongly disagree,’ ‘disagree,’ ‘neither agree nor disagree,’ ‘agree,’ and ‘strongly agree’ respectively. The total scores were transformed into scores out of 100, with 0 being the least favorable QoL and 100 being the most favorable (Cruz et al., 2011; Harper et al., 1998). The questionnaire provides a brief, comprehensive, and multilingual measurement of QoL (Berwick et al., 1991). As a sizable portion of the population were more comfortable using Malay language, a validated Malay version of the multidimensional WHOQOL-BREF questionnaire was adopted (Cheung et al., 2019; Hasanah et al., 2003; Skevington et al., 2004; Iqbal et al., 2020).\n\nThe DASS-21 questionnaire\n\nThe DASS-21 is a 21-item questionnaire used in assessing the level of self-perceived depression, anxiety, and stress in participants aged 14 and above (Nordin et al., 2017). It is the shorter version of the DASS-42 questionnaire and consists of three domains: depression, anxiety, and stress. Each domain is allocated seven questions, with a Likert scale ranging from zero to three (Lovibond et al., 1995). The points on the scale indicate level of agreement to a given statement, with point 0 indicating ‘never,’ 1 ‘sometimes,’ 2 ‘often,’ and 3 ‘almost always.’ The Malay version of this questionnaire was adequately translated with high validity and internal reliability (Ramli et al., 2009). The DASS-21 requires a shorter time to complete compared to DASS-42 and no special training is required (Musa et al., 2007; Oei et al., 2013).\n\nThe IBM Statistical Package for Social Sciences version 27.0 was used for data analysis. The qualitative variables included age, gender, ethnicity, marital status, educational level and occupation. The quantitative variables included the WHOQOL and DASS-21 scores. Both WHOQOL-BREF and DASS-21 scores were expressed as mean ± standard deviation. Pearson’s correlation was used to compare the scores of the DASS-21 and WHOQOL domains. A p value ≤ 0.05 was considered statistically significant. There were no participants with missing data as all questions in the online form were compulsory.\n\nThis study received Institutional Review Board approval from Taylor’s University Center for Research Management (HEC 2019/058). All participants were provided with detailed information about the purpose of the study. Written informed consent was obtained from the participants before completion of the online questionnaire. The consent was obtained for participation as well as for the publication of the survey data. Information collected from participants was treated in confidence, and the anonymity of the participants was maintained throughout.\n\n\nResults\n\nA total of 180 individuals participated in the study (see Underlying data (Patil, 2021)). The average age of respondents was 42.1 ± 11.4. The majority of the respondents were females (82.2%), more than 80% of them were Malays, 62.2% were married, 78.3% had secondary level education, and 46.1% were working (Table 1).\n\nTable 2 shows the summary of the scores obtained in the four domains of QoL and the depression, anxiety, and stress subscales. The average scores for the four QoL domains were 66.36 ± 14.27 (physical), 66.96 ± 15.56 (psychological), 64.52 ± 20.38 (social) and 59.28 ± 17.23 (environment). The psychological domain showed the highest scores while the opposite was observed in the environment domain. The DASS-21 scores were highest for stress (5.66 ± 4.59), followed by anxiety (4.91 ± 4.35) and depression (4.66 ± 4.60).\n\nStatistically significant negative associations were observed between all subscales of DASS-21 and all domains of QoL (Table 3) with the exception of the environment domain. The correlation between the QoL domains and DASS subscales were highly significant (p < 0.001).\n\nAs shown in Figure 1, the strongest correlation was observed between depression and the psychological domain (r = −0.520, p < 0.001) followed by stress and psychological domain (r = −0.496, p < 0.001).\n\nThe scores for depression showed a strong negative correlation in comparison with the social domain scores (r = -0.448, p < 0.001) and the scores for stress had a correlation coefficient of −0.427, p < 0.001) (Figure 2).\n\nCorrelation between the social domain scores of Quality of Life and the depression, anxiety, and stress scores.\n\n\nDiscussion\n\nNotwithstanding the lack of agreement with previous literature by Gan and Hue (2019), our results have shown that the average score for the DASS-21 was highest for the stress subscale. This is also contrasting with a study by Basudan et al. (2017) who described a higher level of anxiety compared to depression and stress among dental students in Saudi Arabia. Baum, Garafalo & Yali (1999) found that low socioeconomic status was reliably associated with chronic stress.\n\nThe scores for the environment domain were found to be the lowest, indicating that participants felt they had inadequate physical living conditions. This differed from a similar study by Wan Puteh et al. (2019) where poor health status (physical health) was found to be the determinant of low QoL. However, a Malaysian study by Zainal et al. (2012) offers a possible explanation for our findings, as it presents evidence that poor housing conditions can negatively affect the quality of life of the urban poor.\n\nThere was a statistically negative association between all subscales of DASS-21 and all domains of QoL. This supports another Malaysian study by Bujang et al. (2015) which found that all psychological aspects of DASS-21 have a negative impact on the community's QoL. The highest clinical relevance was observed between the psychological domain and depression. This is expected, as meta-analysis by Lorant et al. (2003) found that those in low socioeconomic groups have a higher probability of being depressed. This resonated with findings reported by Gan and Hue (2019) where depression was significantly associated with psychological domains.\n\nEttman et al. (2020) reported a three-fold increase in depression prevalence during the pandemic compared to before. This could be explained by the ground-to-halt economy, restricted social activities, compromised health, sleep disturbances and disruption to normal routine (Bignardi et al., 2020; Fountoulakis et al., 2021; Gaidhane et al., 2020; Majumdar et al., 2020; Rehman et al., 2021). Furthermore, social isolation due to lockdown is another risk factor for mental health issues (Banerjee & Rai, 2020). In addition, the robust causal relationship between life’s stressful events and major depressive episodes further explains the high stress scores during the pandemic lockdown (Breslau & Davis, 1986; Hammen, 2005; Tafet & Nemeroff, 2016).\n\n\nConclusions\n\nThis study found that there is a significant negative association between depression, anxiety, stress and the perceived QoL with the strongest correlation being observed between depression and the psychological domain. Clinical relevance was also found between psychological and social domains of QoL with all of the DASS-21 subscales. This warrants further investigation to determine the causation and nature of this relationship in the context of B40 communities. In addition, the scope of this study needs to be widened to cover other B40 communities in other parts of the country to understand the pattern of association between QoL and the mental health subscales. Similarities and differences in the patterns could assist in planning strategies and measures to address potential associations. Undeniably, the COVID-19 lockdown has left a significant mark on the QoL and mental health status of individuals, adding further stressors to an already disadvantaged and vulnerable population. Therefore, poor QoL and the potentially devastating mental health consequences associated with the pandemic need to be addressed. Further research will pave the way for interventions to be proposed and executed effectively, and these interventions should be replicable in all B40 communities. Vulnerable populations require particular focus so that sustainable change can be implemented to help them through this uncertain season and beyond.\n\n\nLimitations\n\nThe survey could not be done face-to-face due to COVID-19 lockdown restrictions. The sample size was small and the findings cannot be generalized to all of the B40 communities in Malaysia. Additionally, due to the cross-sectional nature of our study, the causal relationship between QoL and depression, anxiety and stress could not be established. There was an unequal distribution of sociodemographic factors whereby the majority of the participants were females (more than 80%) and Malays (87.2%).\n\n\nData availability\n\nHarvard Dataverse: PPR Seri Pantai_ Association between QoL and mental health. https://doi.org/10.7910/DVN/NKPXYL (Patil, 2021).\n\nThis project contains the following underlying data:\n\n- PPR Data_final_160321.ods (PPR survey data).\n\n- PPR DATA SYNTAX.sps. (syntax for data analysis).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nContributions\n\nGan Sing Joo\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nDaniel Mahalingam Owen Devan\n\nRoles: Investigation, Methodology, Resources, Supervision, Formal Analysis, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nChuah Shao Qi\n\nRoles: Investigation, Methodology, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nSapna Shridhar Patil\n\nRoles: Conceptualization, Project Administration, Funding Acquisition, Resources, Software, Supervision, Validation, Visualization, Data Curation, Writing – Original Draft Preparation, Writing – Review & Editing.", "appendix": "Acknowledgements\n\nThe authors would like to thank Associate Professor Dr Karuthan Chinna for his valuable help in data analysis. We would also like to express our gratitude to Dr Jo Ann Andoy Galvan (Senior Lecturer and Community Medicine Posting coordinator), Professor Rusli Bin Nordin (Former Head of School), Assoc Prof Dr Yeong Chai Hong (Associate Professor), Dr Wong Yin How (Senior Lecturer) from Taylor’s University and Associate Professor Halyna Lugova from the National Defence University Malaysia for their guidance and support. This work would not have been possible without the immense efforts and help from the community leaders of PPR Seri Pantai. Finally, we would like to thank all the participants who have spared their time to take part in this study.\n\n\nReferences\n\nAli K, Mufti U, Sharma G, et al.: A Cross-Sectional Study to Assess the Quality of life, Depression, Anxiety and Stress Levels after 45 Days COVID-19 Lockdown. Int J Curr Res Rev . 2020; 12(22): 108–114. 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Publisher Full Text" }	[ { "id": "122017", "date": "15 Feb 2022", "name": "Gajanan D Velhal", "expertise": [ "Reviewer Expertise Epidemiology", "Communicable & Non communicable diseases", "Nutrition", "Health Planning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article has attempted to study the association between quality of life and mental health in an underprivileged urban community in Malaysia during the period of mobility restrictions imposed to curb COVID-19 transmission.\nThe authors have attempted to explore an extremely relevant topic in the wake of the current situation with imposed restrictions, loss of financial security, and the need to sustain and survive during a crisis that is both medical and humanitarian in nature.\nAlthough the study has been performed only in a single B40 community, the results justify the need to conduct such studies in several such communities to assess the extent of the problem and address the emerging issues. The authors possibly could look into the causal relationship between mental health and quality of life by adopting analytical type of study design for a better insight.\nIs the work clearly and accurately presented and does it cite the current literature? The work has been presented in an understandable manner, the language is easy to comprehend and the literature review conforms to the requirement of being recent.\n\nIs the study design appropriate and does the work have academic merit? For the given objective, the design of the study looks appropriate. The work of the authors highlights the need to address significant issue affecting health and well-being.\n\nAre sufficient details of methods and analysis provided to allow replication by others? The methodology used for this study has been detailed out clearly and the analysis is aligned to the objectives. The survey instruments used have been described in adequate detail and the data collection technique has been clearly described.\n\nIf applicable, is the statistical analysis and its interpretation appropriate? The statistical analysis is clearly mentioned and the results reflect the performed analysis. The interpretation of the obtained results is in line with the requirements.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes the source data is available.\n\nAre the conclusions drawn adequately supported by the results? The conclusions are drawn in line with the observations made by the authors. The recommendations made by the authors in the context of the pandemic highlight the further research and strategies required to address the problem.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "251391", "date": "06 Mar 2024", "name": "Dr. Richard Mottershead", "expertise": [ "Reviewer Expertise Life story and narrative research. Post-crisis research", "emancipatory research", "psychiatric and mental health 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\nFirstly, I would like to thank the authors for allowing me to review their study on the association between QoL and mental health in an underprivileged urban community in Malaysia during the period of mobility restrictions in the covid 19 pandemic.\n\nThe study provides an important insight into an underprivileged community and their lived experiences during the recent pandemic. The impact on QoL during these restrictions is of importance to provide understanding for future humanitarian crisis. The study also allows for comparisons with other ethnographic groups creating a wider perspective. This is a well-composed and structured article. The authors appear to have a creditable background and research experience which gives the article credibility. 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. I think that the authors are correct not to generalize and to highlight the subjectivity of examining one sample within a Malaysian urban community. However, the data does provide useful insight and knowledge into the lived experience of those experiencing humanitarian challenges.\n\nThe methodology and methods are clear and would allow for the study to be replicated. Ethical approval is clearly listed as is the data collection technique used and provides transparency to the reader.\n\nThe conclusion is clear, concise and informative with recommendations that would suggest areas for future research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-693
https://f1000research.com/articles/10-692/v1	29 Jul 21	{ "type": "Research Article", "title": "PhD students in life sciences can benefit from team cohesion", "authors": [ "Zsófia Viktória Vida", "István Péter Járay", "Balázs Lengyel", "Zsófia Viktória Vida", "István Péter Járay" ], "abstract": "Background: Scientific progress during doctoral studies is a combination of individual effort and teamwork. A recently growing body of interdisciplinary literature has investigated the determinants of early career success in academia, in which learning from supervisors and co-authors play a great role. Yet, it is less understood how collaboration patterns of the research team, in which the doctoral student participates, influences the future career of students. Here we take a social network analysis approach to investigate this and define the research team as the co-authorship network of the student. Methods: We use the Hungarian Scientific Bibliography Database, which includes all publications of PhD students who defended theses from the year 1993. The data also include thesis information, and the publications of co-authors of students. Using this data, we quantify cohesion in the ego-network of PhD students, the impact measured by citations received, and productivity measured by number of publications. We run multivariate linear regressions to measure the relation of network cohesion, and publication outputs during doctoral years with future impact. Results: We find that those students in life sciences, but not in other fields, who have a cohesive co-author network during studies and two years after defence receive significantly more citations in eight years. We find that the number of papers published during PhD years and closely after the defence correlates negatively while the impact of these papers correlates positively with future success of students in all fields. Conclusions: These results highlight that research teams are effective learning environments for PhD students where collaborations create a tightly knit knowledge network.", "keywords": [ "co-authorship network", "academic success", "scientific impact", "scientific career" ], "content": "Introduction\n\nAs international competition in science accelerates, there has been a growing interest in the determinants of individual success in academia (Sinatra et al., 2016; Clauset et al., 2017; Fortunato et al., 2018). A general notion is that success breeds success because recognized publications open new opportunities for funding and collaboration (Bol et al., 2018). This has directed attention to young scholars because achievements at early stage might generate future success (Wang et al., 2019). The relation between scientific collaboration and success has been investigated for some time (de Solla Price & Beaver, 1966; Luukkonen et al., 1993; Melin & Persson, 1996; Katz & Martin, 1997; Sonnenwald, 2007; Hood & Wilson, 2001; Sarigöl et al., 2014). It is especially useful to understand early career success because young scholars need mentors to learn from and are more likely to stand out in case they work with successful supervisors (Sekara et al., 2018; Ma et al., 2020; Li et al., 2019). Although early careers might learn from more people, it is less understood how future success of early career researchers depend on the team they work with during their early career.\n\nTeams in scientific research are gaining dominance across all fields (Wuchty et al., 2007; Ziman, 1994). Research teams typically include postdocs, graduate and undergraduate students who collaborate with the principal investigator and other seniors of the group (Mali et al., 2012). Such collaborations can be observed in co-authorship that is frequently used to map collaboration networks in teams (Beaver, 2001; Glänzel & Schubert, 2004) and are thought to influence success of projects (Uzzi & Spiro, 2005). Two counteracting mechanisms are important in this respect. On the one hand, projects can create more novelty in the case where co-authors have not collaborated before because they can combine diverse expertise (De Vaan et al., 2015; Vedres, 2017; Zeng et al., 2021). On the other hand, team cohesion generated by shared co-authors, strong and persistent collaboration, trust and previous success can provide an environment in which knowledge sharing are efficient (Uzzi & Spiro, 2005; Aral & Van Alstyne, 2011; Mukherjee et al., 2019). Thus, the question, whether early career researchers benefit more from diverse than from cohesive teams, is important because striving for novelty in scientific research and efficient learning are difficult to achieve at the same time.\n\nIn this paper, we take a social network analysis approach to investigate co-authorship networks of early career researchers. To quantify diversity and coherence in the collaboration network of students and across the author teams they belong to, we apply the network constraint measure developed by Burt (1992, 2000, 2001). This measure ranges between 0 and 1: high values around 1 show that co-authors of the PhD student work frequently together; low values around 0 show that the PhD student works with co-authors who are otherwise not collaborating with each other. This measure has been very widely used to capture diverse knowledge access through connections in various contexts including creative industries (Juhász et al., 2020), innovation (Tóth & Lengyel, 2021) and to capture the role of network cohesion in knowledge transfer (Reagens & McEvily, 2003; Tortoreillo et al., 2012).\n\nOur empirical case concerns researchers who have had a successful defense in any Hungarian doctoral school between 1993 and 2010. Our data contains information on the dissertation, including the scientific field and year of defense, and bibliometric information data comes from publication records of egos and their co-authors. We estimate the accumulated number of citations at the eighth year following defense, which gives us a simple measure of success at the end of the early phase of academic career (Van Balen et al., 2012).\n\nCross-sectional linear regressions with year and scientific field dummies show that the number of papers published until the second year after the defense correlates negatively with accumulated citations, but the impact of these papers correlates strongly with future impact. This finding indicates that thorough work focusing on a few but important papers is a much better strategy than producing many papers during doctoral studies. We find that in case of life science students, both the number of co-authors and most importantly the constraint measure correlates positively with future impact. These latter two co-efficients are not significant for other scientific fields. These results provide new evidence that PhD students in life-sciences can benefit from working in a cohesive research team probably because these provide a good learning environment.\n\n\nMethods\n\nThe ad-hoc ethical committee of the Library and Information Centre, Hungarian Academy of Sciences discussed the use of personal data for the purpose of the research project “Team cohesion of PhD students in life sciences”, and approval to use the data was given. Consent from the participants to use their data was waived by the committee. The Library and Information Centre of the Hungarian Academy of Sciences as data handler of the Hungarian Scientific Bibliography Database is authorized to use the data for scientific research as per law 1994/XL and 10/2021.(II. 24.) regulation decreed by the President of the Hungarian Academy of Sciences.\n\nAll other data used in the study was publicly available.\n\nWe combine two data sources to collect information about early-career scholars. Data on doctoral defenses have been collected from www.doktori.hu, an openly available collection of all successful PhD theses defended in Hungarian doctoral schools starting from 1993, the year when the PhD system was introduced in the country. We downloaded data from the website in January 2017. This data contains 16,151 Hungarian PhD students who defended their theses until that date. Information provided includes the ID and name of every PhD student, the title of their thesis, the year of defense, scientific area, and the name of supervisors. Our second data source is the Hungarian Scientific Bibliography Database (MTMT) that contains the scientific publications’ metadata of all active Hungarian researchers.\n\nThe two databases can be matched on the individual student level. A total of 23% of students were matched between the doktori.hu database and MTMT data using their student IDs. The rest of the students were matched by hand using name and scientific field. We could identify 60% of the PhD students in the MTMT data. The number of PhD students who could be matched with an MTMT profile was 9,415 and is illustrated in Figure 1A by the year of defense. In our regression exercise, we focus on the future impact of PhD thus restrict the analysis to the 2,061 PhD students who defended theses in the 1993-2010 period.\n\nA. The number of PhD students by year in www.doktori.hu (light blue) and the successfully identified PhD students in the MTMT database (navy blue). Data includes all students who defended between 1993 and 2017 but the analysis will focus on those who defended in the 1993-2010 period. B. The number of publications by the Hungarian PhD holders (year of defenses between 1993-2010) and their co-authors between 1990 and 2019.\n\nBibliometric data was downloaded from MTMT by the data handler colleague of the Hungarian Academy of Sciences after the identification of PhD students in MTMT. This happened in two steps. First, we downloaded all 272,954 publication records of the identified 9,415 PhD students in 2017. Then, we identified 20,139 co-authors of PhD students in MTMT and downloaded their publication records in 2020. This final bibliometric dataset contains records of 1,205,184 papers published by 43,485 authors between 1990-2019. Note that only those authors are included who are affiliated with Hungarian institutions and must have registered on MTMT. There are around 50,000 MTMT accounts altogether, meaning that our data collection has covered around 86% of the total scientific community in the country. Figure 1B illustrates the number of all publications from the entire career of those PhD students who defended between 1993 and 2010 and from their co-authors. The data processing and analysis was carried out in R (version number 4.0.0).\n\nMeasuring scientific success, especially individual scientific performance is a complex problem. Traditionally, it is based on production (publication) numbers, scientific impact (citation numbers) and structural measurements for example the network characteristics of authorship (Van Balen et al., 2012; Glänzel et al., 2019). However, the raw citation number depends on several factors, such as the year of publication, research field, document type (e.g. research article, review article or proceedings), and journal characteristics (e.g. frequency of occurrence, number of articles in the journal). For example, it is easy to see that the earlier an article has appeared, the more citations it could receive. The citation habits are different in individual research fields, so to compare two citation measures we must do it in the same research area. The various document types use different number of references. Thus, the comparison is more accurate if it is made within the same document type. Moreover, the journal characteristic also can cause a bias on raw citation numbers. The solution for these problems is using normalized citation numbers.\n\nIn our case the MTMT database contains only raw citation numbers in the year when we downloaded the data, in 2020 (Figure 2 illustrates the publication and citation distributions over the examined period). To handle this problem, we compared each PhD holder in two-year periods, in a cumulative way using the year of their PhD defense as a starting point. We compared PhD holders by their research field. The MTMT database contains document types as articles, books, others, but we were unable to distinguish research articles and review articles. Therefore, we did not consider the document types. Figure 3A shows the cumulative number of citations of the examined PhD students, while Figure 3B shows the cumulative number of their publications.\n\nA. The distribution of number of publications by Hungarian PhD holders (year of defense 1993-2010) between 1990 and 2019. B. The distribution of number of citations between 1990 and 2019 by Hungarian PhD holders (year of defense 1993-2010) in 2020.\n\nA. Cumulative number of citations by the Hungarian PhD holders (year of defense 1993-2010) in 0-10 years after defense. B. Cumulative number of papers by Hungarian PhD holders (year of defense 1993-2010) in 0-10 years after their defense.\n\nTo answer our research question, whether cohesive or diverse co-authorship network structure favors the success of a young researcher, we analyzed the weighted and dynamic ego-networks of PhD students. Such networks were generated from the publication records. These ego-networks include the PhD student in the center (ego), to which co-authors (alters in the ego-network terminology) are connected. Links are undirected but weighted by the number of co-authored papers. The networks are dynamic, such that we add new collaborators and new links to the ego-network of individual PhD students as new papers are published, but do not delete ties over the years. Since we have access to the publications of co-authors, the links between alters contain those publications that were not authored by the PhD student.\n\nCohesive networks are dense and include strong, high-bandwidth ties (Aral, 2016). That is, co-authors frequently publish with each other. Such network structures are thought to capture an environment, in which shared work experience and developed trust facilitate learning from peers. In cohesive networks knowledge transfer is faster and more efficient such that the PhD student can learn complex knowledge easier (Reagens & McEvily, 2003). On the contrary, diverse networks, in which co-authors have not worked with each other but with the PhD student, capture an environment that provides the student with diverse capabilities of co-authors. In such networks, innovation and novel combination is more likely (Burt, 2001). In case the student can integrate distinct pieces of knowledge, diverse networks might help them to publish papers with high degree of novelty.\n\nWe used Burt (2000) constraint indicator that characterizes ego-networks in the cohesive-diverse continuum using the formula:\n\nwhere pij and piq is the number of papers that PhD student i has co-authored with colleagues j and q, and pqj is the number of papers that j and q has co-authored without i. The indicator takes high values in case co-authors publish intensively together and low values are produced when co-authors do not publish together.\n\nAs the size of ego-networks grow, the probability that co-authors are connected might decrease, which has often been found in co-author networks (see for example Tóth & Lengyel, 2021). Thus, one must consider the degree of PhD students as well as their number of co-authors.\n\nThe distributions of degree and constraint are depicted in Figure 4. As expected, these two indicators change the opposite direction. The number of relations rise in time (Figure 4A) which is obvious because we used a cumulative ego network and did not erase former co-authorships. We can also see an increase in the distribution of degree in time, which means that while some researchers could evolve their co-authorship networks after their PhD, others had a narrowed scientific network. The distribution of constraint slightly decreases (Figure 4B), and the median of constraint also falls in time. The cause is that the size of ego networks grows in time and those PhD holders who get more and more co-authors have also more diverse collaboration network.\n\nA. The distribution of degree in cumulative ego networks of the Hungarian PhD holders (year of defense 1993-2010) between 1990-2019. B. The distribution of constraint in cumulative ego networks of the Hungarian PhD holders (year of defense 1993-2010) between 1990-2019.\n\nWe calculated further measures that might be also used to characterize cohesion and diversity in ego-networks. Betweenness centrality quantifies diversity in the network of PhD students by measuring the number of shortest paths in the network that go through the ego. The higher betweenness centrality of the ego the more diversity in the network. Global clustering quantifies the fraction of closed triangles in the network among all possible triangles, while network density measures the fraction of observed ties among all possible ties with the ego. The higher these measures the higher cohesion in the ego-network.\n\nTable 1 reports Pearson correlation coefficients between network parameters at the second and eight year after PhD defense. As expected, we find a negative correlation between degree and all other network indices. Constraint is strongly correlated with network density. We have run alternative regression specifications with the network measures listed in Table 1 but only found significant results for constraint.\n\nTwo years (below diagonal) and eight years (above diagonal) after Hungarian PhD holders defense (year of defense 1993-2010).\n\nOur data enables us to capture impact of publications as a snapshot in 2020 by the total number of citations received until then. This allows for cross-sectional specification, in which we can compare students who finished in the same year and consider publications that they produced until a certain year after defense. This way, we can avoid the problem that earlier publications have more time to collect citations.\n\nTo answer the question whether cohesive co-authorship networks of PhD students during their studies help their future success, we estimate the number of accumulated citations (CITi,t+8) of student i to the paper that they published until the 8th (t+8) year following defense at year t with the following equation:\n\nwhere CITi,t+2 denotes citations to papers published until the second year after defense, PAPi,t+2 and are papers published until the second year after defense and ∆PAPi,t+8 is the number of papers published between the second and eight year after defense, DEGi,t+2 is the degree, and CONi,t+2 is the constraint measure of the student’s co-author network, θi is scientific area-specific fixed-effect, ti is year dummies and εi is the error term.\n\nTo estimate Eq.2, we used ordinary least squares (OLS) linear regression models. The scientific area fixed effects are specified by research fields of doctoral schools. These latter refer to 54 categories of research fields defined by the Hungarian Accreditation Committee (HAC, 2018): exactly one research field has been assigned to each doctoral school. All variables are log-transformed.\n\n\nResults\n\nTable 2 reports results of an OLS regression of estimating Eq.2. In columns 1-3, we estimate citations to papers that were published until the 8th year following defense with variables that capture publications and co-authorship until the 2nd year following defense. We introduce variables in a stepwise manner such that a baseline model is run in column 1 and network variables are introduced in columns 2 and 3.\n\nOLS regressions with year and scientific field fixed effects and robust standard errors.\n\n* p < 0.1;\n\n p < 0.05;\n\n* p < 0.01.\n\nThroughout the models, we found a very strong positive correlation between CITt+2 and CITt+8 that is a trivial relation but has importance in our empirical exercise. Because citations are collected for all publications in 2020, CITt+8 includes CITt+2 However, the very high correlations also mean that most of the citations at the end of the early career stage are received to the publications that were published during or closely after PhD studies. PAPt+2 is negatively correlated while ΔPAPt+8 is positively correlated with the dependent variable. These findings suggest that due to accumulation of citations, the best strategy for PhD students is to produce a few but high impact papers that will help them to collect citations in their early career.\n\nIn column 2, we introduce degree that leaves correlations of other covariates almost unchanged. DEG is positively correlated with CITt+8 suggesting that the number of co-authors facilitates citations. Note that there might be various mechanisms at play; citations might grow with the number of co-authors because they can also cite the paper or spread the word, and alternatively, the project and the PhD student can gain from working with and learning from many collaborators.\n\nConstraint is positively correlated with CITt+8 Controlling for DEG, the number of publications, and including year and scientific field dummies, CON quantifies the extent to which co-authors of the PhD student have collaborated in publications that are published until the second year after the defense of the student. Our finding suggests that such cohesive ego-networks are beneficial for PhD students. Because we also control for the citations to papers, this finding confirms that PhD students benefit the most from working in cohesive collaboration networks because these create efficient learning environments.\n\nCorrelations of independent variables indicate that the models are not violated by multicollinearity. The highest value of the Pearson correlation coefficients is ρ = 0.41 between DEG and PAPt+2 We document the correlation between DEG and CON in Table 1 (ρ = 0.61), but the inclusion of these variables together is conceptually motivated as we describe before. Further, the inclusion of CON in Model 3 does not substantially influence the coefficient of DEG.\n\nIn Table 3, we report full regression models decomposed into four big scientific areas such as Science, Life Science, Engineering, Social Science. To achieve these scientific areas, we have grouped the 54 scientific fields (as per National Accreditation Committee). We found that DEG and CON is significant for the Life Science subsample while DEG only is weakly significant in Engineering. Thus, cohesive research environment is important for Life Science students and less for students in other fields.\n\nOLS regressions with year and scientific field fixed effects and robust standard errors.\n\n* p < 0.1;\n\n p < 0.05;\n\n* p < 0.01.\n\n\nDiscussion\n\nIn this study we examined the success of students who defended theses in Hungarian doctoral schools between 1990 and 2010 by looking at their publication records and accumulated citations in 2019. Our bibliometric database also contains the PhD students’ publications and their co-authors’ publications between 1990 and 2019. We analyzed whether cohesive or diverse co-author network structure gives a better chance to a young researcher to stand out in terms of citations eight years after defense. Linear regression models suggest that those students who participate in cohesive collaboration networks, receive significantly more citations at the end of their career. This result highlights the need for strong collaborations and effective learning environment during doctoral studies. However, our results regarding the structure of co-author networks are specific to Life Science students. Thus, cohesion is mostly important in areas where new knowledge is produced in teamwork.\n\nThe present paper contributes to a growing literature, in which studies try to determine factors that support the future success of young researchers. Li and co-authors (2019) demonstrate that those students who publish with top scientists had a greater chance of being more successful 20 years later. Moreover, this effect is more important in the case of PhD students affiliated with a less prestigious PhD school. Sarigöl et al. (2014) illustrate a similar phenomenon: a paper gets more citations if its’ authors are central in the large co-author network of their field. We add to this discussion by studying the co-author ego-networks of PhD students. Our findings confirm that the structure of the group collaboration matters for the future academic career of students.\n\nWe also find that those students are more successful, measured in citations, who focus on few papers. These results are robust across all large scientific fields. By concentrative efforts into a small number of publications, students can achieve higher quality papers that might be accepted to better journals. Because citations typically demand several years to accumulate, students need high-impact papers at the beginning of their career to stand out later when they are at the end of the early-career stage. This can help them in research proposals and thus facilitate academic careers in the long run.\n\n\nData availability\n\nRaw data, as accessed through the Hungarian Scientific Bibliography Database, are not publicly available due to privacy considerations of the authors. Access can be requested through the Library and Information Centre of the Hungarian Academy of Sciences; contact details can be found on the library’s website https://www.mtmt.hu/kapcsolat. Access to the data will be provided under the following conditions: the researcher presents legitimate research purposes to the ad-hoc ethical committee of the Library and Information Centre of the Hungarian Academy of Sciences and after the committee’s positive decision, signs a non-disclosure agreement.\n\nZenodo: Data and Code for the manuscript PhD students in life sciences can benefit from team cohesion, https://doi.org/10.5281/zenodo.5129288 (Vida et al., 2021).\n\nThis project contains the following underlying data:\n\n- Phd_data.csv (contains anonymized author ID, network and publication variables after 2-4-6-8 years of PhD defense, and discipline of PhD thesis).\n\n- Phd_regressions_code.R (analysis code used in the study).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors acknowledge the kind help of Gusztáv Ladányi in collecting the bibliometric data and Dániel Horváth in matching the bibliometric data with thesis defense data.\n\n\nReferences\n\nAral S: The future of weak ties. 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Press; 1994." }	[ { "id": "90722", "date": "19 Aug 2021", "name": "Koen Frenken", "expertise": [ "Reviewer Expertise scientometrics", "innovation 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 presents an interesting study on team cohesion and its effects on PhD students’ success based on a novel dataset. While the paper is well-written, I list suggestions for further development and three textual issues.\nEmbedding in the literature: There is a sizeable literature by now on the (collaborative) conditions that support PhD students. I list below some references. I do not suggest that you have to integrate all these studies in your paper, but rather that you read the papers and see which ones are relevant to your study. Taking some of these papers into account would better embed your study in the current literature and also better highlight what your contribution is1-15.\n\nLimitations: You explain well your data and methodology but some of the limitations are not well spelled out. First, you look only at PhD students who finished by writing the thesis. Hence, the ones who did not finish, arguably the least successful ones, are left out. This means that - in a way - you “sample on the dependent variable” in the sense that your dependent variable is a success variable, while you analyse only those who successfully defended the thesis. I do not see this as a major issue, but I think it should be highlighted as a limitation.\nSecond, on page 4 you report that 60 percent of the PhD students could be identified in the MTMT dataset. Here, I expected more information why the remaining 40 percent could not be matched.I understand from page 5 that the co-author network of a PhD student that is collected, comprises only of authors in MTMT, which I reckon excludes foreign co-authors. Please clarify. And, if my comment is correct, you could also highlight this as a data limitation\nThird, it is further also unclear whether the “raw citation numbers” in the MTMT database include only citations by authors in the MTMT. Again, please clarify. And, if my comment is correct, you could also highlight this as a data limitation.\n\nResults: page 8, table 1: why do you report a correlation matrix including multiple variables that are NOT included in the regression analysis later on? Rather uncommon. Instead, I suggest you show first a table with the descriptive statistics only of the variables included in the regression analysis and then also a correlation matrix, again, only for the variables included in the regression analysis later on.\npage 9, table 2: You do OLS regressions but your dependent variable is a count variable which is most probably also skewed. Please look into this again. Most probably, you have to do Poisson or Negbin regressions instead. (see e.g.: Cameron, C., Trivedi, P.K., 1998. Regression analysis of count data. Cambridge University Press, New York.)\n\nText: page 3: “, but the impact of these papers correlates strongly with future impact” -> I do not understand this sentence\npage 5: what do you mean by “raw”\npage 10 “its’ ” should be “its”\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? Yes", "responses": [] }, { "id": "171447", "date": "12 May 2023", "name": "Yi Bu", "expertise": [ "Reviewer Expertise Scientometrics", "bibliometrics", "scholarly communication" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper studies performance of PhD students in life science by examining a couple of bibliometric-wise indicators, e.g., publications, citations, and co-authorship network-related measurements. Generally, this paper presents some interesting findings. Yet, the dataset is only limited to Hungary, which may limit its generalisation to more countries with diverse culture, research environments, and regulations.\nThe usage of citations seems to be unfair because the authors show the total number of citations received until the dataset was downloaded. This is not incorrect methodologically but such a comparison needs to be normalized (by, for instance, publication year, document type, and discipline [here I meant to say domains under life sciences]).\nSome additional basic descriptive statistics of the ego-centered networks should be offered.\nNot sure but is there any collinearity issue in the regression model? This should be examined prior to the regression model(s).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": [] } ]	1	https://f1000research.com/articles/10-692
https://f1000research.com/articles/10-691/v1	29 Jul 21	{ "type": "Case Report", "title": "Case Report: A giant fecaloma revealed by severe aspiration pneumonia and urinary retention", "authors": [ "Elhem Mchirgui", "H Elloumi", "Imen Ganzoui", "Wissem Triki", "H Elloumi", "Imen Ganzoui", "Wissem Triki" ], "abstract": "Fecaloma is an accumulation of hardened impacted stool typically occurring in the sigmoid colon and rectum. It mainly affects elderly and bedridden patients suffering from chronic constipation and can be revealed by different signs. We report a case of 74-year-old female, with anorexia, Alzheimer’s disease, and chronic constipation, who was admitted to the emergency department with complaints of dyspnea and anuria. Clinical examination showed fever, Glasgow Coma Scale score of 13/15, tachycardia with a blood pressure of 100/50 mmHg, polypnea with hypoxia, foci of crepitant rales in pulmonary auscultation and a tender hypogastric mass with mild diffuse abdominal tenderness. Digital rectal examination revealed hard fecal material. Computed tomography (CT) images demonstrated bilateral pulmonary parenchymal condensation and a huge heterogeneous fecaloma in the sigmoid colon and rectum compressing the bladder. Based on these findings, the diagnosis of giant fecaloma causing aspiration pneumonia and urinary retention was retained. Manual disimpaction and bowel enemas were done but they were unsuccessful and surgical treatment was refused. Ultimately the patient died due to septic shock. Early diagnosis should be made to relieve symptoms and prevent complications.", "keywords": [ "Fecaloma", "Elderly", "Constipation", "CT scan", "Complication", "Mortality." ], "content": "Introduction\n\nFecaloma, a rare cause of fecal impaction, is an accumulation of hardened impacted stool typically occurring in the sigmoid colon and rectum and affecting, especially, elderly patients.1–5 Symptoms of fecaloma are non-specific, dominated by constipation, abdominal pain, abdominal distention, nausea and vomiting.3–5 The diagnosis is made through abdominal computed tomography scan (CT scan), X-rays or abdominal ultrasound.1,2 Fecaloma is often managed conservatively by manual disimpaction, laxatives and enemas. However, endoscopic or surgical interventions may be mandatory especially when it is refractory to conservative treatment or complicated.4 Fecaloma is also associated with increased morbidity and mortality.3,4 We report a case of a giant fecaloma revealed by severe aspiration pneumonia and urinary retention.\n\n\nCase report\n\nA 74-year-old north-African female presented, in March 2021, to the emergency department of our hospital with dyspnea for one week and anuria for one day. The patient had a three-year history of Alzheimer’s disease, anorexia and chronic constipation. For one year, she had been bedridden after a fall. On initial evaluation, she was cachectic, had a Glasgow Coma Scale score of 13/15, a temperature of 38.5°C, a heart rate of 110 beats per minute, blood pressure of 100/50 mmHg and a respiratory rate of 22 breaths per minute with a saturation of 83%. On physical examination, the patient had foci of crepitant rales at both pulmonary bases and mild diffuse abdominal tenderness with a tender hypogastric mass on palpation. Digital rectal examination revealed hard stool impaction.\n\nThrough blood testing, the patient had white blood cells at 5800/mL; hemoglobin at 9.2 g/dL; C-reactive protein at 312 mg/l; urea at 1mg/l; creatinine at 129 μmol/l; natremia at 133 mmol/l; potassium at 4 mmol/l and chloremia at 96 mmol/l. Her blood gas showed respiratory alkalosis with severe hypoxemia at 60 mmHg. Thoraco-abdominal CT scan revealed left pulmonary parenchymal condensation and liquid esophageal stasis (Figure 1a) suggesting aspiration pneumonia (Figure 1b) and a huge heterogeneous fecaloma measuring 10 × 12 cm in the sigmoid colon and rectum (Figures 2a and 2b) associated with stercoral stasis in the remaining colon and compressing the bladder (Figure 2a). The patient was tested for coronavirus disease 2019 (COVID-19) using a polymerase chain reaction test, due to the pandemic context and the clinical presentation of dyspnea, hypoxemia and pneumonia on the CT scan. The test was negative.\n\nChest computed tomography with an axial (a) and coronal (b) cuts, revealing esophageal stasis (a, Black line) and pneumonia (b, Blue cross).\n\nAbdominal computed tomography with an axial (a) and coronal (b) cuts revealing a distension of the rectum and sigmoid colon by a giant fecaloma of 10 × 12 cm (a, Blue arrow) going up to the left hypochondrium (b, Red line). The bladder is pushed forward and to the left (a, Blue cross).\n\nThe diagnosis of giant fecaloma causing urinary retention and aspiration pneumonia was retained. A Foley catheter was introduced into the urinary bladder bringing back 500 mL of concentrated urine. Intravenous antibiotics was prescribed with oxygen therapy by facial mask. A conservative treatment of the fecaloma with manual disimpaction and bowel enemas was practiced but was unsuccessful. Surgical treatment was discussed with surgeon and anesthesiologist teams, and it was considered to have high operatory risk. Her family was informed but they refused the surgery. The patient's condition deteriorated, and she died on the third day of hospitalization from septic shock.\n\n\nDiscussion\n\nTo the best of our knowledge, a huge fecaloma complicated by aspiration pneumonia has never been reported in the literature. Although it is an exceptional complication, physicians should consider it as a possible cause of pneumonia in older patients with chronic constipation. In our patient, treatment by manual disimpaction and bowel enemas was tried but it was inefficient. An endoscopic treatment could be an interesting alternative but was not available in our institution, so was a limitation in the therapeutic management in this case. The surgery was rejected because of high operatory risk. A fatal outcome may be the consequence of delayed extraction of the fecal impaction by life-threatening complications, as in our case.\n\nFecaloma is a hard stool impaction in the rectum and/or colon that cannot be evacuated spontaneously.3,4 It typically occurs in the rectum and sigmoid colon. Cecal and small bowel localizations are rarely reported.6,7 Fecaloma is typically seen in elderly patients, particularly in those bed bound and institutionalized and more common in women.3–5 Chronic constipation, frequently seen in elderly and bedridden patients, plays an important role in the development of fecaloma1,3,4,8 Other common risk factors reported in the literature were neuropsychiatric diseases and use of constipating medication like antiepileptic, antidepressant and opioid therapy.1,2,7,9–12 Fecaloma was also associated with Chagas disease, Hirschsprung's disease, inflammatory and neoplastic diseases, scleroderma, diabetic neuropathy, and anorectal malformations.2,4,7\n\nThe fecaloma’s symptoms are various and non-specific. The most common complaints are constipation, abdominal pain, abdominal distention, nausea and vomiting.3–5 Fecaloma can be also be revealed by rectal bleeding, urinary retention and respiratory insufficiency, febrile status, impaired consciousness, altered general condition7,13,14 Physical examination can demonstrate an abdominal distension, tenderness or mass on palpation and a hard mass in the rectum in digital rectal examination.7,10,14 In our case, respiratory signs including hypoxemia and pneumonia were the predominant features. The fecaloma was also responsible of urinary retention with renal failure. Fecaloma diagnosis is made radiologically through X-rays, barium enema, abdominal ultrasound, or abdominal CT scans.1,2\n\nComplications associated with fecal impaction are estimated at 40.6% of cases.3 Some of these complications can be life threatening.7,14 Colonic complications, secondary to the effect of the fecaloma on the intestinal wall or lumen, leads to several injuries like bowel obstruction,2,15 Intussusception,16 stercoral ulcer,1,11 stercoral colitis8 or toxic megacolon leading to colonic perforation and fecal peritonitis and bleeding2,3,7 Otherwise, the fecal mass can compress adjacent anatomical structures and lead to hydronephrosis,12,14,17 nerve compression and deep vein thrombosis.2\n\nThe fecaloma’s management is sometimes challenging. Therapeutic options include conservative, endoscopic and surgical treatments. The conservative treatment of fecalomas, especially those located in the rectum, consists of manual disimpaction of the fecal mass associated with the administration of laxatives and enemas to soften the hardened stool.12–14 Oral lavage with polyethylene glycol solution in sometimes helpful in case of proximal fecal impaction.4 Successful management of fecaloma by an endoscopic fragmentation of fecal material in the distal colon has been recently reported.9,11 When conservative treatment fails, surgical intervention is required to prevent complications. Surgery should be indicated first-line in case of giant fecaloma, and when the patient’s general condition is poor or peritonism signs are present.4,10,18 Fecaloma formation must be prevented in high risk patients with laxatives, fibers and water intake.4 Some authors propose routine abdominal radiological exploration for these patients to prevent the development and irreversible complications.1,2\n\nWhen it is not treated promptly with the best treatment method, fecaloma can be life-threatening.7 In fact, patients presenting with fecal impaction are considered to be at high risk for serious morbidities and mortality reported in 40.6% and 21.9% patients, respectively.3 The prognosis is particularly worse in elderly patients and in those with heart or neuropsychiatric diseases and chronic renal failure.4,5\n\n\nConclusion\n\nFecaloma commonly reported in elderly patients suffering from chronic constipation, should be considered as a serious condition. Early diagnosis should be made to relieve symptoms and prevent complications which can be often serious. The diagnosis should be suspected when there are signs of organ compression, especially in elderly patients with chronic constipation. Aspiration pneumonia can be one of the fecaloma’s complications.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the family of the patient.", "appendix": "References\n\nHuang W-C, Huang T-Y, Chen P-J, et al.: Fecaloma impaction and stercoral ulcer. Adv Dig Med. 2020; 7(3): 166–169. Publisher Full Text\n\nGil AG, Liu Q, Ho S: Giant Fecaloma Causing Large Bowel Obstruction: A Case Report. Gastro Med Res. 2020; 5(1). GMR.000602. Publisher Full Text\n\nSommers T, Petersen T, Singh P, et al.: Significant morbidity and mortality associated with fecal impaction in patients who present to the emergency department. Dig Dis Sci. 2019; 64(5): 1320–1327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSerrano Falcón B, Barceló López M, Mateos Muñoz B, et al.: Fecal impaction: a systematic review of its medical complications. BMC Geriatr. 2016; 16: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHalawi HM, Maasri KA, Mourad FH, et al.: Faecal impaction: in-hospital complications and their predictors in a retrospective study on 130 patients. Colorectal Dis Off J Assoc Coloproctology G B Irel. févr . 2012; 14(2): 231–236. PubMed Abstract \| Publisher Full Text\n\nWang BT, Lee SY: Cecal fecaloma: A rare cause of right lower quadrant pain. Eur J Radiol Open. 2019; 6: 136–138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlakaj F, Hamza A, Bicaj B, et al.: Giant fecaloma mimicking large tumor of the abdomen: A case report. Forensic Sci Int Rep. 2020; 2: 100108. Publisher Full Text\n\nAbasin T, Din MN: Stercoral colitis due to massive fecal impaction: a case report and literature review. Radiol Case Rep. 2021; 16: 1946–1950. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhosh G, Shah S, Maltz C: A case of a giant fecaloma. Clin Gastroenterol Hepatol. 2018; 16(4): e48. PubMed Abstract \| Publisher Full Text\n\nKhan MA, Dar HA, Shah AH, et al.: Fecaloma presenting as huge abdominal mass. JGH Open. 2020; 4(2): 294–295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoraveji S, Siddiqui AD: Unique Endoscopic Therapy of Colonic Obstruction Caused by a Fecaloma: 1631. Off J Am Coll Gastroenterol ACG. 2018; 113(Supp 938).\n\nTu K-C, Kuo J-R: Fecaloma causing megacolon and bilateral hydronephrosis. Formos J Surg. 2020; 53(2): 70. Publisher Full Text\n\nCurro G, Lazzara C, Latteri S, et al.: Supergiant fecaloma as manifestation of chronic constipation. Il G Chir. 2017; 38(1): 53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOzlu DN, Seker KG, Seker YC, et al.: Postrenal Acute Renal Failure Due to Giant Fecaloma-related Bilateral Hydronephrosis: A Case Report and Brief Literature Review. Cureus. 2020; 12(4). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalayci T, Genel IDH: A Rare Ileus Etiology: Giant Fecaloma. Causapedia. 2018; 7(3): 164–168.\n\nKhan Z, Darr U, Renno A, et al.: Transient descending colocolonic intussusception due to a large fecaloma in an adult. ACG Case Rep J. 2017; 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoo N, Lee HS: Acute Hydronephrosis owing to A Giant Fecaloma in an Older Patient. Ann Geriatr Med Res. 2020; 24(3): 223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTchangai B, Alassani F, Tchaou M, et al.: Staged Surgery for Giant Fecaloma Complicating Idiopathic Megacolon. Open J Gastroenterol. 2016; 6(12): 418–422. Publisher Full Text" }	[ { "id": "101553", "date": "21 Dec 2021", "name": "Hyung Seok Lee", "expertise": [ "Reviewer Expertise Interventional Nephrology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report deals with complications caused by chronic constipation that is frequently encountered but often overlooked in the elderly.\nComplications caused by giant fecaloma can occur in various ways by compressing or displacing adjacent organs as well as direct harm, such as bowel wall perforation. In particular, this case report presents that if fecaloma is not timely treated, it can lead to a rare complication, such as aspiration pneumonia, which has useful clinical implications for other practitioners.\nThis article is well written and contains enough information. Therefore, I have no further comments on this article.\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": "264316", "date": "03 May 2024", "name": "Noboru Misawa", "expertise": [ "Reviewer Expertise constipation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper entitled “Case Report: A giant fecaloma revealed by severe aspiration pneumonia and urinary retention”, the authors has reported a case of a giant fecaloma revealed by severe aspiration pneumonia and urinary retention. I would like to add a comment.\nMajor comment\nIn this report, do the authors think that aspiration pneumonia was caused by fecaloma? If so, the authors need to be more specific about the mechanism. The authors stated that in the Discussion, “Fecaloma diagnosis is made radiologically through X-rays, barium enema, abdominal ultrasound, or abdominal CT scans”. However, more specific information should be given on diagnostic methods for the assessment of faecal masses in the recto-colon. In particular, the recent report on ultrasound assessment of faeces in the rectum should be mentioned.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-691
https://f1000research.com/articles/10-447/v1	04 Jun 21	{ "type": "Research Article", "title": "Evolution of human respiratory virus epidemics", "authors": [ "Nash Rochman", "Yuri I Wolf", "Eugene V Koonin", "Yuri I Wolf" ], "abstract": "Background: It is often assumed that pathogens evolve towards reduced virulence, but counterexamples abound. Faced with a new pathogen, such as SARS-CoV-2, it is highly desirable to be able to forecast the case fatality rate (CFR) and overall disease burden into the future. Considerable effort has been invested towards the development of a mathematical framework for predicting virulence evolution. Although many approaches accurately recapitulate complex outcomes, most rely on an assumed trade-off between CFR and infection rate. It is often impractical to empirically validate this constraint for human pathogens. Methods: A compartment model with parameters tuning the degree to which symptomatic individuals are isolated and the duration of immunity is constructed and evaluated at both short timescales and at equilibrium (when it exists). Results: We reveal kinetic constraints where the variation of multiple parameters in concert leads to decreased CFR and increased pathogen fitness, whereas independent variation of the parameters decreases pathogen fitness. Smallpox, SARS-CoV-2, and influenza are analyzed as diverse representatives of human respiratory viruses. We show that highly virulent viruses, such as smallpox, are likely often constrained by host behavior, whereas moderately virulent viruses, such as SARS-CoV-2, appear to be typically constrained by the relationship between the duration of immunity and CFR. Conclusions: Evolution of human respiratory epidemics appears to be often kinetically constrained and a reduction in CFR should not be assumed. Our findings imply that, without continued public health intervention, SARS-CoV-2 is likely to continue presenting a substantial disease burden. The existence of a parameter regime admitting endemic equilibrium suggests that herd immunity is unachievable. However, we demonstrate that even partial isolation of symptomatic individuals can have a major effect not only by reducing the number of fatalities in the short term but also by potentially changing the evolutionary trajectory of the virus towards reduced CFR.", "keywords": [ "SARS-CoV2", "influenza virus", "smallpox virus", "epidemic modeling", "case fatality ratio" ], "content": "Background\n\nThe case fatality rate (CFR) and infection rate largely determine the survival of both a pathogen and its host. Constraints imposed by the host behavior and pathogen biology prevent independent variation of these rates. Therefore, trends in virulence evolution can be predicted only through understanding these constraints. Although comprehensive models of the evolution of virulence capable of describing complex environments have been developed,1–5 most studies to date impose constraints assumed from first principles and lacking experimental or empirical validation.6,7 Most commonly, a trade-off function is assumed8,9 between the infection rate and the CFR. It is important to note that different definitions of virulence can lead to dramatically different predictions.10 In this work, we are primarily concerned with long-term trends and consider the CFR exclusively (not the rate of instantaneous mortality or other measures of virulence).\n\nIndividual hosts harboring high numbers of pathogen are more likely to die than those harboring lower numbers, but they also shed pathogen at increased rates and therefore could infect a greater number of new hosts.11 However, this straightforward picture is complicated by a landscape of opposite evolutionary outcomes.12 For example, vaccines that alleviate symptoms but still admit infection have been suggested to have induced selective pressures which favor the evolution of strains with increased virulence in malaria13,14 and decreased virulence for diphtheria and pertussis,15 likely due to differences in the cost of toxin production.16 This demonstrates how predictions of specific outcomes useful for informing public health intervention cannot be generalized across pathogens. The infectivity–virulence trade-off has been demonstrated for malaria.14 Although this could be true more generally, the existing data are scarce, and demonstrating this trade-off for most human pathogens is extremely difficult, due to the impracticality of comprehensive contact tracing. Many strategies to replace the explicit use of this trade-off have been discussed.11 One approach is to construct realistic models of within-host pathogen dynamics.17,18 This approach provides the ability to make nuanced predictions about specific pathogens and environmental conditions, but requires a deep understanding of the pathogen biology, which is often lacking, especially in the case of newly emergent pathogens. Thus, general models that avoid the use of this constraint as a parameter and without explicit descriptions of within-host pathogen dynamics continue to prove useful.\n\nTowards this goal, we explore a range of epidemiological outcomes for human pathogens modelled using available data on well-characterized respiratory viruses. Our principal aim is to chart a “global phase space”, indicating where endemic equilibrium is possible and to establish the bounds of potential evolutionary trajectories within this phase space without leaving that endemic equilibrium. We do not, however, explicitly conduct an invasion analysis. The ability of an emergent strain to invade depends on many factors that are not considered in the present model and the existence of a path within the global phase space does not guarantee that invasion along that path is possible or probable for any particular virus. Conversely, if a path in the global phase space leads out of endemic equilibrium, this suggests that invasion along that path is impossible. Through this approach, we are able to identify broad evolutionary constraints on pathogen evolution.\n\nThe apparent inverse relationship between the time during which the host is asymptomatic but infectious and the CFR,19–27 as well as the difficulty of vaccination against lower-CFR viruses, such as influenza, relative to higher-CFR viruses, such as smallpox, indeed imply the existence of broad, intrinsic constraints that could link the duration of immunity and CFR. We demonstrate that, in addition to trade-offs, the evolution of CFR is often subject to “kinetic constraints” that prevent CFR reduction by imposing a barrier to increasing the total number of infected hosts, our measure of pathogen fitness. This is analogous to an energetically favorable chemical reaction with a high activation energy.\n\nConsider a simple, reversible chemical reaction between reactant, R, and product, P. When sufficient energy, A1, is applied, R is converted to P. This is the activation energy of the forward reaction. Now consider that P is much more stable than R so that P cannot be converted back to R unless 10×A1 energy is applied. This is the activation energy of the reverse reaction. Given sufficient energy and time, the system will reach an equilibrium in which the quantity of P is 10-fold higher than the quantity of R. However, if insufficient energy is applied, less than A1, the system will remain almost wholly composed of R. Under these conditions, the reaction is kinetically constrained. Now suppose that P is much less stable than R so that P is converted back to R whenever 0.1×A1 energy is applied. In this case, even given sufficient energy and time, the system will remain in a state in which the amount of R is at least 10-fold greater than the amount of P. Under these conditions, the (forward) reaction is thermodynamically constrained.\n\nThe evolution of pathogen life history traits may be described as a system of reversible reactions where the “stability” of each set of life history traits is estimated by the total number of infected hosts at endemic equilibrium. The evolution of decreased CFR would be thermodynamically constrained if it were impossible to both increase the total number of infected hosts and decrease CFR. Additionally, the evolution of decreased CFR could be kinetically constrained by many barriers to invasion, which might vary among pathogens and even among host populations for the same pathogen. We sought to identify global kinetic constraints for CFR evolution, where there exists a parameter regime, in which the total number of infected hosts is higher and CFR is lower, but accessing this regime requires simultaneously modifying multiple parameters, some of which might be determined by host behavior. In other words, there may be an evolutionary path to decreased CFR, but it is narrow, even without the consideration of specific barriers to invasion. We show that, for high-CFR viruses such as smallpox, the relationship between the infection rate and CFR is likely a kinetic constraint, rather than an actual trade-off, whereas for intermediate-CFR viruses, such as SARS-CoV-2, there is a kinetic constraint between immunity and CFR. Analysis of such constraints could open avenues for prediction of epidemic outcomes and quantitative validation of such predictions.\n\n\nMethods\n\nThe introduction of a novel pathogen into a host population is likely to result in one of three outcomes. 1) The number of fatalities is large enough for the pathogen to wipe out the host population (as a result, the pathogen itself also goes extinct) (Figure 1A). 2) The number of infections is large enough, whereas the number of fatalities is small enough, such that the pathogen creates a bottleneck in the susceptible host population. With all hosts either infected or immune, the pathogen is eliminated from the host population (Figure 1B). 3) The number of infections is small enough such that a bottleneck in the susceptible population is avoided and long-term persistence within the host population is possible if the number of infections is not too small and the basic reproduction number is greater than one, R0 > 1 (Figure 1C). If the basic reproduction number is less than one, disease-free equilibrium is achieved. These three outcomes can occur on vastly different timescales, with a host wipeout or susceptible bottleneck occurring much faster than the rate at which equilibrium is approached. Throughout this work, we simulate each epidemic until one of these three outcomes is reached. With R0 > 1, a state of stable endemic equilibrium can be reached where the fraction of the host population susceptible to infection remains constant (up to fluctuations). However, if the pathogen-associated fatality exceeds the birth-rate of the host population, the host–pathogen relationship becomes unsustainable in the long term. Such an unsustainable relationship would pose an intense selective pressure on the host population, likely resulting in the extinction of the pathogen through the modification of host behavior or the emergence of resistant hosts.\n\nA-C. Cartoons representing the three potential outcomes for an epidemic beyond trivial disease-free equilibrium. Lines indicate total number of hosts, shaded areas indicate the fraction of hosts which are not susceptible. A. Host extinction. B. A bottleneck in the susceptible population. C. A balance between infections, CFR, and birthrate. D. Host interaction, quarantine, and isolation. E. Probability of Infection Following Host Contact (Infectivity). F. CFR. G. Immunity.\n\nWhich of these courses an epidemic follows, largely depends on the balance of four factors. 1) The frequency of host–host interaction, with or without isolation of infected individuals or prophylactic quarantine (Figure 1D). 2) The likelihood of an uninfected host to become infected after interacting with an infected host (Figure 1E). 3) The CFR of the pathogen, that is, the likelihood that an infected host will die as a result of infection (Figure 1F). 4) The duration of host immunity post infection (Figure 1G). Although the effect of tuning each of these parameters often appears obvious, for example, decreasing the frequency of host-host interaction almost always decreases the number of infections - many counterintuitive observations become apparent. For example, decreasing the frequency of host-host interaction under conditions that would otherwise lead to a bottleneck in the susceptible population (Figure 1B) can result in long term persistence within the host population which, avoiding pathogen extinction, ultimately increases the total number of infections sustained over time. In an effort to better delineate how the host-pathogen relationship varies across the space of these factors, we constructed the following model.\n\nHosts are assigned one of the four possible states: 1) immune, I; 2) susceptible, S; 3) asymptomatic, A; and 4) symptomatic or “clinical”, C. New hosts are assumed to be born susceptible at a rate, kB, and a baseline death rate, kD, is assumed constant across all compartments. Susceptible hosts can be infected by coming into contact with either asymptomatic or clinical hosts. Asymptomatic hosts either recover at rate, kR, or progress to the clinical compartment at rate, kP. Clinical hosts either recover at rate, kR, or die due to the pathogen at rate, kDV.\n\nIn the model, as written, recovery confers permanent immunity with probability, α. This is done as a mathematical convenience. Under many if not most circumstances, it is more realistic to assume recovery confers immunity which is gradually lost at a constant rate, kL. At endemic equilibrium, one may solve for the parameter kL=1−αkRA+C/I (see Appendix A in the extended data28) as a function of α; however, the dynamics approaching equilibrium are different for the two formalisms. Thus, when analyzing short-term dynamics, we assume that only a negligible portion of the population has lost immunity and fix α=1.\n\nThe population is assumed to be well mixed, with the exception of the clinical compartment, a fraction 1−β of which is isolated and cannot infect susceptible hosts. Note this is equivalent to modelling a decreased contact rate for the entire clinical compartment. The rate at which susceptible hosts become infected is the triple product of the rate of contact between hosts, the fraction of the population (not isolated) which is infected, and the probability of infection upon contact: kcontactA+βCI+S+A+βCPinfect. For simplicity, we consider the product, kI≡kcontactPinfect, which depends on both host behavior and pathogen biology. Throughout the text, we consider Pinfect=1 and describe variation in kI as variation in kcontact. Lower values of Pinfect would not change the bounds on the global phase space or the qualitative trends displayed in any figures; however, they would correspond to higher values of host contact rates than those that appear on the figure axes. This yields the system of ordinary differential equations (Figure 2A):\n\nWith two infected states (asymptomatic and clinical) and isolation, ISAC is a simple model within the range of models29–31 that have been developed in response to the SARS epidemic.\n\nShort-term dynamics can be inferred from this system of equations, to establish whether a bottleneck in the size of the susceptible population (Figure 1B), or the entire population representing a host crisis (Figure 1A), occurs. For the analysis of dynamics within this period, we assume that only a negligible portion of the host population has gained and subsequently lost immunity, setting α=1. To study this case, we explicitly simulate an epidemic beginning with an initial condition where the size of the infected compartments is five orders of magnitude lower than the value corresponding to the endemic equilibrium, in order to capture realistic dynamics for the emergence of a new pathogen. The basic reproduction number, R0, for this model can be derived through the construction of next generation matrices32 (see Appendix B in the extended data):\n\nShort-term dynamics are sometimes determined simply by the R0, value and when R0 < 1, the pathogen will go extinct. When R0 > 1, a wide range of dynamics are possible including short term bottlenecks and long-term endemic equilibrium.\n\nIn many cases when R0 > 1 and a bottleneck is avoided, we are interested in examining the stationary points for the above system of equations. Unique, stable stationary points correspond to endemic equilibrium. While uniqueness and stability have been proven for a very broad class of compartment models,33,34 more generally, this can be numerically demonstrated for any stationary point as is done throughout this work. At endemic equilibrium, the fraction of the total population, N, in each compartment, X, is constant: XN′=x′−xn′=0 (we denote the fraction of each compartment with lowercase letters). Endemic equilibrium corresponds to a stable stationary point for this system of equations. We represent values corresponding to endemic equilibrium with a “”, to reflect this. We do not assume a constant size of the total population; however, the results of this simplifying assumption are presented in Appendix C (extended data). More generally, n′=kB−kD−ckDV which yields:\n\nWe are primarily interested in establishing the fraction of hosts that are infected i∗+c∗ and the number of deaths caused by the pathogen per year kDVc∗ at endemic equilibrium and, generally, aim to algebraically solve this system of equations for the relative size of each compartment. This system can be solved to yield a fourth order polynomial with respect to c (we used the MATLAB symbolic toolbox (RRID:SCR_001622),35 an open-source alternative is Julia, see Appendix D in extended data); however, it is cumbersome enough that a numerical solution appears preferable and establishing opportunities for linearization is desirable.\n\nEndemic equilibrium requires a constant or growing population, limiting the total number of deaths due to infection that can be sustained by the host population per unit time and, accordingly, provides an upper size bound for the clinical compartment. kB−kDkDV≥c∗;kB−kDkR≥ckDVkR. For human populations and pathogens, it is reasonable to assume that the birth rate is much lower than the recovery rate, kB,kDkR≪1, yielding the limit: c∗kDVkR≪1. Conversely, populations with higher birth rates admit endemic equilibrium for more virulent pathogens, and even among human populations, the host population size plays a critical role in the determination of whether acute pathogens persist.18\n\nThus, for an endemic equilibrium to exist, either the clinical compartment has to be very small or the death rate due to the virus has to be very low compared to the recovery rate (again, considering the opposite limit, when the birth rate is high, parameter regimes exist where neither endemic equilibrium is reached nor does either the host population or the virus go extinct). This allows us to linearize the model with respect to either the size of the clinical compartment, for pathogens with high CFR, or the ratio of the death rate to the recovery rate, for low CFR pathogens. For the high CFR case, we solve the above system of algebraic equations to first order in c∗ (e.g. c∗2+c∗∼c∗, where by definition 0<c∗<1, see Appendix E in the extended data). This linearized model admits an analytic solution for endemic equilibrium, given a sufficiently large α corresponding to at least partial immunity whenever R0>1. In this case, the additional constraint kP>kR+kDV, which largely holds for the pathogens considered here, is applied for convenience. For the low CFR case, we solve the above system of equations to first order in kDVkR (see Appendix F in the extended data). Here the stability of the solution depends on the parameters (it does not belong to the class of models referenced above). Both the stability and the general solution for the critical point are calculated numerically (see Appendix G in the extended data); however, analytic forms for endemic equilibria in the stricter limit c∗kDVkB≪1 are provided as well.\n\nParameters are referenced against those selected to represent three respiratory pathogenic viruses: smallpox, SARS-CoV-2, and influenza representing a range of phenotypes (Table 2)19–27 in agreement with the ranges reported for each parameter by the selected references. We emphasize, however, that given the generality of the model, we are unable to make specific predictions for any of these viruses in a defined host population. We instead contrast the results for “smallpox-like” (high CFR), “SARS-CoV-2-like” (intermediate CFR), and “influenza-like” (low CFR) viruses. Here, kB and kD are fixed whereas kI is varied; however, we will emphasize that throughout the text, we consider Pinfect=1 and describe variation in kI as variation in kcontact. kR and kP are fit to an estimated disease course for a host which is asymptomatic and infectious for the time tP=1/kP, and symptomatic and infectious for the time tR=1/kR before recovering. For simplicity, death due to infection is assumed to occur only during the symptomatic and infectious phase: kDV=CFR1−CFRkR. Smallpox is modelled with a CFR of 30%, mean time to recovery 1 week, and no asymptomatic and infectious period. SARS-CoV-2 is modelled at a CFR of 1%, 1 week recovery period, and 3 days asymptomatic and infectious. Influenza is modelled at a CFR of 0.05%, 1 week recovery period, and 3 days asymptomatic and infectious. Smallpox infection is assumed to confer permanent immunity. SARS-CoV-2 and influenza infections are assumed to confer immunity for one year on average. A constant birthrate of 2.5 births per 2 people over 100 years and death rate of one death per person over 100 years is assumed.\n\n\nResults\n\nEndemic equilibrium, where an unchanging fraction of the host population remains infected in the long term, is bounded within a range of (host) contact rates (Figure 2B). When the mean time between contacts is too long and kI is too low, kI=kR+kD+kP1+βkPkR+kD+kDV−1, R0 < 1, the pathogen goes extinct, and disease-free equilibrium, where there is no remaining pathogen, is reached. Likewise, when contacts are too frequent, a bottleneck in the susceptible population occurs (Figure 1B, here assumed to drop down to 10% of the total population), and the pathogen goes extinct. For some viruses, such as smallpox-like and SARS-CoV-2-like viruses, but not influenza-like viruses, a range of contact rates exists where the fraction of the infected population at endemic equilibrium is large enough so that the death rate exceeds the birth rate and the host–virus relationship is unsustainable in the long term, resulting in population decline without decreased pathogen CFR or modified host behavior. This range is very narrow for smallpox-like viruses but notably broad for SARS-CoV-2-like viruses (Figure 2B, not shown to scale) encompassing a wider range of host behavior than endemic equilibrium. The existence of this region in the parameter space implies that herd immunity, without vaccination, might be impossible to reach, at least, in some segments of the parameter space. In the middle range of contact rates admitting endemic equilibrium (Figure 1C), the decreased fraction of the infected population for all three examined viruses is offset by increasing CFR leading to an increased death rate. Under these model assumptions, the yearly death rate for a SARS-CoV-2-like virus is approximately 6-times that of an influenza-like virus.\n\nA. Cartoon of the compartment model. Note that, in the model, permanent immunity is gained with probability α for mathematical convenience. Alternatively, temporary immunity can be gained with a probability of 1 and lost at rate kL. Although the dynamics are different, the corresponding endemic equilibria can be identified by expressing kL as function of α and compartment frequencies. B. Disease free equilibrium (gray), endemic equilibrium (striped), and unsustainable (red) regions for three analyzed viruses over a range of host contact rates. The unsustainable region for smallpox-like viruses is narrow and not shown to scale. C. For endemic equilibrium (contact rates correspond to the midrange shown in B), the fraction of the host population infected and the death rate. Note that, representing equilibrium, the choice of timescale (years) is arbitrary.\n\nTo examine an expanded two-dimensional phase space, we allowed the CFR to vary from 10% to 100% for smallpox-like viruses, with no asymptomatic spread and permanent immunity, and from 0% to 10%, for SARS-CoV-2-like and influenza-like viruses, with asymptomatic spread and temporary immunity (Figure 3). CFR, host–host contact rate, and the duration of immunity, which is explored in the next figure, are the three most relevant parameters for the study of human pathogenic respiratory viruses apparent from the observed variability among known pathogens (Table 2). However, it is important to acknowledge the effects of the rate of recovery on the global phase space as well. Throughout the text, we assume a 1-week recovery period. In a supplemental figure (see extended data), we display the results for two additional values (3 days/2 weeks, Figure S1. A/B). The dependency on CFR is remarkably similar across all three recovery regimes, but as one would expect, slower rates of recovery correspond to lower host-host contact rates required to achieve endemic equilibrium.\n\nThe CFR and the contact rate were varied. For variable recovery rate, see Figure S1. Disease-free equilibrium (gray), endemic equilibrium (striped), unsustainable (red), and host population crisis (loss of 90%, dark red) regions are shown. For CFR > 10%, smallpox-like features are assumed. For CFR<10% SARS-CoV-2/influenza-like features are assumed (these viruses are only distinguished within the model by CFR). Lines within the region bounding endemic equilibrium indicate contours for the percentage of the population infected (a* + c)100. Darker color corresponds to higher values. Two regions subject to kinetic constraints are outlined.\n\nAs the CFR increases, both threshold contact rates, corresponding to R0 = 1 and to the bottleneck in the susceptible population respectively, increase and the range admitting endemic equilibrium narrows. Across much of the phase space, the host-pathogen relationship is unsustainable, and at very high CFR, the total host population falls below 10% (dark red) in the initial phase of the epidemic, signaling possible extinction at short timescales. The contours within the region corresponding to the endemic equilibrium indicate the total size of the infected host population (which is proportional to the size of the viral population and constitutes our measure of virus fitness). Within this region, increasing contact rate and decreasing CFR increases the size of the infected population. At extremely high CFR, the gradient points primarily in the direction of decreasing CFR, and at low contact rates, the gradient points primarily in the direction of increasing contact rate.\n\nThroughout most of the region in this phase space corresponding to endemic equilibrium for smallpox-like viruses (Figure 3), moving down and to the left increases the size of the infected population and suggests evolution towards decreased CFR. However, this is not the case at the top-right corner representing viruses with extremely high CFR and extremely high contact rates (or alternatively viruses with a much higher likelihood of infection following host contact). Such, hypothetical, viruses would have an unsustainable relationship with the host population if CFR decreased (and infected hosts were less likely to die before interacting with uninfected hosts) and are, in a sense, kinetically constrained, likely, by the host behavior. The population size of such viruses would dramatically increase if the CFR was reduced but would remain in endemic equilibrium only if contact rates or the rate of infection simultaneously decreased. Smallpox evolution could be similarly kinetically constrained. On the phase diagram for high-CFR viruses, smallpox, with a CFR of 30%, is located near the triple point for host populations with high contact rates where the regions of disease-free equilibrium, endemic equilibrium and unsustainability meet (highlighted in Figure 3). To increase the size of the infected population, both CFR and contact rate must decrease, whereas decreasing only the CFR ultimately results in disease-free equilibrium when such a virus enters new host communities, due to a bottleneck in the size of the susceptible population.\n\nThe corresponding triple point located at the boundary between influenza-like and SARS-CoV-2-like viruses lacks this property. For these viruses, decreasing CFR increases the size of the infected population throughout the parameter space. However, the boundary highlighted within the SARS-CoV-2-like diagram (Figure 3) represents a different kinetic constraint, this one, between immunity and CFR. While decreasing CFR below 10% minimally affects the threshold contact rates (R0 = 1 and susceptibility bottleneck), varying the duration of immunity has a dramatic impact on the phase diagram (Figure 4A). As the duration of immunity decreases, with α=0.01 corresponding to approximately 1.5 years at the phase boundary, the endemic equilibrium region shrinks substantially.\n\nA. Phase diagram of a SARS-CoV-2-like virus varying CFR and contact rate. Red lines indicate the boundary between endemic equilibrium and unsustainable for α ∈ {0.01,0.045,0.08} regions corresponding to approximately {1.5,7,13} years of immunity, respectively. B. The fraction of the population infected, (a* + c*), vs the duration of immunity for a hypothetical virus with a CFR of zero (other parameters matching influenza and Sars-Cov-2) and the maximum admitted contact rate for endemic equilibrium. C. Peak death rate at the height of an epidemic with contact rate corresponding to the maximum value admitting endemic equilibrium given β = 1 and varying β (decreasing β corresponds to increasing isolation). Death rate is scaled by the maximum value for each virus.\n\nAs is apparent from the analytic solutions given in the extended data and illustrated for a hypothetical virus with a CFR of zero (other parameters matching influenza and Sars-Cov-2) and the maximum admitted contact rate (Figure 4B), decreasing the duration of immunity dramatically increases the size of the infected population at endemic equilibrium across the parameter regimes. While typically viewed from a host-centric perspective, immunity is almost always necessary for the maintenance of endemic equilibrium and thus required for maintaining large viral populations over long time scales. Consider a SARS-CoV-2-like virus near the highlighted boundary in Figure 3. Suppose this virus acquires an adaptation enabling immune evasion and thus decreasing the mean duration of immunity post infection. The size of the infected population will increase and, being near the boundary, the host–pathogen relationship will become unsustainable. This is another example of a kinetic constraint. Decreasing both CFR and the duration of immunity increases the size of the infected host population. However, maintaining a stable host–pathogen relationship and large numbers of infected hosts in the long term requires that the reduction in CFR is proportional to the reduction in immune duration. Otherwise, the overall death rate for the host population might increase despite a reduction in pathogen CFR. Notably, decreasing the host–host contact rate moves the population farther from this boundary in the phase space and alleviates this kinetic constraint. Therefore, even if host–host contact rates cannot be reduced enough to break endemic equilibrium by reaching R0 < 1, it’s possible that a modest reduction can change the evolutionary trajectory of the pathogen from one of stagnant or increasing CFR to one of decreasing CFR.\n\nOne way a host population can effectively decrease the rate of infection is through the isolation of symptomatic individuals. Isolation decreases death rate at the peak of the epidemic (Figure 4C) and can prevent an epidemic entirely by driving R0 below 1, which requires β<kR+kD+kPkI−1kR+kD+kDVkP. For pathogens with substantial asymptomatic or pre-symptomatic spread (small kP), this approach might not be feasible. Although isolation narrows the range of contact rates that admits endemic equilibria or unsustainability (Figure 5A), the death rate at endemic equilibrium varies little with decreasing β and may even increase in the case of SARS-CoV-2-like viruses. Notably, however, SARS-CoV-2-like viruses are particularly sensitive to changes in β such that a modest decrease in β, that is, increased isolation, can change the long-term outcome from endemic equilibrium to disease-free equilibrium. Furthermore, despite a 30-fold difference in CFR, both smallpox-like viruses and SARS-CoV-2-like viruses lead to the death of approximately 0.1% of the host population per year at endemic equilibrium. This highlights how pathogens with low or intermediate CFR can cause as many fatalities as a pathogen with a much higher CFR if allowed to reach endemic equilibrium.\n\nA. Disease free equilibrium (gray), endemic equilibrium (striped), and unsustainable (red) regions for three viruses over a range of host contact rates. Red region for smallpox-like viruses is narrow and not shown to scale. Bars are ordered by decreasing β ∈ {0,0.25,0.5,0.75,1}. B. Death rate at endemic equilibrium (contact rates correspond to the midrange shown in A). Bars are ordered by decreasing β ∈ {0,0.25,0.5,0.75,1}. Note that, representing equilibrium, the choice of timescale (years) is arbitrary.\n\n\nDiscussion\n\nWe show here that CFR evolution in human pathogenic respiratory viruses can often be kinetically constrained. However, these constraints represent only the most general limitations on potential evolutionary trajectories. We did not conduct an invasion analysis, and many paths that apparently are not constrained in the global phase space (Figure 3) are inaccessible under specific conditions. The accessibility of these trajectories depends on both the global bounds to endemic equilibrium discussed here and the gradient of pathogen fitness. We consider the total number of infected hosts as our measure of pathogen fitness, which is only viable for establishing long-term trends. Different measures of fitness are required to determine barriers to invasion.36\n\nNonetheless, even these most general constraints likely play key roles in shaping the host–pathogen relationship of important human respiratory viruses. A reduction in CFR increases the average amount of time an infected host spends in the clinical compartment. This increases the total number of new infections that result from contact with each infected host. For smallpox-like pathogens with high CFR in communities with frequent contact, this can create a bottleneck in the size of the susceptible population, resulting in disease-free equilibrium (that is, extinction of the virus) when not accompanied by a decrease in the rate of infection. The rate of infection is determined by both the probability of infection given host–host contact and the host–host contact rate. Under these conditions, if the probability of infection following host contact were internally constrained by CFR, this would not constitute a fitness trade-off and could facilitate host adaptation. On the other hand, high host–host contact rates could externally constrain the rate of infection making reduction in CFR costly to the virus. Evolution of the smallpox virus shows a steady pattern of gene losses that likely lead to increasing infectivity and virulence.37,38 This evolutionary trend appears to be compatible with the conclusion that high CFR, smallpox-like viruses are unlikely to evolve towards decreasing CFR due to constraints imposed by the host behavior. Similar constraints have likely led to the emergence of acuteness for human bacterial respiratory pathogens of the genus Bordetella.18\n\nFor SARS-CoV-2-like pathogens with moderate CFR, evolution towards decreased CFR can be kinetically constrained by the relationship between CFR and the duration of immunity. Decreasing the duration of immunity increases the size of the infected population and the overall death rate which can make the host–pathogen relationship unsustainable. The existence of a large region of the phase space corresponding to unsustainable or kinetically constrained moderate-CFR viruses implies two distinct forms of host response over two different timescales. Over long timescales, unsustainable viruses are likely to face extinction due to the elimination of the susceptible host population. Although, in principle, this could occur via extinction of the entire host population, the emergence of host resistance is likely. Over short timescales, especially for modern human populations, the emergence of an unsustainable virus, such as SARS-CoV-2, may be considered societally unacceptable, leading to drastic measures that result in a major reduction in host–host contact rates. Both trends likely contribute to the paucity of moderate CFR human respiratory viruses which are subject to this kinetic constraint between immunity and CFR.\n\nPerhaps paradoxically, immune evasion could incur a fitness cost for the pathogen and even lead to its extinction due to the host response. However, some level of immune evasion is required, among host populations with a low birthrate, to maintain any state of endemic equilibrium in the case where lifelong immunity is conferred against individual strains and the duration of immunity is determined by antigenic drift rather than the decline of immunity itself. Evolution towards decreased CFR is uncertain in this case, and a better understanding of internal genomic constraints7 could help predict the effects of immunomodulation. This is important when assessing the impact of novel or imperfect vaccination which can lead to counterintuitive results.15,39,40 Diversification related to immune evasion commonly enables the maintenance of large virus populations over long time scales, as is the case for influenza.41–43 In the case of SARS-CoV-2, immune evasion, diversification, and host adaptation of SARS-CoV-2 have already been demonstrated.44–46 Furthermore, products of virus genes that are specifically found in pathogenic beta-coronaviruses have been implicated in immunomodulation,47 suggesting that the virus adapts to maintain an endemic equilibrium in this way. Although the present model cannot predict whether SARS-CoV-2 will become more or less virulent, our results do suggest that evolution of its CFR is kinetically constrained such that the region of the parameter space where reduced CFR could evolve is small. On the other hand, we show that even modestly decreasing the host–host contact rate can alleviate this kinetic constraint and promote CFR reduction.\n\nDuring both the ongoing SARS-CoV-2 pandemic and the first SARS-CoV-1 epidemic, stringent public health measures were taken to limit infection, extending beyond isolation of symptomatic individuals and into the quarantine of asymptomatic, and likely uninfected, contacts.22,31 Although only a crude depiction of the nuanced dynamics underlying SARS-CoV-2 infections, the analysis presented here suggests that isolation and quarantine are particularly effective towards changing the long-term outcome for viruses with moderate CFR and high probability of infection following host contact, such as SARS-CoV-2. The phase diagram for SARS-CoV-2-like viruses is sensitive to the parameter β which reflects the isolation of symptomatic individuals (and additionally asymptomatic carriers who have tested positive). The evolution of the epidemic for such viruses is dominated by disease-free equilibrium or an unsustainable host–virus relationship. Endemic equilibrium is possible only in a narrow parameter range and is therefore unlikely. Nonetheless, the existence of this range suggests that herd immunity is unlikely in the absence of vaccination and would amount to an extreme number of fatalities. Our analysis shows that, while highly amenable to public health intervention, SARS-CoV-2-like viruses can be expected to contribute to a substantially higher death toll than influenza-like viruses, comparable instead to that of a smallpox-like virus, over a protracted period.\n\nWe should emphasize one last time that our aim in this study was to use a maximally general model to chart the global phase space of human respiratory viruses. We did not use any explicit trade-off functions or specify within-host pathogen dynamics. Given the generality and simplicity of this approach, these results cannot be immediately leveraged to predict the outcome of specific epidemics for specific host populations. Such prediction requires an invasion analysis, taking into account many additional factors.5,17,18 Furthermore, throughout this work, we discuss ways in which host–pathogen interactions affect evolutionary outcomes. It is also important to acknowledge that environmental influences, perhaps most prominently the stability of the exposed pathogen,48 can additionally constrain the evolutionary outcome of an epidemic. Additionally, the model presented here is deterministic and assumes a large, well-mixed host population. Finite size effects for small or spatially segregated host populations can additionally affect epidemic outcome,2 resulting in substantial stochasticity.\n\n\nConclusions\n\nHuman respiratory epidemics often evolve under kinetic constraints. These constraints can prevent the reduction in CFR for both high- and moderate-CFR viruses. The incorporation of these constraints can assist in the interpretation of classical model results for epidemics where some parameters, such as host–host contact rate, are unknown. We show that SARS-CoV-2-like viruses are unlikely to reach a state of endemic equilibrium; however, the potential for such equilibrium implies that herd immunity without vaccination may be impossible. At equilibrium, moderate-CFR viruses can cause as many fatalities as high-CFR viruses with both SARS-CoV-2-like viruses and smallpox-like viruses leading to death of about 0.1% of the population per year. However, even partial isolation of symptomatic individuals can have a major effect not only by reducing the number of fatalities in the short term but also by potentially changing the evolutionary trajectory of the virus towards reduced CFR. Such simple public health interventions can continue to dramatically decrease the forecasted cost of SARS-CoV-2 over both the short and long term.\n\n\nAuthor contributions\n\nConceptualization and Formal Analysis: NDR, YIW, and EVK\n\nWriting – Original Draft Preparation: NDR and EVK\n\nWriting – Review & Editing: NDR, YIW, and EVK", "appendix": "Acknowledgements\n\nThe authors thank Koonin group members for helpful discussions.\n\n\nData availability\n\nZenodo: Extended Data for ‘Evolution of human respiratory virus epidemics’ https://doi.org/10.5281/zenodo.4818157. 28\n\nThe project contains the following underlying data:\n\n• Appendix.pdf\n\n• FigureS1.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nGandon S, Day T: The evolutionary epidemiology of vaccination. J R Soc Interface. 2007; 4(16): 803–17. 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Publisher Full Text\n\nZhang Y, Zhang J, Chen Y, et al.: The ORF8 protein of SARS-CoV-2 mediates immune evasion through potently downregulating MHC-I. bioRxiv. 2020.\n\nKorber B, Fischer WM, Gnanakaran S, et al.: Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell. 2020; 182(4): 812–27. e19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan Y, Schneider T, Leong M, et al.: Novel immunoglobulin domain proteins provide insights into evolution and pathogenesis of SARS-CoV-2-related viruses. MBio. 2020; 11(3). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalther BA, Ewald PW: Pathogen survival in the external environment and the evolution of virulence. Biol Rev. 2004; 79(4): 849–69. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "87356", "date": "25 Jun 2021", "name": "Claus O. Wilke", "expertise": [ "Reviewer Expertise mathematical modeling", "evolutionary biology", "infectious disease" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this contribution is very well written and seems reasonable. I have no major concerns, but I do have a few comments:\nThe authors write \"as well as the difficulty of vaccination against lower-CFR viruses, such as influenza, relative to higher-CFR viruses, such as smallpox\". I don't believe that difficulty of vaccination is directly linked to CFR, except maybe in the sense that there's less economic incentive to develop a vaccine if CFR is low.\n\nTable 1 looks a bit weird. Maybe use two columns?\n\nWhile the paper is overall well written, in the end I find it difficult to figure out what exactly the authors' conclusion is. I think the problem lies in the term \"kinetically constrained\", which is used throughout the manuscript but never actually explained. In the end, evolution of CFR comes down to the existence (or lack thereof) of transmission-mortality trade-offs, and I think you're trying to say that these trade-offs can be shaped by the rate constants of the various \"reactions\" in your model. If so, can you say this explicitly? And also, would this be at all surprising? Can you make any stronger or more specific statements?\n\nYou may find this paper helpful as a demonstration of how one can talk about this topic in very clear and understandable terms (Bull et al. (20141)).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6928", "date": "29 Jul 2021", "name": "Eugene Koonin", "role": "Author Response F1000Research Advisory Board Member", "response": "Response to Claus Wilke: Overall, this contribution is very well written and seems reasonable. I have no major concerns, but I do have a few comments. Response: We thank the reviewer for their kind remarks. The authors write \"as well as the difficulty of vaccination against lower-CFR viruses, such as influenza, relative to higher-CFR viruses, such as smallpox\". I don't believe that difficulty of vaccination is directly linked to CFR, except maybe in the sense that there's less economic incentive to develop a vaccine if CFR is low. Response: We agree with the reviewer that economic incentive is an important consideration which we did not mention in the previous version and have added in the revision. With this sentence, however, we intended to highlight the fact that lower CFR viruses are less likely to provide permanent immunity which in turn complicates vaccination strategies. This has been clarified in the revision. Table 1 looks a bit weird. Maybe use two columns? Response: We apologize if the former layout reduced the legibility of Table 1. We have reformatted the table as suggested. While the paper is overall well written, in the end I find it difficult to figure out what exactly the authors' conclusion is. I think the problem lies in the term \"kinetically constrained\", which is used throughout the manuscript but never actually explained. In the end, evolution of CFR comes down to the existence (or lack thereof) of transmission-mortality trade-offs, and I think you're trying to say that these trade-offs can be shaped by the rate constants of the various \"reactions\" in your model. If so, can you say this explicitly? And also, would this be at all surprising? Can you make any stronger or more specific statements? Response: We regret the confusion and have further expanded our discussion of kinetic vs. thermodynamic constraints in the revised version. Regarding the point that we are, “trying to say that these trade-offs can be shaped by the rate constants”, this is certainly the case, and we agree with the reviewer that this is to be expected. In this work, however, we highlight the opposite. There are very broad trade-offs (or in some instances the lack of trade-offs), which are largely independent of model parameters and shared across broad regimes within the life history characteristic phase space of all human respiratory viruses. We infer these very general evolutionary constraints without the use of an explicit trade-off function and without specifying an intra-host evolutionary model. This results in a “10,000 foot view” of the landscape, very general but not highly precise. In the original manuscript, we were cautious not to overstate the predictive value of our approach for specific epidemic outcomes. We thank the reviewer for this comment and acknowledge that these efforts may have resulted in unnecessary ambiguity in the text. We have strengthened and reiterated the statement of the principal conclusions throughout the revised version. You may find this paper helpful as a demonstration of how one can talk about this topic in very clear and understandable terms (Bull et al. (2014)). Response: We thank the reviewer for bringing this work to our attention. We agree that it is highly relevant, most interesting and very clearly written. We have appropriately cited this work." } ] }, { "id": "86917", "date": "07 Jul 2021", "name": "Kim Sneppen", "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 paper investigates the sustainability of steady state (endemic states) of various diseases. Given the paper is a rather simple modification of a SIRS model, the paper is a remarkable difficult read. Thus I may easily be missing something due to my difficulty in following the logic.\nThe title mention evolution, but I failed to find what is evolving, probably because I could not grasp the true meaning of the kinematic constraints, i.e. what mechanism among the virus should want to drive it towards a steady state endemic state, given that the virus tend to be selected to maximize its offspring from generation to generation? A more clear explanation of this potentially important new scale of evolutionary selection would be helpful.\n\nOverall, I am uncertain about the steady state approach to the diseases, as endemic like diseases often comes in pulses, like smallpox in the late 18th century, or perhaps like standard influenza epidemics (with all their caveats on mutations and partial cross-immunity).\n\nIn more detail, I am sorry, but do not get the logic of Fig. 1. Clearly, a disease can eventually go extinct because either the transmission rate is to low, or so high that one fast reach a state where herd immunity makes further propagation impossible. In between these extremes there will be some endemic state, supported by susceptible from birth or from loss of immunity from previously infected people.\n\nTable 2 does not give a hint about real differences in infection rates between smallpox and coronavirus. Apart from that then the parameters from SARS-CoV-2 is misleading, as serial interval and infectious period each is about 5 days.\n\nFig. 2 nicely outlines the model, but then in panel B,C) presents data using a y-axis that quantifies kI(?) in contact days? What about infectivity, some diseases like smallpox have a R0 of about 20, while other like SARS have R0 between 2 and 5. How does the y-axis in Fig. 2 relate to these very different scenarios?\n\nFig. 3 is again a hard figure to read? How can smallpox softly transit from SARS-Cov-2. I do not get how increased case fatality rate changes the regime of the sustainable endemic state? Also, the disease free equilibrium at short contact times presumably reflects situations where the first epidemic was so brutal that herd immunity was more than obtained? I don't understand the unsustainable red region? Why larger for larger CFR?\n\nFig. 4 is difficult given the arbitrariness of all parameters. What is the point of 3 sub-figures, what could I as a reader learn?\n\nFig. 5 investigates mitigation, something that has little to do with endemic states (presumably we would not like to have a permanently mitigated society?). I also failed to locate whether the mitigation fraction beta only included reduction in the symptomatic transmission? For SARS-CoV-2 there is a lot of asymptomatic transmission, but not for SARS.\n\nIn conclusion, the authors say that SARS-CoV-2 cannot reach a state of endemic equilibrium. They might be right, but one might as well imagine that disease immunity decline sufficiently fast that it could be constantly circulating in some mild form. Also, I cannot see how its increasingly many mutant variants would make such a conclusion robust.\n\nIn the results section of the abstract the authors state: \"We reveal kinetic constraints where the variation of multiple parameters in concert leads to decreased CFR and increased pathogen fitness, whereas independent variation of the parameters decreases pathogen fitness. Smallpox, SARS-CoV-2, and influenza are analyzed as diverse representatives of human respiratory viruses. We show that highly virulent viruses, such as smallpox, are likely often constrained by host behavior, whereas moderately virulent viruses, such as SARS-CoV-2, appear to be typically constrained by the relationship between the duration of immunity and CFR.\" This is beyond my understanding: smallpox could not really be constrained (or could not when it recurrently broke out in cities in the 18th century). SARS-CoV-2 was in contrast contained by human mitigation, and had little directly to do with CFR.\n\nA minor point is the strange discussion of reversible chemical reactions in a paragraph on page 2. It seems to assume that energy is directly proportional to transmission rates. Also its relation to the rest of the manuscript is unclear.\n\nOverall, the paper presents a worthwhile investigation into an important phenomenon. However, it is very difficult to read and evaluate given its unclear assumptions (and complicated sentences). I would encourage a major rewrite, emphasizing which few (hopefully) parameters that are important, and with new figures that more clearly illustrate the main point.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "6927", "date": "29 Jul 2021", "name": "Eugene Koonin", "role": "Author Response F1000Research Advisory Board Member", "response": "Response to Kim Sneppen: The paper investigates the sustainability of steady state (endemic states) of various diseases. Given the paper is a rather simple modification of a SIRS model, the paper is a remarkable difficult read. Thus I may easily be missing something due to my difficulty in following the logic. Response: We apologize for any confusion and hope that our revised presentation is substantially clearer. The title mention evolution, but I failed to find what is evolving, probably because I could not grasp the true meaning of the kinematic constraints, i.e. what mechanism among the virus should want to drive it towards a steady state endemic state, given that the virus tend to be selected to maximize its offspring from generation to generation? A more clear explanation of this potentially important new scale of evolutionary selection would be helpful. Overall, I am uncertain about the steady state approach to the diseases, as endemic like diseases often comes in pulses, like smallpox in the late 18th century, or perhaps like standard influenza epidemics (with all their caveats on mutations and partial cross-immunity). Response: This study is primarily concerned with very long-term evolutionary outcomes. The reviewer makes the important point, which has been emphasized in the revised version that, in reality, a pathogen rarely reaches a true steady state. Outbreaks are almost always spatially and/or seasonally dependent, and the fraction of a host population that is infected fluctuates significantly over time. However, even diseases like Influenza or Smallpox that are or were subject to periodic epidemics or pandemics, during which the number of infected individuals is much higher than average, both are or were endemic to the human population such that a predictable number of cases would be sustained on average within a year or a decade. This study largely focuses on such averages as emphasized in the revision. In more detail, I am sorry, but do not get the logic of Fig. 1. Clearly, a disease can eventually go extinct because either the transmission rate is to low, or so high that one fast reach a state where herd immunity makes further propagation impossible. In between these extremes there will be some endemic state, supported by susceptible from birth or from loss of immunity from previously infected people. Response: Figure 1 was introduced to provide context for a broad audience. The reviewer is correct that the content of this figure may be obvious for an expert reader. Indeed, the logic is described correctly in the reviewer’s comment. We still think that this figure is useful for a reader unfamiliar with specifics of evolutionary epidemiology, and under the current circumstances, we expect there will be many such readers of this paper. Table 2 does not give a hint about real differences in infection rates between smallpox and coronavirus. Apart from that then the parameters from SARS-CoV-2 is misleading, as serial interval and infectious period each is about 5 days. Response: The reviewer is correct that Table 2 does not list the differences in the rates of infection. This is because the rate of infection is the triple product of the rate of contact between hosts, the fraction of the population (not isolated) which is infected, and the probability of infection upon contact. Notwithstanding the variability in the probability of infection upon contact, which itself depends on the viral load of the infected individual and the nature of contact, substantial variability of the rate of contact between hosts makes it extremely challenging to report globally meaningful rates of infection for pathogens, and especially, respiratory pathogens. We agree that this table could be misleading considering the large values reported for the time symptomatic, which does not reflect the fact that hosts may be symptomatic but no longer infectious. This has been clarified in the text. SARS-CoV-2 is modelled with a 7 day period, during which the host is infectious and symptomatic, preceded by a 3 day asymptomatic and infectious period. This was the consensus prediction at the time this manuscript was initially prepared and we have acknowledged in the revised text that, given its recent identification, the best fit parameter values for SARS-CoV-2 are subject to change. We do not model the serial interval because, like the rate of infection, it depends on the rate of contact. Fig. 2 nicely outlines the model, but then in panel B,C) presents data using a y-axis that quantifies kI(?) in contact days? What about infectivity, some diseases like smallpox have a R0 of about 20, while other like SARS have R0 between 2 and 5. How does the y-axis in Fig. 2 relate to these very different scenarios? Response: We thank the reviewer for their appreciation of this figure and regret any possible confusion. As discussed in the previous comment, the rate of infection is the triple product of the rate of contact between hosts, the fraction of the population (not isolated) which is infected, and the probability of infection upon contact. kI, as listed in Table 1, is the product of the rate of contact and the probability of infection upon contact: kcontact*P (infect). In principle, both may vary, so for simplicity, we study this compound parameter, which can increase as a result of either increasing P(infect) or increasing kcontact. As P(infect) is unitless, kI has units of 1/time and varying kI is equivalent to varying the time between contact, which is what we report. While this was stated in the original version where the model is initially described, we acknowledge the presentation could use improvement and apologize for any confusion. We have added additional clarification on the nature of kI and the units of the y-axis in the figures. Additionally, we note that R0 depends on the rate of host contact and so will also vary across communities and over time for a single pathogen. Fig. 3 is again a hard figure to read? How can smallpox softly transit from SARS-Cov-2. I do not get how increased case fatality rate changes the regime of the sustainable endemic state? Also, the disease free equilibrium at short contact times presumably reflects situations where the first epidemic was so brutal that herd immunity was more than obtained? I don't understand the unsustainable red region? Why larger for larger CFR? Response: We regret the confusion. The reviewer is completely correct that smallpox cannot smoothly transit from SARS-CoV-2. This is evident in Figure 3 because there is no path from the smallpox-like regime to the SARS-CoV-2-like regime that does not exit endemic equilibrium. The solid line in the plot indicates that to the right of the line, the pathogen confers permanent immunity and to the left of the line, temporary immunity. The dashed line represents a smooth transition between the SARS-CoV-2-like regime and the Influenza-like regime, which are both modelled to produce temporary immunity. This has been clarified in the revised version. The duration of immunity is the third key parameter that is varied in Figure 4A. Increased CFR changes the regime of the sustainable endemic state because it changes the balance between births and deaths in the population. An endemic pathogen with a high CFR results in a greater number of mortalities per year for the host population. The reviewer is correct that disease-free equilibrium at short contact times reflects a selective bottleneck in the host population as illustrated in Figure 1B. This has been emphasized in the revised version. However, it should be noted that a selective bottleneck at low CFR does not necessarily represent an outbreak resulting in significant mortality. The unsustainable red region represents the case where endemic equilibrium is possible but eventually, because the number of deaths sustained by the pathogen exceeds the birth rate, the host population will either go extinct or must adapt in some way to avoid the pathogen. This represents an unsustainable host-pathogen relationship. This region is larger for larger CFR because the number of deaths sustained by the population increases with increasing CFR. Fig. 4 is difficult given the arbitrariness of all parameters. What is the point of 3 sub-figures, what could I as a reader learn? Response: We respectfully suggest that the parameters are not arbitrary. Figures 3 and 4A are critical to understanding the life history phase space that is available human respiratory viruses. Our choice of parameter exploration in the initial submission was motivated by the range of parameters observed for pathogenic human respiratory viruses. Additionally, where possible, analytic dependencies on the size of the clinical population at endemic equilibrium are presented in the Appendix. These pathogens span a wide range of CFR, and host-host contact rates can vary significantly among human populations. We additionally vary the amount of time a host is both asymptomatic and infectious although we do not allow this parameter to vary freely because that would have been implausible from a biological standpoint: hosts infected with high CFR viruses are not known to spend significant time in an asymptomatic and infectious state. We additionally explore the full range of immune duration (parametrized by 0<=alpha<=1) for SARS-CoV-2-like and Influenza-like viruses. This parameter largely determines the region of the phase space that corresponds to unsustainability, and representative values are shown in red curves in figure 4A. This covers the CFR (k_DV), the rate of progression (k_P), the rate of contact/probability of infection (k_I), and the rate at which immunity is lost (k_L). We discuss the importance of small birth rate, and a small difference between k_B and k_D (deathrate not specific to the pathogen), for interpreting our results and linearization of the model requires that value to be small. These rates are expected to vary significantly less among human populations than pathogen-specific rates and we submit are not critical to vary. Lastly, we devote the final figure to studying the effects of the parameter beta representing isolation of symptomatic individuals. Thus we hope we have demonstrated to the reviewer that we effectively surveyed the entire parameter space admitting endemic equilibrium for the pathogens of interest. Additionally, although we continue to maintain that the parameters fully explored in the main text constitute those of greatest relevance given the known variability among human respiratory viruses, we acknowledge that a demonstration of the dependence on k_R is useful for the reader as well. This is available as a supplementary figure. We agree that panels B and C are perhaps less critical to understanding the key results of this study, but still contain information central to what is discussed in the text. Specifically, panel B demonstrates that the size of the viral population at endemic equilibrium decreases with an increasing duration of immunity. Panel C demonstrates the reduced cost of an epidemic resulting in reducing the contact rate among symptomatic hosts and is a segue into the final figure. Fig. 5 investigates mitigation, something that has little to do with endemic states (presumably we would not like to have a permanently mitigated society?). I also failed to locate whether the mitigation fraction beta only included reduction in the symptomatic transmission? For SARS-CoV-2 there is a lot of asymptomatic transmission, but not for SARS. Response: We regret any confusion. Mitigation may refer to either the isolation of symptomatic individuals or the quarantine of asymptomatic, and likely uninfected, contacts. While we discussed this in the original version, this has been emphasized in the revised version. We only model the former – isolation of symptomatic hosts and would argue that in that sense we already live in a “permanently mitigated society”. Such mitigation has a dramatic effect on the range of host-host contact rates which admit endemic equilibrium as displayed in Figure 5A. The reviewer is correct that the death rate at endemic equilibrium is insensitive to mitigation. This is displayed in Figure 5B. The reviewer is also correct that asymptomatic transmission for SARS-CoV-2 is more substantial than it was for SARS. We discuss how high CFR pathogens are generally subject to less asymptomatic transmission than those with lower CFR. In conclusion, the authors say that SARS-CoV-2 cannot reach a state of endemic equilibrium. They might be right, but one might as well imagine that disease immunity decline sufficiently fast that it could be constantly circulating in some mild form. Also, I cannot see how its increasingly many mutant variants would make such a conclusion robust. Response: We agree that the wording in the abstract was somewhat overstated, so we have amended it in the revision. In the main text we state, “We show that SARS-CoV-2-like viruses are unlikely to reach a state of endemic equilibrium; however, the potential for such equilibrium implies that herd immunity without vaccination might not be achievable”. We do not intend to state SARS-CoV-2 “cannot” reach a state of endemic equilibrium. The reviewer suggests that the duration of immunity may decline sufficiently fast so that the pathogen could circulate in some mild form. This implicitly assumes that, in addition to a reduction in the duration of immunity, SARS-CoV-2 would also evolve towards a reduction in CFR. This is precisely what we discuss. Regarding the emergence of novel variants, we think it is important to emphasize that antigenic drift is a major driver of endemic equilibrium, and furthermore, that all SARS-CoV-2 variants still firmly reside in the SARS-CoV-2-like region of the global phase space. We have clarified this point in the revised text. In the results section of the abstract the authors state: \"We reveal kinetic constraints where the variation of multiple parameters in concert leads to decreased CFR and increased pathogen fitness, whereas independent variation of the parameters decreases pathogen fitness. Smallpox, SARS-CoV-2, and influenza are analyzed as diverse representatives of human respiratory viruses. We show that highly virulent viruses, such as smallpox, are likely often constrained by host behavior, whereas moderately virulent viruses, such as SARS-CoV-2, appear to be typically constrained by the relationship between the duration of immunity and CFR.\" This is beyond my understanding: smallpox could not really be constrained (or could not when it recurrently broke out in cities in the 18th century). SARS-CoV-2 was in contrast contained by human mitigation, and had little directly to do with CFR. Response: We regret any confusion. In this study, we discuss both evolutionary constraints as well as the reduction in the rate of contact for symptomatic hosts, containment. We demonstrate that the evolution of smallpox-like viruses is likely constrained so that the CFR of smallpox remained high throughout the entire period over which it was endemic to human populations – well in line with the reviewer’s comments. We do not suggest that smallpox was historically contained; however, we do demonstrate that due to the relatively few asymptomatic transmission events associated with smallpox, in principle, containment may be possible for a modern society. Furthermore, we would argue that the CFR of SARS-CoV-2 played a key role in its containment. Should a novel virus have emerged with a much lower CFR, containment strategies would likely not have been pursued at all. A minor point is the strange discussion of reversible chemical reactions in a paragraph on page 2. It seems to assume that energy is directly proportional to transmission rates. Also its relation to the rest of the manuscript is unclear. Response: This discussion serves to introduce the concepts of kinetic and thermodynamic constraints to endemic equilibrium analogous to kinetic and thermodynamically limited chemical reactions. We believe that this analogy is illustrative of the evolutionary trajectories of viruses on multidimensional fitness landscapes and include this discussion in an effort to make the study more approachable to a broad audience. As is the case with Figure 1, we acknowledge that an expert reviewer may find this discussion unnecessary or perhaps even distracting. We apologize for any confusion and have further expanded the explanation of the idea of kinetic vs. thermodynamic constraints in the revised version in an effort to better connect this illustration to the topic at hand. Overall, the paper presents a worthwhile investigation into an important phenomenon. However, it is very difficult to read and evaluate given its unclear assumptions (and complicated sentences). I would encourage a major rewrite, emphasizing which few (hopefully) parameters that are important, and with new figures that more clearly illustrate the main point. Response: We thank the reviewer for their comments. We have substantially revised and amended the text in an effort to clarify the presentation; however, we have elected to maintain the original figures." } ] } ]	1	https://f1000research.com/articles/10-447
https://f1000research.com/articles/10-372/v1	11 May 21	{ "type": "Research Article", "title": "The discrepancy between Clove and Non-Clove Cigarette Smoke-Promoted Candida albicans Biofilm Formation with precoating RNA-aptamer", "authors": [ "Boy Muchlis Bachtiar", "Basri A. Gani", "Astri Deviana", "Nastiti Rilo Utami", "Anissa Dien Andriyani", "Endang Winiati Bachtiar", "Boy Muchlis Bachtiar", "Basri A. Gani", "Astri Deviana", "Nastiti Rilo Utami", "Anissa Dien Andriyani" ], "abstract": "This study explores the influence of precoating aptamer (Ca-apt1) on C. albicans viability while the fungus was growing in the presence of exposing condensed cigarette smoke (CSC), prepared from clove (CCSC) and non-clove (NCSC) cigarettes, for 48 h. Using qPCR, we found that mRNA expression of adhesion-associated genes (ALS3 and HWP1) was impaired by precoating C. albicans yeast cells with the aptamer. Conversely, the gene transcription was upregulated when aptamer-uncoated yeast was pre-treated with either CSC. In addition, by analysing the result of MTT ([3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay, we found that the presence of added CCSC or NCSC in growth medium for 48 h was significantly enhanced C. albicans biofilm development. However, the presence of precoated aptamer was significantly impaired biofilm development accelerated by the NCSC. The inhibitory effect of the Ca-apt1 was not dependent on the precoated aptamer (1 and 10%). Interestingly, we noted that the enhancer effect of treated CCSC was no longer effective when the yeast had been precoated with 10% aptamer tested. Additionally, light microscopy analysis revealed that precoating aptamer alleviates morphological changes of C. albicans (from yeast to hypha formation) that are enhanced by adding CCSC or NCSC in the growth medium. In conclusion, these results suggest that administration on Ca-ap1 exhibits a significant protective effect on CSC-induced biofilm formation by C. albicans.", "keywords": [ "Cigarette Smoke", "Candida albicans", "Biofilm", "RNA-aptamer", "ALS3 and HWP1." ], "content": "Introduction\n\nCandida albicans is a normally harmless inhabitant of the oral cavity. However, unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is an opportunistic pathogen. The fungus’s behaviour correlates with its ability to grow in distinct morphogenic states, including budding yeast or blastospores, pseudo hyphae, and true hyphae1–3. This morphogenic transition from yeast to hypha form is important for the pathogenesis of C. albicans, is dependent on how the fungus cell responds to the environmental cues4. As shown in the literature, cigarette smoke is one factor that can aid and accelerate this transformation5–7, and there is a clear association between oral candidiasis and smoking habit8.\n\nSmoking is a social habit in many countries9, and it is an important public health problem, including in Indonesia10.\n\nHowever, despite the extensive research exploring the deleterious role of a smoking habit on oral microorganisms, there is little information about the effect of non-conventional tobacco, i.e., clove cigarettes, on the virulence attributes of C. albicans. By searching the literature, we found that, like conventional cigarettes, the clove cigarette, commonly known as kretek, is the most popular11 in Indonesia. It is also a source of numerous toxicants, and they have a potential implication in the oral ecosystem12, which may also influence oral Candida pathogenicity13. The unique aspect of kretek is the dried clove buds it contains14 which have never been identified in a conventional cigarette. The involvement of clove cigarette smoke condensate (CCSC) on the biofilm formation of C. albicans remains obscure. Given that cigarette smoke contains many toxicants12, it is important to explore the involvement of CCSC on the growth and morphogenesis of C. albicans, as this fungus is the most implicated oral pathogen in the clinical setting.\n\nDuring the last several years, aptamers, either single strain DNA or RNA, and against different microorganism species, have become the focus of growing interest. In an earlier study, we reported an anti-Candida activity of an RNA-aptamer (Ca-apt1) against the fungus while growing as biofilm15. Here, we evaluated the aptamer's beneficial properties against C. albicans biofilm formation induced by cigarette smoke. Particularly, the aptamer's ability to restrict the transition of fungus phenotype from yeast to hyphal form.\n\n\nMethods\n\nIn this study, we used a clinical isolate that we collected previously from the denture surface of denture wearer subject15 and selected by using CHROMAgar16, while the reference strain (C. albicans ATCC 10231), which was used as a targeted aptamer ligand15, was needed for the evaluation and validation of this experimental study. Briefly, all microorganisms were taken from stock cultures frozen in 15% glycerol at -80°C and sub-cultured onto yeast peptone agar plates (1% yeast extract, 2% peptone, 2% glucose;YPD) or when indicated in yeast nitrogen base medium, supplemented with 50 mM glucose (YNB).\n\nThe synthetic oligonucleotide used in this study was RNA-aptamer (Ca-apt 1) that was obtained from the systemic evolution of ligands by exponential enrichment (SELEX) method15.\n\nIn this study, condensed smoke cigarettes (CSC) were generated from Indonesia's non-filtered clove cigarette (kretek) and imported non-clove cigarettes (Figure 1) that we purchased from a local tobacco outlet. The CSC, either from a clove cigarette (CCSC) or a non-clove cigarette (NCSC), was prepared by smoking five cigarettes that were smoked to an in-house smoking device5 and concentrated in 100 mL of 0.09% Sodium Chloride solution. The resulting condensate smoke solution was further sterilized by filtration through a Millipore filter (0.22 µm). The influence of CCSC/ NCSC-containing YNB (pH was adjusted to 7.2) on the viability of aptamer-precoated C. albicans in the preformed biofilm was assessed colorimetric (MTT) assay and kept at 4°C until use.\n\nA and B are Clove and non-clove cigarette, respectively.\n\nTo determine that the inhibition effect of the tested aptamer in the early biofilm development stage was independent from the biofilm biomass maturation, we separated biofilm development into adhesion and growth phases. Initially, by analysing the mRNA expression of adhesion-associated genes (ALS3 and HWP1), we wanted to confirm if the inhibition event was due to the lessened ability of C. albicans to switch its phenotype from yeast to hypha form. To do this, the test aptamer was first preincubated with C. albicans yeast cells before the fungus was inoculated into microplate wells. Further, we did an analysis of mRNA expression of adhesion-associated genes (ALS3 and HWP1) after 90 min incubation time. The transcription level of these genes was also measured after C. albicans was exposed with either cigarette smoke, but without precoating aptamer. This was done to evaluate CSC's involvement in the transition C. albicans morphogenetic, from yeast or blastospore to hyphae form. All the procedures used as described in a previous study17. Briefly, RNA isolation, purification, and reverse transcription of cDNA were conducted using TRIzol™ Reagent (Invitrogen Life Technologies, Carlbad, California, United States) followed by reverse transcription using the TaqMan® Reverse Transcription Reagents (Applied Biosystems, Waltham, Massachusetts, USA). The resulting cDNA (1 µg) was amplified by qPCR with specific primers used in our previous study18. The qPCR analysis was performed in ABI StepOnePlus™ Real-Time PCR Systems (Applied Biosystems) with Platinum™ SYBR™ Green qPCR SuperMix-UDG (Invitrogen). The qPCR cycling conditions consisted of a 10-minute initial denaturation at 95°C followed by 40 PCR cycles of 15 seconds at 95°C and one minute at 60°C. The formula of fold change 2-ΔΔCt was used to calculate the relative mRNA expression, which was compared with that of the housekeeping gene, 18S rRNA. The mRNAs gene (ALS3 and HWP1) expressed by C. albicans without bound aptamer or without CSC exposure were used as a control, set at one.\n\nTo test the effectivity of CSC, with and without precoating aptamer, on C. albicans biofilm formation, we used a biofilm assay that was performed as previously described18. Briefly, 100 µL containing 1.8 X 105 yeast cells of C. albicans (counted by using a haemocytometer), from overnight culture in YPD broth was aliquot into 96-well microtiter plates containing mixture of 70% of fresh yeast nitrogen base (YNB; sigma-Aldrich) and 30% condensate smoke (vol/vol). The pH of the mixture was adjusted to neutrality (7.0) using 1M NaOH. This was done to define that the pH of the mixture does not play a role in modulating biofilm formation.\n\nThe aptamer was prepared by separating it in two different concentrations (1ng/µL and 10 ng/µL) in buffer15, prior to being added into separate wells, and the plates were incubated at 37°C in 5% CO2 in air for 90 min with gentle shaking. Candida albicans biofilm with mix medium added instead of aptamer was used as a negative control. To promote biofilm formation, the adhered cells were washed twice with sterile Phosphate Buffer Saline (PBS), C. albicans were further grown in a medium (150 µL) containing growth medium without aptamer. The culture period was further lengthened to 48-h. The extent of biofilm formation estimated using the semi-quantitative MTT (3-(4,5-dimethylthiazol-2,5-diphenyltetrazolum bromide) reagent (Sigma-Aldrich, St. Louis, MO, USA). The absorbance was measured spectrophotometrically at 450 nm with 620 nm as the reference wavelength for this assay. The results expressed as OD450/620, and were correlated with cellular metabolic activities within the biofilm. The assay was done in triplicate, repeated two times independently. Moreover, the percentage of fungal damage was calculated based on data obtained in MTT assay, using the formula: 100X [(1- ODCandidaCSC+apt / ODCandida alone)]. Biofilm formation on the bottom of microtiter well plates was qualitatively observed using an inverted light microscope.\n\nFollowing incubation for 90 min or 48-h, C. albicans cultures were observed microscopically and its morphology images were analysed qualitatively.\n\nStatistical analysis was performed with GraphPad PrismTM (version 9.00) software (GraphPad Software, Inc., San Diego, California, USA). The two-way analysis of variance (Two-way ANOVA) with Geisser-Greenhouse correction was used to verify the significant level of response between and within groups comparison. Each experiment was carried out in triplicate wells and repeated at least twice, independently. A P < 0.05 value was considered statistically significant.\n\n\nConsent statement\n\nWritten informed consent was obtained from participants for use of their data.\n\n\nEthical approval\n\nThe samples were collected, after informed consent was obtained from all the participants15, in accordance with the approved protocol of the Bioethics Committee of the Faculty of Dentistry, Universitas Indonesia (protocol number 020950818). The protocol conformed to the criteria of the Helsinki Declaration and the good clinical practical guidelines of the International Council on Harmonization.\n\n\nResults\n\nTo understand the underlying relationship between precoating aptamer (Ca-apt1) and C. albicans morphogenesis at molecular level, we firstly compared the expression profile of two adhesion-associated genes (ALS3 and HWP1). To do this, the test aptamer was preincubated with C. albicans yeast cells before the biofilm was allowed to form. Further, we combined quantitative analysis of mRNA expression of these genes and qualitative microscopic images to describe the adhesion event. The result showed, that after 90 min, the expression of ALS3 by the adherence cells were proportionally reduced by ≈ 8% and ≈ 4%, for aptamer concentration 1% and 10%, respectively. A similar trend was noted in either C. albicans strain used. We noted that the reduction of HWP1 expression was higher than ALS3 under different concentrations of Ca-apt1. In either C. albicans strain, this gene downregulated was > 50% (Figure 2A and B). Further, the microscopic images show that at the attachment phase (90 min), the biofilm consisted of both yeast cells and blastospore that adhered to the surface, and those cells adhered to other cells attached to the surface (Figure 2C–E).\n\nThe upper panel shows the effect of precoating aptamer on ALS3 and HWP1 expressed by C. albicans in preformed biofilm (90 min incubation time) were analysed by qPCR. The mRNA expression levels were normalized relative to the control (yeast cells without aptamer precoating), which was set to one for each gene to determine the fold change in expression of genes in C. albicans clinical isolate (A) and the ATCC 10231 (B). The results are expressed as the mean and standard deviation (SD) of triplicate experiments and repeated two times independently. Significantly higher downregulation in the expression of mRNA (P < 0.05) in the presence of precoating aptamer. The lower panel shows C. albicans yeast cell (that only shown in clinical isolate) without precoating aptamer (C), and those cells were precoated with 1% (D) and 10% (E) of Ca-ap1 and visualized by using light microscopic images at X 200 magnification.\n\nNext, we tested the mRNA expression of ALS3 and HWP1 on the biofilm formation promoted by either cigarette smoke condensate (CSC) after 48-h incubation time without precoating aptamer. As shown in Figure 3A and B, by comparing CCSC and NCSC, we observed that Candida adhesion was due to the presence of CCSC that subsequently increased the transcription level of hypha-associated gene (HWP1), compared to the biofilm induced by NCSC-added growth medium (p< 0.05). For the adhesion-related gene (ALS3), the transcription level was comparable when induced by either CCSC or NCSC-treated growth medium or growth medium only (control/ unexposed C. albicans). We noted that both C. albicans strain (clinical isolate and ATCC) showed a similar pattern. Figure 3C–E show the result provided by the microscopic analysis. After a 48-h time period, the hyphal form was more abundant in CCSC-treated growth medium than those in NCSC-treated biofilm.\n\nThe qPCR analysis of ALS3 and HWP1 genes is shown after the preformed biofilm (90 min incubation) was treated by growth medium supplemented with different CSC (CCSC or NCSC). The mRNA level of each gene was normalized to that of 18S rRNA, while the expression level of the control (untreated biofilm) was set to one for each gene to determine the fold change in the expression of each targeted gene in clinical isolate (A) and the ATCC strain (B) of C. albicans. Significantly higher upregulation of mRNA expression of HWP1 induced by CCSC than NCSC (P < 0.05). Data are expressed as the mean and standard deviation (SD) of triplicates from two separate experiments. The CCSC and NCSC are clove and non-clove cigarettes smoke condensate, respectively. The lower panel shows the 48-h biofilm formation that only shown in clinical isolate; the control (C) and those biofilms treated with CCSC (D) and NCSC (E), respectively. All the biofilms are visualized by using light microscopic images at X 200 magnification.\n\nNext, we tested the impact of different concentrations of precoating aptamer (1% and 10%) on C. albicans biofilm formation. For this, biofilms were developed in CCSC/NCSC-containing YNB (pH was adjusted to 7.2), and the quantification of biofilm cell (MTT assay) was evaluated after the biofilms reach the maturation time (48-h). This method was done, because we wanted to determine whether the precoating aptamer could adversely affect C. albicans biofilm formation after treating with the biofilm enhancers (CCSC or NCSC). Our data found that in comparison to the control, 1µg/ µL aptamer concentration of precoated yeast cells was sufficient to reduce biofilm cell, by about 10%, and 40 %, as observed in CCSC and NCSC-treated biofilm, respectively. At this maturation state, the viable cells detected within this biofilm were significantly reduced (p< 0.05), because of the increased aptamer concentration by 10% in the pre-adherence phase of the biofilm (Figure 4A–B). Further, the effect of CCSC or NCSC on the transition of yeast to hypha morphology in pre-adhered cells was visualized microscopically. We observed that when the fungus cells were precoated with aptamer without exposed with CCSC or NCSC and incubated for 48-h, the pre-formed biofilms were dominated by blastospores, as compared to the control cells (uncoated cells), which showed that the adhered cells were dominated by dense hyphae form (Figure 5 A–B). This qualitative effect was also dependent on the concentration of the aptamer tested (Figure 5C–F).\n\nThe effect of CCSC (A) and NCSC (B) exposure on biofilm formation of C. albicans, which had been precoated with different concentration of Ca-apt1, evaluated after incubation for 48-h. The relative biofilm formation compared to biofilm without the precoating aptamer (control) was calculated. Data represent the mean and standard deviation (SD) of three biofilms grown on two separate occasions. Asterisks denote statistically significant differences between the effect of cigarette smoke condensate determined by MTT assay, p < 0.05. The CCSC and NCSC are clove and non-clove cigarette smoke condensate, respectively.\n\nThe pictures show biofilm of C. albicans derived from clinical isolate, without either precoating aptamer or added CSC (A), C. albicans with precoated aptamer but untreated with either CSC/ CCSC or NCSC (B), C. albicans precoated with 1% aptamer, CCSC (C) and NCSC (E), and 10% aptamer with CCSC (D) and NCSC (F). All microscopic image show at X 200 magnification.\n\n\nDiscussion\n\nThe current study attempted to get further insight into clove cigarettes' effect on non-mammalian eukaryotes cells, taking the fungus C. albicans as a model system. Initially, as a means to determine that the restriction effect of C. albicans to switch its phenotype from yeast to hypha form was due to the presence of the precoating aptamer on yeast, we separated biofilm development into adhesion and growth phases. The result of the transcription assay during the initial stage of biofilm formation indicated that the aptamer involvement in reducing the expression of adhesion and hypha-associated gene (ALS3 and HWP3)19, and the transcription profile was in line with the increased concentration of the aptamer, but not on C. albicans strains. This result suggests that the aptamer (Ca-apt1) might bind to a common chemical structure on the fungal yeast cell, probably glucans and chitins20 that form the basic cell wall scaffold21. We presume that this aptamer may interfere with the adherence mechanism where these molecules are involved21.\n\nOur study is different from most in vitro studies, which use an antibody that recognizes cell-wall proteins, that are commonly related to the host immunological status22,23. In this study, the precoating aptamer (Ca-apt1) does not directly recognize the cell wall proteins as an antibody would. In this way, we assumed that based on the ALS3/HWP1 profiles explained above, the obvious virulence factors (morphology transformation from yeast to hypha) were more likely to be the main effect of precoating Ca-apt1. This assumption was supported by microscopic observation that at the attachment phase (90 min time point) the morphology of C. albicans consisted of both yeast cells and blastospore only. This result confirmed the transcription assay that the aptamer involvement in reducing adhesion and hypha-associated gene (ALS3 and HWP3)19 was in line with the increased concentration, but not on C. albicans strains. Likewise, this qualitative data exhibited a significant potentiate of Ca-apt1 in modulating biofilm-associated genes, as well as C. albicans morphogenesis, at the early step of biofilm formation.\n\nAlthough the pattern was similar, in general, the biofilm formation (90 min time point) treated with CCSC resulted in a higher viable cell than its NCSC counterpart, as we observed that after being treated for 48-h by CCSC or NCSC. Different cellular response of C. albicans was noted, both by analysing the gene (ALS3 and HWP1) profiles and observing microscopically. At this maturation biofilm state, the induction of hypha-associated gene (HWP1) was significantly different, but not for the gene's adhesion (ALS3). We noted that the transcription level of HWP1 by either C. albicans strain was higher than ALS3 only when the preformed biofilm was treated with CCSC. This result indicates that unlike ALS3, the transcription level of HWP1 was dependent on the source of the condensate smoke used. The effect of condensate smoke on C. albicans cells viability in biofilm development does not relate to C. albicans strain. We assumed that different tobacco components, specifically the principal component alkaloid of tobacco (nicotine), between kretek and conventional cigarettes24,25, may have a different effect on C. albicans when the fungus was growing in distinct environmental conditions that trigger the hyphal growth. Additionally, the HWP1 product (Hwp1) is a hypha-association protein commonly expressed on germ tube26,27. Indeed, this result can be considered after 90 min, when germ tube was induced28. The upregulation of HWP1 when the biofilm is maturing (48-h) is a survival pathway used by C. albicans to resist, or be tolerant against, the effect of chemical toxic-containing CCSC, by modulating the hypha form.\n\nIt has been reported that, like NCSC, CCSC comprises a high number of dangerous alkaloid chemical compounds, the most abundant of these being nicotine24. The other chemical compound in the gas phase of cigarette smoke is nitrogen (N2)29. The nicotine in smoke yields from CCSC is equally delivered as by NCSC30. However, the nitrogen content in CCSC is lower than found in conventional cigarettes/NCSC31. The most distinguished particle-phase, which is only found in CCSC, is eugenol14. Eugenol is a phenylpropanoid compound reported to have antimicrobial activity against planktonic cells of C. albicans and sessile cells within C. albicans biofilms32.\n\nIn contrast to the eugenol, the presence of nicotine in cigarette smoke promotes a high level of adhesins leading to the increased adherence of C. albicans33 on a solid surface, as found in this study. Hence, it has the potential to increase biofilm formation by C. albicans. Considering that eugenol impairs the growth of C. albicans, we assume that under the experimental condition set in this study, C. albicans sensed eugenol in CCSC, and the presence of nitrogen helped it to grow. Since we grew the fungus in a hypha conditioning medium, the result obtained in this study indicates that by comparing with NCSC, the lower nitrogen in CCSC is the likely reason for the augmentation of C. albicans biofilm formation33–35 enhanced by the CCSC. As we evaluated microscopically, more hypha in 48-h biofilm was observed in response to the presence of CCSC than NCSC.\n\nAs shown in the literature, cigarette smoke exposure (CSC) has a potential adverse health effect on the oral ecosystem36, and the growth rate of C. albicans increases when the fungus is grown in the presence of non-clove cigarette smoke condensate/NCSC5. Indeed, cigarette smoke is an important predisposing factor for oral candidiasis34. Although kretek/clove cigarette smoke condensate (CCSC) and NCSC contains many toxicants, they have some different chemical constituents30,31, which may lead to the differences in Candida cell behaviour that we observed in this study.\n\nWe further address whether the precoating aptamer could adversely affect the preformed biofilm formation's viability when treated with CCSC or NCSC. The experiment was done to confirm that, on the one hand, experimental C. albicans biofilm formation promoted by cigarette smoke (CCSC and NCSC) could be induced. On the other, that the precoating aptamer is needed for the success of this model. In this way, the discrepancy between CCSC and NCSC on C. albicans susceptibility was measured using a MTT reduction assay. This is a reliable test for an indirect method to quantify biofilm cell numbers37,38. It has been demonstrated that when growing as a biofilm, the metabolic activity of C. albicans increases over time39. However, another factor (the aptamer) was added in the current study. Here, we observed that the control group (C. albicans of untreated condensate cigarette smoke/ CSC, without precoating aptamer (Ca-apt1) remained growing as a biofilm throughout the experimental time period set in this study. This result may suggest that C. albicans used the tobacco compound as a nutritional source, as aromatic hydrocarbons in cigarette smoke can be converted by the fungus34.\n\nFurther, we found that after treating with NCSC for 48-h, the lowest concentration of tested aptamer (1ng/µL) was enough to reduce the cell viability > 50%. This indicates that the effectivity of NCSC as an accelerator of biofilm formation5 was significantly suppressed. Hence, it affirms that the anti-adhesion effect of the aptamer on biofilm development is enhanced by conventional cigarette smoke. Surprisingly, aptamer-precoated C. albicans behaves differently upon exposure to growth medium-containing CCSC. We noted that at one ng/µL of the aptamer, significant cell growth changes were not observed compared to the control (uncoated aptamer). However, at 10 ng/µL, the aptamer could inhibit the biofilm cell growth until C. albicans reached a steady state at a 48-h time period, more and less at a similar rate to those cells exposed by NCSC. The result was supported by the light microscopy data, in which we observed that when the fungus was exposed with the minimal concentration (1 ng/µL) of CCSC, the composition of 48-h-old biofilms consisted of pseudo hypha, almost similar to the control (C. albicans without pre-coated aptamer). We also observed that when the concentration of the tested aptamer was increased by 10-fold, it prevented the cell's viability and successful germination of the adherence cells, resulting in scant biofilms, and more yeast cells were observed microscopically in treated groups (CCSC and NCSC exposure). For NCSC, we observed that regardless of the tested aptamer's concentration in precoating step, the NCSC-treated biofilms were predominantly in the blastospore or yeast form compared to the control. After 48-h exposure, many of these biofilm cells were unbudded and had an unelongated morphology. At the end of the experiment time, both C. albicans strains, which belonged to the treated groups (C. albicans with precoating aptamer), showed a similar phenotype response to either CCSC or NCSC exposure. Our result contradicted a previous report that demonstrated that the clinical isolate showed less biofilm growth activity than the laboratory strain40. We reasoned that the similar trend found in either C. albicans strain used in this study is probably because the clinical isolate and the ATCC strain used in this study have a similar karyotype as it has been previously reported41.\n\n\nConclusion\n\nRegardless of the mechanism involved, this study demonstrated that both CCSC and NCSC have a potentiate to enhance biofilm formation by C. albicans. By exposing either cigarette smoke prepared from clove and non-clove cigarettes, C. albicans, either clinical isolate or the reference strain, shows a similar trend. However, the pattern of biofilm formation was different. Besides, this study clearly indicates that the aptamer (Ca-apt1) had an impact on the degree of cell adhesion and its concentration affected the profile of biofilm formation at the maturity phase. However, the underlying mechanisms remain to be investigated at molecular level\n\n\nData availability\n\nFigshare: Underlying data for ‘The discrepancy between Clove and Non-Clove Cigarette Smoke-Promoted Candida albicans Biofilm Formation with precoating RNA-aptamer’, DOI 10.17605/OSF.IO/WF2KB42\n\nThis project contains the following underlying data:\n\nData for Figs 2-4 CCSC-2021 F1000 Aptamer Cigarete.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\nLicense: CC0 1.0 Universal", "appendix": "References\n\nSudbery P, Gow N, Berman J: The distinct morphogenic states of Candida albicans. Trends Microbiol. 2004; 12(7): 317–24. PubMed Abstract \| Publisher Full Text\n\nAkpan A, Morgan R: Oral candidiasis. Postgrad Med J. 2002; 78(922): 455–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoll DR: Candida commensalism and virulence: the evolution of phenotypic plasticity. 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Oral Dis. 2005; 11(5): 268–73. PubMed Abstract \| Publisher Full Text\n\nGunasegar S, Himratul-Aznita WH: Nicotine enhances the thickness of biofilm and adherence of Candida albicans ATCC 14053 and Candida parapsilosis ATCC 22019. FEMS Yeast Res. 2019; 19(2). PubMed Abstract \| Publisher Full Text\n\nArcavi L, Benowitz NL: Cigarette smoking and infection. Arch Intern Med. 2004; 164(20): 2206–16. PubMed Abstract \| Publisher Full Text\n\nHawser S: Adhesion of different Candida spp. to plastic: XTT formazan determinations. J Med Vet Mycol. 1996; 34(6): 407–10. PubMed Abstract\n\nKuhn DM, Balkis M, Chandra J, et al.: Uses and limitations of the XTT assay in studies of Candida growth and metabolism. J Clin Microbiol. 2003; 41(1): 506–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChandra J, Mukherjee PK, Leidich SD, et al.: Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res. 2001; 80(3): 903–8. 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PubMed Abstract \| Publisher Full Text\n\nBachtiar EW: The discrepancy between Clove and Non-Clove Cigarette Smoke-Promoted Candida albicans Biofilm Formation with precoating RNA-aptamer. 2021. http://www.doi.org/10.17605/OSF.IO/WF2KB" }	[ { "id": "86552", "date": "29 Jun 2021", "name": "Samira Shirooie", "expertise": [ "Reviewer Expertise Pharmacology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the introduction, mention the genes ALS3 & HWP1 and their roles.\nIn results, in the first headline was written \"Evaluation of the mRNA levels of HWP1 and YWP1 in pre-coating aptamer C. albicans\" that must be corrected to \"Evaluation of the mRNA levels of HWP1 and ALS3 in pre-coating aptamer C. albicans\" .\nThe data of the control group should be shown in figures 2A and 2B.\nWrite abbreviations in separate part before introduction.\nIn figures 3 A & B, compare the CCSC and NCSC groups with their control group not with the another control group, for example compare CCSC for HWP1 expression level with the control group of HWP1 not with ALS3.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "6870", "date": "12 Jul 2021", "name": "Boy Muchlis Bachtiar", "role": "Author Response", "response": "Reviewer #1 1. In the introduction, mention the genes ALS3 & HWP1 and their roles. Author’s response (AR): We have mentioned the suggestion, and it can be seen at the end of paragraph one of the introduction section, as follows; ...In this regard, the presence of nicotine in cigarette smoke promotes C. albicans growth rate and adhesion, in which, a higher level of expression of genes related to adherence, such as ALS3 and HWP1 have been reported9. 2. In results, in the first headline was written \"Evaluation of the mRNA levels of HWP1 and YWP1 in pre-coating aptamer C. albicans\" that must be corrected to \"Evaluation of the mRNA levels of HWP1 and ALS3 in pre-coating aptamer C. albicans. AR: Yes, you right. Thank you. Accordingly, we have corrected the name of the gene. 3. The data of the control group should be shown in figures 2A and 2B. AR: As suggested, the control groups have been added in figures 2A and 2B. Thank you. 4. Write abbreviations in separate part before introduction. AR: We only use abbreviations or acronym for they have been widely known in scientific or academic communication. 5. In figures 3 A & B, compare the CCSC and NCSC groups with their control group not with another control group, for example compare CCSC for HWP1 expression level with the control group of HWP1 not with ALS3. AR: Thank you for this important suggestion. As suggested, we have deleted the double asterisks, as it can be confusing. Thank you." } ] }, { "id": "87553", "date": "30 Jun 2021", "name": "Zamirah Zainal-Abidin", "expertise": [ "Reviewer Expertise Pharmacology", "Oral Microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article reported a study on the effects of two types of cigarette smoke condensates (CSC), namely the clove CSC (CCSC) and non-clove CSC (NCSC) on the formation of biofilm by C. albicans from clinical isolate and ATCC 10231. This study also investigated the effects of pre-coating RNA aptamer, Ca-apt1 in modulating the gene expression and phenotypic changes of C. albicans cells that have been exposed to the abovementioned types of SCC, with the result of modulation is concentration-dependent.\nThe authors found that C. albicans cells of both clinical isolates and ATCC strains, precoated with different concentrations (1 ng/µL and 10 ng/µL) of aptamer (Ca-apt1) showed downregulation of adhesion-associated genes, i.e., ALS3 and HWP1, where the relative fold changes of the expression of the studied genes were Ca-apt1 concentration-dependent. This finding was substantiated by a microscopic study of the formation of the biofilm by C. albicans, compared with the ones without Ca-apt1.\n\nThe same approach of the investigation was adopted to investigate whether Ca-apt1 confers a protective effect on the CCSC and NCSC induced biofilm formation by C. albicans. The results demonstrated that CCS-induced biofilm formation was mitigated in the presence of Ca-apt1. The authors proposed that Ca-apt1 modulates the morphogenesis of C. albicans growing as a biofilm, as indicated by the predominance of blastospores in the treated Ca-apt1 biofilm, relative to the untreated control.\n\nSince adhesion is the crucial step in biofilm formation, the authors suggested that the C-apt1 may bind to a common chemical structure of the C. albicans cells that may play a vital role in the adherence of the fungus to a substrate. This suggestion is indeed intriguing, which warrants further studies on the anti-adhesion mechanism of Ca-apt1.\nThe most specific comments based on the sections in the manuscript are as follows:\nTitle: Capitalization of the title needs to be consistent.\nAbstract: In the sentence “Interestingly, we noted that the enhancer effect of treated CCSC was no longer effective…”. As described in the Methods section, the CCSC did not undergo any form of treatment in this study. It is recommended to rephrase this sentence to present the actual form of the CCSC used in this study.\n\nResults: 1. The first subheading should be reporting on ALS3 and HWP1 instead of HWP1 and YWP1.\n2. For Figures 2A and B, it is recommended that the Y-axes be standardized in both graphs, with a maximum relative fold change = 1.0. The comparison between the two strains in terms of inhibition of biofilm formation may then be emphasized. The indication of the dual asterisk () in the graphs should also be described. In the subtitle, use 1 ng/µL and 10 ng/µL rather than 1% and 10%. The authors may also describe Figure 2C-E in terms of comparison to the abundance of adherent C. albicans cells attached to the surface between the 3 treatments.\n3. In Figure 4, it is recommended that the Y-axes are standardized in both graphs, with a maximum percentage of control = 100. The comparison between the two strains in terms of reduction of the pre-formed biofilm can then be highlighted.\n\nDiscussion: 1. In paragraph 4, in this study, the comparison in terms of chemical contents between NCSC and CCSC is not reported. The concentrations of the common chemical compounds may be different among cigarette manufacturers. Therefore, it is recommended to provide relevant information on the concentrations of CSC chemicals in cigarettes used specifically for this study.\n2. In the final paragraph, the statement “Hence, it affirms that the anti-adhesion effect of the aptamer on biofilm development is enhanced by conventional cigarette smoke”. The authors should point out the results in this study or cite references that compared the formation of C. albicans biofilms without CSC to support that statement.\n3. In the statement “...with the minimum concentration (1 ng/μL) of CCSC...”, it is supposed to be aptamer.\nConclusion: In the statement “…C. albicans, either clinical isolate or the reference strain, shows a similar trend.” The authors will need to elaborate a little more on what trend they are referring to e.g., gene expression?\nOther comments: The manuscript is well written, with minor grammatical errors and some typos, which can be easily corrected by giving it a manuscript editing round-up.\nIn general, the authors have shown in this study the potential of Ca-apt1, an RNA aptamer in modulating virulence factors of C. albicans, one of the opportunist microorganisms within the oral cavity. In addition, the authors also have shown that cigarette smoke condensates can exacerbate the growth of C. albicans biofilms and how adding Ca-apt1 has mitigated this effect. I hope the authors will further investigate the underlying protective mechanism of aptamer at both genotypic and phenotypic levels, against the exacerbation of biofilm formation and pathogenicity of C. albicans when they are exposed to cigarette smoke, as well as adopt a tissue culture or animal model appropriate for investigation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6871", "date": "12 Jul 2021", "name": "Boy Muchlis Bachtiar", "role": "Author Response", "response": "Reviewer # 2 The most specific comments based on the sections in the manuscript are as follows: Title: Capitalization of the title needs to be consistent. Author’s response (AR):We agree. Thank you. As suggested, we have corrected the capitalization of the title. Abstract: In the sentence “Interestingly, we noted that the enhancer effect of treated CCSC was no longer effective…”. As described in the Methods section, the CCSC did not undergo any form of treatment in this study. It is recommended to rephrase this sentence to present the actual form of the CCSC used in this study. AR: Thank you for this important correction. Hence, we changed the word to make clear the meaning, as follows; “…we noted that the enhancer effect of added CCSC was no longer effective when the yeast had been precoated with 10 ng/μL aptamer tested”. 3. Results: 3.1. The first subheading should be reporting on ALS3 and HWP1 instead of HWP1 and YWP1. AR: Yes, you right. Thank you. We have changed the name of the gene, as suggested. 3.2. For Figures 2A and B, it is recommended that the Y-axes be standardized in both graphs, with a maximum relative fold change = 1.0. The comparison between the two strains in terms of inhibition of biofilm formation may then be emphasized. The indication of the dual asterisk () in the graphs should also be described. In the subtitle, use 1 ng/µL and 10 ng/µL rather than 1% and 10%. The authors may also describe Figure 2C-E in terms of comparison to the abundance of adherent C. albicans cells attached to the surface between the 3 treatments. AR: As suggested by the reviewer, we have standardized the Y-axes in both graphs, where the maximum relative fold change has been set to 1. Moreover, as corrected by the reviewer#1, the dual asterisks were removed. The typos in the subtitle, 1 and 10%, has been changed to 1 and 10 ng/ µL. Lastly, we described the figure 2C-E, where the abundance of adherent C. albicans between the 3 treatments was compared. Thank you. 3.3. In Figure 4, it is recommended that the Y-axes are standardized in both graphs, with a maximum percentage of control = 100. The comparison between the two strains in terms of reduction of the pre-formed biofilm can then be highlighted. AR: yes. We agree. Accordingly, we changed the T-axes in both graphs with a maximum percentage of the control set at 100. Thank you. 4. Discussion: 4.1. In paragraph 4, in this study, the comparison in terms of chemical contents between NCSC and CCSC is not reported. The concentrations of the common chemical compounds may be different among cigarette manufacturers. Therefore, it is recommended to provide relevant information on the concentrations of CSC chemicals in cigarettes used specifically for this study. AR: Thank you for the recommendation, and we agree. Thus, as suggested, in paragraph 4, we added a relevant information regarding the smoke chemical in CCSC and NCSC used in this study. 4.2. In the final paragraph, the statement “Hence, it affirms that the anti-adhesion effect of the aptamer on biofilm development is enhanced by conventional cigarette smoke”. The authors should point out the results in this study or cite references that compared the formation of C. albicans biofilms without CSC to support that statement. AR: We agree, and we have revised the sentence, and added references needed. It can be seen in the revised version of this manuscript; the revised sentence as follows: “Hence, it affirms that the anti-adhesion effect of the aptamer on biofilm development is enhanced accelerated by conventional cigarette smoke5, 7.” Thank you. 4.3. In the statement “...with the minimum concentration (1 ng/μL) of CCSC...”, it is supposed to be aptamer. AR: We agree, and we have corrected the typo. Thank you. 5. Conclusion: In the statement “…C. albicans, either clinical isolate or the reference strain, shows a similar trend.” The authors will need to elaborate a little more on what trend they are referring to e.g., gene expression? AR: We agree. Thus, we revised the sentence to elaborate the meaning of “trend” specifically. Hence, the new sentence is now becoming : “...shows a similar trend in its capacity to form biofilm.” 6. Other comments: The manuscript is well written, with minor grammatical errors and some typos, which can be easily corrected by giving it a manuscript editing round-up. In general, the authors have shown in this study the potential of Ca-apt1, an RNA aptamer in modulating virulence factors of C. albicans, one of the opportunist microorganisms within the oral cavity. In addition, the authors also have shown that cigarette smoke condensates can exacerbate the growth of C. albicans biofilms and how adding Ca-apt1 has mitigated this effect. I hope the authors will further investigate the underlying protective mechanism of aptamer at both genotypic and phenotypic levels, against the exacerbation of biofilm formation and pathogenicity of C. albicans when they are exposed to cigarette smoke, as well as adopt a tissue culture or animal model appropriate for investigation. AR: Thank you. Hence, we added the reviewer’s suggestion at the end of the conclusion." } ] } ]	1	https://f1000research.com/articles/10-372
https://f1000research.com/articles/8-1751/v1	14 Oct 19	{ "type": "Software Tool Article", "title": "A strategy for building and using a human reference pangenome", "authors": [ "Bastien Llamas", "Giuseppe Narzisi", "Valerie Schneider", "Peter A. Audano", "Evan Biederstedt", "Lon Blauvelt", "Peter Bradbury", "Xian Chang", "Chen-Shan Chin", "Arkarachai Fungtammasan", "Wayne E. Clarke", "Alan Cleary", "Jana Ebler", "Jordan Eizenga", "Jonas A. Sibbesen", "Charles J. Markello", "Erik Garrison", "Shilpa Garg", "Glenn Hickey", "Gerard R. Lazo", "Michael F. Lin", "Medhat Mahmoud", "Tobias Marschall", "Ilia Minkin", "Jean Monlong", "Rajeeva L. Musunuri", "Sagayamary Sagayaradj", "Adam M. Novak", "Mikko Rautiainen", "Allison Regier", "Fritz J. Sedlazeck", "Jouni Siren", "Yassine Souilmi", "Justin Wagner", "Travis Wrightsman", "Toshiyuki T. Yokoyama", "Qiandong Zeng", "Justin M. Zook", "Benedict Paten", "Ben Busby", "Giuseppe Narzisi", "Valerie Schneider", "Peter A. Audano", "Lon Blauvelt", "Peter Bradbury", "Xian Chang", "Chen-Shan Chin", "Arkarachai Fungtammasan", "Wayne E. Clarke", "Alan Cleary", "Jana Ebler", "Jordan Eizenga", "Jonas A. Sibbesen", "Charles J. Markello", "Erik Garrison", "Shilpa Garg", "Glenn Hickey", "Gerard R. Lazo", "Michael F. Lin", "Medhat Mahmoud", "Tobias Marschall", "Ilia Minkin", "Jean Monlong", "Rajeeva L. Musunuri", "Sagayamary Sagayaradj", "Adam M. Novak", "Mikko Rautiainen", "Allison Regier", "Fritz J. Sedlazeck", "Jouni Siren", "Yassine Souilmi", "Justin Wagner", "Travis Wrightsman", "Toshiyuki T. Yokoyama", "Qiandong Zeng", "Justin M. Zook" ], "abstract": "In March 2019, 45 scientists and software engineers from around the world converged at the University of California, Santa Cruz for the first pangenomics codeathon. The purpose of the meeting was to propose technical specifications and standards for a usable human pangenome as well as to build relevant tools for genome graph infrastructures. During the meeting, the group held several intense and productive discussions covering a diverse set of topics, including advantages of graph genomes over a linear reference representation, design of new methods that can leverage graph-based data structures, and novel visualization and annotation approaches for pangenomes. Additionally, the participants self-organized themselves into teams that worked intensely over a three-day period to build a set of pipelines and tools for specific pangenomic applications. A summary of the questions raised and the tools developed are reported in this manuscript.", "keywords": [ "Hackathon", "Pangenome", "Graph Genome", "RNAseq", "Structural Variant" ], "content": "Introduction\n\nThe current human reference genome, GRCh38 (Schneider et al., 2017), derives from a draft sequence that was constructed from a handful of individuals (Lander et al., 2001) likely of African and European ancestries (Reich et al., 2009). Today, GRCh38 captures a limited amount of additional genetic variation by providing alternative sequence representations (“alt loci”) for complex or highly variable regions, such as the SMA and MAPT loci on chromosomes 5 and 17, respectively (Schneider et al., 2017), whose sequence is derived from additional DNA samples. However, analyses of other individual human genome assemblies from Europeans (Ameur et al., 2018; Audano et al., 2019; Kidd et al., 2010; Levy et al., 2007; Wheeler et al., 2008), East Asians (Audano et al., 2019; Kidd et al., 2010; Li et al., 2010; Seo et al., 2016; Shi et al., 2016), South Asians (Audano et al., 2019; Kitzman et al., 2011), Amerindians (Audano et al., 2019) and Africans (Audano et al., 2019; Kidd et al., 2010; Li et al., 2010; Sherman et al., 2019) have still revealed a substantial amount of genomic information not represented in the reference assembly. Indeed, large re-sequencing projects showed an extensive human genetic diversity, even within the genomic content captured in the reference sequence (Bycroft et al., 2018; 1000 Genomes Project Consortium et al., 2010; Mallick et al., 2016). Although GRCh38 is the most complete human reference to date, it is not clear how to construct a linear reference that can capture diversity and address population biases that impact analysis (Brandt et al., 2015; Degner et al., 2009).\n\nThe diploid structure of human DNA is not currently represented in the current reference model, which is instead an arbitrary linear combination of different haplotypes (i.e., a mosaic) from multiple individuals. A human “pangenome” is a representation of all genomic variation observed in human populations (Computational Pan-Genomics Consortium, 2018). In this context, a pangenome is a more comprehensive representation of genetic diversity than an individual diploid genome or a reference comprised of linear chromosomes built from multiple individuals, such as GRCh38. By extension, pangenomics encompasses approaches that utilize a pangenome reference. Pangenomics is designed to address the limitations of current standards, such as reference bias during the identification of genomic variants, population stratification and admixture, or ancestry-specific functional variants—among others, which impact evolutionary, agricultural and health genetics research. For example, reference bias in the sequence alignment to GRCh38 (excluding its alt loci) reduces our ability to correctly genotype regions that are likely to significantly diverge from the reference chromosome representations—e.g. immune regions such as the major histocompatibility complex (MHC) and killer cell immunoglobulin-like receptors (KIR), and the CYP2D6-8 loci involved in drug metabolism (Dilthey et al., 2015). Alignment around indels becomes more challenging as their size increases with soft-clipping being preferred over split-read alignment (Garrison et al., 2018; Narzisi et al., 2014). Variants cannot be identified within regions completely missing from the reference sequence, many of which have been recently identified to be common across individuals (Taliun et al., 2019). Although bias and missing sequence may still persist in a pangenome, their effects should be substantially less, and may even be ameliorated by adding new content to the framework. In addition to these issues with the current reference, several studies using long reads have reported an average of ~20,000 structural variants (SV) per human genome, most of which fall within repetitive elements and segmental duplications (HGSVC) (Audano et al., 2019; Chaisson et al., 2015). Many of these SVs intersect genes and regulatory elements, harbor transposable elements, and affect gene expression (Audano et al., 2019; Chiang et al., 2017). Although they are largely inaccessible to short-read sequence with current methods, these variants can be more easily re-identified using a pangenome (Chen et al., 2019; Hickey et al., 2019). Complex loci that harbor multiple repeats are also quite challenging to detect and genotype by aligning reads to a linear reference. Important disease-linked repeats, such as the CAG repeat in the HTT gene that causes HD and the CAG repeat in ATXN8 that causes Spinocerebellar ataxia type 8 (SCA8), are both flanked by other polymorphic repeats making them particularly difficult to accurately genotype. Sequence graphs offer again a general and a more flexible approach to handle these complex loci (Dolzhenko et al., 2019).\n\nThe International HapMap Consortium defines a haplotype as “a particular combination of alleles along a chromosome” (International HapMap Consortium, 2005). A diploid individual has two haplotypes for any given genomic sequence—up to the complete genome itself—since it inherits a set of homologous chromosomes from each parent (Crawford & Nickerson, 2005). At the population level, there may be more than two haplotypes for any given sequence. The definition of haplotype will vary in the scientific literature depending on discipline-specific questions and applications (Hoehe, 2003). For evolutionary and population geneticists, haplotype may be short for haplotype block, which is a group of alleles that are inherited together across multiple generations and results from recombination and selection; the arrangement and length of haplotype blocks will inform about past population history (Wang et al., 2002). For medical geneticists, haplotype may represent a functional haplotype at the gene level, i.e. genetic markers linked to a disease-associated allele in so-called linkage disequilibrium, or LD (Slatkin, 2008). For livestock and crop breeders, a haplotype may be the minimal genomic region that influences a trait of interest (Hayes, et al., 2013; Qian et al., 2017). Whatever the definition of a haplotype, haplotypic information can simultaneously provide clues about population history and disease or trait association (Martin et al., 2018).\n\nToday’s widespread use of short-read sequencing provides easy access to genotypes but does not necessarily directly inform about the parental origin of each allele. However, the real power of haplotypes resides in phasing, which is the assignment of a given combination of alleles to each homologous chromosome (Browning & Browning, 2011). Beyond the methodological challenge of phasing genomes (Choi et al., 2018), the two haploid sequences in a diploid genome cannot be captured simultaneously in one linear sequence. However, a genome graph representation of a pangenome provides a spatial framework to embed multiple haplotypes at once and preserve phasing information (Paten et al., 2017). This property of graph representations of a genome is critical. At the gene scale, phasing information can be used to recognize compound heterozygosity, whereby the two homologous copies of a gene are each affected by a distinct recessive mutation (Snyder et al., 2015). Phenotype prediction depends heavily on the ability to distinguish point mutations or deletions between chromosomes (Cirulli & Goldstein, 2010; Tewhey et al., 2011), making the retention of phasing information fundamental for the interpretation of results in a personalised medicine setting. Other applications of phasing include the inference of past population demographic history by looking at the distribution and size of haplotype blocks along chromosomes (Schiffels & Durbin, 2014). Variant imputation also depends heavily on the availability of phasing information and becomes a key approach in large cohort studies with missing genotypes (Das et al., 2018). Finally, the sequencing of fetal cell-free DNA in maternal plasma is a very promising way to study fetal genomes in a non-invasive manner. However, it is first essential to phase haplotypes from at least one of the parents (Fan et al., 2012; Kitzman et al., 2012).\n\n\nMethods\n\nHere we describe the data sets and graph construction techniques used during the codeathon, as well as the pipelines and software that were developed.\n\n\nImplementation\n\nTo establish protocols to build pangenomic graphs from chromosome-level and ultra-long assemblies, we constructed graphs using the human reference genome GRCh38.p13, CHM1 cell-line data, and two primate references: chimpanzee (PanTro PTRv2; Clint; GenBank assembly accession GCA_002880755.3) and Sumatran orangutan (PonAbe3 PABv2; Susie; GenBank assembly accession GCA_002880775.3). Additionally, we built human-only graphs using the human reference genome (GRCh38.p13; GenBank assembly accession GCA_000001405.28) and the Japanese reference genome (JG1; available at https://jmorp.megabank.tohoku.ac.jp/201902/downloads/).\n\nWe used three different methods to build the graph and explore the potential limitations and advantages of each method. The first two methods allow us to explore evolutionary questions, such as ancestral states, large structural variations between groups, and complex gene genealogies. We first created graphs based on sequences from chromosome 21 from GRCh38 (CM000683.2), Clint the chimpanzee (CM009259.2), Susie the Sumatran orangutan (CM009283.2), and CHM1 (AC244111.3, AC244144.2, AC244518.2, AC245051.3, AC245314.2, AC246819.2, AC255431.1, AC256301.1, AC277730.1, AC277802.1, AC277887.1). The first method used minimap2 (v2.16-r922) (Li, 2018) with the parameter preset asm5 to do an all-vs-all alignment of the sequences. Next, we used seqwish (6e4fe705;) to induce a graph in GFAv1 (Graphical Fragment Assembly) format, later converted to VG format (Garrison et al., 2018) for further investigation. The second method used Cactus (Paten et al., 2011), a method designed to build genome graphs of different taxa while accounting for the phylogenetic relationship between the organisms included. The generated Cactus graph in HAL format was converted to VG format using hal2vg for mapping and visualisation. In addition, we used SibeliaZ (Minkin & Medvedev, n.d.) to build a graph from chr1 of JG1 and GRCh38.\n\nFinally, we designed a prototype of a graph coordinates system based on previously proposed ideas (Rand et al., 2017) that streamlines the incorporation of new haplotypes into the graph, while preserving a structure that is retro-compatible with the GRCh38 linear reference coordinates (Figure 1). Such a coordinate system offers a host of advantages, as it allows easier surjection/projection of graph coordinates onto the linear reference coordinates. It also streamlines variant discovery and improves annotation portability.\n\nA) Example of a GFA file (https://github.com/GFA-spec/GFA-spec) that represents a reference genome and one alternate haplotype. Nodes, represented by lines starting with ‘S’, have a name in the second column and a nucleotide sequence in the third column. Edges, represented by lines starting with ‘L’, connect nodes whose sequence appears adjacent to each other in one of the haplotypes. The node names appear in the second and fourth columns, and the orientations appear in the third and fifth columns. B) A path file accompanying the GFA file includes paths for the reference genome and haplotype 1. The haplotype name is in column 2 and the sequence of nodes and their orientations are in column 3. The nucleotide sequence for any haplotype can be resolved by reading out the sequence for each node in the path. C) Visualization of A using path labels from B. The red path represents ref1, while the blue path represents haplotype ref1@h1.\n\nOur work modifies vg (Garrison et al., 2018; Hickey et al., 2019) to create a fast and efficient read mapper. During the codeathon, we have improved a prototype minimizer-based mapper by adding a faster clustering function to cluster minimizer hits and hit extension logic for handling clusters that have no good full-length gapless alignment (Figure 2).\n\nInput reads are scanned for minimizers, which are searched against a precomputed minimizer index of the graph reference. Minimizer hits for sufficiently rare minimizers are located in graph space, and the hits for all minimizers are clustered. The clusters are extended gaplessly, with a tolerance for mismatches. If a cluster produces a single full-length gapless extension, it is output as the alignment. Otherwise, partial gapless extensions are chained together by performing alignments of the intervening sequences and graph paths that connect them.\n\nThe clustering algorithm has been improved by reducing the amount of data copying in the clustering implementation. Alignments may be output from the extender if chaining is not necessary. Additionally, we have devised an improved algorithm for comparing sets of clusters.\n\nWe also implement hit chaining which allows us to deal with crossovers and indels. When the extender cannot find a full-length gapless extension of the read alignment to some haplotype with below a threshold number of mismatches, where it previously would leave the read unaligned, it will instead now compute maximal unambiguous-path exact matches between the read and the graph’s embedded haplotypes and feed them to an extension step. The extension step will trace out the haplotype segments that could connect between those matches, perform gapped alignment of the relevant read sequence against each, and take the best for each possible connection. Then the resulting multipath alignment will be linearized into an optimal gapped single-path alignment for the read.\n\nWe also sought to test a plant model, using chr 10 of maize (Zea mays) by composing a graph using vg construct and comparing it to a graph created with minimap2 and seqwish (Figure 3). Unfortunately, we were unable to perform the read mapping portion of this experiment, because we were unable to index the graph produced with minimap2 and seqwish for mapping. We thus aborted the experiment at that step.\n\nAfter constructing graphs with vg construct and with minimap2 and seqwish, we sought to simulate reads from the vg construct graph, align them to the minimap2/seqwish graph with our faster, better short read mapper with hit chaining, and then to evaluate the mapper’s accuracy based on the simulated reads’ original and realigned positions along corresponding positional paths in the two graphs.\n\nWe could not index the minimap2/seqwish graph for mapping because it contained extremely large snarls, with hundreds of thousands of net graph nodes. One of the indexes we needed to produce, the distance index, which is used for identifying nearby seed hits for clustering, requires doing an all-against-all distance computation on the net graph of each snarl, and that process tried to allocate more memory to hold its result than we had provisioned on our machine. We believe that the graph we generated, shown in Supplemental Figure 1 (available as Extended data) as an odgi visualization, was pathologically complex and intractable, because we did not remove spurious, short alignments from the minimap2 output. The intractability of this graph precluded further analysis.\n\nUsing known variants and haplotypes during mapping of RNA sequencing (RNA-seq) data have shown to be important for reducing reference bias and thus improving downstream analyses. Reference bias is known to negatively impact estimation of allele-specific expression (Degner et al., 2009) and variant-aware mapping is one of the best ways to mitigate this problem (Castel et al., 2015). Furthermore, it has been shown that inference of gene expression in the highly polymorphic MHC can be improved by using the alternative reference haplotypes during mapping (Lee et al., 2018). A few variant-aware methods for mapping of RNA-seq reads exist, including GNSAP (Wu & Nacu, 2010) and Hisat2 (Kim et al., n.d.). Hisat2 is similar to vg in that it is also based off of a graph representation.\n\nWe wanted to test whether we could also use vg to map RNA-seq reads to a graph containing both known variants, splice-junctions and haplotype-specific transcript paths. We called this a spliced variation graph. We further wanted to show that we could use the reads mapped to the graph to get unbiased estimates of allele-specific transcript expression. The pipeline would serve as a proof of concept for a graph based approach for inferring allele-specific transcript expression when an individual's haplotypes are available, similar to the personal genome approach (Rozowsky et al., 2011).\n\nMutation rates vary across the genome with certain hotspots associated to accessible regions as well as other genomic features. This is also discussed in the presence of the gene duplication where in a single copy gene case the mutations are rare due to the selection pressure. However, in cases in which there are two or more copies of the gene this selection pressure is reduced, and higher mutation rates are possible for at least one copy of the gene.\n\nTo assess the presence SNPs inside of SVs, we constructed a graph genome in vg (Garrison et al., 2018) to incorporate the SVs found in a recent Cell paper (Audano et al., 2019). This highlights one application where graph genomes might provide improved insight over traditional mapping approaches. To assess this we used SNP calls for HG002, a gold standard in genomics reported to be present based on the Genome In A Bottle (GIAB) consortium (Zook et al., 2016). We compared the power of vg over short Illumina reads and Pacific Biosciences (PacBio) Circular consensus sequencing (CCS) reads and PacBio continuous long reads (CLR). Subsequently we extended our project to additional samples, focusing on the assessment of mutation rates inside common SVs between the Caucasian and African populations. This revealed changes in mutation rates when looking at tandem duplications between the flanking and the affected regions. It would be interesting to scale this project further for larger cohort samples to assess the mutation rate across multiple samples and ethnicities. This would lead to a broader understanding if SVs are indeed the driver for certain phenotypes, or if the variations within the SVs are more likely to be impacting the phenotypes.\n\nThe code to generalize this analysis for larger cohorts such as the 1000 Genomes Project or Simons Genome Project samples is available on GitHub.\n\nLinear genomes currently rely on genomic intervals as a core formalism for annotation but it is difficult to generalize this formalism to reference graphs. A genomic interval corresponds to a path through a graph. However, if we restrict the annotation to one path in the graph, the alternate alleles in the graph are not included in the annotation. We argue that connected subgraphs are a more appropriate formalism for annotating genome graphs. Using a new core formalism for annotation necessarily means that infrastructure to manipulate it does not yet exist. We need stable and exchangeable representations of the data, software support, and analysis tools to make the formalism useful for practitioners. We have developed a proof-of-concept system for projecting linear reference annotations onto genome graphs and utilizing them in downstream visualizers and analyses. The standard file format, named gGFF, has been defined on GitHub and code to manipulate and use this file format has been included in vg. We also developed a tool for performing utility operations on gGFF files, such as intersection and union.\n\nA common use of annotations is generating gene or transcript-level counts of RNAseq read mappings for differential expression analysis. We have implemented an example RNA-seq quantification pipeline using a graph constructed from GRCh38 ch21 and variants from the 1000 Genomes Project. We converted this to a splice site-aware graph with vg rna. The next step would be to map RNA-seq reads to this graph and estimate coverage per base-pair using vg pack and gene-level quantification computed using GENCODE 29 annotation.\n\n\nOperation\n\nThe software should run on most Linux installations. Interested parties are encouraged to clone the GitHub repository and follow the workflow/instructions provided for the individual implementations of the Use cases listed below. Pull requests and contacting the authors is strongly encouraged.\n\n\nUse cases\n\nGraph genomes can be used for inference of extension and phasing from sparse information derived from SNP chips and RNA-seq. They can also be used to infer allele specific expression on an individual level.\n\nThere are also regions—outside the alternative loci regions that are defined for GRCh38—that can’t easily be reduced to a single reference, and assembling each individual’s genome will likely be implausible for the foreseeable future.\n\nAdditionally, we are developing methods to represent variation in the clinically important MHC and to look for similarities in populations.\n\nIn theory, having clusters of haplotypes within and across populations will allow us to efficiently determine the relationships of proximal and distal phenotype-relevant events.\n\nTaking these points together, a pangenomic graph will likely result in a reduction in the “total cost of ownership of genomes”; i.e. people can use information derived from graphs instead of remapping to a linear genome over and over again, creating novel .bams/.vcfs ad infinitum.\n\nRepresenting haplotype information in reference genomes is beneficial in increasing mappability and reducing bias. The major concerns for representing haplotypes in the existing reference genome are the alteration of coordinates, redundant representation, and ambiguous sequence inference. Our proposed notation tackles these issues with the following design philosophies:\n\n1- The haplotype contigs are coordinated and defined as an add-on outside the extant reference genome coordinates. This allows the set of haplotype contigs to be updated separately, and the inclusion of haplotype sequence does not alter the underlying reference genomic coordinates. This design also allows the user to include fix patches [i.e. updates that correct errors or add sequence associated with gaps in the reference sequence; (Schneider et al., 2017)] in the graph or to recreate custom sequence using their haplotype of interest.\n\n2- Each haplotype and nested haplotype are defined as a unique segment based on the reference genome or the closest haplotype; therefore, the number of bases that need to be stored for each haplotype sequence is minimalized.\n\n3- Each haplotype can be uniquely represented using GFA-like notation that can track back into the node storing specific sequence for each haplotype.\n\nOur proposed model allows nodes and edges represented in the GFA to change without changing the sequence corresponding to each haplotype (Figure 4).\n\nAdding additional haplotype from A to B. The existing sequence and coordinates remain the same even though the nodes and edges change.\n\nSeveral new plant genomes have recently been sequenced and build upon the previously produced model plant assemblies, providing a foundation for research and end-use applications in agriculture. Crop plants form the foundation for the world's natural food and textile resources, and plant breeding efforts are often focused on improving several quality traits. A graph-based sequence-centric view of genomes sets the stage for facilitating key decisions that can be made to improve crop infrastructure.\n\nDiversity in plants comprises an array of genome types with regard to species identity, genome size, chromosome number, and ploidy level. Pangenome studies have commenced for many of the model plant species, such as Arabidopsis thaliana (flowering plants) (Clark et al., 2007), Medicago truncatula (legumes) (Miller et al., 2017; Zhou et al., 2017), and Brachypodium distachyon (grasses) (Gordon et al., 2017), due to the attractive attributes of their small genomes and short generation-times. Likewise, pangenome studies have commenced on their corresponding larger cousins, which include crop plants of economic importance such as crucifers, soybean, and wheat (Montenegro et al., 2017), respectively. These previous pangenome studies used highly developed sequence analyses, but not a graph-based approach. Several pangenome-related papers appear to be in preparation for other important plant species (e.g., maize); whether they all use graph-based methods remain to be seen. The exercise of testing graph-based sequence views will help formulate use-case scenarios.\n\nImmediate uses for graphs of plant genomes would be to validate hypothetical evolutionary tree diagrams assigned to species, and perhaps address instances where species are proposed to be ancient polyploids, or to gauge genome changes in current polyploid genomes. RNA-Seq methods may also be matched against graph-based maps to quantify expression from the genomes. For instance, it would be interesting to assess whether nutritional- or medicinal-related trait changes can be tracked to genomic structural variation using graph-based methods targeted on key metabolic pathway-associated genes. The tracking of highly repetitive transposon-initiated events may also explain some of the alterations observed in different genome species and their evolutionary consequence resulting in gene duplication, rearrangements, and the like. Use of graph-based methods to map out highly variable regions may also provide strategies toward implementing targeted engineering of species, or assist in classic breeding strategies where known attributes are known to structurally exist. Similarly, many wild ancestor lines are sought to bring in new gene function to serve as sources for disease resistance, quality traits, and nutrition; their inclusion in the graph will enable an understanding of their contributions on the whole genome scale. The construction of pangenomes by graph-based methods, and the subsequent visualization of these graphs therefore appear likely to have a valuable role in the future of agricultural improvements.\n\nGraphs can also be used to validate and benchmark analytical methods. For example, in order to test the RNA-seq mapping performance of vg we created a spliced variation graph of chr21 using the rna submodule in vg (see WDL pipeline for more details). We used variants from the NA12878 individual in the 1000 Genomes Project (1000 Genomes Project Consortium et al., 2015) and transcripts from the GENCODE v29 annotation (Frankish et al., 2019). The paired-end RNA-seq reads were simulated using RSEM (Li & Dewey, 2011) from the haplotype-specific transcripts generated from vg rna. vg’s two mapping algorithms map and mpmap were able to align 71.6% and 73.8% of the simulated reads with a mapping quality of at least 30, respectively. This is similar to the value observed for Hisat2 using the same data. We also tested both algorithms on graphs only consisting of exonic sequence. Using these graphs, the performance increased slightly (1.5 to 2%). Due to a lack of time we were not able to finish the second part of the pipeline that involved estimating allele-specific expression from the mapped reads.\n\nThis is very much a work in progress. For example, all splice-junctions and variations present in the reads were also present in the graph. In addition, due to time constraints we only used the number of mapped reads as a proxy of performance and did not assess whether the reads were correctly mapped. These issues will need to be addressed in future benchmarks in order to get a more accurate estimate of vg performance on spliced variation graphs.\n\nThe MHC, located on human chr6, is a region highly enriched for genes and variation, including the human leukocyte antigen (HLA) which is involved in immune system function. Genetic associations between variants in this region involve different diseases, including autoimmune diseases. MHC haplotypes differ substantially, making it challenging to map reads from this region and call variants with conventional methods on a linear reference. We sought to generate a base-level accurate, fully phased, diploid assembly of the MHC of GIAB HG002 (NA24385, Ashkenazi son). The only previous studies producing fully phased, contiguous diploid assemblies for the MHC involved the NA12878 genome with PacBio reads (non-CCS) (Jain et al., 2018; Koren et al., 2018). In this work, we use newer PacBio CCS and ultralong Oxford Nanopore reads, along with 10x Genomics data, to produce and carefully evaluate a targeted MHC diploid assembly for a second individual from GIAB.\n\nThe data for this work relied on sequencing results from three different PacBio CCS libraries with average read lengths of 9 kb and 13 kb for the Sequel I chemistry and 11 kb for the Sequel II chemistry, and each dataset having ~25 to 30X coverage. We also used “ultralong” data from Oxford Nanopore Technology (ONT) with total coverage of 16X (4X coverage by reads > 100 kb), and Promethion ONT data with total coverage of ~40X (~6X coverage by reads > 100 kb). We also used 10x Genomics data for phasing. HG002 reads were extracted from the MHC (HLA1/HLA2) region on GRCh37/hg19 chr6:28,477,797-33,448,354. Illumina data for the Ashkenazi father (HG003, NA24149) and mother (HG004, NA24143) from this trio was also used to bin the CCS reads by haplotype. The HLA typing reports for HG002/HG003/HG004 were generated at Stanford Blood Center on December 16, 2016.\n\nThe first data processing step involved finding reads from each haplotype mapped to MHC regions. An initial inspection of the HG002 MHC region occurred on the whole-genome de novo assembly of trio binned reads produced using the CCS data. The MHC region initially appeared to be well-assembled, with 1 contig derived from the father and 2 contigs derived from the mother, but further inspection revealed that the results were not coherent and that some of the haplotypes may possibly having been compressed. A second approach used 15 kb PacBio CCS reads that were mapped to the MHC and then selecting for each haplotype. A local de novo assembly of these reads resulted in 10-15 contigs with many gaps between, although the assembly was close to the full length of the MHC. PacBio CCS reads were processed with Whatshap v0.19 to generate a phased VCF, which was then used to partition CCS reads by haplotypes. Reads for each haplotype were assembled independently into contigs that were then aligned to 10x Genomics Supernova assembled contigs to generate scaffolded CCS contigs for the diploid assembly. This diploid assembly was then used as the input for vg to build a genome graph via all versus all alignment (by Minimap2) followed by seqwish.\n\nThe CCS and ONT long reads were aligned to the genome graph to confirm the diploid assembly using the PedMEC phasing pipeline (Garg et al., 2016). In addition, phasing of HLA typing results in the diploid MHC assembly were also checked against the independent HLA typing results from Stanford Typing Lab, based on the proband phased haplotypes derived from the typing results of the parents (HG003 and HG004), as shown in Table 1 (the parents’ typings are not phased).\n\nWe will continue to explore ways graph-based analyses could be used to benchmark methods used to characterize the MHC. It will be important to identify if these haplotypes can be represented in standard VCF files with respect to the primary GRCh37/38 references in GIAB benchmark sets, or whether existing benchmarks will need new representations and benchmarking tools. Although vg can project haplotypes into a VCF file with respect to the primary reference, it remains to be determined whether this is compatible with current benchmarking tools for small variants and structural variants. Other future work will entail examining whether fully phased diploid assembly is possible in other more complex, yet medically important regions, such as those of the killer-cell immunoglobulin receptor and spinal muscular atrophy.\n\n\nConclusion\n\nOngoing improvements in sequencing technology and diminishing costs make the generation of high-quality genome assemblies from diverse populations possible in a way today that could only have been imagined during the Human Genome Project (HGP). These new data are likely to form the basis for a new pangenome representation for the reference assembly that includes a graph, but they also raise many as-yet unanswered questions. We must consider the sample content, data/file formats that will be used, graph construction algorithms, how relevant metadata about quality and content will be communicated to users, and whether and how changes will be managed and tracked. New tools and validation sets must be built and community education will be essential, as will long-term curation, as is currently performed by the Genome Reference Consortium for the HGP reference. Ensuring the reference assembly remains a fair resource, accessible to users world-wide is also critical, and for the first time, some ethical and privacy concerns around the reference may need to be addressed. The new software developed here provide a preview of the use cases and potential for a new pangenome reference and play an important role in developing answers to these many questions.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: The Human Pangenome. https://doi.org/10.17605/OSF.IO/24K9N (Busby & Biederstedt, 2019).\n\nFolder ‘images’, contained within folder ‘Giraffe’ contains odgi.png (Supplemental Figure 1). This file is an odgi visualization of the Zea mays chr10 minimap2/seqwish graph for two species. The pink and purple bars at the top represent regions of the linearized graph that are visited by each species’ chromosome path. The black lines forming an impenetrable morass below the bars represent adjacencies between graph nodes. This graph has pathologically high connectivity.\n\nThis file is available under the MIT license.\n\n\nSoftware availability\n\nFor graph building and observing the GRCh38 path through a primate graph, source code and directions can be found here: https://github.com/NCBI-Hackathons/TheHumanPangenome/tree/master/DS\n\nFor ultra-fast read mapping to graph structures, source code and directions can be found here: https://github.com/NCBI-Hackathons/TheHumanPangenome/tree/master/Giraffe\n\nCode for converting from gff3 annotations to graph annotations can be found here: https://github.com/NCBI-Hackathons/TheHumanPangenome/tree/master/annotation\n\nWDL pipeline for mapping of RNA-seq data to spliced variant graphs can be found here:\n\nhttps://github.com/NCBI-Hackathons/TheHumanPangenome/tree/master/RNA\n\nCode for assessing structural variants with graphs can be found here:\n\nhttps://github.com/NCBI-Hackathons/TheHumanPangenome/tree/master/SV\n\nCode used to graph the MHC region can be found here:\n\nhttps://github.com/NCBI-Hackathons/TheHumanPangenome/tree/master/MHC\n\nArchived source code is available at: https://doi.org/10.17605/OSF.IO/24K9N (Busby & Biederstedt, 2019).\n\nLicense: MIT License.", "appendix": "Acknowledgements\n\nCertain commercial equipment, instruments, or materials are identified to specify adequately experimental conditions or reported results. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "55187", "date": "29 Oct 2019", "name": "Anna Kuosmanen", "expertise": [ "Reviewer Expertise Bioinformatics", "method development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes the results of the first pangenomics codeathon. The purpose of the codeathon was two-fold, to propose technical specifications and standards for a usable human pangenome and to build tools for genome graphs.\nThe traditional representation of a reference genome is a set of linear sequences (chromosomes), with possibly additional alternative sequences to capture variations. An alternative to a linear reference genome is a \"pangenome\", a representation of all genomic variation observed in a population. Pangenomes are modeled as graphs in this article. The article discusses the benefits of a pangenome reference over the traditional reference, and describes several software tools and pipelines for pangenomics applications.\nThe authors explain very well the limitations of the linear reference genome, and describe how a pangenome graph reference would be superior. And it is great that the Conclusions section also raises important non-technical matters related to pangenomes, such as privacy concerns.\nThe tools and pipelines described in the article build on VG, with some of them being very much work in progress, as is natural for the results of a codeathon. All the tools and pipelines, as well as the data used in the codeathon, are described in detail, and additionally all the code is available on github, allowing for easy replication of the development. Github also has detailed instructions on the use of the tools/pipelines and examples of the output.\nThe article organization is at times confusing. The methods section consists of two parts: Implementation and Use cases, with the topics of Use cases and Implementation overlapping. But each category also has topics which are not in the other. The distinction between these two categories isn't clear either, as depending on the topic the data and/or methods descriptions can be found in one or the other (e.g. in \"graph coordinate system\", data and methods are described in Implementation, and \"RNA-seq mapping\" has all the data and methods in \"Use cases\").\nIn my opinion the article is scientifically sound, but it could use some re-structuring for better readability.\nMinor comments:\nFor building the graphs you describe the first two methods in detail, but for third you simply say \"In addition, we used SibeliaZ to build a graph...\". The third method could use a sentence or two about it too.\n\nFigure 1: I found this slightly confusing that there's a GFA file that has one path (\"P\") line, and then there's \"a path file accompanying the GFA file\" with two path lines (of which one is in the former).\n\nIn section \"Pipeline for mapper evaluation on maize graphs\", it is unclear what is the goal of this experiment till you look at Figure 3. The first sentence of the section also sounds odd, like there are words missing (\"We also sought to test a plant model...\"), and the wording of \"comparing graphs\" is in conflict with Figure 3 text.\n\nTypo in GSNAP (\"GNSAP\") in section \"Mapping RNA sequencing data to variant graphs\".\n\nIn section \"Use case: Producing a fully phased diploid assembly of the HG002 MHC region\", what kind of data is 10X Genomics data?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "55209", "date": "12 Nov 2019", "name": "Robert A. Beagrie", "expertise": [ "Reviewer Expertise Genomics", "epigenetics", "gene regulation", "human genetics", "sequence variation." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLlamas, Narzisi, Schneider et al. present the results of a pangenomics codeathon held at UCSC this March. Specifically, they detail their progress towards creating a useable human pangenome and a set of fast and reliable software tools for manipulating and working with pangenome graphs. They compare three different methods for building a pangenome graph (Minimap/Seqwish, Cactus and Sibeliaz), suggest a prototype graph coordinates system to facilitate comparison/conversion to linear reference genomes, improve short-read mapping by vg, evaluate their graph genome performance in various use cases and provide tools for annotating pangenome graphs. Useable graph genomes that incorporate known human genetic diversity would be an incredibly useful resource for a wide range of fields, so the work presented certainly should be of broad interest. Overall, the authors have made good progress on a number of fronts, especially given the limited time available during a codeathon, however I think they could do a better job of justifying their design choices, summarising their findings and outlining necessary future steps.\n\nThe manuscript starts by comparing three methods for building pangenome graphs to “explore the potential limitations and advantages of each method”. I fully agree that determining the best currently available method is an important first step towards a human pangenome and progress has been made towards this goal. However, the SibeliaZ part of the pipeline built a graph using a different chromosome from the other two, which will make future comparisons much more complicated. The authors do not reach the stage where they can draw conclusions about the limitations or advantages of the different methods, but at a minimum, they should outline the future steps that would need to be taken to decide on a “best” method.\n\nThe authors propose a new graph co-ordinate system as an extension of the GFA file format, where the major difference seems to be an additional file listing the alternative haplotypes. I do not fully understand the explanation of why the new format is an improvement over GFA. The idea seems to be to allow haplotypes to be updated “separately”, yet in Figure 4 both the GFA file and the additional haplotype file need to be altered to add a new haplotype, so what is the advantage of the separate haplotype file? Whilst the coordinates of the reference do not change from Fig 4a to Fig 4b, the coordinates of haplotype 1 do. Would it not be important to maintain co-ordinates for all previously defined haplotypes when adding in new variants?\n\nIn summary, the selection and application of software tools and methodological approaches is scientifically sound, the questions addressed are important and interesting and the manuscript does a great job of explaining the potential benefits of a pangenome graph representation over traditional linear genomes. However, the manuscript needs some rewriting to clearly articulate what the authors have learned about best practices for constructing pangenomic graphs and what they see as the next important steps on the path to constructing a high-quality human pangenome.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1751
https://f1000research.com/articles/10-193/v1	08 Mar 21	{ "type": "Opinion Article", "title": "Embodied listening and coupling", "authors": [ "Bruno Jactat" ], "abstract": "Current approaches to listening are built on standard cognitive science, which considers the brain as the locus of all cognitive activity. This work aims to investigate listening as phenomena occurring within a brain, a body (embodiment), and an environment (situatedness). Drawing on insights from physiology, acoustics, and audiology, this essay presents listening as an interdependent brain-body-environment construct grounded in dynamic systems theory. Coupling, self-organization, and attractors are the central characteristics of dynamic systems. This article reviews the first of these aspects in order to develop a fuller understanding of how embodied listening occurs. It introduces the mind-body problem before reviewing dynamic systems theory and exploring the notion of coupling in human hearing by way of current and original analogies drawn from engineering. It posits that the current use of the Watt governor device as an analogy for coupling is too simplistic to account for the coupling phenomena in the human ear. In light of this review of the physiological characteristics of the peripheral auditory system, coupling in hearing appears more variegated than originally thought and accounts for the diversity of perception among individuals, a cause for individual variance in how the mind emerges, which in turn affects academic performance. Understanding the constraints and affordances of the physical ear with regard to incoming sound supports the embodied listening paradigm.", "keywords": [ "listening", "language learning", "teaching", "cognitive science", "embodiment", "situatedness", "dynamic systems theory", "coupling." ], "content": "Introduction\n\nAs a second language instructor of French, I have often noted hearing difficulties among my students. I am interested in the fact that bodily functions, such as hearing, may interfere with or, conversely, help with learning a new language. Does the body actually have an influence on how the mind functions? Proponents of embodied cognition say it does and claim that the mind does not operate independently, neither from its hosts, the brain and body, nor from its environment. If this is the case, then I am curious about what a review of the human auditory system can tell us about its influence on shaping the mind.\n\nThis paper proposes that the minds of learners are impacted by the shape, size, position, fabric and functions of their physical ears, and that variability among these features accounts for diversity of minds and, by extension, variance in learning capacities. I introduce the term embodied listening here to refer to the fact that listening, as a cognitive trait in humans, is both constrained and facilitated by these bodily features.\n\nWhile examining literature related to embodiment, I have found that some current metaphors used to describe embodied cognition are too simplistic. Dynamic systems theory is often used as a framework to explain how complex systems emerge and operate, such as embodied minds. Within this paradigm, coupling is a characteristic that defines how two separate elements such as the body and mind work in sync. An engineering device known as the Watt governor (see section Dynamic systems theory), is used to analogize the notion of coupling but it does so in a way that fails to capture the delicate sophistication of coupling in the hearing organ. This paper discusses these limitations and introduces the elastomer coupling device as an alternative analogy that can account for the ear’s complexity, the interactions between the different ear parts and the interactions between the environment, the physical ear and the mind.\n\nFinally, this work is meant to provide new insights to educators who, as myself, are faced with the task of helping students learn, whether a new language or any subject that requires them to lend an ear. Ideally, it can provide us with clues as to what we can do as educators to better accommodate bodily differences.\n\n\nTopic overview\n\nThe embodied mind thesis posits that mechanisms underlying cognition are shaped not only by the brain but by the entire body. Furthermore, mind and body are not perceived as split entities. Indeed, cognitive scientists who advocate embodiment agree that the mind is not just operating between the ears, but that physical features of the whole body play a substantial role in the development and functioning of the mind. Shapiro refers to this as the conceptualization theme in embodiment: “The properties of an organism’s body limit and constrain the concepts an organism can acquire” (2019, p.4). Broadly speaking, this means, for example, that an elephant’s brain and body bring forth a mind that has little to do with the type of mind that emerges from a human brain and body. More narrowly, and more usefully, this also suggests that structural variances in the body and brain within humans generate individual differences in the mind. Since the body contributes to the overall functions of the mind through its perceptual, sensory-motor, and affective experiences with the environment, then language also, as a constituent of cognition, is equally important in the mind’s development and ongoing processing (Lakoff et al., 1999). It logically follows that academic performance is impacted by a person’s bodily mechanisms. For example, sensory processing disorders are known to cause, among other things, cognitive and academic difficulties among people on the autistic spectrum or with ADHD, and, more generally, in various degrees among people of all ages and conditions (Geffner & Ross-Swain, 2018). As pertains to typical language learning and processing, numerous studies have reported relationships between bodily processes and language, substantializing embodied theories, notably in cognitive linguistics. In this framework, language is learned bottom-up by individuals who interact with language through their bodily and worldly experiences. Situated language use, experienced with and through a body, provides the grounding for memorizing and schematizing useful linguistic features, a process that has been well-documented by proponents of usage-based theories of language (Tomasello, 2003; Tomasello, 2009).\n\nIf, according to the usage-based theory of language, the interactions a language user has with the environment and body matter in the building of linguistic skills, then perception must play an important role in the amount and quality of information perceived and processed. In terms of auditory perception, a review of the auditory system will shed light on its influence on learning. Research spanning fields including acoustics, audiology, anatomy, physiology, and neuroscience will prove useful to this investigation.\n\nSince my inquiry addresses issues that are pertinent not only to researchers working in the cognitive sciences (especially language learning) but also to the boots-on-the-ground teacher trainers, language instructors, and professors, whose training might not have familiarized them with the specialized terminology associated with the fields mentioned above, I will either provide definitions for these terms or, better, will use the more common term when available (e.g. ear canal instead of external auditory meatus).\n\nAlthough those scientific fields provide evidence for the embodied approach, I will include an additional framework that has often been applied in embodied research to describe the interactions between brain, body, and environment, namely dynamic systems theory. This article discusses coupling, one of the three key components of dynamic systems — coupling, self-organization, and attractors — and argues that coupling, as seen in the ear, leads to an updated understanding of listening as embodied and situated. Coupling is envisaged as the interactions between the different bone and flesh structures of the ear (or peripheral auditory system) which are schematically made up of the outer ear (ear flap, ear canal, and eardrum), middle ear (three little bones called the hammer, anvil, and stirrup) and inner ear (the cochlea, a tiny conch-like structure).\n\nAs mentioned above, some of the analogies currently used to substantiate the embodied position have proven quite attractive. However, I will also discuss some of their short-comings and provide alternatives in support of embodied listening.\n\nTo my knowledge, the term embodied listening has not been used in the field of cognitive linguistics. I principally use this term to refer to the fact that some of our conceptual systems, notably those related to language, are grounded in the way the physical ear processes incoming auditory inputs and subsequently, how these are processed by the higher-level auditory operations of the peripheral and central nervous systems.\n\nBy limiting this inquiry into listening to the level of perception, the present work addresses a fundamental concern in language learning: why some lower-proficiency listeners fail to process auditory information at the ascending sensory (“bottom-up”) level and thus fail in comprehension. Field explains that even if those listeners were capable of higher level (“top-down”) cognitive operations, such as inferring meaning, “they cannot employ them if they have to focus heavy attention on decoding, nor can they if there is insufficient decoded material to provide a basis for constructing meaning” (2019b, p. 309). This statement underscores the difficulties a learner is faced with when not capable of efficiently perceiving and processing aural inputs. Field’s observation also points to an area of research in language learning that needs to be addressed more substantially in order to better understand what can be done about it, namely what causes differences in decoding among learners. The present review uses the embodiment paradigm to look at how discrepancies between lower and higher proficiency listeners originate from variances in physical features.\n\nIn the end, the implications of this view of listening highlight the uniqueness of every individual’s cognitive capacities and reveal some of the reasons why each human being perceives the world differently. Ultimately, we will see how the wide variety of inclinations with which one reacts to and interacts with the world accounts for the incredible diversity of our minds.\n\n\nThe mind-body split in listening\n\nSince French mathematician, physician, and philosopher René Descartes formulated his theory of mind in 1637 (Descartes, 1637, p.17), Western thought has been pervaded by the view that mind and body have fundamentally distinct natures and are ontologically split. On the one hand, mental properties experienced by a private self (subjectivity) include consciousness (e.g. perceptions, emotions) and intentionality (e.g. beliefs, desires). On the other hand, human physical properties observable by all (objectivity) include all matter of sizes, weights, shapes, colors, motions, etc. The relationship between these two sets of properties constitutes the mind-body problem (Robinson, 2020). At the core of this dilemma, lies two main interrogations: what is the exact nature of these two entities (ontological question)? Also, do they influence each other and, if so, how (causal question)?\n\nTo override this conundrum, behaviorist scholars have adopted a materialist view, holding the mind to be an irrelevant abstraction to explain the rational activities of human beings. For them, mental states are but an extension of physical states. For instance, this view posits that the laws of stimulus-response alone explain the development of language (Skinner, 1957).\n\nConversely, cognitivists consider the body to be irrelevant in explaining the working of the mind. Cognitivism assumes that the acquisition of mental structures arises from a determined internal matrix, a conception known as innateness. In the case of language, this innate matrix is referred to as the Universal Grammar, a grammar shared by all cultures and languages that arises naturally in spoken or sign language (Pinker, 2003). In short, this theory posits that inner symbolic language (mental symbols) translates outer symbolic language (words and sentences) and thus creates meaning. These symbols are arbitrary and amodal (i.e. not linked to any particular sensory modality), and their relationships are but a network of abstract symbols that do not have physical referents to the body or the world (De Vega et al., 2012). Stated differently, abstract symbols are correlated to other abstract symbols, which are further associated with other symbols and so forth. As an example of this entanglement, imagine if someone who has never been to Japan were to ask: “What is the city of Nara like?” And, if they then received the answer: “It’s like Kyoto but greener.” Having no referent experience of being in Kyoto, this answer is wholly ineffective in helping the questioner form an image of what the city of Nara might be like. It would take a much lengthier description to provide a nebulous idea of what the former imperial capital of Nara is like1. This abstract system of symbolic interactions faces a dead-end referred to as the symbol grounding problem. Harnad (1990) analogizes this to trying to learn Chinese with a Chinese/Chinese dictionary as the only source of available information: “The trip through the dictionary would amount to a merry-go-round, passing endlessly from one meaningless symbol […] to another […], never coming to a halt on what anything meant” (1990, p. 339). In other words, the learning would not be grounded into bodily and worldly experience. Conversely, the embodiment theory emphasizes that meaning-making in the brain is grounded in the sensorimotor experiences of the body (embodiment) interacting with the environment (situatedness), and in so doing provides a way out of the circularity of the above-mentioned symbolic system. Namely, this theory sees information given by the senses as being at the core of how we think, understand, and feel, conceiving of thought and language as the reactivation of these sensorimotor experiences as mental simulations (Barsalou, 2008). In order to form a more precise idea of what Nara is like, a first step might be to show photographs or a video depicting it. The sensorimotor experience of looking at the photographs and talking about them or watching a travel video and hearing related sounds and explanations provides the grounding for associations made with the word “Nara.” Ultimately, the best way to know what “Nara” stands for, is to travel there and bathe in the sensorimotor sensations of the old city.\n\nHaving noted these trends, most research around consolidating the embodied approach still relies heavily on analyzing how meaning arises in the mind through methods such as debating the philosophical implications of such a view (cognitive linguistics, philosophy of mind) or scrutinizing the brain (neurosciences). For instance, new scientific findings in neurosciences have shown how listening to words activates sensorimotor areas of the cortex, a finding that demonstrates how perceptual processes such as listening are not isolated but embedded in the experience of one’s body interacting with the outside world. Thus, listening to words such as kick, lick, or pick, or phrases such as press the piano pedal, bite the banana, or pick up the pen activate the motor areas of the brain that respectively control leg, mouth, and hand movements (Aziz-Zadeh et al., 2006). Although fascinating breakthroughs of this sort have been made in neurosciences, the field has yet to produce an account of how meaning-making through listening arises via embodiment, namely the physical devices of audition, including the ear, auditory cortex, and the circuitry between them. My intention here is to examine these physical devices and the role they play in embodied listening while considering the support they give to the embodied approach more generally.\n\nAnother point I would like to draw attention to is that cognitivism, which primarily looks to information processing and symbol manipulation to explain cognition, has led many current scholars to adopt a disembodied theory of mind and language. In fields related to education, this Cartesian view of listening is widespread. Perusing current books on listening in linguistics (see Ashcraft & Tran, 2010; Brown & Brown, 2011; Buck, 2001; Cutler, 2012; Field, 2010; Field, 2019a; Field, 2019b; Flowerdew et al., 1994; Flowerdew & Miller, 2005; Goh, 2014; Lynch, 2009; Nation & Newton, 2020; Richards, 2008; Rost & Wilson, 2013; Rost, 2016; Vandergrift & Goh, 2012; Wilson, 2008), one is hard-pressed to find any material on the physical nature of listening, with the exception of Rost2. Listening is entirely restrained to the sphere of the mind, with not the slightest concern for its anatomical and physiological dimensions. As a consequence, even the most recent publications concerning listening skills that are targeted toward teachers, teacher trainers, language testers, and textbook authors are largely based on the cognitivist paradigm (Bailey, 2020; Cauldwell, 2013, Cauldwell, 2018; Conti & Smith, 2019; Nemtchinova, 2020; Ockey & Wagner, 2018; Sepulveda, 2012). The overall effect is that many educators’ ideas and teaching agendas are pervaded with the belief that “since listening is cognitive in nature, there is no need to look at its physical features”. However, as I will show, exploring the workings of the human body engaged in listening will shed new light on how learners hear, listen, and construct meaning as well as provide insight into how educators can take bodily factors into account when teaching listening. Embodied listening can provide a cohesive account of auditory perception, processing skills, and meaning-making skills and help bridge the Cartesian divide. The scope of this article only allows for a limited investigation, and I will consequently confine this study to the exploration of the peripheral auditory system. A more exhaustive examination of this perspective by way of exploring, for example, the central auditory system (e.g. auditory nerve, brainstem and brain), an endeavor I intend to achieve in a sequel article, will further substantiate the cause for embodied listening.\n\nBefore continuing, it is worth recalling the distinction between hearing and listening. Hearing refers to the reception of sounds from the open-field (e.g. speech, music, natural sounds, traffic, etc.) or emanating from within the body (e.g. digestion, the heartbeat, or disabling signals such as tinnitus). On the other hand, listening refers to higher-order cognitive processes including attention, interpretation, and response. Succinctly, hearing is “a process of perceiving sound,” while listening is “a process of making sense of those sounds for the purpose of communicative action” (Bodie & Wolvin, 2020, p.295), and, we should add, for the purpose of learning and making sense of the world. In order to focus on embodied listening by exploring the ear’s functions, I will limit the upcoming review and discussion to the anatomical and physiological aspects of the peripheral auditory system (the external and middle ear). This review is grounded in the hearing processes of perception, which form the foundation of the higher processes of listening and are a crucial part of the rich interplay of operations that go into meaning-making.\n\n\nDynamic systems theory\n\nOne framework often used to understand embodied cognition is dynamic systems theory. Thelen and Smith provide a helpful summary: “The term dynamic systems, in its most generic form, means systems of elements that change over time. The more technical use, dynamical systems, refers to a class of mathematical equations that describe time-based systems with particular properties.” (2007). This framework has often been applied to a variety of fields as a metatheory, used for describing living and nonliving systems that exhibit continuous change and re-organization over time. Applications have been found in fields such as in meteorology, for explaining cloud formation, or in biology, for, among other things, characterizing behavior in ant colonies (Gordon, 2010). Dynamic systems theory has proven a robust theoretical framework to study the processes underlying transformations in complex systems. These changes are difficult to model because they result from interactions between the diverse elements that make up a given system/organism, whether it concerns a single system/organism (i.e. a cell) or multiple systems/organisms (i.e. our galaxy). When observing atmospheric physics or insect behavior, we can see that, despite the seemingly chaotic interactions of the constituents of a given system, organized patterns emerge in the overall system. Understanding complex systems as dynamic systems will also serve our investigation into cognitive science (Spivey, 2008; Thelen & Smith, 2007).\n\nAmong the various central characteristics of dynamic systems, three are observable in cognitive science, thus supporting the claim that cognition is embodied and situated: the phenomenon of coupling (or interdependent cycles of causation), the self-organizing principle, and the attractors theory. I draw from the theory of coupling to provide evidence for the claim that listening, as a constituent of cognition, is naturally embodied and situated.\n\n\nCoupling\n\nThe definition of coupling found in Merriam-Webster’s online dictionary reads as follows:\n\n1. the act of bringing or coming together: pairing. Specifically: sexual union;\n\n2. a device that serves to connect the ends of adjacent parts or objects;\n\n3. the joining of or the part of the body that joins the hindquarters to the forequarters of a quadruped;\n\n4. a means of electric connection of two electric circuits by having a part common to both3.\n\nThis fundamental idea of two constituent parts being connected to work together has been further developed in dynamic systems theory. In terms of cognitive science, which observes and tries to describe the complex interactions underlying the workings of the mind, the idea of coupling is meant to characterize how two systems, such as the brain and body or the body and environment, for instance, interact. A brief review of how this notion first emerged will help us grasp the point made by the proponents of embodiment and situatedness.\n\nThe phenomenon of coupling was first noted in 1665 by Dutch astronomer and physicist Christiaan Huygens (1673), who observed that two of his pendulum clocks, hung next to each other, synchronized into coupled oscillation after about 30 minutes (Strogatz, 2003, p.106–109). The motion of the two pendulums converged until they swung with the same period and amplitude but in opposite directions. Huygens discovered the mechanism responsible for the sympathetic motion of the pendulums: the small vibrations of the wooden beam on which the clocks were hanging. The behavior of Huygens’ pendulums can be captured in a mathematical formula, in which the behavior of one pendulum also includes a term that describes the behavior of the other pendulum, a basic tenet of dynamical systems theory. Coupling might then be observed in the motions of any dyad that demonstrates such properties: two metronomes sitting on a board (Pantaleone, 2002), the mating flashes of fireflies (Buck & Buck, 1966), or even your own footsteps falling in step with others, a phenomenon which the fictional Welton Academy English teacher John Keating demonstrated to his pupils in the movie Dead Poets Society when he had his students walk in a circle around the classroom until they all unwittingly began marching in sync (Weir, 1989).\n\nHuygens is also credited with the invention of centrifugal governors, a mechanical device in windmills that regulates the distance and pressure between millstones (Hills, 1996). In the case of embodied cognition, scholars refer to the phenomenon of coupling as it was observed in an adaptation of the centrifugal governor for a steam engine by James Watt in 1788: the flyball governor (Van Gelder, 1995). Until this innovation, controlling the speed of a steam engine was problematic. A schematized explanation of the device’s functions is shown in Figure 1: as engine speed increases, the governor rotates at a faster pace and the balls swing out, closing the throttle valve to regulate the rate of steam entering the cylinders, and thus reducing and controlling the speed of the engine. The point of interest here is that the behavior of the valve and flyball governor are constantly coupled; they display completely synchronized motions. They are interlocked in a fully autonomous and auto-controlling feedback mechanism, characteristic of the coupling phenomenon.\n\nThe output shaft from the locomotive’s rail wheel drives the flywheel of the governor into rotation. As speed picks up, the flyballs move outward and up pulling on the bell crank lever that controls the aperture of the throttle valve. As the valve closes, less steam reaches the engine reducing its speed.\n\nPhilosopher Tim van Gelder (1995), a proponent of dynamicism in cognitive science argues that this device is a more suitable metaphor for modeling human cognition than the classical computer analogy and sense-model-plan-act framework that is used by cognitivists (see also Brooks, 1991). Since its introduction as an analogy to study and explain cognition, debate over the Watt governor has not abated, and the limitations and merits of this analogy are still being discussed (Baltieri et al., 2020; Bechtel, 1998; Beer, 2000; Beer & Williams, 2015; Chemero, 2009; Eliasmith, 1997; Seth, 2014; Shapiro, 2019).\n\nFor the purposes of this article, I will compare this model with the understanding of hearing science, notably the processes underlying the first physiological stages of hearing, which I describe in detail below. However, first it will be fruitful to address the issues that arise when we adopt this device as an analogy for embodied listening: namely the lack of flexibility and absence of adaptability.\n\nThese limitations surface when comparing instances of causation and coupling between the device’s parts on the one hand, and between the brain, body, and environment on the other. Shapiro summarizes the way some scholars use the governor as an analogy for the brain-body-environment connection: “The circle of causality present in the governor involved components such as the throttle valve, the flyballs, and the flywheel. The circle of causality from which cognition emerges comprises the brain, the body, and the environment” (2019, p.156). Yet, this parallelism is somewhat imperfect: it assumes that the relationship between the mechanical parts of the governor and the causality that binds them together is comparable to the relationship and the binding between the brain, body, and environment. One problem with this metaphor is that the biological constituents (such as the brain, body, and other biological organisms found in the environment), as well as the constituents of the sub-systems (e.g. the parts of the ear), are not rigidly locked into one another as in the flyball governor. In light of a review of the ear’s functions, the analogy to Watt’s governor begins to break down because in the governor the constituent parts are mechanically locked, a condition that is not comparable to the characteristics of the human ear. On the contrary, the ear is constituted of flexible interdependent parts, as I will show when discussing transmission and adaptive properties. Sound is not transmitted as is but undergoes various transformations from one part of the ear to the next. This is due to the existence of adaptive constraints (the nature of the body) and adaptive demands (imposed by the environment) that call for versatile and flexible accommodative behaviors, a far cry from the rigid nature of the mechanical device. However, as I will demonstrate, coupling needs not be so steely. Pliability can better capture modes of interaction between systems and within physiological sub-systems such as the ear.\n\nThe Watt governor bears abstract similarities to embodied listening but should not be taken as exhaustively representing coupling in its multiple forms. Such an oversimplified analogy risks offering a distorted if not amputated understanding of coupling. Thus, we should consider an alternative device.\n\nStaying in the field of engineering, a more compelling model of coupling might be that of a device made of various shafts joined in a flexible manner to account for adaptive interactions between systems and within systems. A device that can serve as an analogy for coupling should allow its constituent parts to interact with some degree of adaptability and autonomy as with elastomer connection hubs found in machines between two shafts and appropriately called flexible couplings (made of an elastic material such as rubber). Such couplings serve as both transducer and buffer4.\n\nA flexible coupling is a mechanical part used to connect two shafts that allows one axis to drive the other with equal torque (spinning velocity); they are thus commonly used in rotary motion applications in machinery (such as vehicles, oil drills, DIY tools, pumps, packaging machinery, rolling mills, etc.) The primary function of these shaft couplings is to transfer power from a driving end to a driven end, such as between a motor and a propeller in an outboard or, conversely, between a propeller and a generator as in a wind turbine (Figure 2). Moreover, in engineering, coupling devices also serve several other purposes: to connect varying shaft diameters to one another, to accommodate varying degrees of misalignment, to alter the vibration characteristics of rotating parts, to reduce noise, to reduce the transfer of shock loads from shaft to shaft, and to disconnect when overload occurs. The latter characteristics introduce protection, and this is always better achieved by flexible couplings than rigid ones.\n\nCompared with the rigid connections of the flyball governor, the characteristics of flexible interfaces allow for a more faithful illustration of what might be happening between the complex systems of the brain, body, and environment. Within any of those systems, there are coexisting sub-systems. Although all interconnected, different body parts have specific modus operandi, with the ear’s main purpose being hearing. The ear is likewise constituted of various “shafts,” transducing acoustic energy from the air to bones, from those bones to liquid, and from those fluids to the nervous system circuity, and then all the way up to the auditory cortex in the brain. The ear is also comprised of built-in devices that protect the driven “shaft” components (i.e. eardrum) from the driving “shaft” components (i.e. ear canal). As with Watt’s governor, there are parallels between the properties of flexible couplings and the basic tenets of embodiment, especially in that coupling precludes the rigid and constrained properties seen in the governor. This model of interconnected shafts enriches the notion of coupling by introducing a less deterministic approach suitable to the adaptive development and behavior of biological systems. Coupling between systems can thus be examined at various levels, as in Table 1.\n\nAlthough the Watt governor is used to make a case for coupling at the broad level of a system of systems, coupling characteristics can be found at all levels. This article focuses mainly on the sub-system of the ear to substantiate the design of an embodied listening construct. The main conclusion drawn from the analogy of Watt’s governor with the mind, is that the brain, body, and environment operate in sync, like Huygens’ clocks, and that the mind emerges from those interactions. This ontological view of the mind is quite appealing when making a case for embodiment and situatedness but can also be misleading when we contemplate the incredible complexity of the numerous stages of auditory processing, which are not nearly as straightforward as Watt’s governor.\n\nFrom now on, I will consider coupling in relation to the ear in much more detail. Table 2 synthesizes the main analogies that can be drawn between a flexible coupling and the ear.\n\nTransmission and adjustment account for the processes whereby a device accepts energy from a source in one form and transforms it into a different form as it transfers it to another device. The recipient will act in turn as an emitter and send the energy to another recipient and so on until it reaches its destination (e.g. in the case of the ear, the auditory cortex in the brain is the endpoint). Transmission and adjustment are co-occurring operations. Adjustment comprises amplification (anatomical and physiological) and inhibition or dampening (filtering irrelevant sound or noise whether external or internal).\n\nInstances of coupling can be observed at several stages in the operations constituting sensory processing: during collection, transmission, and transduction (Figure 3).\n\nSignal (acoustic) → Collection: Air vibrations (from open field) → Transmission: Sensory Stimulus (from air to bone to liquid) → Transduction (into electrical impulses) → Processing: Integration (subcortical centers) / Sensation / Perception (auditory cortex) → Action (or reaction).\n\nTo appreciate the subtleties of coupling, a review of the transmitting and adaptive properties of the anatomical ear is warranted6. How does sound travel from the outside open field to the brain? First, I will describe the transmission “shafts” that make up the various anatomical parts of the ear. Then I will review how they interact through adaptive processes (in section Adjustment below). Four major transmission processes are explained: A) from air to air; B) from air to bone; C) from bone to liquid; and D) from liquid to electrical signal.\n\n(A) From air to air (from open field to outer ear)\n\nThe first “hub” and “shaft” are the pinna7, the concha, and ear canal that make up the outer ear (see Figure 4). Sound waves are first funneled by the pinna (or auricle), the outmost visible part of the ear, into and through the auditory canal. The outer ear amplifies and dampens airborne sound energy, leads it to the eardrum, and causes it to vibrate.\n\n(B) From air to bone (from outer ear to middle ear)\n\nThe eardrum “hub” connects the outer ear “shaft” to the middle ear “shaft,” or ossicular chain, allowing for the transformation of airborne acoustic energy into bone conducted vibrations (in audiology this is referred to as air-conduction and bone-conduction). The eardrum induced vibrations are conveyed behind the eardrum to the middle ear “shaft” consisting of the three smallest bones found in the human body: the hammer, anvil, and stirrup (scientifically labeled as the malleus, incus, and stapes).\n\n(C) From bone to liquid (from middle ear to inner ear)\n\nThese three ossicles serve as an intermediary bone conduction “shaft” between the outer ear (air conduction) and the inner ear (liquid conduction). They allow for the efficient transfer of energy from one element (air) to another (fluid). The stapes, shaped like a stirrup, pushes into a small oval window that opens onto the cochlea (the snail-like pea seen in Figure 4). The tread of the stapes oscillates against the window displacing the fluids inside of it.\n\n(D) From liquid to electrical signals (from inner ear to nervous system)\n\nLymph fluids fill the spiraled tubes of the cochlea. When the fluids are moved by the stirrup bone, they cause undulation of the basilar membrane, a thin tissue that divides the cochlear tubes into parallel corridors. According to the frequency of the sound, laminal waves appear on the membrane, which sweep the hairs sitting atop the rows of neurons that line the cochlear tubes. Hair cells then convert these waves (mechanical energy) into electrical pulses (graded receptor potentials), which travel along the auditory nerves so they can be integrated by sub-cortical networks and then processed and perceived by the auditory cortex (for a more detailed account see Musiek & Baran, 2020).\n\nFrom this brief overview of how acoustical energy is translated into electrical nerve signals, we can see that, although sensory processing happens within milliseconds, it is a tiered process that transforms energy via conductors made of various materials (including air, bone, liquid, and nerves). Thus, coupling in the sense of Watt’s governor is not happening. Rather, the auditory process is the result of a plurality of adapted coupling events.\n\nAdjustment processes include both amplification properties (exacerbating relevant sound) and inhibitive properties (filtering irrelevant noise whether external or internal) of the outer ear, the eardrum, and the ossicles.\n\nNow, with an idea of how acoustic energy is transported to the nervous system in place, let us examine the adaptive processes that occur along the transducing line of operations. These adaptive processes are hallmarks of embodiment, the notion that the mind forms through repeated interaction with its environment by perceiving it with the senses. Adaptation means that there are constraints and opportunities offered to the nervous system by the very structure of the body that is transducing the information. Because sensory inputs are preprocessed by the body itself (Chiel & Beer, 1997) the signal that is sent to the nervous system is an adapted version of signals from the environment. In the same way that a rubber coupling can influence how energy is transferred between two shafts because of its inherent physical properties (material, shape, size, thickness, etc.), so do the physical properties of the ear influence the quality and quantity of acoustic information that will be transduced through the anatomical shafts all the way to the brain. A description of the relevant morphology and physiology of the outer, middle, and inner ear structures will help us understand how this occurs.\n\nBefore describing these adaptations, notice that the mind also acts upon the body and environment through its unique perceptions and the actions that the mind takes in conjunction with the body in the world, creating a feedback loop that can toggle the perceptual mechanisms (top-down mechanisms). Since this occurs in the central auditory system (the nervous system), it is beyond the scope of the present paper8. A look at adjustments made by the peripheral auditory system (the anatomical ear) will suffice to demonstrate that listening as a cognitive construct is pre-processed by the body and should therefore be regarded as embodied. I will now explain the adaptive properties shared by the different “shafts” and “hubs” of the peripheral auditory system, serving as either amplifiers or filters or both when transporting vibrations to the central auditory cortex.\n\n(A) The pinna: its shape, size, and angle affect collection of sound and thus its perception\n\nObviously, animal ears come in a wide variety of shapes and sizes. Take the pointed ears of fennec foxes, the great flaps of an elephant’s ears, or the ears of a mole, just tiny holes hidden under its fur. Different ears serve different functions among species but most capture different swathes of sound, accounting in part for the variability in hearing ranges. Less obvious but equally significant, varying shapes among human ears likewise capture varying degrees of acoustic information (Shaw, 1974).\n\nThe first “hub” serving as an interface between the open field sound environment and the “shaft” or conduit to carry airborne signals to the eardrum is the outer, C-shaped flap of the ear, called the pinna. The very shape of the human ear has consequential effects on how we hear (Spagnol et al., 2012).\n\nWhen sound reaches the outer ear, not all sounds are gathered equally and sent through the ear canal. Because of the intricate ridges and depressions on the surface of the pinna, frequencies are not evenly amplified and filtered. In fact, the pinna acts as an amplifier for mid-range frequencies, as a filter at low frequencies, and as a direction-dependent filter for higher frequencies in order to enhance spatial perception (Musiek & Baran, 2020, p. 51).\n\nWavelengths of frequencies the size of or smaller than the pinna will be more readily funneled into the ear canal. In addition, the swirly shape of the pinna acts as a resonator to enhance mid-range frequencies between 2000 Hz and 7000 Hz (Ballachanda, 1997), the range typical of human voices. These folds and recesses enhance sounds that are meaningful to humans, leaving other pitches untouched or reducing background noise usually found in lower frequencies.\n\nIndeed, lower frequencies with wavelengths larger than the pinna more readily spill around the ear. The size of the auricle is directly correlated with the size of the frequencies that can be captured. Take an elephant: their ears can detect frequencies lower than any known to land mammals (O’Connell-Rodwell, 2007). With their large, fanned-out, and movable pinna, they can pick up infrasound not detectable by the human ear. Variations between species are salient and well known; similarly, anatomical differences in the outer ear alter the external ear transfer function from one person to the next (Shaw, 1974). Big ears, small ears, flat, flabby, or more cartilaginous ears already account for some of the variability in individual sound perception.\n\nBecause the pinna is at an angle turned slightly to the front, the ear gathers more information from the soundscape a person is facing rather than from what is behind them. As humans usually face each other when speaking this is a fitting adaptation. The ear flaps have the effect of somewhat dampening how much background noise and other sounds arriving from behind are able to reach the ear canal, a phenomenon known as the pinna shadow effect. This discrepancy in humans’ capacity to pick up sounds behind and in front of them also allows for enhanced detection of sound sources. This is why people with behind-the-ear (BTE) hearing aids have their ability to determine where a sound is coming from negatively impacted. In this instance, the pinna shadow effect is impaired, and the hearing-aid user is, therefore, less able to distinguish between front and back (Van den Bogaert et al., 2011). People with flatter ears could also conceivably experience difficulties with sound location, even though they are able to pick up sounds from behind better. On the other hand, cupping the hand behind the ear to concentrate sounds coming from a source in front, boosts mid to high frequencies by as much as 8 dB — a 250% amplitude — and attenuates similar sound spectrums originating from the back by up to 9.5 dB — about 300% (Barr-Hamilton, 1983). These variations highlight the difference it might make to have flattened cauliflower ears or to have protruding ones like Will Smith’s. In short, the shape, size, and angle of the pinna affect the collection of sound into the ear canal and thus influence perception, that is to say, the way one experiences surrounding soundscapes.\n\nThe overall pinna effect (sound filtering and localization) is so profound on hearing, that some hearing aid manufacturers are now moving away from BTE devices to engineering receivers placed directly inside the ear canal, making it capable of utilizing the pinna effect unique to each user. They know how influential the body is on perception and thus cognition. Indeed, each individual brain has been perceiving sound shaped by the person’s unique pinna since the day they were born, building mental models of the ambient soundscapes. So, when receivers are placed behind the ear flange, most of the neuro-acoustic benefits developed by the brain from years of experience with sound is lost. Therefore, as Groth et al. remark, in-canal hearing aids allows the auditory system to “organically select, separate, and integrate sonic features delivered via our ears” (Groth et al., 2020; p.17). Instead of “reconstructing” sound from behind the ear, this ecological perspective underscores the advantages of using the existing embodied listening profile of the user to enhance smoother sound processing and intelligibility\n\n(B) The ear canal: its shape (twists and turns), size (diameter and length), and material affect transmission of sound and thus perception.\n\nBehind the pinna that acts as a “hub,” lies the first anatomical “shaft,” the external auditory meatus, more commonly known as the ear canal. Because of its cylindrical shape, it acts as a resonator, altering the acoustic signal that it carries to the eardrum. This membrane is compliant to the incoming waves, but, because it closes the end of the tube it also reflects a minute part of the energy back into the conduit, accounting for the resonance in the ear canal. It is a bit like blowing into the top of a glass bottle: because it is closed at its base the bottle will resonate at a particular pitch according to its shape. Likewise, the ear canal resonates in a particular way because of its size and shape. It acts as a quarter-wave resonator, that is, it boosts wavelengths that are 4 times longer than its own length. Because the average ear canal length in adults is approximately 2.5 to 3 cm long, it will enhance wavelengths of 10 to 12 cm. These wavelengths fall into the 3000 to 4000 Hz range (Dallos, 1973). Unsurprisingly, the human voice is mostly distributed within that bandwidth.\n\nAs a consequence of the morphology of the outer ear, which is comprised of the pinna, concha and ear canal, the spectrum of sound delivered to the eardrum emphasizes frequencies useful for vocal communication in humans. Conversely, lower and higher frequency ranges are de-emphasized. This means that “the signal that reaches the eardrum is not the same signal that is being delivered by the sound source” (Musiek & Baran, 2020, p. 55). The outer ear is partial to what is deemed as the most important sound to humans, that is, the voices of their fellow human beings\n\n(C) The eardrum (or tympanic membrane): its shape, size, angle, stiffness, and thickness affect transduction of sound and thus perception.\n\nThe second “hub” serving as an interface between the ear canal “shaft” (outer ear) and the ossicular chain “shaft” (middle ear) is the eardrum. This “hub” serves as a transducer, converting sound energy arriving through the ear canal into mechanical energy by way of the handle of the hammer bone (malleus), which is attached against the back of the eardrum.\n\nAlthough often compared to the taut surface of a drum, the eardrum is not flat but cone-shaped, with the rim facing the ear canal and the tip of the cone (umbo) protruding about 2mm back into the middle ear cavity (Alvord & Farmer, 1997). It is not a coincidence that cone-shaped membranes are also used in home stereo equipment and speaker system diaphragms. Woofers have large flexible membranes for low frequencies, squawkers have medium-sized membranes for midrange frequencies, and tweeters have small stiff membranes for high frequencies. Technically, this makes sense, as separating membranes into three types enables better concentration of sound direction and power for each bandwidth. By contrast, humans have only one membrane, the size and tension of which does not allow for it to vibrate at the full spectrum of arriving frequencies. Since the membrane cannot operate large displacements, it does not vibrate to low-frequency sounds very well (as does a woofer, with its large supple skin to allow it to generate low sounds). The eardrum’s mass inertia also limits the transfer of high-frequencies (which the small stiff membrane of the tweeter does well). Thus, energy transfer in the eardrum is most efficient in the range of 800 to 6000Hz (Emanuel & Letowski, 2009, p. 162). In short, the human ear works best as a squawker.\n\nFurthermore, the dimensions and thickness (number of layers) of the membrane show considerable individual variation as well as dependence on age and sex (Graham et al., 1978). Hearing scientists agree that there is no such thing as an “average human eardrum” (Van der Jeught et al., 2013). In other words, membrane stiffness differs among individuals, resulting in a high degree of variability when sound energy is converted into mechanical energy through the three small bones in the ear.\n\nAny changes in the dimensions of the pinna, concha, ear canal, or eardrum structures will consequently alter the characteristics of received sound before they reach the brain. Variation is usually inherited, but distortions can be incurred by, for example, physical injuries, occlusion (ear mold), or ageing — conditions that will affect what a person hears, what they become used to hearing, and, ultimately, their overall perception of their acoustic surroundings.\n\nTo put all these factors into perspective, we can refer to Shaw’s (1974) compilation of data on the combined effects of sound amplification and dampening by the outer ear, as seen in Figure 5. Shaw demonstrates that the head and body also play a non-negligible role in sound diffraction and reflection, which can be seen in curves 1 and 2. As discussed above, the pinna has a strong intensifying role but mostly at the level of the concha (the doorway to the ear canal, seen in curve 3). The helix and antihelix curvatures of the pinna have a lesser distorting effect (curve 4). But none of these structures have as prominent an effect as the ear canal and eardrum (curve 5). The greatest average pressure gain as a result of all these combined effects is between 2000 and 3000 Hz (curve T), the bandwidth humans use most for speech intelligibility. The ear resonance is improved at its peak at 17 dB around 2700 Hz, an amplification of about 700%.\n\n(From Shaw, 1974, with permission from Springer Science and Business Media.)\n\nAlthough the workings of the external ear account for a great deal of variability in what humans capture from their acoustic environment and for the particular emphasis given to speech sounds, this is but the first step in sound transmission. Let us look now at what happens in the middle ear.\n\n(D) The ossicular chain: its shape, size, angles, and flexibility affect transduction of sound and thus its perception\n\nThe middle ear “shaft” looks like a lever made of the three bones commonly known as the hammer, anvil, and stirrup. This bony “shaft” cumulates functions of conduction, protection, transduction, and amplification.\n\nIts main role is to ensure that air-conducted vibrations from the outer ear are properly transferred and transformed into liquid-conducted vibrations in the inner ear by means of mechanical energy. The hammer handle set in the back of the eardrum receives sound-induced vibrations from the tympanic membrane, and, in conjunction with the anvil, causes the stirrup’s footplate to oscillate against the oval window of the cochlea, where encased fluids are stirred. Because sound, as it exists in the air, does not naturally penetrate fluids, the ossicular chain acts as a coupling device between air and liquids. As an analogy, take the experience of going underwater in a swimming pool: all surrounding sounds are suddenly dampened. This is because airborne sounds bounce off the surface of the water. For sound to effectively penetrate an aqueous environment, an adaptation is necessary. The ossicular chain provides this conversion by means of the three mechanisms known in audiology as “ossicular coupling.”\n\nOssicular coupling occurs through three simultaneous mechanisms: i) area difference ratio (eardrum to stirrup’s footplate); ii) lever action (hammer to anvil); and iii) curved membrane effect (eardrum to hammer) (Figure 6). I explain each of these mechanisms below.\n\n(i) Area difference ratio (eardrum to stirrup’s footplate)\n\n(A) Most sound vibrations are transduced through the ossicles to the oval window. (B) Residual sound is transferred directly by the eardrum into the tympanic cavity and slightly affects both windows. The spiraled cochlea ducts are elongated and modified in this view.\n\nBecause the area of the eardrum (45mm²) is much larger than the tread of the stirrup (3.2mm²), sound vibration that strikes the eardrum is pressed down into the much smaller surface of the footplate, increasing pressure while reducing speed and displacement, and thereby transforming the mechanical energy into hydraulic energy. Since the surface of the eardrum is around 17 times larger than the oval window, the sound pressure is condensed, leading to an amplification of about 25 dB.\n\nTo relate to how this works, Emanuel & Letowski (2009, p.161) analogize this mechanism to a pair of snowshoes: because they are large and flat, a person’s body weight can be dispersed over the wide surface of the snowshoes, allowing a person to walk over unpacked snow. If a person puts on regular boots, which have soles with a smaller surface area, then the same body weight will likely push them through the loose snow. The stapes is like a pair of regular boots: it pushes in and out of the oval window, pressurizing the liquid behind the window into hydraulic action.\n\n(ii) Lever action (hammer to anvil)\n\nAs seen in Figure 7, the handle of the hammer is 1.3 times as long as the anvil. This mechanism produces an action that converts pressure from the hammer into higher pressure by way of a short lever action at the tip of the longest side of the anvil. To understand how this works, we can draw an analogy to a playground seesaw (Figure 8): if we were to slide the plank over the fulcrum so that one end of the plank was longer than the other, it would make it easier for a child to lift the weight of an adult. Similarly, as a result of the unequal lengths of the bones, the hammer is able to hoist the anvil with greater ease and power.\n\nArea difference between the surface of the eardrum and the stirrup plate fitted onto the oval window of the cochlea. Lever action between the hammer and the anvil.\n\nFirst-class lever.\n\n(iii) Curved membrane effect (eardrum to hammer)\n\nThe third phenomenon in ossicular coupling is a result of the eardrum’s position in the middle ear cavity. The Eustachian tube (see Figure 4) equilibrates the air pressure in the middle ear with that of the external atmospheric pressure, permitting the eardrum’s membrane to sit in its most natural and neutral position. But, as air pressure hits the eardrum from the ear canal, it creates a depression between both sides of the diaphragm, and, as a result, the eardrum makes a buckling motion, which increases the force fourfold, focusing the sound waves onto the hammer bone (Figure 9).\n\nBuckling of the eardrum.\n\nThe coupled motion of the tympanic membrane and ossicles, including the stapes footplate, generate a combined magnification in pressure of about 45 times (an increase of 30 to 35 dB). This mechanical thrust delivers enough power to efficiently transfer air-borne sound vibrations into fluid-borne vibrations. Ossicular coupling provides us with an exemplar of how coupling in the ear is adaptive, constraining certain sounds and boosting others. Moreover, it highlights the variegated features of coupling: overall, the coupling of open-field sound with a sound-image in the cortex is the result of multiple cascading instances of coupling, each bearing distinctive modes of operation as illustrated in the examples above.\n\nIncidental to ossicular coupling, there is a second pathway by which the middle ear transmits energy to the cochlea called acoustic coupling. Some of the energy that hits the lower part of the tympanic membrane is not transferred to the ossicles but reverberates directly into the air-filled cavity of the middle ear (where the Eustachian tube connects). This has only a slight effect on the two windows placed at each end of the perilymph filled corridors of the cochlea (see Figure 6). Yet, Peake and colleagues have shown that “a loss in ossicular coupling is consistent with the cochlea responding only to the pressure difference at its oval and round windows (i.e. acoustic coupling)” (Peake et al., 1992, p.17), and this might be one of the only ways for some people to still capture sound cues instead of becoming completely deaf when ossicular coupling fails.\n\nAnd, indeed, the efficiency of this ossicular mechanism can be compromised when the ossicles are misshapen or displaced (or nonexistent), either by birth defects or even minor physical traumas, such as a Q-tip injury. Most often injury occurs when the middle ear is deeply affected by repeated otitis media with effusion, especially in children under 2 years old. The lasting negative consequences of such illness on cognition and academic performance have been amply confirmed (Williams & Jacobs, 2009).\n\nThat being said, even among individuals with “normal” ears (no pathology nor malformations) we can detect significant differences of up to -25 dB (a 6 to 7-fold variance in volume intensity) in the hearing response curves. Dallos (1973) noticed that the shape of auditory threshold curves (the string of points at which given sounds are perceptible) is contingent on the sound transmission performances of the outer and middle ear. Goode (Goode, 1997, p17) suggested that even slight variances, both in the stiffness of the eardrum (Zwislocki, 1975) and the lever ratio contributing to the force of amplification in the ossicles (Brenkman et al., 1987), account for the large perceptual differences noted between individuals, actually calling sensitive ears in the top 10% “golden ears” and in the bottom 10% “tin ears” (in Emanuel & Letowski, 2009, p.162).\n\n\nDiscussion\n\nIf it is possible to represent coupling between the brain, body, and environment through the Watt governor, the same model should also be an edifying way to describe coupling in each of the lower-tier systems, such as the body itself (system) or the ear (sub-system). This is precisely what dynamic system theory endeavors to achieve: to provide a model that can describe any dynamic system, from the macro to the micro-level. However, from the above examination of sound transmission processes in the sub-system of the ear, we can see that coupling at this level is not as rigid as the Watt governor model might suggest. Coupling is not a straightforward process but involves various alignments, where some information is attenuated and other information is amplified. A more appropriate analogy to describe coupling for a sub-system, such as the ear, might be the elastomer coupling devices found in between shafts in machinery, which are similar to the buffer and amplification properties of the ear. Yet, when equating the Watt governor mechanism or the flexible shaft-to-shaft mechanism to the brain-body-environment system, a literal-minded thinker might take it as over-constrictive, but a more figurative-minded reader will see its value in the way it illustrates a concept. The above inquiry into the multifaceted nature of coupling exhibited by the ear makes for a more nuanced understanding of the very concept of coupling. By itself, the analogy of the governor does not render the diversity of coupling features to be found in the ear. However, the combination of various analogies for differing instances of coupling, such as the flexible coupling hubs, shafts, musical instruments, audio-speakers, snowshoes, the seesaw, and other metaphors paint a more vivid and variegated picture on the canvas of dynamic systems theory.\n\nAt this point, one might object that coupling, outfitted in such motley apparel, has nothing to do with dynamic systems theory since it does not fit with the idea of non-linearity so typical of systems displaying dynamic properties. So far, coupling in the ear has indeed been depicted as a linear shaft-to-shaft operation, arrayed in manifold coupling mechanisms. However, this approach does not in any way rule out the co-occurrence of non-linear phenomena. Both linear and non-linear approaches are complementary to shedding light on the complexity of coupling in a dynamic system. Coming back (one last time) to the Watt governor and the engine it is part of, we can observe both linear and non-linear phenomena. In the following description, note how which is used to translate linear causality from one mechanical part to another (an asynchronous action) and as is used to convey the idea of non-linear causality as in coupling (a synchronous action): shoveling coal in the boiler increases the steam load, which speeds up the engine, which entrains the horizontal flywheel as it drives the central shaft of the governor to rotate faster, as it propels the flyballs on the arms to move outward and upwards, as it causes the arms to pull down on the sliding ring, as it moves the bell crank lever, as it reduces the opening of the butterfly valve which decreases the amount of steam which lowers the speed of the engine. The overall operations are generated through mechanisms displaying both linear and non-linear traits.\n\nWhat about the ear? Research in hearing science has paved the way to provide non-linear models of the acoustic and structural coupled systems of the ear (Ihrle et al., 2013). Briefly put, these scientists explain how sound transfer is affected as the resting position of the middle ear architecture alters when receiving sound. The eardrum is affected by the elasticity of both the ear canal in front of it and the tympanic cavity behind it: both chambers are dynamical elastic bodies, not static entities as described in anatomical books. This means that each structure influences others, a distinctive property characteristic of a dynamic system. This study shows that, for example, the pressure differences encased in the cavities on both sides of the eardrum affect its position, while the complex geometry of the eardrum itself further influences the motions of the ossicular chain. The overall effect is additional modulation of how sound is transferred through the middle ear. From the point of view of physics, the transfer of acoustic energy is not just deterministic (linear) but also probabilistic (non-linear).\n\nHaving observed this, we can turn back to the main discussion, which aims at explaining how coupling, from both a linear and non-linear perspective, helps us see that sound perceived by the brain after its transfer through various internal channels does not exactly correspond to the sound emitted from a given source. This supports the claim that the building blocks of the mind are influenced by the very nature of the body (adaptive constraint), from which acoustical input (adaptive demand) — or for that matter any sensory input — is collected and transferred to higher levels of integration in the brain.\n\nFurthermore, coupling effects that are important to human variability can also be found between the cochlea and the auditory nerves that send signals to the brain. This will be explored in a sequel article, when describing self-organizing systems. For the time being, what emerges from a close inspection of the coupling functions in the ear, is that given the premise that the mind’s development is contingent on perception, which itself varies according to physical properties, the mind’s configuration must at least be partially reliant on the body’s anatomy and physiology, thus making a case for embodied listening and, by extension, embodied cognition.\n\nAs we have seen, specific anthropometric features of people, i.e. size, shape, position (angle) of any of the anatomical parts of the outer, middle, and inner ear9, vary widely depending on age, gender, and genetic factors and have cascading influences on cognitive performance. Moreover, audiologists have concluded that the specific resonance characteristics of these parts vary greatly as a result of these factors. Because auditory thresholds are different in individuals (i.e. the sounds a person can perceive at a given volume), this can do a lot to explain the varying responses each person will provide to incoming auditory information. Indeed, the way a person experiences the soundscape around them may be said to be unique. We usually take for granted that nearly everyone hears the same way (save for our elders and others with hearing loss). However, we often fail to realize to what extent there exists considerable variance in listening ability among the people we interact with on an everyday basis. Whether a teacher is faced with a class full of kids, teenagers, or adults, they would do well to realizing that no two sets of “ears” are the same and to empathize with the fact that each individual has a different perceptual system, which impacts the formation and the workings of their mind. Academic achievement can be affected positively, merely by virtue of possessing “golden” ears, or negatively, through no fault of one’s own, as a consequence of having “tin ears.” If, from the outset, individual differences prevail at the physical level, thereby impacting academic outcomes, the role of educators in trying to mitigate these inequalities and provide for more equitable learning opportunities becomes even more important.\n\nIn closing, we should consider the following conclusions:\n\n1. Coupling in hearing is variegated: both linear and non-linear coupling phenomena make allowances for an overall description of how sound is processed by the outer and middle ear;\n\n2. Hearing is a pre-perceptual mechanism: it impacts listening through the adaptive constraints imposed by the ear’s anatomy and physiology;\n\n3. Anatomical variability in individuals is the norm: by extension, what a person perceives and how they respond, for example in terms of academic achievement, can be surmised as at least partially resultant of such structural singularities;\n\n4. Listening is embodied: the structure of the ear creates constraints and opportunities for the emergence of the mind.\n\nFinally, the above review of the external and middle ear mechanisms sheds light on the fact that a constrained definition of coupling alone, as previously discussed, cannot account for the complexity of auditory processing. And we have only glimpsed the tip of the iceberg. In sequel articles, we will delve into the waters of auditory integration and explore how it gives rise to further sophistication, which both substantiates dynamic systems theory as a means of understanding the emergence of an embodied mind and adds nuance to this framework. I will also demonstrate how further embodiment features, as well as environmental factors, help explain large variations in listening from one individual to the other. These insights should be of use to educators, who are trying to understand what they can do to provide their learners with better listening strategies and opportunities. This will be made possible by incorporating the body and environment into the general picture of an embodied and situated listening paradigm, a project that is under development.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nThe author thanks Michael Larson who deftly polished the text and Joseph Shaules for his insightful feedback on improving this article. I am also grateful for the time and efforts from the editors and the reviewers. Warm thanks goes to Natsumi Tsuchida for the care she put into drawing the figures.\n\n\nFootnotes\n\n1 This analogy was inspired by an original example in Shapiro (2008). Reilly et al. have also offered a similar anecdote (Reilly et al., 2016, p.1004).\n\n2 Michael Rost (2016) wrote an opening chapter on neurological processing in his book, Teaching and Researching Listening. A stand-alone piece, it is not meant to tie into the following chapters which all describe processes from the standard cognitive viewpoint — an eloquent illustration of the mind-body split that pervades the field. The author draws our attention to this fact in the concluding sentence of the chapter: “Though the contemporary study of neurolinguistics often focuses on the cognitive aspects of information processing and manipulation of symbols, we should bear in mind that meaning-making is fundamentally ‘embodied’ in our physical and emotional capacities, […]” (p.15). The essence of this cautionary comment is that Rost acknowledges the embodied stance as having value in the quest to further understand listening phenomena.\n\n3 Coupling. (2015). In Merriam-Webster.com. Retrieved November 4, 2020, from https://www.merriam-webster.com/dictionary/coupling\n\n4 Couplings even include, for some applications, an automated release system (mechanically separating shafts from one another). Linking this mechanism to engagement and disengagement processes of attending to acoustic signals provides a potentially powerful analogy especially in relation to overload effects. Despite these promising parallels, they will not be drawn out in the present study.\n\n5 The acoustic reflex depends on the activation of higher-level functions within the central auditory system. However, this article focuses on the peripheral auditory system and is not meant to offer an overview of the topic.\n\n6 Once again, subsequent articles will explore the interactions within the central auditory system (the auditory nervous system) and its implications in the emergence of the mind. Here, I only consider the peripheral auditory system.\n\n7 A Latin derivative that means ‘fin,’ as in the fins of a fish. However, the pinna is not closely related to fish fins in terms of evolutionary biology. On the other hand, our hands are.\n\n8 This is mainly processed by outer-hair cells, a type of neuron found in the cochlea and coordinated by higher-level functions that will not be discussed here.\n\n9 The cochlea of the inner ear also differs in shape and size from one individual to the next. 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The Nervous System Human Communication and its Disorders. 1975; 3." }	[ { "id": "81098", "date": "06 Apr 2021", "name": "Curtis Kelly", "expertise": [ "Reviewer Expertise neuroscience of learning", "predictive processing and embodiment" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPros & cons: + like the way that this is written for teachers as well as academics, as I think most papers should be.\n+ Finally, someone looking at something specific (listening) from the bigger view (systems).\n? Is listening the right term? Or is it hearing?\n- If space is a problem, I am not sure we need that much history of coupling, initial parts could be condensed. Then again, the main purpose of this paper seems to be making a metaphor that helps us understand the mechanical and neural mechanisms of listening, which is done with coupling. Therefore, I think the paper title would better prepare the reader if it were something like “The Mechanics of Embodied Auditory Perception: Using the Coupling Devices as a Metaphor.\n+ I like the way the paper posits the steps or auditory processing from signal to action, coving both mechanical and neural activity.\n- I’d like even just a little bit on the processing part because theories like predictive processing say that auditory sensation is passed down from higher cortical regions rather than just processed through input, ie, transmission is two-way, but it seems this is the next paper.\n+ In neuroscience, we are finding more and more that the brain builds advanced concepts from real life experiences which are used metaphorically. That making such a metaphor is the purpose of this paper is kind of exciting, and something we will see more of in the future.\nEven though a lit review, In the section just before “Coupling,” I’d like to see a couple of paragraphs outlining the mechanics of listening/hearing, not just embodiment, and then proposing that we might get a better understanding of that process by looking at coupling.\n+Writing quality is superb.\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": "6961", "date": "28 Jul 2021", "name": "Bruno Jactat", "role": "Author Response", "response": "Professor Kelly, my sincere gratitude goes to you for taking the time to review this long article. The length of the text might indeed have been shortened or better yet made into two shorter articles. My purpose here was 1) to revise the current metaphor used to illustrate the notion of ‘coupling’ within Dynamic Systems Theory, and 2) to use the updated metaphor to underline how variegated coupling appears in hearing and listening, and by extension highlight the uniqueness of individuals, how no two pair of ears are identical. Therefore the bulk of the text which could be considered as two endeavors put into one. Since this article serves as a foundation to sequel papers—that will delve more into the listening processes you aptly describe as comprising functions such as ‘predictive processing’ (“…auditory sensation is passed down from higher cortical regions…”)—I have made only minor changes to slightly shorten the text (two paragraphs have been deleted). Moreover, your comment about revisiting the title hits the nail on the head: Indeed, listening has been barely encroached on within this paper as the contents rather lay the foundations of hearing mechanisms. I have accordingly changed the title to: “Mechanics of the Peripheral Auditory System: Foundations for Embodied Listening Using Dynamic Systems Theory and the Coupling Devices as a Metaphor.” Your suggestion of adding “a couple of paragraphs outlining the mechanics of listening/hearing, not just embodiment” would definitively help the reader. As I have already succinctly described the difference in the paragraph right before the ‘Dynamics system theory’ section, and not willing to further lengthen the text, I will make good use of your suggestion in the upcoming paper and furthermore develop the distinction there. Once again, thank you very much for your positive comments which encourage me to pursue my research and reflections on these topics." } ] }, { "id": "87992", "date": "05 Jul 2021", "name": "Brian J. Birdsell", "expertise": [ "Reviewer Expertise Embodied cognition", "creativity", "and metaphor theories" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 explores embodied listening from an embodied cognition and dynamic systems perspective and how it may affect listening performance by foreign language learners. The paper is well-written and well-researched and contributes to both the fields of cognitive science and second language acquisition. A large portion of the paper is an in-depth analysis of the anatomy of the ear (particularly the external and mid-ear mechanisms) and the auditory process of making meaning from sounds. The author argues for using an elastomer coupling analogy, as opposed to the more commonplace Watt governor mechanism analogy, to illustrate the coupling between the different auditory mechanisms. In the end, the author then points out the wide variability of the ear apparatus among individuals and individual learners resulting in some having “golden” ears and others “tin” ears and this directly impacts academic outcomes in the target language.\nThe paper successfully places the auditory process within a dynamic systems framework, but I would have liked to have seen the author elaborate in more detail “embodied listening” and how it fits within the larger framework of embodied cognition. Few would disagree that the auditory systems between individuals are highly variegated, but how does one proceed to measure this variance and how does this directly affect one’s cognitive performance on a given listening task? In short, can we actually measure this variability between those with “golden” as opposed to “tin” ears? Moreover, how does this “embodied listening” proposal fit into other theoretical approaches within the embodied cognition literature. For example, one possible avenue to further explore is an area of research called “body-specificity hypothesis” (Casasanto, 2009).1 The main argument here is that body specific preferences, in this case, being either right or left-handed, has conceptual consequences in how one perceives abstract concepts like good and bad. For right handers, good is typically spatially viewed (through gestural movements) to be situated on the right side and left for bad. In contrast, for left handers this is the opposite. This suggests that the dominant hand affects how one conceives these evaluative abstract concepts. Specifically, how does auditory variation affect cognitive performance? Or how does auditory variation result in divergent interpretations from the same auditory stimuli?\nIn regards to language learning, particularly a second language, the ideas presented in this paper bring up a number of important points. First, variation is the norm and likely is the result of exposure to the L2; age of exposure; but as presented in this paper, physiological characteristics of the auditory system. How can teachers know whether a breakdown in listening should be contributed to a lack of exposure to the sounds of that language or some possible physiological variation with the auditory channel? Some practical guidelines for language teachers would be beneficial here. Secondly, this paper also seems to support the theoretical view that all communication is “good enough” (Ferreira, Bailey, & Ferraro, 2002).2 and instead of viewing concepts and categories as stable constructs, it is more fruitful to view them as dynamic and ad hoc (Cassanto & Lupyan, 2015)3 and highly variable dependent on context and individual variation, both at the experiential level, but also physiological level.\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": "6962", "date": "28 Jul 2021", "name": "Bruno Jactat", "role": "Author Response", "response": "Professor Birdsell, thank you for your concise and clear summary of my work as well as the valuable feedback and pointers you have kindly provided. The notion of “embodied listening” indeed remains to be developed as this paper focused principally on hearing rather than listening. As another reviewer suggested, the title being misleading, it needs to be revisited. To that effect, the article now comes with a new title: “Mechanics of the Peripheral Auditory System: Foundations for Embodied Listening Using Dynamic Systems Theory and the Coupling Devices as a Metaphor.” The sequel paper will definitively delve into “embodied listening” in more detail. I appreciate your suggestion to frame it into the larger topic of embodied cognition, a piece of advice I will follow on in the next paper. Casanto’s “body-specificity hypothesis” is one of those frameworks that can likewise be explored in auditory processing. Research over decades show that humans have a side-dominant ear, just like we have a dominant hand and eye. This in turn does guide behaviors and has practical implications in the classroom. I will actually be addressing these issues, such as how the right ear generally makes more sense of language than the left (known as the Right Ear Advantage—REA). Your questions regarding how auditory perception is influenced by body specific preferences will be developed when considering listening, as ear-preference is linked with the individualized neuro-architecture of brain hemispheres. Practical guidelines will be offered to teachers once those issues addressed. As an example, simply asking students in class to hold their smartphones to their ear, usually points to their dominant ear. By asking students to seat themselves in the classroom so that their dominant ear is turned toward the teacher is thought to help understanding. Teachers should also place their own desk slightly to the left side of the room when facing the class as there are statistically more right-eared people than left-eared. Too often desk position is aleatory and simply placed opposite the classroom entrance. Similar guidelines will be drawn out in a sequel paper. Because there was a paragraph right before the “Topic Overview” section that didn’t hold its promise of providing such cues, I have taken it out altogether. I have looked at the references you suggested regarding ‘Good-Enough Representations’ and will include these views in the future, as they reflect the top-down processes in listening that will be discussed. Let me reiterate my sincere gratefulness for your review and the stimulating questions that you have brought up. They will definitively help guide my research and reflections to come." } ] } ]	1	https://f1000research.com/articles/10-193
https://f1000research.com/articles/9-1470/v1	16 Dec 20	{ "type": "Research Article", "title": "The effects of the glycaemic control on the severity of the delirium in the advanced phase of Alzheimer’s disease", "authors": [ "Antonio Martocchia", "Marta Scarienzi", "Pietro Prunas", "Enrico Bentivegna", "Mauro Cacciafesta", "Paolo Martelletti", "Giorgio Sesti", "Marta Scarienzi", "Pietro Prunas", "Enrico Bentivegna", "Mauro Cacciafesta", "Paolo Martelletti", "Giorgio Sesti" ], "abstract": "Background: Behavioural and psychological symptoms of dementia (BPSD) and delirium are common in advanced phases of Alzheimer’s disease (AD). Methods: Thirty-eight moderate-severe AD patients were enrolled (n=16 affected by type 2 diabetes). Each patient received a comprehensive geriatric assessment (CGA) (including evaluation of BPSD and frailty), and a complete metabolic evaluation (including the measurement of the glycated haemoglobin, HbA1c). Results: Both the hyper- and hypo-glycaemic extremes of the glycaemic spectrum worsened BPSD, but delirium was more susceptible to hypoglycaemic events. The severity of delirium was significantly related to cognitive function (r = -0.585, p<0.001) and frailty (r = +0.440, p<0.05). Conclusions: The measurement of HbA1c was useful for evaluating the risk of delirium in relationship to glycaemic control and nutritional status.", "keywords": [ "Marigliano-Cacciafesta-polypathology-scale", "glycaemic control", "Alzheimer’s disease", "delirium" ], "content": "Introduction\n\nBehavioural and psychological symptoms of dementia (BPSD) and delirium are common in the advanced phases of Alzheimer’s disease (AD), whereas cognitive decline is prevalent in the first phases of the disease.\n\nBoth the hyper- and hypo-glycaemic extremes of the glycaemic spectrum may worsen cognitive functions in AD patients1–3, and nutrition plays an important role in the development of the delirium in frail older patients4.\n\nThe aim of this preliminary report was to evaluate the relationship between delirium and glycaemic control in the advanced phases of AD.\n\n\nMethods\n\nThe study involved human participants with metabolic syndrome in aging and it was performed in accordance with the ethical standards of the local institutional committee (S. Andrea Hospital, ID number 67910) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent for participation and evaluation of data was obtained from the individual participants included in this study or from their caregivers, where ability to consent was affected by delirium.\n\nWe randomly (systematic sampling) enrolled 38 elderly patients (n = 11 males and n = 27 females) from the outpatients of the S. Andrea Hospital of Rome, in 2019. From their medical records, the patients met the criteria for probable AD dementia proposed by the National Institute on Aging-Alzheimer’s Association (NIA-AA)5 (Mini Mental State Examination (MMSE) score of 12.4+7.3, mean + standard deviation, in the moderate-severe stage of the disease). Prior to inclusion, subjects with other diseases, such as territorial infarction, intracranial haemorrhage, brain tumour, hydrocephalus, or severe white matter hyperintensities (WMH) were excluded from the study. Sixteen of the patients (16/38, 42% of the sample) were affected by type 2 diabetes (T2DM, according to 2020 American Diabetes Association, ADA, criteria)6, undergoing treatment with oral glucose lowering agents (n = 13) or with insulin (n = 3).\n\nEach patient received from our group: a) a comprehensive geriatric assessment (CGA), including the evaluation of the Activity of Daily Living (ADL), the Instrumental Activities of Daily Living (IADL), the Cumulative Illness Rating Scale (CIRS) and the Marigliano-Cacciafesta Polypathology Scale (MCPS)7,8; b) an anthropometric evaluation, including height, weight, body mass index (BMI), waist circumference, Mini Nutritional Assessment (MNA)9; c) a behavioural and psychological examination, using the Cornell’s scale, the Clinical Dementia Rating scale (CDR), the Neuropsychiatric Inventory (NPI) and the Confusion Assessment Method (CAM, with CAM-severity)10–12. An assessment of glucose metabolism, including fasting glycaemia and insulinaemia (excluded in insulin-treated patients), glycated haemoglobin (HbA1c) and insulin resistance by the means of the HOMA-index (glycemia mg/dl x insulinaemia µU/mol /405) (excluded in insulin-treated patients) and an evaluation of renal function, measuring creatinine and glomerular filtration rate (GFR) estimated using the Chronic Kidney Disease Epidemiology Collaboration formula (CKD-EPI), was performed using a clinical chemistry analyzer (c16000 Architect System, Abbott Laboratories). A continuous glucose monitoring (FreeStyle Libre system) was used in selected patients with a high risk of hypoglycaemic events, or with great variability of glycaemia during the day.\n\nFor the statistical analysis, carried out using Primer of Biostatistics Version 7, one-way analysis of variance (ANOVA) was used for the evaluation of the differences between the groups of patients. The relationship between two variables was examined by the means of regression analysis. A p<0.05 was assumed as significant. Data are presented as mean + standard deviation.\n\n\nResults\n\nThe clinical characteristics of the 38 AD patients with and without T2DM are described in Table 113.\n\nMMSE, Mini Mental State Examination; ADL, Activity of Daily Living; IADL, Instrumental Activities of Daily Living; MCPS, Marigliano-Cacciafesta Polypathology Scale; CIRS, Cumulative Illness Rating Scale; SI, severity index; BMI, body mass index, MNA, Mini Nutritional Assessment; CDR, Clinical Dementia Rating scale; CAM, Confusion Assessment Method; CAM-S, CAM-severity; NPI, Neuropsychiatric Inventory; HbA1c, glycated haemoglobin; GFR, glomerular filtration rate; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration formula; M, male; F, female.\n\np<0.05, p<0.01, **p<0.0001.\n\nThe MMSE scores were inversely and significantly related to CAM-S score (r = -0.585, p<0.001), while MCPS scores were positively related to the CAM-S score (r = 0.440, p<0.05) (Figure 1a and 1b).\n\na) The relationship between MMSE and CAM-S scores. b) The relationship between MCPS and CAM-S scores. MMSE, Mini Mental State Examination; CAM-S, Confusion Assessment Method-severity; MCPS, Marigliano-Cacciafesta Polypathology Scale.\n\nThe regression analysis between HbA1c and MMSE score showed a concave downward “∩” parabola (not significant) with apex at HbA1c of 10.5% and MMSE of 13.0. The regression analysis between HbA1c and the psychometric BPSD scales (Cornell, NPI and CAM-S) showed a “U” shape (concave upward parabola) (Cornell p<0.05, NPI<0.01 and CAM-S p<0.0001) (Figure 2). The apices of the parabolas were at HbA1c of 6.9%, 7.6% and 10.3%, the levels that minimized the Cornell, NPI and CAM-S scores, respectively (Figure 2). Therefore, the mean HbA1c that minimized all the behavioural disorders was 8.3%.\n\nContinuous glucose monitoring in selected patients (n = 3) showed that, even if HbA1c level was within the normal range, either a great variability in glycaemia during the 24 hours or repeated hypo-glycaemic events during the night (up to 26 hypoglycaemic events in a single patient) were present (Figure 3).\n\nA) Patient A, 81 years, MMSE=1.5, NPI=77, Cornell=13, CAM-S=17, HbA1c=6.6%, glucose mean 150 mg/dl, above/in/under the interval 56/43/1%, event of low glucose=0, mean duration=0. B) Patient B, 77 years, MMSE=13.7, NPI=30, Cornell=12, CAM-S=4, HbA1c=5.5%, glucose mean 72 mg/dl, above/in/under the interval 0/16/84%, event of low glucose=26, mean duration=296 min. C) Patient C, 82 years, MMSE=10.5, NPI=52, Cornell=18, CAM-S=11, HbA1c=5.7%, glucose mean 94 mg/dl, above/in/under the interval 7/54/39%, event of low glucose=4, mean duration=281 min.\n\n\nDiscussion\n\nIn our preliminary study, polypathology and frailty was associated with the severity of delirium as demonstrated by using the MCPS scale in correlation with the CAM-S scale.\n\nTo the best of our knowledge this is the first report about the association of the MCPS and CAM-S scale, whereas the MCPS was previously evaluated by our group in patients with comorbidities and frailty8.\n\nGlycaemic control was associated with modification of BPSD and delirium in the advanced phases of AD. In particular, delirium (as indicated by the CAM-S score) was more susceptible to the glycaemic control in its low range. As considered by point 7 (Metabolism and Nutritional state) of MCPS, high glucose levels increase the score of the MCPS scale (+25) only in conditions of diabetes mellitus decompensation, with significant risks of development of delirium.\n\nGlycaemic control modulated the appearance of BPSD and delirium with a polynomial U-shaped curve, with both the hyper- and hypo-glycaemic extremes of the glycaemic spectrum worsening BPSD. The regression equations showed that HbA1c levels between 6.9–10.3% (mean 8.3%) minimized BPSD, in agreement with International Diabetes Federation (2020)14 and ADA (2020)6 guidelines for managing T2DM in older people. As matter of fact, in healthy older subjects (with few coexisting chronic illnesses, intact cognitive and functional status) a reasonable HbA1c goal is <7.5% (with fasting or preprandial glucose of 90–130 mg/dl), whereas in complex/poor health patients (with more chronic illnesses, moderate-to severe cognitive impairment or >2 ADL dependencies) the reasonable HbA1c goal rises to <8.5 (with fasting or preprandial glucose of 100–180 mg/dl) to avoid possible overtreatment, hypoglycaemic events and fall risk.\n\nThe HbA1c value of 6% corresponds to a mean glucose of 126 mg/dl, with 95% prediction limits of 100–152 mg/dl, which includes the lower goals for glycaemia (100–180 mg/dl) suggested by ADA standard of care15–17 in older frail patients with moderate-severe dementia. More than half of our patients (22/38, 58%) showed HbA1c levels lower than 6%.\n\nThe reduction of one point (%) of HbA1c (from 6.8% to 5.8%) was associated with an increase of +2.2 points in Cornell, +7.0 points in NPI and +0.2 in CAM-S scores. Because of the parabolic profiles of the curves, another reduction of one point of HbA1c (from 5.8% to 4.8%) resulted in an exponential increase of BPSD scores (+5.6 Cornell, +14.4 NPI and +2.6 CAM-S).\n\nThe variability of glycaemia seemed to be associated with greater BPSD in AD patients, although it was not possible to carry out a statistical analysis because of the few number of patients in the sample.\n\nIn conclusion, the measurement of HbA1c in elderly AD patients with advanced dementia (with and without T2DM) is recommended in order to evaluate glycaemic control (and nutritional status). The reduction of mean glycaemic levels (due to malnutrition in non-diabetic patients or overtreatment in diabetic patients) should be avoided by means of a multidimensional approach.\n\n\nData availability\n\nDANS EASY: The Glycaemic Control and the Severity of the Delirium. https://doi.org/10.17026/dans-xq4-58fq13.\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\nRizzo MR, Marfella R, Barbieri M, et al.: Relationships between daily acute glucose fluctuations and cognitive performance among aged type 2 diabetic patients. Diabetes Care. 2010; 33(10): 2169–2174. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSakurai T, Kawashima S, Satake S, et al.: Differential subtypes of diabetic older adults diagnosed with Alzheimer's disease. Geriatr Gerontol Int. 2014; 14 Suppl 2: 62–70. PubMed Abstract \| Publisher Full Text\n\nYaffe K, Falvey C, Hamilton N, et al.: Diabetes, glucose control, and 9-year cognitive decline among older adults without dementia. Arch Neurol. 2012; 69(9): 1170–1175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosted E, Prokofieva T, Sanders S, et al.: Serious consequences of malnutrition and delirium in frail older patients. J Nutr Gerontol Geriatr. 2018; 37(2): 105–116. PubMed Abstract \| Publisher Full Text\n\nMcKhann GM, Knopman DS, Chertkow H, et al.: The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement. 2011; 7(3): 263–269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmerican Diabetes Association (ADA): Standard of medical care in diabetes-2020. Diabetes Care. 2020; 43: S1–S212. Publisher Full Text\n\nLinn BS, Linn MW, Gurel L: Cumulative illness rating scale. J Am Geriatr Soc. 1968; 16(5): 622–626. PubMed Abstract \| Publisher Full Text\n\nMartocchia A, Frugoni P, Indiano I, et al.: Screening of frailty in elderly patients with disability by the means of Marigliano-Cacciafesta polypathology scale (MCPS) and Canadian Study of Health and Aging (CSHA) scales. Arch Gerontol Geriatr. 2013; 56(2): 339–342. PubMed Abstract \| Publisher Full Text\n\nGuigoz Y, Vellas B, Garry PJ: Assessing the nutritional status of the elderly: The Mini Nutritional Assessment as part of the geriatric evaluation. Nutr Rev. 1996; 54(1 Pt 2): S59–S65. PubMed Abstract \| Publisher Full Text\n\nAlexopoulos GS, Abrams RC, Young RC, et al.: Cornell Scale for Depression in Dementia. Biol Psychiatry. 1988; 23(3): 271–284. PubMed Abstract \| Publisher Full Text\n\nCummings JL, Mega M, Gray K, et al.: The Neuropsychiatric Inventory: comprehensive assessment of psychopathology in dementia. Neurology. 1994; 44(12): 2308–2314. PubMed Abstract \| Publisher Full Text\n\nInouye SK, van Dyck CH, Alessi CA, et al.: Clarifying confusion: the confusion assessment method. A new method for detection of delirium. Ann Intern Med. 1990; 113(12): 941–948. PubMed Abstract \| Publisher Full Text\n\nMartocchia A: The Glycaemic Control and the Severity of the Delirium. DANS. 2020. http://www.doi.org/10.17026/dans-xq4-58fq\n\nDunning T, Sinclair A, Colagiuri S: New IDF Guideline for managing type 2 diabetes in older people. Diabetes Res Clin Pract. 2014; 103(3): 538–540. PubMed Abstract \| Publisher Full Text\n\nGlyconverter.tk: Glycated hemoglobin converter. Reference Source\n\nNathan DM, Kuenen J, Borg R, et al.: Translating the A1C assay into estimated average glucose values. [published correction appears in Diabetes Care 2009;32:207] Diabetes Care. 2009; 31(8): 1473–1478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuenen JC, Borg R, Kuik DJ, et al.: Does glucose variability influence the relationship between mean plasma glucose and HbA1c levels in type 1 and type 2 diabetic patients? Diabetes Care. 2011; 34(8): 1843–1847. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "85330", "date": "17 May 2021", "name": "Sandra Sigala", "expertise": [ "Reviewer Expertise Clinical pharmacology", "translational 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 Ms entitled \"The effects of the glycaemic control on the severity of the delirium in the advanced phase of Alzheimer’s disease\" by Martocchia and coll is very interesting and gives support to the evidence that glycemic control in the frail elderly is important in slowing cognitive degeneration/Alzheimer's onset.\nAuthors highlighted as well the importance in these patients of a comprehensive clinical evaluation, which includes both biochemical and functional values.\nThe main limit of the study is the sample size, but this point has been taken up and discussed by the Authors themselves.\nI have only minor points to underline:\nThe first sentence in the Introduction needs a reference\n\nCharacters in the Figure legend need to be unified - Figure 3 is hard to read\n\nThroughout the Ms, please correct typos and the language style\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6949", "date": "28 Jul 2021", "name": "Antonio Martocchia", "role": "Author Response", "response": "Dear Reviewer Prof Sandra Sigala, We really appreciated your review, discussing the importance of a comprehensive clinical evaluation in frail elderly with Alzheimer’s disease. We agree that the limit of study is the sample size: we completed the sentence in the manuscript in the Discussion: “The variability of glycaemia seemed to be associated with greater BPSD in AD patients, although it was not possible to carry out a statistical analysis because of the few number of patients in the sample (a limit of the study)”. We added the reference (1) and modified the first sentence: “Behavioral and psychological symptoms of dementia (BPSD) and delirium are common in the advanced phases of Alzheimer’s disease (AD) and worsen with disease severity, whereas symptoms of cognitive decline may be prevalent in the first phases of the disease (1)”. We unified the characters in the figure legends. We modified the figure 3. We corrected typos and the language style throughout the Ms. Yours sincerely Antonio Martocchia, MD PhD" } ] }, { "id": "89381", "date": "13 Jul 2021", "name": "Takahiko Nagamine", "expertise": [ "Reviewer Expertise psychopharmacology", "emergency medicine", "diabetes" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper analyzes HbA1c (blood glucose control status) and delirium in patients with Alzheimer's disease. It is interesting to note that both high and low blood glucose levels increase the risk of delirium. The paper is clinically significant because it shows that HbA1c as a predictor of delirium may be valid. If possible, two points should be considered: one is to conduct a study with separate analysis for the presence or absence of diabetes mellitus, and the other is to design the study to take into account the effects of antipsychotics and sleeping pills on blood glucose and delirium.\nAlzheimer's disease is caused by a decrease in neurotransmission in the acetylcholine system. The central acetylcholine nervous system may be less responsive to glucose loading. Older patients with Alzheimer's disease may occasionally experience hypoglycemia. This is due not only to the decline in acetylcholine and other neurotransmitters in Alzheimer's disease itself but also to antipsychotic drugs used for delirium 1.\nThe level of HbA1c is also affected by the type of hypoglycemic drug. For example, concomitant use of insulin secretagogues (e.g., sulfonylureas) and dopamine blockers can cause hypoglycemia and delirium-like disturbances in consciousness 2.\nTo avoid the effect of diabetic drugs, it is recommended to divide Alzheimer's disease into two groups, one with comorbid diabetes and the other without diabetes, and study the relationship between HbA1c and delirium in each group. Delirium is influenced by Alzheimer's disease, but also by medications used to treat insomnia (e.g., benzodiazepine receptor agonists), so it is advisable to adjust for medications that have a risk of inducing delirium as a background factor3.\n\nEven though there are confounding factors, the study of HbA1c as a predictor of delirium is very unique and a good perspective from the point of view that Alzheimer's disease is called diabetes of the brain.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6950", "date": "28 Jul 2021", "name": "Antonio Martocchia", "role": "Author Response", "response": "Dear Prof Takahiko Nagamine, We agree with the evidence about HbA1c as a predictor of delirium in Alzheimer’s disease and with the interest about the effects of the presence/absence of diabetes mellitus or concurrent therapies (antipsychotics and sleeping pills, as well as insulin secretagogues, e.g. sulfonylureas, and dopamine blockers) on HbA1c and delirium. In our sample of AD patients with diabetes, one subject was treated with repaglinide (we added a sentence in the text in methods). The percentage of use of drugs (antipsychotics, sleeping pills, antidepressants, antidementia, antiparkinsonian agents, mood stabilizing medications) in AD patients with or without diabetes, were respectively: 37%, 19%, 19%, 6%, 19%, 6%, and 32%, 18%, 23%, 9%, 9%, 9% (differences not significant) (we added a sentence in the text, results and discussion). One AD patients with diabetes was treated with zolpidem. We added the suggested references in the bibliography’s list. Yours sincerely, Antonio Martocchia, MD PhD" } ] } ]	1	https://f1000research.com/articles/9-1470
https://f1000research.com/articles/10-675/v1	28 Jul 21	{ "type": "Clinical Practice Article", "title": "Functional and non-functional types of adrenal tumors: a case series", "authors": [ "Dwiki Haryo Indrawan", "Fauriski Febrian Prapiska", "Syah Mirsya Warli", "Bungaran Sihombing", "Ginanda Putra Siregar", "Dwiki Haryo Indrawan", "Syah Mirsya Warli", "Bungaran Sihombing", "Ginanda Putra Siregar" ], "abstract": "Adrenal gland masses could be classified into functional, malignant, or benign. An adrenal cortical adenoma is one of the most common incidentalomas found with either functional or non-functional type. Pheochromocytoma is a neural crest cell origin tumor associated with catecholamine production. A classic triad of headache, sudden episodic perspiration, and tachycardia marked a pheochromocytoma. We report three patients with adrenal tumors. First, a 52-year-old woman with complaints of pain in the left flank suggests a left kidney tumor. The patient has an increased blood pressure intraoperatively. Adrenal cortical adenoma was found postoperatively. The second case is an Indonesian male 27-year-old with pain in the upper right abdomen. Intraoperative, the patient also has an escalation in blood pressure. Antihypertensive drugs are also used in this patient. Postoperatively, a pathology result of pheochromocytoma was revealed from this patient. The third case, adrenal myelolipoma, was suspected in a 48-year-old male and underwent surgery because of tumor growth. Later, a histopathological examination revealed myelolipoma of the adrenal. Management of adrenal tumor should be done individually based on each patient. In the first and second cases, blood pressure was unstable intraoperatively and was managed using several drugs, and was stable at follow-up. In the third case was no hemodynamic problem. In the case of an adrenal tumor, management tailoring should be based on the individual patient.", "keywords": [ "Adrenal cortical adenoma", "adrenal tumor", "pheochromocytoma", "adrenal myelolipoma" ], "content": "Introductions\n\nThe adrenal gland is a unique retroperitoneal endocrine organ that is different if compared with other retroperitoneal structures in embryological, anatomical, and their role in homeostasis. Tumors of the adrenal gland can be classified as functional or non-functional. The tumors may arise from the adrenal gland or could be secondary lesions. They may be benign or malignant.1,2 Tumors of the adrenal gland are relatively common. The prevalence of adrenal gland tumors is three to ten percent of the human population.3 Adrenal tumor sometimes found as an incidentaloma, which means the tumor more than 1 cm in size was found during an imaging study with indications other than the adrenal condition, but this excludes the patients who were done an imaging study during cancer work-up. Adrenal tumors are known to be one of the most commonly found incidentaloma.4–8\n\nAdrenal masses could be classified into functional, malignant, or benign masses. In functional terms, adenoma causing Conn’s of Cushing’s syndrome, pheochromocytoma, aldosteronoma, and adrenal carcinoma were included. While malignant were comes from metastases, carcinoma, lymphoma, or neuroblastoma. Last but not least, benign masses consisted of non-functioning adenoma, angiomyolipoma, cysts, and hemorrhage.9 Pheochromocytomas are neural crest cell tumors and linked with catecholamine production.10 The initial examination, usually using ultrasound as the diagnostic modality, could be detected in 90% of cases as an enlargement of the adrenal gland.11 The founding could be variable from solid to mixed cystic and solid to cystic.12 Pheochromocytomas patients often present with hypertension, tachycardia, headaches, palpitations, diaphoresis, chest pain, anxiety, and even weight loss. Around 10% of the patients do not have hypertension as their primary symptom.13\n\nAn adrenal cortical adenoma is one of the most common incidentalomas found.13 An adenoma mass should be grouped into functioning and non-functioning adrenal adenomas. It should be noted that functioning and non-functioning adenomas could not be classified using imaging techniques.14 Adenomas show a uniform hypoechoic mass relative to the fat. Adenomas are relatively small mass made them be difficult to be found during an ultrasound examination.9\n\nThe treatment for adrenal tumor requiring surgery usually because of the treatment failure in functional adrenal masses or a malignant tumor for either primary adrenal cortical carcinoma or solitary metastasis from nonadrenal sources, such as lungs, breasts, kidneys, and melanomas.15 Today, laparoscopic adrenalectomy is the first choice for adrenal disorder surgical procedures. But, there are some conditions that are requiring open adrenalectomies, such as patients with adrenal carcinoma, large pheochromocytoma with blood pressure that may be hard to control, and patients that are requiring a simultaneous abdominal procedure.16 But the absolute contraindications for adrenalectomy are extensive metastatic disease, uncorrected coagulopathy, and severe cardiopulmonary disease that precludes anesthesia.14 The authors report two cases of adrenal glands tumors requiring open adrenalectomy. This study was conducted with surgical CARE checklist guideline as a guidance.15\n\n\nCase 1\n\nA 52-year-old Indonesian female presented with pain on the left flank. Pain has been felt for six months. Hematuria was found. History of diabetes was found with the highest fasting glucose reported was 159 mg/dL, hypertension was found with a regular blood pressure of about 170/100 mmHg—patient with a history of percutaneous cardiac intervention four months ago. The patient was referred to our Regional Referral Hospital with a left adrenal tumor. The patient also has depression diagnosed by a psychiatrist having 23-score in Beck Depression Inventory-II (BDI II) questionnaire. It has been experienced by the patient two years ago, initiated by the death of her husband. The patient also felt a problem in her marriage, as she never felt intimate with her husband from the first day of their marriage. The patient was diagnosed with an adrenal tumor for the last five years, and since then, she has felt useless in her life. From the radiologic examination, we found a mass from the suprarenal organ, suggestive of an adrenal tumor as a conclusion (Figure 1). Thus, we used the left adrenal tumor as a working diagnosis.\n\nAdrenalectomy was performed for the left adrenal tumor. During the operation, unstable blood pressure was noted and could increase over 200 mmHg. Then the anesthesiologist used several medications to lower the blood pressure immediately. The problems with her blood pressure did not persist after the surgery. The highest blood pressure after adrenalectomy was 130/80 mmHg. Post adrenalectomy mass was sent to the pathology department in order to perform a pathology examination (Figure 2). The result came within one week after the procedures and revealed a picture of tumor cells forming nests is largely separated by fibrous fibers by an invasion of blood vessels. Tumor cells with rounded and oval nuclei are enlarged, rough chromatin, protruding nuclei, cytoplasm partly eosinophilic, partly clear, and bubbly. Abnormal mitosis is easy to find. The stroma consists of infiltrated fibrous connective tissue, with a conclusion of adrenal cortical adenoma.\n\nDuring the follow-up, the patient shows a normal condition, with normal blood pressure (maximum 125/80 mmHg) but the fasting glucose still not yet within the normal value.\n\n\nCase 2\n\nWe were reporting an Indonesian male 27-year-old with pain in the upper right abdomen. The pain has been experienced by the patient since one month ago and aggravating within this week. A mass was felt by the patient on the upper right abdomen. No hematuria was reported, no passing stone, no history of hypertension and diabetes. From physical diagnosis, we found a positive ballottement test on the right flank. Other physical examinations were within normal limits.\n\nDuring the preoperative preparation, blood pressure was within the normal limit. An initial complete blood test showed normal hemoglobin (12.3 g/dL), white blood cells (9,800/μL), and thrombocyte (319,000/μL). Normal glucose analysis was found, different from our first case.\n\nThe radiological examination found an adrenal tumor (Figure 3). Based on the radiological examination, the right adrenal tumor was suspected as our diagnosis.\n\nRight adrenalectomy was performed on December, 7th 2017. We found a bulging mass from the posterior. Then we opened the white line of Toldt. The next step was releasing the mass from lateral, superior, inferior, and the last, media. Anterior pedicels and branches from the vena cava and aorta towards the right adrenal were then ligated and cut. Intraoperatively, several antihypertensive drugs were used by the anesthesiologist to maintain the blood pressure at a lower level. Normal blood pressure was preserved after removal of the adrenal gland.\n\nThe mass on the right upper abdomen was inspected by the pathology department on December, 8th 2017, and after the next five days, a result was reported, which was a pheochromocytoma (Figure 4). The follow-up was done, and the patient did not show any problem with the blood pressure, blood glucose, and the complete blood count was normal.\n\n\nCase 3\n\nWe were reporting a 48-year-old male who came to urology clonic referred from digestive surgery consultant with an intraabdominal tumor suspected with adrenal myelolipoma. The mass was felt to increase in size for the last one month. No pain on palpation, no history of hematuria, cloudy urine, or passing stone. The patient also complained of fatigue and shortness of breath. On examination, hemodynamic was within normal limits. On urological examination, ballotement was found on the right flank without any tenderness. From computed tomography scan, an upper right abdomen mass was found, pushing liver to the superior and kidney to the inferior (Figure 5). The mass sized 12.8×10×10 cm, suspected of adrenal myelolipoma.\n\nWe performed a tumor removal for this patient (Figure 6). Intraoperatively, the hemodynamic remained stable. Based on this finding, we could rule out pheochromocytoma. Later, a histopathological examination revealed adrenal preparations appear to be the proliferation of mature fat cells with a round, oval, eccentric, smooth chromatin nucleus, numerous cytoplasm and clear, between groups of fat cells appear to group hematopoietic cells, at certain focus looks interstitial bleeding (Figure 7). No signs of malignancy were found in this preparation, the impression of Myelolipoma of Adrenal.\n\n\nDiscussion\n\nFrom this case series, we reported two cases of adrenal tumors, with one case of adrenal cortical adenoma and one case of pheochromocytoma. Adenomas are the most common benign tumors from the adrenal gland. From our first case, an adrenal cortical adenoma was found associated with diabetes, hypertension, and depression. This case is suggestive of a functional adrenal cortical adenoma. The glucocorticoids’ production from the adenoma could increase hypercortisolism, or known as Cushing syndrome.17\n\nCortisol production from benign adenomas often results from a unilateral hyperplastic mass, although a bilateral one might also happen. Systemic manifestations from hypercortisolism are central obesity, dyslipidemia, or hypertension.17 In our patient, we found hypertension without dyslipidemia or central obesity. On the other hand, one patient may come to clinical practice with a high blood glucose level, and when screened for hypercortisolism, he/she may have a subclinical Cushing syndrome.18 In our patient, we have a high blood glucose level, which becomes normal after adrenalectomy. Similar results were stated by Midorikawa and Mitchel. They found that an adrenalectomy procedure may improve glucose control together with hypertension.19,20 Cushing syndrome may also be related to depression. Labeur et al. also stated this manifestation in their study.21\n\nPheochromocytoma is a tumor of the catecholamine-producing cells of the adrenal medulla. The classic presenting sign of pheochromocytoma patients is paroxysmal hypertension, while the other could demonstrate persistent high blood pressure and normotensive.17 There is a classic hallmark triad of pheochromocytoma, headache, sudden episodic perspiration, and tachycardia.22 In our case, we failed to find any clinical manifestation, which has been stated earlier. Our patient has normal blood pressure without headache, perspiration, or even tachycardia. This might be similar to a study from Adler et al. They said as many as 20% of pheochromocytoma patients could present with no symptom at all.23\n\nIntraoperatively, both of our cases showed similar symptoms with a spike in blood pressure. This manifestation has also been reported by Kakoki et al.24 However, after the adrenalectomy procedure, the patients did not sustain any elevated blood pressure. This also happened during the follow-up.\n\nAdrenal myelolipoma is a rare, non-functional, and benign neoplasm of the adrenal gland.25 Because of the rarity, in the past adrenal myelolipomas, were found during the autopsy. Nowadays, because of radiological studies such as ultrasonography, computed tomography, and magnetic resonance imaging, incidentaloma has become more commonly found.26 The management of this tumor is usually conservative because most of them are asymptomatic. However, surgical intervention is suggested in a large tumor (larger than six cm).27 So, the management is based on the size and the symptoms of the tumor.28\n\n\nConclusions\n\nWe report a case series with three cases of adrenal tumor. The first case is an adrenal cortical adenoma suggestive a functional type. The second case is a pheochromocytoma without the classic hallmark. Both cases have unstable intraoperative blood pressure and have to be stabilized using several medications. During the follow-up, normotensive was achieved in both patients. The third cases have no hemodynamic disturbance in preoperative, intraoperative, and postoperative. In case of adrenal tumor, management tailoring should be based on individual patient.", "appendix": "Acknowledgement\n\nThe authors would like to thank everyone who supported this study. Special thanks are given to Adam Malik General Hospital and Universitas Indonesia which fully supported the authors during writing period.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patients.\n\n\nReferences\n\nMunver R, Yates JK, Degen MC: Surgical Anatomy of the Adrenals. In: Campbell-Walsh Urology. 11th Ed.Philadelphia: Elsevier; 2016; 1519–1527.\n\nPalter V, Devon K, Hallet J, et al.: Tumours of Adrenal Glands. In: Wright F, Escallon J, Cukier M, et al. (eds). Surgical Oncology Manual. Cham: Springer; 2016.\n\nElse T, Kim AC, Sabolch A, et al.: Adrenocortical Carcinoma. Endocr Rev. 2014; 35(2): 282–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdrenal UR: Surgery, Basic Science and Clinical Evidence. In: Norton JA, Bollinger RR (eds). Springer; 2001.\n\nLinos DA: Adrenal incidentaloma (adrenaloma). Hormones (Athens) . 2003; 2: 12–21. PubMed Abstract \| Publisher Full Text\n\nBulow B, Jansson S, Juhlin C, et al.: Adrenal incidentaloma - follow-up results from a Swedish prospective study. Eur J Endocrinol . 2006; 154: 419–423. PubMed Abstract \| Publisher Full Text\n\nAnagnostis P, Karagiannis A, Tziomalos K, et al.: Adrenal incidentaloma: a diagnostic challenge. Hormones (Athens) . 2009; 8: 163–184. PubMed Abstract \| Publisher Full Text\n\nKanagarajah P, Ayyathurai R, Manoharan M, et al.: Current concepts in the management of adrenal incidentalomas. Urol Ann . 2012; 4: 137–144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConder G, Rendle J, Kidd S, et al.: A-Z of Abdominal Radiology. Cambridge, UK: Cambridge University Press; 2009. Reference Source\n\nMittendorf EA, Evans DB, Lee JE, et al.: Pheochromocytoma: advances in genetics, diagnosis, localization, and treatment. Hematol/Oncol Clin North Am . 2007; 21: 509–525. PubMed Abstract \| Publisher Full Text\n\nKidneys HM, Glands A: Ultrasound Teaching Manual Hofer. Matthias, Th ieme . 1999; 37–50.\n\nBowerman RA, Silver TM, Jaffe MH, et al.: Sonography of adrenal pheochromocytomas. AJR Am J Roentgenol . 1981; 137: 1227–1231. PubMed Abstract \| Publisher Full Text\n\nPinto A, Barletta JA: Adrenal tumors in adults. Surg Pathol Clin. 2015; 8: 725–749. PubMed Abstract \| Publisher Full Text\n\nFarrugia FA, Martikos G, Surgeon C, et al.: Radiology of the adrenal incidentalomas: Review of the literature. Endocr Regul. 2017; 51: 35–51. PubMed Abstract \| Publisher Full Text\n\nAgha RA, Fowler AJ, Saeta A, et al.: The SCARE Statement: Consensus-based surgical case report guidelines. Int J Surg. 2016 Oct; 34: 180–186. PubMed Abstract \| Publisher Full Text\n\nLim SK, Rha KH: Surgery of the adrenal glands. In: Wein AJ, Kavoussi LR, Partin AW, et al., editors. Campbell-Walsh urology. 11th ed.Philadelphia: Elsevier; 2015. p.1577.\n\nVaughan ED: Open Approaches to The Adrenal Gland In: Smith JA, Howards SS, McGuire EJ, et al. (eds). Hinmans’s Atlas of Urologic Surgery. 3rd ed. Philadelphia: Elsevier. 2010. p. 1103–1110.\n\nKutikov A, Crispen PL, Uzzo RG: Pathophysiology, evaluation, and medical management of adrenal disorders. In: Wein AJ, Kavoussi LR, Partin AW, et al., editors. Campbell-Walsh urology. 11th ed. Philadelphia: Elsevier; 2015. p.1535–1547.\n\nCatargi B, Rigalleau V, Poussin A, et al.: Occult Cushing’s syndrome in type-2 diabetes. J Clin Endocrinol Metab . 2003; 88(12): 5808–5813. PubMed Abstract \| Publisher Full Text\n\nMidorikawa S, Sanada H, Hashimoto S, et al.: The improvement of insulin resistance in patients with adrenal incidentaloma by surgical resection. Clin Endocrinol (Oxf) . 2001; 54(6): 797–804. PubMed Abstract \| Publisher Full Text\n\nMitchell IC, Auchus RJ, Juneja K, et al.: “Subclinical Cushing’s syndrome” is not subclinical: improvement after adrenalectomy in 9 patients. Surgery. 2007; 142(6): 900–905, discussion 905.e1. PubMed Abstract \| Publisher Full Text\n\nLabeur M, Arzt E, Stalla GK, et al.: New perspectives in the treatment of Cushing’s syndrome. Curr Drug Targets Immune Endocr Metabol Disord. 2004 Dec; 4: 335–342. PubMed Abstract \| Publisher Full Text\n\nBravo EL, Tagle R: Pheochromocytoma: state-of-the-art and future prospects. Endocr Rev . 2003; 24(4): 539–553. PubMed Abstract \| Publisher Full Text\n\nAdler JT, Meyer-Rochow GY, Chen H, et al.: Pheochromocytoma: current approaches and future directions. Oncologist . 2008; 13(7): 779–793. PubMed Abstract \| Publisher Full Text\n\nKakoki K, Miyata Y, Shida Y, et al.: Pheochromocytoma multisystem crisis treated with emergency surgery: A case report and literature review. BMC Res Notes. 2015 Dec 9; 8: 758. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeng C, Jiang H, Ding Q, et al.: Adrenal myelolipoma: a mingle of progenitor cells? Med Hypotheses. 2013; 80: 819–822. PubMed Abstract \| Publisher Full Text\n\nWani N, Kosar T, Rawa I, et al.: Giant adrenal myelolipoma: incidentaloma with a rare incidental association. Urol Ann. 2010; 2(3): 130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNabi J, Rafiq D, Authoy F, et al.: Incidental detection of adrenal myelolipoma: a case report and review of literature. Case Rep Urol. 2013; 2013: 1–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamirez M, Misra S: Adrenal myelolipoma: To operate or not? A case report and review of the literature. Int J Surg Case Rep. 2014; 5(8): 494–496. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "128202", "date": "11 Apr 2022", "name": "Christina Pamporaki", "expertise": [ "Reviewer Expertise Adrenal diseases" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the article by Indrawan et al. the authors describe a case series of three patients with adrenal masses. In particular, they describe a case with adrenal cortical adenoma, a second case with pheochromocytoma and a third with an adrenal myelolipoma.\nI regret to say, that the article should not be approved for indexing. The article has scientific misinformation. For instance, the authors indicate that the usual examination for the pheochromocytoma is the ultrasound or that only 10% of patients with pheochromocytoma do not present hypertension, whereas the truth is that many patients with pheochromocytoma have normal blood pressure levels (see reference list and attached document for examples) and the diagnostic imaging of choice after the biochemical confirmation of a pheochromocytoma is the CT (or MRI in certain cases). Another example is the part where the authors indicate that adrenalectomy is totally contradicted in cases of synchronous metastatic disease. That is also misleading. In many cases disease debulking has been associated with better survival for some patients.\nThe authors do not describe in detail the physical examination, or the diagnostic procedures followed in daily routine care for adrenal lesions (e.g., concentrations of plasma or urinary metanephrines, steroids, dexamethasone suppression test, assays used for the metabolites, upper cut offs of reference intervals). The discussion does not include information or conclusions based on findings that could add to our understanding of the disease processes, diagnosis or treatment.\nThe article will require copy editing. In addition, the article adds little on what is already known in the literature and lacks novelty.\n\nIs the background of the cases’ history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the conclusion balanced and justified on the basis of the findings? No", "responses": [ { "c_id": "8213", "date": "20 May 2022", "name": "Fauriski F Prapiska", "role": "Author Response", "response": "Thank you for your thoughtful remarks. We appreciate the time and effort that you have dedicated to provide feedback on our manuscript and are grateful for the comments on our paper. We would like to address a few topics that have been raised. We thank the reviewer for pointing this out. USG is utilized as an initial diagnostic technique, not as a gold standard. According to Campbell-Walsh-Wein Urology, Computerized Tomography Scan is the gold standard in adrenal imaging. We appreciate the reviewers insightful suggestion. However, as stated by Zuber, Kantorovich, and Pack in the metabolism Clinics of North America Journal, roughly 5% to 15% of pheochromocytoma patients have normal blood pressure. Once again, we thank you for the time you put in reviewing our paper. Best regards, Fauriski F Prapiska (Corresponding author)" } ] }, { "id": "301636", "date": "23 Sep 2024", "name": "Vipula Kolli", "expertise": [ "Reviewer Expertise adrenal gland", "myelolipomas", "adrenal rest tissue tumors", "endocrinology", "CAH" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nF1000 Clinical Practice Article Functional and non-functional types of adrenal tumors: a case series.\nMINOR COMMENTS:\nThe authors of this article have reported three cases of adrenal mass tumors requiring an open adrenalectomy including one adrenal myelolipoma. They also reported that two out of three cases had intraoperative increased blood pressure and blood pressure was stabilized during the follow-up.\nThis article is reviewing about three articles and their differential diagnosis. Sometimes, diagnosing an adrenal mass can be challenging if the lesions doesn’t readily show typical imaging features.\nStrengths: Authors have successfully described the whole history of the patients. Weakness: Authors should have provided a review of literature of radiological and hematological findings and diagnoses of adrenal masses in the discussion section.\n\nADDITIONAL INFORMATION:\nIn this study authors described the three cases of adrenal tumors. However, little bit more patient information including symptoms and values can help in understanding the cases more. Proofreading should be done throughout the manuscript and discussion should be expanded.\n\nIs the background of the cases’ 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 conclusion balanced and justified on the basis of the findings? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-675
https://f1000research.com/articles/10-673/v1	28 Jul 21	{ "type": "Research Article", "title": "Metronomic oral vinorelbine doublet chemotherapy with carboplatin in treatment of advanced lung cancer: a feasibility and safety study", "authors": [ "Maria Kandi", "Peter Meldgaard", "Marianne Marquard Knap", "Peter Meldgaard", "Marianne Marquard Knap" ], "abstract": "Background: Non-small cell lung cancer (NSCLC) is globally one of the most common forms of cancer. Palliative treatment is a delicate balance against toxicity and survival. Using small frequent doses of chemotherapy, metronomic regimens have been hypothesized to maintain or even improve efficacy while achieving a lower treatment-related toxicity. The mechanism is thought to result from a more continuous exposure of the tumour cells to the drugs. Treating NSCLC, this study addresses the feasibility and tolerability of carboplatin in combination with 12 weeks of daily metronomic vinorelbine. Method: Patients were included over a period of ten months. All patients had biopsy-verified incurable NSCLC and were candidates for first line chemotherapy (PD-L1<50% and no targetable mutations). This open label, non-randomized prospective safety and feasibility study was investigator initiated. Patients received up-to four cycles of standard dose carboplatin AUC 5 every third week in combination with 12 weeks of metronomic oral daily Navelbine® (20/30 mg). Patients were evaluated by CT scans after end of treatment and then every 8 weeks (+/- 1 week) until progression. Results: A total of 20 patients were included. Male/female-ratio was 4/16. Age ranged from 49-83 with a median of 70.5 years. Majority had adenocarcinoma (95%). Two patients withdrew their consent within a week. 18 patients were included in safety analysis. 13 received all four cycles. Grade 1/2 toxicity was frequently seen and included fatigue 13 (72%), diarrhoea 13 (72%), constipation/congestion 13 (72%). Grade 3 toxicities were dyspnoea 2 (11%), nausea 3 (17%) and fatigue 3 (17%). Two (11%) had grade 4 toxicity with neutropenic fever, both recovered. No grade 5 toxicity was detected. Conclusion: In treatment of NSCLC this study is the first addressing the regimen of carboplatin in combination with daily metronomic vinorelbine. We conclude that doublet chemotherapy with daily vinorelbine is safe and feasible.", "keywords": [ "NSCLC", "Oral vinorelbine", "Feasibility", "Metronomic", "Chemotherapy", "Palliation", "Doublet regimen", "Daily" ], "content": "Background\n\nNon-small cell lung cancer (NSCLC) is the second most common cancer worldwide.1 In Denmark 4,658 new cases were diagnosed yearly from 2012 to 2016.2 The treatment goal for patients with metastatic NSCLC is to relieve symptoms and prolong survival while maintaining the best possible quality of life. Palliation, therefore, needs to be balanced against survival and toxicity. In most countries platinum based combination chemotherapy has been the standard first line treatment in NSCLC for decades, and in Denmark platinum has been used in combination with vinorelbine in NSCLC (www.dolg.dk).\n\nWhen chemotherapy is given according to conventional principles, the treatment is typically given in high doses every three weeks, as the goal is to maximize the therapeutic outcome. However, because of the toxicity, there is a need for treatment-free intervals between the cycles, which could provide space and time for the development of resistant clones and regrowth of the tumour cells.3 Furthermore, the immunosuppressive effect of the chemotherapy can lead to infections and the need of antibiotic therapy and hospitalization and can ultimately be fatal.\n\nCompared to classical cytotoxic chemotherapy, metronomic treatment regimens may have a complementary mechanism of action, targeting the tumour vasculature, thus counteracting tumour regrowth that may occur between chemotherapy cycles; in addition, metronomic chemotherapy suppresses regulatory T cells and induces the maturation of dendritic cells, thereby leading to an anti-tumour immune response.4,5 Metronomic regimens, in which small, frequent doses of chemotherapy are administered, have been suggested to lower treatment-associated toxicities.6 The high frequency of administration aims to expose tumour cells continuously to the drug, preventing recovery between cycles and possibly improving tumour control.7 Preclinical studies, as well as clinical observations, suggest that metronomic regimen increases the effect of a drug, not only by a direct effect on cancer cells, but also by activating endothelial cells in the tumour vasculature and thereby the tumour anti-angiogenesis. By giving smaller, but more frequent, doses of the drug, higher dose intensity, without corresponding side effects, is obtained.8\n\nIn a randomized phase II study, conducted in NSCLC patients, toxicity and efficacy between intravenous and oral administered vinorelbine, seemed comparable, and safety profiles of both formulations appeared to be qualitatively similar.9 Easy oral administration of vinorelbine, allows for flexible treatment schedules including a more frequent and metronomic dose application, and makes it an obvious candidate for metronomic studies.\n\nMany studies have focused on comparing the efficacy of different drugs,10 but little interest has been given to the posology.11–16 Phase II studies with weekly intravenous vinorelbine 25-30 mg/m2 as first line therapy to advanced breast cancer patients showed response rates of 41-50%, and acceptable toxicity profile.11–14 A previous phase I dose finding study in pre-treated NSCLC patients showed that metronomic daily oral vinorelbine as monotherapy, given in cycles of four weeks with one week of drug holiday, was safe and feasible in recommended dose level of 30-40 mg daily.17 Platinum-based doublet chemotherapy in a phase I dose finding study determined that cisplatin 85 mg/m2 every third week in combination with oral vinorelbine 60 mg three times weekly was feasible and active in advanced NSCLC.16 Upon registration of a drug there is often limited knowledge of the best dosing and there could be much to gain in this field. Metronomic chemotherapy is not yet well validated in the clinic, and the optimal dosage and regimens are yet to be identified.\n\nThere are no published studies with daily oral vinorelbine in combination with carboplatin for treatment of patients with advanced non-small cell lung cancer. The current study aims to test a daily dosage of vinorelbine 20/30 milligram (mg) in combination with carboplatin focusing on the safety aspect and tolerability. If this outcome is achieved this dosing schedule would be usable in future randomised combination studies with carboplatin including maintenance arm with vinorelbine in lung cancer treatment.\n\n\nMethods\n\nThis study was investigator-initiated and designed as a prospective non-randomized, open-label, single site, safety, and feasibility study. It was planned to recruit 20 patients. Patients with incurable NSCLC, who were chemo-naïve and candidates for 1st-line chemotherapy (PD-L1 < 50%), were included. Patients included in the study received standard dose of carboplatin AUC 5 every 3rd week and metronomic oral Navelbine® (20/30 mg) daily for 12 weeks. After four cycles of chemotherapy, if partial response or stable disease, patients with non-squamous carcinoma received pemetrexed as maintenance chemotherapy according to national standard treatment recommendation (www.dolg.dk). Patients were followed by computer tomography (CT) scans for evaluation every second month and evaluated according to Response Evaluation Criteria In Solid Tumours (RECIST).18 The study design is shown in Figure 1. Each patient received first cycle as carboplatin AUC 5 day 1 and Navelbine® 20 mg daily in first 3 weeks, and if there was acceptable toxicity, the dose of Navelbine® was escalated to 30 mg daily. A maximum of four cycles were delivered. For safety reason the first six patients enrolled had World Health Organisation (WHO) performance status (PS) 0-1. The next fourteen patients included were allowed to have PS 0-2.\n\nThere was incorporated an interim analysis in the study. We proceeded with the analysis when the 10th patient had received 2 cycles (6 weeks) of treatment or if 4 patients experienced an unacceptable toxicity (UT; see definition in appendix A in extended data in figshare datafile19), despite the dose reduction of Navelbine and carboplatin or in case of any grade 5 toxicity.\n\nDose adjustment plan and definition: A dose adjustment of chemotherapy was permitted according to treatment modification guidelines at the Department of Oncology, Aarhus University Hospital (see guidelines for treatment modification in appendix A in extended data in figshare datafile.19). Dose adjustment was allowed in the case of significant haematological and/or non-haematological toxicity. Generally, dose adjustment and/or treatment delay was decided at the respective visit based on symptoms, physical examination and haematological values (see Dose Adjustment section, appendix A in extended data in figshare datafile19).\n\nPatients were instructed to report any toxicity that occurred between cycles or in between visits. Moreover, during the entire treatment period patients kept a diary. They registered dose and time of taking Navelbine and brought their diary on every visit. The patients were asked to register any adverse event in the diary. There were scheduled five visits during the 12 weeks of treatment. Regarding antiemetics there were no restrictions. Metoclopramide was given in addition to the standard antiemetics (oral prednisolone 75 mg day 1, 50 mg day 3-4 and 25 mg day 5; aprepitant 125 mg day 1, 80 mg day 2-3 and ondansetron 16 mg day1-2), when treating with a standard carboplatin and vinorelbine regimen. Significant effort has gone into using the same antiemetics across patients in the study.\n\nSafety assessment: Symptoms and haematological values were measured at baseline and then every third week during the four cycles. Additional haematological values were measured at day 12 ± 2 during the first cycle to register any haematological side effects. If haematological toxicity occurred the patient was contacted by telephone.\n\nAll toxicities were graded according to the Common Terminology Criteria for Adverse Events (CTCAE version 4.03), during the first 84 days (12 weeks) of the treatment period. Toxicity was registered as patient-reported outcome (via online records/AmbuFlex®), every 3rd week during the treatment period. The focus was on the following: febrile neutropenia, neutropenia (reported only by study-nurse or physician according to blood tests), any fever, hospitalization, vomiting, diarrhoea, stomatitis, anorexia, dyspnoea, weight loss, thromboembolic events and any other adverse events. All unacceptable toxicities (UTs) such as febrile neutropenia, thrombocytopenia or any non-haematological grade 3 and 4 toxicity were defined (see unacceptable toxicities (UTs), appendix A in extended data in figshare datafile19).\n\nThis study was designed and performed according to the fifth version of the Declaration of Helsinki, the protocol, ICH-GCP guidelines and national laws. The protocol was approved by the Research Ethics Committee case no.: 1-10-72-187-17; the Danish Data Protection Agency (Datatilsynet) case no.: 1-16-02-741-17; the national Medical Products Agency no.: 2017083768; with EUDRACT no: 2017-000659-23; https://www.clinicaltrialsregister.eu/ctr-search/search?query=2017-000659-23, and monitored by the GCP unit, Aarhus university, Denmark.\n\nPatient records and case report forms obtained during the study, were archived by the oncology department and at the Clinical Research Unit, Aarhus University Hospital, for at least 5 years after the completion. The study was conducted respecting the patient’s physical and mental integrity and privacy. The investigator ensured that each study patient was fully informed about the nature and objectives of the study and possible risks associated with participation. The investigator and the co-investigators obtained written informed consent from each patient before any study-specific activity was performed.\n\n\nStatistical methods\n\nThe objective of this study is to evaluate the safety and feasibility of a combination of carboplatin AUC 5 every 3rd week with vinorelbine 20/30 mg daily during 12 weeks in patients with advanced or metastatic NSCLC. Assuming overall sample size of around 200 patients in a future randomised phase II study with the same study design we will need about 10% of overall sample size to examine safety and feasibility. In this study we estimated that 20 patients would be sufficient to give the information about the safety and feasibility of metronomic therapy in combination with carboplatin, when using the Simon’s Two-Stage for phase II clinical trial.20\n\nContinuous data is summarized with the following items: frequency, median, (if n ≥ 3), range and mean. Characteristics of the disease at diagnosis and demographic data at baseline was tabulated on all included patients.\n\nOverall survival (OS) for the whole group was calculated using the Kaplan-Meier method. Progression free survival was calculated from date of inclusion until progression. OS was measured from the date of inclusion just before starting the first line chemotherapy until the date of death from any cause or the last date the patient was known to be alive. OS analyses were carried out using STATA version 16 (Stata, RRID:SCR_012763); an open-access alternative is R (https://www.r-project.org/).\n\n\nResults\n\n20 patients with inoperable or disseminated, pathologically proven NSCLC, from May 2018 to February 2019, were included. The median age was 70.5 years (range 49-83). 19 patients (95%) had adenocarcinoma. Patient characteristics are shown in Table 1. Two patients withdrew their consent within one week; one decided not to receive any palliative treatment; the other decided to receive standard treatment, as the patient could not cope with protocol requiring an extra blood sample during the first cycle. These two patients are only included in the intent-to-treat population in the overall survival analyses.\n\n* PD-L1, Programmed Cell Death Ligand 1.\n\n18 patients were evaluable for toxicity analysis as shown in the flowchart in Figure 2. These eighteen patients received a total of 62 cycles of carboplatin in addition to daily oral vinorelbine. The median dosage of oral vinorelbine received per patient was 1780 mg (range 260-2370 mg). 13 patients received all four cycles of chemotherapy (see appendix B, fig 5 in extended data in figshare datafile21). One patient received only three cycles, as the physician clinically suspected progression, although the following evaluating CT-scan showed stable disease. Four patients received only 1-2 cycles of chemotherapy; one patient had clinical progression (without verifying CT scan); one patient had decline in PS and was terminated from study (see flowchart, Figure 2); two patients had clinical disease progression during the treatment time and performed an evaluation CT scan in advance.\n\nThe majority of patients had grade 1 or 2 toxicity. Frequent toxicities (any grade) included fatigue 13 (72%), diarrhoea 13 (72%), constipation 13 (72%), nausea 9 (50%) and dyspnoea 12 (67%). Some patients had more than one toxicity. Grade 3 toxicities were dyspnoea 2 (11%), nausea 3 (17%) and fatigue 3 (17%) as shown in toxicity Table 2. These mentioned toxicities led to a brief break in daily vinorelbine intake and dose reduction according to dose adjustment guidelines (in appendix A in extended data in figshare datafile).19 Two patients (11%) had febrile neutropenia as a grade 4 toxicity: one patient recovered after three weeks and continued the treatment in reduced dose; the other patient recovered after six days, but did not receive any treatment hereafter, according to investigator’s decision, as the PS disqualified the patient from any further treatment (exacerbation of patient’s chronic obstructive lung disease). Both febrile neutropenia events occurred at day 12 and 13 after the first cycle, respectively. None of the patients included in the study had cumulative haematological toxicity. No patients in the study had grade 5 toxicity.\n\nSix (46%) of the thirteen patients who received all four cycles did not have reduction in vinorelbine doses (20/30 mg), even though the total dosage varies per patient from 2200-2370 mg, as treatment cycle period varied from 19-23 days. Five patients received a reduced dosage of vinorelbine from the second cycle (stayed at 20 mg without dose escalation to 30 mg): one patient had a single rise in alanine aminotransferase (ALT) grade 2 at first cycle; one had diarrhoea grade 2; one had fatigue and weight loss grade 3; one had dose interruption in first cycle because of febrile neutropenia grade 4 and started the second cycle at 20 mg (a dose reduction) and one patient continued at 20 mg in all treatment periods, because of the patient’s age, 80 years (physician’s decision). Two patients received full doses of chemotherapy up to the third cycle but had an interruption of 3-5 days in vinorelbine intake at the end of the fourth cycle, because of fatigue and nausea grade 2. Four patients did not receive more than 1 to 2 cycles due to degrading in PS and/or disease progression as mentioned above.\n\nThe total dose of vinorelbine administered per patient per cycle and response characteristics is shown in appendix B, fig 5, in extended data in figshare datafile.21\n\nMedian follow-up time was 22.1 months (95% CI: 18.9 – 25.2 months). At the end of the study, four patients were alive. Sixteen patients had eligible progression free survival (PFS) data for analysis. Median PFS was 4.8 months (95% CI: 4.3 – 5.2 months). Median overall survival was 14.1 months (95% CI: 5.9 – 18.6 months) for the entire population, see Figure 3. Five patients (28%) out of 18 patients had partial response (PR), and six patients (33%) had stable disease (SD). Five patients (28%) had progressive disease (PD) in evaluation scans. One of these patients had regression in target lesion but was considered PD because of newly developed malignant pleural exudate (as marked with asterisk in the waterfall plot, Figure 4). Two patients could not go through evaluation with CT scans, but developed clinical symptoms of progression and decline in PS. Response rate (RR) in this feasibility and safety study was 61% (11 out of 18 patients).\n\n*Patient had regression in evaluable target lesion, but CT scan showed a new lesion:\n\nmalignant pleural exudate.\n\n\nDiscussion\n\nThis study showed that metronomic oral vinorelbine 20/30 mg daily in combination with carboplatin AUC 5 was safe and feasible. The most frequent toxicities such as fatigue, diarrhoea, and constipations have been manageable.\n\nOral vinorelbine as metronomic monotherapy has been evaluated, using different dose regimens, such as fractionated regimen (day 1, 3 and 5) and daily intake.22–26 In patients with NSCLC the daily intake of oral vinorelbine up to 40 mg, as monotherapy, was well-tolerated.22,23 In a study, Tufman et al. showed no relevant accumulation in the pharmacokinetic analysis, using vinorelbine as monotherapy in a daily dosing of 30-40 mg.15 Oral vinorelbine is rapidly absorbed at 80 mg/m2 (Tmax 1.4 ± 0.7 hours) and shows a bioavailability of 43% ± 14%.8 The absorption is not affected to a significant extent by food.27\n\nOther studies have focused on metronomic oral vinorelbine as combination chemotherapy. A dose finding phase I doublet chemotherapy study of oral vinorelbine 50 mg 3 times weekly in combination with cisplatin 80 mg/m2 (day 1 every 3 weeks) showed no haematological toxicities. With escalation of vinorelbine up to 70 mg 3 times weekly plus 85 mg/m2 cisplatin per cycle, grade 3 or 4 toxicities were dose limiting.16 The Hellenic Oncology Research Group (HORG), in a phase II study, evaluated the safety and efficacy of metronomic vinorelbine in combination with cisplatin as first line treatment in patients with advanced NSCLC.28 Patients were treated with cisplatin (80 mg/m2) in combination with oral metronomic vinorelbine (60 mg, three times weekly) in cycles of 21 days. They reported myelosuppression as the main adverse event with grade 3 and 4 neutropenia occurring in five (14.3%) and six (17.1%) patients, respectively.28\n\nSeveral breast cancer studies, with metronomic vinorelbine concomitant with capecitabine, have also found that vinorelbine three times weekly is safe and has unchanged efficacy.29–31 Two of these studies indicate that it might be possible to improve both efficacy and safety with daily metronomic therapy.\n\nThere are no published studies of daily oral vinorelbine, without any planed break, in combination with carboplatin for treatment of NSCLC.\n\nIn the current study, we examined whether the doublet chemotherapy with carboplatin every third week concomitant with daily oral vinorelbine in 12 weeks was safe and feasible. Fatigue, constipation, and diarrhoea grade 2 was the most frequent reason for short interruptions in daily intake or dose reduction of vinorelbine to 20 mg daily. We found that the above-mentioned doublet regimen in treatment of NSCLC patients is feasible with both 20 mg daily with escalation to 30 mg daily or only 20 mg daily in the entire treatment period. The observed partial response or stable disease (in 11 out of 18 patients) and disease progression were not correlated with the daily dose of vinorelbine as shown in appendix B, fig 5, in extended data in figshare datafile.21 The study design, carboplatin in combination with daily oral vinorelbine 20 mg (or 20/30 mg) without planned breaks, could be useful in future studies, that includes metronomic doublet regimens.\n\nDuring the first cycle haematological quantities were also measured at day 12 ± 2 and showed no significant reduction in neutrophil counts. Therefore, routine measuring of haematological quantities at day 12 ± 2 in the first cycle may not be necessary. The tolerability profile, with grade 3; fatigue 17%, nausea 17%, grade 4; febrile neutropenia 11% and no grade 5 toxicity, was acceptable and comparable with historical doublet regimen with cisplatin every third week and metronomic vinorelbine 60 mg, 3 times weekly, in first line chemotherapy in NSCLC.28 Progression free survival of 4.8 months in this study was comparable with other studies using platinum based standard doublet treatments with vinorelbine (PFS: 4.2 months) or pemetrexed (PFS: 4.3 months).32\n\nThis study was designed to determine the safety and clinical feasibility and was not powered to conclude on efficacy. Therefore, the trend of higher RR and longer OS is associated with a high level of uncertainty.\n\nIn conclusion, carboplatin in combination with metronomic daily oral vinorelbine 20/30 mg without any pre-planned breaks is a safe and feasible option in the treatment of patients with NSCLC. The toxicity rates, PFS and OS were comparable with standard regimens.\n\n\nData availability\n\nFigshare: Underlying data for ‘Metronomic oral vinorelbine doublet chemotherapy with carboplatin in treatment of advanced lung cancer: a feasibility and safety study’. https://doi.org/10.6084/m9.figshare.14849760.v133\n\nThis project contains the following underlying data.\n\n• F1000Res NavMetroData.xlsx (Complete raw demographic and clinical information for each patient included in this study).\n\nFigshare: CONSORT and TREND checklists for ‘Metronomic oral vinorelbine doublet chemotherapy with carboplatin in treatment of advanced lung cancer: a feasibility and safety study’. https://doi.org/10.6084/m9.figshare.14958543\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThis study was designed, initiated, conducted and analysed under the responsibility of Aarhus University Hospital (AUH), department of oncology. The authors would like to thank the patients for their participation and the families for their support, the staff of the study site and the clinical research unit in department of oncology, AUH, Denmark, all of whom significantly contributed to the success of the study.\n\nThe authors would like to thank Nina Voldby, PhD, MD, pulmonology department, AUH, as a member of steering committee.\n\n\nReferences\n\nSociety T.A.C.: Key Statistics for Lung Cancer.2017. Reference Source\n\nBekæmpelse K: Kort om lungekræft.Reference Source\n\nGregory RK, Smith IE: Vinorelbine--a clinical review. Br J Cancer. 2000; 82(12): 1907–1913. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKerbel RS, Kamen BA: The anti-angiogenic basis of metronomic chemotherapy. Nat Rev Cancer. 2004; 4(6): 423–436. PubMed Abstract \| Publisher Full Text\n\nTorimura T, et al.: Metronomic chemotherapy: possible clinical application in advanced hepatocellular carcinoma. Transl Oncol. 2013; 6(5): 511–519. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWichmann V, et al.: Recent clinical evidence on metronomic dosing in controlled clinical trials: a systematic literature review. Acta Oncol. 2020; 59(7): 775–785. PubMed Abstract \| Publisher Full Text\n\nBarletta G, et al.: Oral vinorelbine in the treatment of non-small-cell lung cancer. Expert Opin Pharmacother. 2014; 15(11): 1585–1599. PubMed Abstract \| Publisher Full Text\n\nMarty M, et al.: Oral vinorelbine pharmacokinetics and absolute bioavailability study in patients with solid tumors. Ann Oncol. 2001; 12(11): 1643–1649. PubMed Abstract \| Publisher Full Text\n\nJassem J, et al.: A multicenter randomized phase II study of oral vs. intravenous vinorelbine in advanced non-small-cell lung cancer patients. Ann Oncol. 2001; 12(10): 1375–1381. PubMed Abstract \| Publisher Full Text\n\nSchiller JH: Current standards of care in small-cell and non-small-cell lung cancer. Oncology. 2001; 61(Suppl 1): 3–13. PubMed Abstract \| Publisher Full Text\n\nFumoleau P, et al.: Phase II trial of weekly intravenous vinorelbine in first-line advanced breast cancer chemotherapy. J Clin Oncol. 1993; 11(7): 1245–1252. PubMed Abstract \| Publisher Full Text\n\nGarcía-Conde J, et al.: Phase II trial of weekly IV vinorelbine in first-line advanced breast cancer chemotherapy. Ann Oncol. 1994; 5(9): 854–857. PubMed Abstract \| Publisher Full Text\n\nTwelves CJ, et al.: A phase II, multicentre, UK study of vinorelbine in advanced breast cancer. Br J Cancer. 1994; 70(5): 990–993. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBruno S, et al.: Phase II trial of weekly i.v. vinorelbine as a single agent in first-line advanced breast cancer chemotherapy. The Latin-American experience. Am J Clin Oncol. 1995; 18(5): 392–396. PubMed Abstract \| Publisher Full Text\n\nTufman A, et al.: Treatment of advanced recurrent NSCLC with daily oral vinorelbine: A phase I trial. J Clin Oncol. 2013; 31(15_suppl): e19108–e19108. Publisher Full Text\n\nPallis AG, et al.: A multicenter phase I trial of metronomic oral vinorelbine plus cisplatin in patients with NSCLC. Cancer Chemother Pharmacol. 2011; 67(6): 1239–1245. PubMed Abstract \| Publisher Full Text\n\nGuetz S, et al.: Metronomic treatment of advanced non-small-cell lung cancer with daily oral vinorelbine – a Phase I trial. Onco Targets Ther. 2017; 10: 1081–1089. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEisenhauer EA, et al.: New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45(2): 228–247. PubMed Abstract \| Publisher Full Text\n\nKandi M: Toxicity and dose adjustment: Definition of Unacceptable Toxicity (UT) and guideline for treatment modification at department of oncology in Aarhus, Denmark.Publisher Full Text\n\nSimon R: Optimal two-stage designs for phase II clinical trials. Control Clin Trials. 1989; 10(1): 1–10. PubMed Abstract \| Publisher Full Text\n\nKandi M: Total dose of vinorelbine administered per patient per cycle and response characteristic.Publisher Full Text\n\nTufman A, et al.: Tumor conference II. Onkologie. 2013; 36(Suppl 6): 14–16.\n\nAddeo R, et al.: Low-dose metronomic oral administration of vinorelbine in the first-line treatment of elderly patients with metastatic breast cancer. Clin Breast Cancer. 2010; 10(4): 301–306. PubMed Abstract \| Publisher Full Text\n\nBriasoulis E, et al.: Dose selection trial of metronomic oral vinorelbine monotherapy in patients with metastatic cancer: a hellenic cooperative oncology group clinical translational study. BMC Cancer. 2013; 13: 263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCamerini A, et al.: Metronomic oral vinorelbine as first-line treatment in elderly patients with advanced non-small cell lung cancer: results of a phase II trial (MOVE trial). BMC Cancer. 2015; 15: 359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKontopodis E, et al.: A phase II study of metronomic oral vinorelbine administered in the second line and beyond in non-small cell lung cancer (NSCLC): a phase II study of the Hellenic Oncology Research Group. J Chemother. 2013; 25(1): 49–55. PubMed Abstract \| Publisher Full Text\n\nBugat R, et al.: The effects of food on the pharmacokinetic profile of oral vinorelbine. Cancer Chemother Pharmacol. 2002; 50(4): 285–290. PubMed Abstract \| Publisher Full Text\n\nKatsaounis P, et al.: Cisplatin in combination with metronomic vinorelbine as front-line treatment in advanced non-small cell lung cancer: a multicenter phase II study of the Hellenic Oncology Research Group (HORG). Cancer Chemother Pharmacol. 2015; 75(4): 821–827. PubMed Abstract \| Publisher Full Text\n\nCazzaniga ME, et al.: Efficacy and Safety of the All-Oral Schedule of Metronomic Vinorelbine and Capecitabine in Locally Advanced or Metastatic Breast Cancer Patients: The Phase I-II VICTOR-1 Study. Int J Breast Cancer. 2014. 2014: p. 769790. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLangkjer S, et al.: First Line therapy of metasttic or locally advanced HER2-positive breast cancer. Update of the HERNATA study. 2013.\n\nBrems-Eskildsen AS, et al.: Metronomic treatment of vinorelbine with oral capecitabine is tolerable in the randomized Phase 2 study XeNa including patients with HER2 non-amplified metastatic breast cancer. Acta Oncol. 2020: 1–8. PubMed Abstract \| Publisher Full Text\n\nBennouna J, et al.: Oral vinorelbine plus cisplatin as first-line chemotherapy in nonsquamous non-small-cell lung cancer: final results of an International randomized phase II study (NAVotrial 01). Clin Lung Cancer. 2014; 15(4): 258–265. PubMed Abstract \| Publisher Full Text" }	[ { "id": "94075", "date": "02 Feb 2022", "name": "Christos Chouaid", "expertise": [ "Reviewer Expertise thoraci 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\nThis study, which concerns the tolerance of the Carboplatinvinorelbine metronomic combination, is well conducted but its clinical impact is very low given the use of anti-PD1 / PDL1 in first line.\nThe authors should at least in a paragraph discussing this limitation in a hexaustic way, cite the studies (i.e. Vergnenegre et al. (20201)) which tested the combination vinorelbine metronomic Immunotherapy and also state what is the clinical impact of their study according to current recommendations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7807", "date": "16 Feb 2022", "name": "Maria Kandi", "role": "Author Response", "response": "Dear Prof. Chouaid, On behalf of the authors I would like to thank you for both your time spent on reviewing our manuscript and the constructive comments. Below you can see our response to your comments: At the time we conducted this safety and pilot study the 1. line treatment of NSCLC with PD-L1 status under 50%, in Denmark, was carboplatin and vinorelbine day 1 and 8 every third week x 4 with optional pemetrexed as maintenance. In February 2020 the national recommendation changed toward giving immunotherapy to non-squamous NSCLC in combination with chemotherapy (carboplatin and pemetrexed) as standard. Ultimo 2020 the national recommendation changed again in favor of triple therapy for all adenocarcinomas regardless of PD-L1 status. This pilot study shows that the combination of carboplatin and metronomic vinorelbine is safe and feasible. The regimen could be used in future studies using combinations of carboplatin + metronomic vinorelbine + immunotherapy (e.g. phase II trials: 1. line carboplatin + metronomic vinorelbine + immunotherapy versus carboplatin + pemetrexed/paclitaxel + immunotherapy in patients with PD-L1 status under 50 %). With kind regards Maria Kandi Corresponding author" } ] } ]	1	https://f1000research.com/articles/10-673
https://f1000research.com/articles/9-5/v1	06 Jan 20	{ "type": "Brief Report", "title": "Independent predictors of comprehensive knowledge of HIV in general population: findings from the Myanmar Demographic and Health Survey (2015-16)", "authors": [ "Kyaw Lwin Show", "Hemant Deepak Shewade", "Khine Wut Yee Kyaw", "Khin Thet Wai", "San Hone", "Htun Nyunt Oo", "Hemant Deepak Shewade", "Khine Wut Yee Kyaw", "Khin Thet Wai", "San Hone", "Htun Nyunt Oo" ], "abstract": "Background: Myanmar has the third highest number of people living with HIV in Southeast Asia behind Indonesia and Thailand. The independent predictors of comprehensive HIV knowledge among general population are not known. Methods: In this nationally representative study, we adopted a cross-sectional design using secondary data from the Myanmar Demographic and Health Survey (2015-16). We included all women and men aged 15-49 years who participated in the survey. We have provided weighted estimates as the analyses were weighted for the multi-stage sampling design. We used modified Poisson regression with robust variance estimates model to identify independent predictors of comprehensive knowledge. Results: Of 17,622 analyzed, 3,599 (20.4%, 95% CI: 19.7, 21.1) had comprehensive knowledge of HIV. Late adolescents, those with less than a high school education, those involved in agriculture and the poorest two quintiles were less likely to have comprehensive knowledge of HIV. Conclusion: In Myanmar, comprehensive knowledge of HIV among the general population needs to be improved and we identified certain independent predictors that could be specifically targeted by the national programme.", "keywords": [ "Cross sectional survey", "demographic health survey", "HIV AIDS knowledge", "risk factors", "SORT IT" ], "content": "Introduction\n\nHuman immunodeficiency virus (HIV) infection is a global epidemic and is the second leading cause of death among infectious diseases after tuberculosis1. During 2017, there were 1.8 million new infections, 37 million people living with HIV and nearly one million acquired immunodeficiency syndrome (AIDS) related deaths2. The right knowledge and a positive attitude towards HIV along with awareness regarding availability of HIV counseling and testing services is a pre-requisite for meeting the first ‘90’ of the UNAIDS ’90-90-90’ targets: by 2020, 90% of people living with HIV should know their HIV status.\n\nMyanmar has the third highest number of people living with HIV in Southeast Asia behind Indonesia and Thailand. In 2015, the prevalence of HIV among adults aged 15–49 years was 0.59%, and 53% of estimated people living with HIV knew their status3,4. The focus of the national AIDS programme is on testing key populations, pregnant women (to reduce mother to child transmission), people with sexually transmitted infections, tuberculosis patients and prisoners3,5. Among young men who have sex with men in Myanmar (2015), having good HIV related knowledge was associated with HIV testing6.\n\nIn Uganda, four in ten women aged 15–49 years from the general population had comprehensive knowledge of HIV7. The Myanmar Demographic and Health Survey (MDHS) 2015–16 reported that one in five respondents had comprehensive knowledge of HIV, three quarters were willing to care a family member with HIV/AIDS, and 29% were ever tested for HIV8. The independent predictors of comprehensive HIV knowledge among the general population have not been analyzed or reported. Therefore, this study aimed to identify the factors associated with comprehensive of HIV knowledge among the general population. Understanding these will aid the programme in taking corrective actions and moving a step closer to attain the first ‘90’ target by 2020.\n\n\nMethods\n\nIn this nationally representative study, we adopted a cross-sectional design using secondary data from the MDHS 2015–16. We included all women and men aged 15–49 years who participated in the survey.\n\nThe Republic of the Union of Myanmar is divided administratively into the Nay Pyi Taw council territory, seven states and seven regions. There are 74 districts and 330 townships. Geographically, states and regions have diversities of plains, delta and hilly regions. The population is over 51 million, of which nearly 70% reside in rural areas9.\n\nThe sampling for MDHS 2015–16 was based on the 2014 census frame and excluded institutional populations (persons in hotels, barracks, and prisons) but included those from internally displaced population camps.\n\nThe survey followed a stratified two stage sample design. The first stage involved selecting clusters that were either a census enumeration area or ward/village tracts. Probability proportional to size sampling was used. Stratification was achieved by separating each state or region into urban and rural areas, each of which formed a separate sampling stratum. A total of 442 clusters (319 rural and 123 urban) were selected independently from total of 30 sampling strata (Figure 1). Second, using systematic random sampling, a fixed number of 30 households were sampled from each cluster. All men aged 15–49 years in every second selected household and all women aged 15–49 years in the selected households were interviewed using the pre-tested Myanmar language questionnaires. They were either residents or visitors who stayed the night before the survey. The response rate among women was 96% and men was 91%.\n\nCensus enumeration area or ward/village tracts.\n\nComprehensive knowledge was considered as ‘yes’ if a person (i) knew about condom use and that limiting sexual intercourse to one partner could prevent HIV and (ii) knew that a healthy looking person could have HIV and (iii) rejected the two most common local misconceptions about the transmission of HIV, which in Myanmar were that HIV could be transmitted through mosquito bites and that a person could get infected with HIV by sharing food with someone who has AIDS.\n\nAll completed questionnaires were entered into the tablets by the field editors after they were edited on paper in the field. Data were re-entered and validated by data processing personnel in Nay Pyi Taw using the CSPro computer package8.\n\nWe analyzed the data extracted from MDHS 2015–16 using STATA (version 12.1 STATA Corp., College Station, TX, USA). We assessed comprehensive knowledge using proportions and 95% confidence intervals (CIs). In the multivariable model to identify independent predictors of comprehensive knowledge (predictive modelling), we used modified Poisson regression with robust variance estimates. We included age, sex and variables with a crude Chi square p-value of <0.2. Before including the variables in the model, we ruled out multicollinearity using variance inflation factor. We assessed the association between socio-economic and demographic factors and comprehensive knowledge (outcome) using adjusted prevalence ratios (aPR) and 95% CI. We first considered programmatically significant association if aPR was ≥1.5 or ≤0.67, and then looked for statistical significance (p<0.05) because MDHS 2015–16 had a large sample size.\n\nWe have provided weighted estimates as the analyses were weighted for the multi-stage sampling design. We used the probability of selection of clusters and households to derive the weights (inverse probability sampling).\n\nWe obtained ethics approval from Ethics Review Committee, Department of Medical Research, Ministry of Health and Sports, Myanmar (Ethics/DMR/2018/163, dated 27 December 2018) and the Ethics Advisory Group of the International Union against Tuberculosis and Lung Disease (The Union), Paris, France (EAG number 38/18 dated 23 August 2018). This study uses existing DHS data and re-analysis was done under the original consent provided by the participants.\n\n\nResults\n\nA total of 17,622 respondents participated in the survey. Their mean age was 31.5 (standard deviation: 9.9) years, with 2,541 (14.4%) being late adolescents. A total of 4,737 (26.8%) were men, 2,181 (12.4%) had no formal education and 3,121 (17.7%) were involved in agriculture (Table 1).\n\ncol %, column percentage; row %, row percentage; PR, prevalence ratio; aPR, adjusted prevalence ratio; CI, confidence interval; HIV, Human Immunodeficiency Virus.\n\nComposite measure that a person (i) knows about condom use and limiting sexual intercourse to one partner can prevent HIV, and (ii) knows that a healthy looking person can have HIV, and (iii) rejects the two most common local misconceptions about the transmission of HIV, which in Myanmar are that HIV can be transmitted through mosquito bites and that a person can become infected with HIV by sharing food with someone who has AIDS.\n\n#Weighted estimates (for multistage survey design) for frequency, proportion and prevalence ratio.\n\n^We first considered programmatically significant association if aPR was ≥1.5 or ≤0.67, and then looked for statistical significance (p<0.05).\n\n^^Adjusted analysis using modified Poisson regression with robust variance estimates, 41 records with at least one variable missing were excluded from the adjusted analysis.\n\nOf 17,622 respondents, 3,599 (20.4%, 95% CI: 19.7, 21.1) had comprehensive knowledge of HIV. On unadjusted analysis, age, education, region, place of residence (urban or rural), occupation and wealth quintile were associated with comprehensive knowledge of HIV. On adjusted analysis, age, education, occupation and wealth quintile were identified as independent predictors.\n\nWhen compared to adults aged 30–39 years, late adolescents were less likely to have comprehensive knowledge of HIV [aPR: 0.60 (95% CI: 0.52, 0.69)]. When compared to those educated to high school level and above, those educated less than high school level were less likely to have comprehensive knowledge, with those with no formal education being 92% less likely [aPR: 0.08 (95% CI: 0.06, 0.11)]. In addition, those involved in agriculture and belonging to the poorest two quintiles were less likely to have comprehensive knowledge (Table 1).\n\n\nDiscussion\n\nThis study from Myanmar investigating the predictors of comprehensive knowledge of HIV among the general population had two strengths. First, we used data from a nationally representative survey. Second, the data were robust as double data entry and validation was done. There were some limitations as well. The study population might include some key affected population that could influence the true prevalence among general population. Residual confounding cannot not be ruled out.\n\nComprehensive knowledge about HIV in the general population was relatively low and this was prominent among late adolescents. This finding is supported by studies from Nigeria (2013) and Democratic Republic of Congo (2013)10. Young people including late adolescents are particularly vulnerable because of high risk sexual behavior and substance use. They lack access to accurate and personalized HIV information and prevention services11.\n\nComprehensive HIV knowledge among those with no formal education was poor. Similar results were also found among Indonesian women (2012)12. This might be linked to not having access to information that is usually available as part of the curriculum and academic activities, resulting in a better understanding of HIV. Moreover, wealth was also a factor that influenced comprehensive knowledge of HIV. People belonging to the poorest two quintiles had poor comprehensive knowledge of HIV, similar to the findings from the Nigerian Demographic and Health Survey (2013)13. Accessing or learning health information could be minimal for those of the poorest quintiles as they might need to engage more with daily work for their living.\n\nThere is a need to target late adolescents and this is possible through school health services. In 2017, the Ministry of Health and Sports issued standardized health messages in the local language for basic health staff. By using these, health promotion activities at the community level should specially be targeted towards late adolescents and socioeconomically disadvantaged people with no formal education.\n\nIn conclusion, comprehensive knowledge of HIV among the general population needs to be improved in Myanmar and we identified certain independent predictors that could be specifically targeted. Further translational health education research should be done on the possible knowledge transfer mechanism for these sub-groups.\n\n\nData availability\n\nThe underlying data for this study is owned by the DHS Program (https://www.dhsprogram.com/data/dataset/Myanmar_Standard-DHS_2016.cfm?flag=0). The electronic data is available from the DHS Program under its terms of use (https://dhsprogram.com/Data/terms-of-use.cfm). Before downloading the data, users must register as a DHS user for reasons laid out on the DHS Program website (https://www.dhsprogram.com/data/Registration-Rationale.cfm) and dataset access is only granted for legitimate research purposes.", "appendix": "References\n\nWorld Health Organization: The top 10 causes of death. 2018; [cited 2018 Jul 11]. Reference Source\n\nUNAIDS: Global HIV & AIDS statistics — 2018 fact sheet. 2018; [cited 2019 May 27]. Reference Source\n\nNational AIDS Program, Department of Public Health, Ministry of Health and Sports, Myanmar: National Strategic Plan on HIV and AIDS, Myanmar 2016-2020. Nay Pyi Taw; 2016. Reference Source\n\nWorld Health Organization: Progress Report on HIV in the WHO South-East Asia Region. World Health Organization. 2016. Reference Source\n\nNational AIDS Program, Department of Public Health, Ministry of Health and Sports, Myanmar: Guidelines for the Clinical Management of HIV infection in Myanmar. Nay Pyi Taw; 2017. Reference Source\n\nPham MD, Aung PP, Paing AK, et al.: Factors associated with HIV testing among young men who have sex with men in Myanmar: a cross-sectional study. J Int AIDS Soc. 2017; 20(3): e25026. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnkunda D, Asiimwe JB: Determinants of comprehensive knowledge of HIV/AIDS among women of the reproductive age (15-49) in Uganda. Int J Community Med Public Health. 2017; 4(10): 3530. Publisher Full Text\n\nMinistry of Health and Sports (MoHS) and ICF, Myanmar: Myanmar Demographic and Health Survey 2015-16. Nay Pyi Taw; 2017. Reference Source\n\nDepartment of Population, Ministry of Labour Immigration and Populatio, Myanmar: The Myanmar Population and Housing Census: Thematic Report on Population Dynamics. Nay Pyi Taw; 2016; 4. Reference Source\n\nGebremedhin SA, Youjie W, Tesfamariam EH: Predictors of HIV/AIDS Knowledge and Attitude among Young Women of Nigeria and Democratic Republic of Congo: Cross-Sectional Study. J AIDS Clin Res. 2017; 08(03). Reference Source\n\nUNAIDS: Report on the global epidemic HIV/AIDS. Geneva, Switzerland; 2002. Reference Source\n\nPradnyani PE, Wibowo A, Mahmudah M: The Effects of Socio-demographic Characteristics on Indonesian Women's Knowledge of HIV/AIDS: A Cross-sectional Study. J Prev Med Public Health. 2019; 52(2): 109-14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFaust L, Yaya S, Ekholuenetale M: Wealth inequality as a predictor of HIV-related knowledge in Nigeria. BMJ Glob Health. 2017; 2(4): e000461. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "61622", "date": "21 Apr 2020", "name": "Adam K. Richards", "expertise": [ "Reviewer Expertise epidemiology of infectious and chronic disease", "design and analysis of complex surveys", "health services research", "health inequity" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 uses data from the 2015-2016 Myanmar DHS to explore factors related to “comprehensive HIV knowledge” in the general Myanmar population. The authors should make it clear that the crude estimates presented in Table 1 combine the sex-specific tables from the full Myanmar DHS report (table1 13.3.1 for women and 13.3.2 for men); the modest novelty of the manuscript as currently written lies in the results modelled in a multivariate analysis of these independent variables.\nMy sense is that the manuscript could be strengthened by making several modest changes as outlined below.\n\nIntroduction First paragraph: it is not at all clear that “the right knowledge and a positive attitude towards HIV… is a pre-requisite… [to meet the 90-90-90 goals”… at least when the definition of HIV knowledge is the one used by the DHS/in this manuscript. It is not unreasonable to propose that at least some of the knowledge domains (specifically, the ‘misperceptions’) may be less relevant when it comes to actual risk behaviors, care-seeking, adherence, stigma etc etc. [see below for additional discussion]\nOutcome The authors should consider presenting results using knowledge of “HIV prevention measures” as an alternative, or perhaps secondary outcome.\nI appreciate that the definition of ‘comprehensive HIV knowledge’ that is applied in other DHS surveys (and is comprised of four elements: one partner/condoms prevent HIV; PLWHA can be healthy; and 2 misconceptions NOT endorsed) though it is worth noting that most DHS surveys also present results for a subset comprosed of the first two of the four items in the comprehensive knowledge, often referred to as ‘HIV prevention measures.’ It would be useful to use this alternative definition, given the somewhat tenuous link between the misconceptions and HIV outcomes of commonly accepted value. Numerous studies demonstrate how individuals are capable of holding multiple beliefs about the etiology of disease while maintaining overall excellent adherence to risk behaviors, testing and treatment. For example, it is possible for TB patients in Bolivia to believe that traditional healers play a valuable role in the treatment of tuberculosis, but this belief does not impede their ability to seek out testing for TB and to strictly adhere to DOT (as long as structural barriers are addressed to enable access to care, payment for testing and treatment etc) – see An Ethnography of Nonadherence: Culture, Poverty, and Tuberculosis in Urban Bolivia. Jeremy A. Greene. Culture, Medicine and Psychiatry volume 28, pages 401–425(2004)1. As the authors likely are aware, it is not uncommon in Myanmar for rural villagers to believe that malaria can be transmitted by eating papaya (or other fruits)… but this does not interfere with their excellent care seeking behavior and adherence to treatment – as evidenced by the impressive results of the large village malaria worker initiatives that have dramatically reduced transmission in endemic areas of Myanmar. Stated more directly, there is a very real chance that it does not matter much whether or not someone believes HIV might be related to mosquitoes or food… as long as they also know that HIV is associated with real risk behaviors (unprotected sex; sharing needles etc), and that their practices align with lower risk behaviors.\nI am not aware of studies demonstrating that misconceptions about HIV (specifically, mosquitoes and sharing food) meaningfully influence HIV risk behaviors, uptake of testing or treatment, HIV-related stigma or other meaningful outcome. If the authors are aware of such studies the might cite them as part of their choice to use the DHS definition of “comprehensive HIV knowledge” over alternative options that carry at least as much meaning – such as knowledge of HIV prevention messages.\nI appreciate that knowledge related to PMTCT of HIV is not the specific emphasis of this brief report. Nonetheless, it is arguably at least as important an indicator of HIV related knowledge in the general population -- as the authors themselves imply in the introduction. (that is, HIV knowledge about the importance of limiting sex partners, condom use etc. is MUCH more valuable among key pops and perhaps migrants than it is among the general population; whereas every woman (and their male partners) who might get pregnant should be aware of HIV testing and treatment during pregnancy..\nExposures The DHS includes questions related to media exposure (print, radio and television; and these answers are often combined into a composite indicator), and it would be valuable to include this in the model, as it might suggest possible means by which the low knowledge of HIV in Myanmar might be ameliorated. The authors should either include one or more of these variables, or explain their choice to omit them.\nDid the authors consider estimating knowledge among important subpopulations who can be classified using DHS data? Unfortunately, information on most high risk HIV behaviors is not available from the DHS, though it may be useful to document HIV knowledge among respondents who are: recently sexually active; reported an STI; engaged in payment for sex; or who lived in particular states/regions. For example, given the extremely high prevalence of HIV among PWID in Kachin and Shan (from the recent IBBS), as well as the relatively common practice of injection drug use in those Northern regions, it is not inconceivable that HIV will become a generalized epidemic in that area; and partners of PWID at are at especially high risk.\nThe lack of information on other HIV risk factors / key populations in the DHS (eg condom use; illicit injection history; same-sex-practices; migration history etc) is a limitation worth noting in the manuscript.\nThe model among adolescents likely should take advantage of the DHS variables specific to this group (ever attended school; attended school in the past year etc).\nStatistical analysis I appreciate and agree with the choice to present prevalence rate ratios (and not odds ratios). In the setting of an outcome of modest prevalence, most would choose to use glm with a binomial distribution and a log link function, and not a robust poisson regression. See for example Martin R Petersen and James A Deddens. BMC Med Res Methodol. A comparison of two methods for estimating prevalence ratios. 2008; 8: 92. The results are unlikely to be qualitatively different using either method, though the authors might explain their choice to the reader (or consider using glm with a binomial distribution)\nModelling choices Stratification / separate models for adolescents The authors appropriately note that HIV knowledge is especially low among adolescents, though it is less clear why this might be the case. A reasonable a priori hypothesis is that the factors associated with HIV knowledge among adolescents are quite different than risk factors among young/middle age adults. [Some authors would even submit a separate manuscript J]. The authors should explain why they chose to estimate a single model for both older adults as well as adolescents. If the authors first conducted stratified analyses and observed similar associations; or if they formally explored the possible presence of interactions between adolescent and older ages, then they should say so. If these were not done, then I strongly encourage the authors to explore possible effect modification among adolescents, and to comment on whether results appear similar.\nInteractions Did the authors consider exploring effect modification by other variables in addition to adolescent vs adults? For example, are SES gradients of similar magnitude in urban and non-urban settings? A brief comment on whether interactions were considered for other/any variables would be useful.\nSupplementary analyses As above, as currently presented the unique novel contribution made by this manuscript is a multivariable/adjusted model. Most associations remain relatively similar in the adjusted model, and the simple analysis sets a somewhat low bar for publication in a peer reviewed manuscript, even as a Brief Report. It would be valuable to augment the manuscript with additional analysis that would require fairly little effort. Here are two straightforward suggestions:\nPredicted probabilities For example, the authors could present absolute predicted probabilities of comprehensive knowledge (or knowledge of HIV prevention practices) for different individuals who do and do not possess factors associated with HIV knowledge: for example, the authors might report the probabilities of HIV knowledge for residents of Myanmar who are:\nhighest wealth quintile; high school or higher education; urban; professional\n\npoorest wealth quintile; no education; nonurban; agricultural worker\nThe authors might calculate predicted probabilities for two groups of adolescents; as well as two groups of adults (4 calculations total. Margins facilitates calculation of the difference between predicted probabilities, the standard error of that difference, confidence intervals etc though it may not be necessary to report that, due to the very large magnitude of the difference apparent on presentation of the point estimates.\nWhat is the potential value of calculated predicted probabilities? First, it provides an intuitive presentation of adjusted estimates on an absolute scale, which complements the information presented on a relative scale (aPRR). Second, it helps to place the findings in context and more directly address several key questions the current manuscript touches on only indirectly. One such question is whether comprehensive knowledge is likely or possible, even when the most favorable conditions exist (ie could any combination of factors produce a predicted probability close to the UNGASS goal of 95% among adolescents?). In addition, presentation of predicted probabilities would create an even more stark contrast for disadvantages groups who comprise a large proportion of the population. This, in turn, might further highlight the important conclusion that efforts to improve HIV knowledge per se (via media, education etc) are unlikely to address the low overall HIV knowledge; and that improving HIV knowledge will require major development and social change. [see also the comment below related to school-based HIV education efforts –calculating predicted probabilities among adolescents who are advantaged in every way, including attendance at school, might provide a rough sense of the magnitude of the impact necessary to achieve targets set by UNGASS].\nPredicted probabilities are easily accomplished in STATA using the ‘margins’ postestimation command (immediately after running the full model regression).\nSummarize SES inequities using concentration index or similar health equity metric The SES gradients (wealth and educational attainment) are very large, independent of eachother, and not explained by observed variables in the model. Methods exist to summarize inequities in health outcomes using tools developed by others, such as the concentration index developed by the World Bank. User-written command concindex is available in STATA that facilitates calculation of the concentration index in the setting of complex survey such as the DHS (using the svy: prefix). I suggest that the authors report two relative CIs for HIV knowledge, using household wealth and educational attainment as the two respective ranking variables.\n\nDiscussion The major finding was already known: that “comprehensive knowledge of HIV,” as defined by the DHS is low in Myanmar; and that the distribution of knowledge demonstrates stark patterning according to axes of power and advantage, such as wealth, education, occupation, urban residence etc. It likely is worth stating that adjusting for the factors included in the multivariable model had little influence on what was documented already (in the DHS report tables).\n\nIf the authors believe that the more strict definition of HIV knowledge (that requires respondents to correctly identify mosquitoes and food as NOT influencing risk of HIV) is in fact important, that it would be fair to say that an overall prevalence of 20.4% is extremely (abysmally?) low. If, however, they believe that in fact it is not crucial that the general population possesses the HIV knowledge specific to these questions, then it may be reasonable to describe this percentage in less stark terms: perhaps simply ‘low’ would suffice; I would avoid saying ‘relatively’ low, as this begs the question ‘relative to what’? the ideal? Other countries? Absolute prevalence of comprehensive knowledge should be reported for each country compared to Myanmar (eg Nigeria, DRC and Uganda). The adolescent figures can be placed in the context of the UNGASS goal to achieve comprehensive knowledge among 95% of adolescents. (!) The SES gradients (wealth and educational attainment) are striking, independent, and are not explained by observed variables included in this model. This likely deserves greater emphasis. Why single out “the poorest two quintiles” for having “poor comprehensive knowledge”… when only 41% possessed comprehensive knowledge even the highest wealth quintile? [hint: predictive margins may be useful here]\nAs above, it would be valuable to report a secondary outcome of the two HIV prevention methods, which results in a slightly higher prevalence 54% among women and 62% among men (p205 in the DHS). It would be valuable to know whether or not the associations appear similar when this more common outcome is used in place of the stricter “comprehensive knowledge” outcome is used (the PRRs will likely be somewhat smaller in magnitude if the denominator/baseline value is larger, though this can be taken into account in your interpretation). The manuscript might contrast HIV Knoweldge in the general population with that reported among key populations (ie from the respective IBBS among PWID and FSW).\nGiven the concentration of HIV risk among key populations – that in Myanmar include migrants, as noted in the more recent strategic plans of MOHS/NAP – the authors could more explicitly state the value of reporting HIV knowledge among the general public. One way to do this is to highlight the emphasis of NAP on PMTCT, and make explicit that the women in the present analysis are women of reproductive age. If a large number of children and adolescents do not attend school (attendance ratios are 83% for primary and only 60% among adolescents) then it is not clear how school-based HIV education would address the gap in HIV knowledge among this vulnerable group.\nAddressing the massive SES gradients in HIV knowledge is an admirable goal, though it’s not at all clear what a “targeted” approach might look like, or whether it makes much sense given the low knowledge overall, and in every subgroup presented – though calculating the predictive margins might help to highlight whether it would be reasonable to omit certain subgroups from a ‘targeted’ campaign.\n\nMinor points Introduction The findings from Uganda belong in the discussion (along with DRC and Nigeria). Methods: the paragraph that begins “Comprehensive knowledge was considered…” should read “present” [drop as ‘yes’]\nResults: second paragraph -- repeating “17,622 respondents” is redundant\n\nFigure 1 – I am not sure what is added by presenting the sampled clusters on a map, in the particular manuscript. Consider mapping the prevalence of the primary outcome of HIV knowledge. Since the crude prevalence of comprehensive knowledge by state/region is already provided in the DHS report (albeit separately for men and women, and in tabulated format); then the authors might present predicted probabilities ‘adjusted’ (standardized) for age, sex and nonurban residence…\n\nIs the work 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": [ { "c_id": "6936", "date": "28 Jul 2021", "name": "Kyaw Lwin Show", "role": "Author Response", "response": "REVIEWER 1 Adam K. Richards, Community Partners International, Berkeley, CA, USA REVIEWER This brief report uses data from the 2015-2016 Myanmar DHS to explore factors related to “comprehensive HIV knowledge” in the general Myanmar population. The authors should make it clear that the crude estimates presented in Table 1 combine the sex-specific tables from the full Myanmar DHS report (table1 13.3.1 for women and 13.3.2 for men); the modest novelty of the manuscript as currently written lies in the results modelled in a multivariate analysis of these independent variables. My sense is that the manuscript could be strengthened by making several modest changes as outlined below. AUTHORS Thank you for your time and kind effort providing comments. We agree with you that novelty of the manuscript lies in the multivariable analysis. As the result of reviewer input, we believe the manuscript has significantly improved. We have added the footnote in the Table 1 “The crude estimates presented here combine the sex-specific tables from the full Myanmar DHS report (table1 13.3.1 for women and 13.3.2 for men)”. REVIEWER Introduction First paragraph: it is not at all clear that “the right knowledge and a positive attitude towards HIV… is a pre-requisite… [to meet the 90-90-90 goals”… at least when the definition of HIV knowledge is the one used by the DHS/in this manuscript. It is not unreasonable to propose that at least some of the knowledge domains (specifically, the ‘misperceptions’) may be less relevant when it comes to actual risk behaviors, care-seeking, adherence, stigma etc etc. [see below for additional discussion]Outcome The authors should consider presenting results using knowledge of “HIV prevention measures” as an alternative, or perhaps secondary outcome. AUTHORS Thank you for your comment. We ran the model with the same method for “knowledge on HIV prevention measures”. We found that compared to those educated high school and above, those educated less than high school were 34% less likely [aPR: 0.66 (95% CI: 0.63, 0.69) : those with no formal education were 63% less likely [aPR: 0.37 (95% CI: 0.33, 0.41)] to have comprehensive knowledge. Other than education, we found no significant differences between categories across variables which is similar to the comprehensive knowledge result. Moreover, the percentage for the specific questions regarding HIV preventive measures and misperceptions were also already presented in the MDHS report. Therefore, we would like to keep current definition of outcome and we would like to compare it with the results of other studies conducted in different countries. REVIEWER I appreciate that the definition of ‘comprehensive HIV knowledge’ that is applied in other DHS surveys (and is comprised of four elements: one partner/condoms prevent HIV; PLWHA can be healthy; and 2 misconceptions NOT endorsed) though it is worth noting that most DHS surveys also present results for a subset comprosed of the first two of the four items in the comprehensive knowledge, often referred to as ‘HIV prevention measures.’ It would be useful to use this alternative definition, given the somewhat tenuous link between the misconceptions and HIV outcomes of commonly accepted value. Numerous studies demonstrate how individuals are capable of holding multiple beliefs about the etiology of disease while maintaining overall excellent adherence to risk behaviors, testing and treatment. For example, it is possible for TB patients in Bolivia to believe that traditional healers play a valuable role in the treatment of tuberculosis, but this belief does not impede their ability to seek out testing for TB and to strictly adhere to DOT (as long as structural barriers are addressed to enable access to care, payment for testing and treatment etc) – see An Ethnography of Nonadherence: Culture, Poverty, and Tuberculosis in Urban Bolivia. Jeremy A. Greene. Culture, Medicine and Psychiatry volume 28, pages 401–425(2004)1. As the authors likely are aware, it is not uncommon in Myanmar for rural villagers to believe that malaria can be transmitted by eating papaya (or other fruits)… but this does not interfere with their excellent care seeking behavior and adherence to treatment – as evidenced by the impressive results of the large village malaria worker initiatives that have dramatically reduced transmission in endemic areas of Myanmar. Stated more directly, there is a very real chance that it does not matter much whether or not someone believes HIV might be related to mosquitoes or food… as long as they also know that HIV is associated with real risk behaviors (unprotected sex; sharing needles etc), and that their practices align with lower risk behaviors. I am not aware of studies demonstrating that misconceptions about HIV (specifically, mosquitoes and sharing food) meaningfully influence HIV risk behaviors, uptake of testing or treatment, HIV-related stigma or other meaningful outcome. If the authors are aware of such studies the might cite them as part of their choice to use the DHS definition of “comprehensive HIV knowledge” over alternative options that carry at least as much meaning – such as knowledge of HIV prevention messages. I appreciate that knowledge related to PMTCT of HIV is not the specific emphasis of this brief report. Nonetheless, it is arguably at least as important an indicator of HIV related knowledge in the general population -- as the authors themselves imply in the introduction. (that is, HIV knowledge about the importance of limiting sex partners, condom use etc. is MUCH more valuable among key pops and perhaps migrants than it is among the general population; whereas every woman (and their male partners) who might get pregnant should be aware of HIV testing and treatment during pregnancy.. AUTHORS Thank you for the detailed comment. We realized the value of having HIV prevention measures as a secondary outcome. However, since our study’s focus was on comprehensive knowledge of HIV and short reports does not allow us to expand the manuscript (maximum 2 tables/figures and 2500 words); we would like to stick to our current outcome. We also found that misconceptions of HIV transmission have been associated with HIV testing in some DHS paper (mentioned below). In this connection, in order to meet the first 90 of UNAIDS 90-90-90 goal, we decided to keep comprehensive knowledge (preventive knowledge + misconceptions) as our outcome of interest. https://www.dovepress.com/what-are-the-determinants-of-misconception-about-hiv-transmission-amon-peer-reviewed-fulltext-article-HIV https://bmcinthealthhumrights.biomedcentral.com/articles/10.1186/s12914-016-0089-8#Tab3 REVIEWER Exposures The DHS includes questions related to media exposure (print, radio and television; and these answers are often combined into a composite indicator), and it would be valuable to include this in the model, as it might suggest possible means by which the low knowledge of HIV in Myanmar might be ameliorated. The authors should either include one or more of these variables, or explain their choice to omit them. AUTHORS Thank you for pointing out this. We included exposure to at least one of the mass media (television, newspaper, radio) at least once a week in the model and added the values in the table 1. REVIEWER Did the authors consider estimating knowledge among important subpopulations who can be classified using DHS data? Unfortunately, information on most high risk HIV behaviors is not available from the DHS, though it may be useful to document HIV knowledge among respondents who are: recently sexually active; reported an STI; engaged in payment for sex; or who lived in particular states/regions. For example, given the extremely high prevalence of HIV among PWID in Kachin and Shan (from the recent IBBS), as well as the relatively common practice of injection drug use in those Northern regions, it is not inconceivable that HIV will become a generalized epidemic in that area; and partners of PWID at are at especially high risk. AUTHORS Thank you for this interesting comment. Because short reports does not allow us to expand the manuscript (maximum 2 tables/figures and 2500 words), we could not cover all the aspects and we decided to estimate knowledge among general population in this paper. We have published a paper regarding HIV testing among STIs which knowledge variable was an independent variable. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310811/ REVIEWER The lack of information on other HIV risk factors / key populations in the DHS (eg condom use; illicit injection history; same-sex-practices; migration history etc) is a limitation worth noting in the manuscript. AUTHORS Thank you. We have stated “The study population might include some key affected population that could influence the true prevalence among general population” as a limitation in our manuscript. LINE 201 of revised manuscript with track changes REVIEWER The model among adolescents likely should take advantage of the DHS variables specific to this group (ever attended school; attended school in the past year etc). AUTHORS Thank you. We agree with your point. We have also analyzed separate models for adolescents and non-adolescents and found similar results as combined one except regional variation prominent among adolescents. In the adolescent model, compared to delta and lowland region, those residing in hilly region (aOR=0.51, 95%CI: 0.34,0.78) and plain region (aOR=0.67, 95%CI: 0.51, 0.88) had significant lower comprehensive knowledge. Considering this is a short report with limited tables and the need for a clear and simple message, we decided against presenting another model for adolescents in this manuscript. We hope this is fine. REVIEWER Statistical analysis I appreciate and agree with the choice to present prevalence rate ratios (and not odds ratios). In the setting of an outcome of modest prevalence, most would choose to use glm with a binomial distribution and a log link function, and not a robust poisson regression. See for example Martin R Petersen and James A Deddens. BMC Med Res Methodol. A comparison of two methods for estimating prevalence ratios. 2008; 8: 92. The results are unlikely to be qualitatively different using either method, though the authors might explain their choice to the reader (or consider using glm with a binomial distribution) AUTHORS Thank you. We first tried a log binomial model. But because of lack of convergence, we proceeded with the modified Poisson regression to estimate the prevalence ratios. We have also mentioned in the manuscript. LINE 141 of manuscript with track changes REVIEWER Modelling choices Stratification / separate models for adolescents The authors appropriately note that HIV knowledge is especially low among adolescents, though it is less clear why this might be the case. A reasonable a priori hypothesis is that the factors associated with HIV knowledge among adolescents are quite different than risk factors among young/middle age adults. [Some authors would even submit a separate manuscript J]. The authors should explain why they chose to estimate a single model for both older adults as well as adolescents. If the authors first conducted stratified analyses and observed similar associations; or if they formally explored the possible presence of interactions between adolescent and older ages, then they should say so. If these were not done, then I strongly encourage the authors to explore possible effect modification among adolescents, and to comment on whether results appear similar AUTHORS Thank you for your comment. We tried the potential interaction across variables (including age category and other variables), included in the model and tested for model selection using AIC/BIC results. However, the models with interaction(s) was not significantly improved compared to the simple model. Therefore, we decided to present the simple model in our manuscript. We have added the information in the manuscript. LINE 148 of revised manuscript with track changes REVIEWER Interactions Did the authors consider exploring effect modification by other variables in addition to adolescent vs adults? For example, are SES gradients of similar magnitude in urban and non-urban settings? A brief comment on whether interactions were considered for other/any variables would be useful. AUTHORS Thank you for your comment. We tried the potential interaction across variables (including age category and other variables), included in the model and tested for model selection using AIC/BIC results. However, the models with interaction(s) was not significantly improved compared to the simple model. Therefore, we decided to present the simple first model in our manuscript. We have added the information in the analysis. LINE 148 of revised manuscript with track changes REVIEWER Supplementary analyses As above, as currently presented the unique novel contribution made by this manuscript is a multivariable/adjusted model. Most associations remain relatively similar in the adjusted model, and the simple analysis sets a somewhat low bar for publication in a peer reviewed manuscript, even as a Brief Report. It would be valuable to augment the manuscript with additional analysis that would require fairly little effort. Here are two straightforward suggestions: Predicted probabilities For example, the authors could present absolute predicted probabilities of comprehensive knowledge (or knowledge of HIV prevention practices) for different individuals who do and do not possess factors associated with HIV knowledge: for example, the authors might report the probabilities of HIV knowledge for residents of Myanmar who are: highest wealth quintile; high school or higher education; urban; professional poorest wealth quintile; no education; nonurban; agricultural worker The authors might calculate predicted probabilities for two groups of adolescents; as well as two groups of adults (4 calculations total. Margins facilitates calculation of the difference between predicted probabilities, the standard error of that difference, confidence intervals etc though it may not be necessary to report that, due to the very large magnitude of the difference apparent on presentation of the point estimates. What is the potential value of calculated predicted probabilities? First, it provides an intuitive presentation of adjusted estimates on an absolute scale, which complements the information presented on a relative scale (aPRR). Second, it helps to place the findings in context and more directly address several key questions the current manuscript touches on only indirectly. One such question is whether comprehensive knowledge is likely or possible, even when the most favorable conditions exist (ie could any combination of factors produce a predicted probability close to the UNGASS goal of 95% among adolescents?). In addition, presentation of predicted probabilities would create an even more stark contrast for disadvantages groups who comprise a large proportion of the population. This, in turn, might further highlight the important conclusion that efforts to improve HIV knowledge per se (via media, education etc) are unlikely to address the low overall HIV knowledge; and that improving HIV knowledge will require major development and social change. [see also the comment below related to school-based HIV education efforts –calculating predicted probabilities among adolescents who are advantaged in every way, including attendance at school, might provide a rough sense of the magnitude of the impact necessary to achieve targets set by UNGASS]. Predicted probabilities are easily accomplished in STATA using the ‘margins’ postestimation command (immediately after running the full model regression). AUTHORS Thank you for this insightful comment. We have included the predicted probabilities across categories in Table 2. REVIEWER Summarize SES inequities using concentration index or similar health equity metric The SES gradients (wealth and educational attainment) are very large, independent of eachother, and not explained by observed variables in the model. Methods exist to summarize inequities in health outcomes using tools developed by others, such as the concentration index developed by the World Bank. User-written command concindex is available in STATA that facilitates calculation of the concentration index in the setting of complex survey such as the DHS (using the svy: prefix). I suggest that the authors report two relative CIs for HIV knowledge, using household wealth and educational attainment as the two respective ranking variables. AUTHORS Thank you for this interesting comment. We ran the analysis for concentration index/concentration curve and found that “The HIV comprehensive knowledge was concentrated among richer people, more educated people and who hold better job title”. However, short reports does not allow us to expand the manuscript (maximum 2 tables/figures and 2500 words); we would like to stick as it is. We hope this is fine. REVIEWER Discussion The major finding was already known: that “comprehensive knowledge of HIV,” as defined by the DHS is low in Myanmar; and that the distribution of knowledge demonstrates stark patterning according to axes of power and advantage, such as wealth, education, occupation, urban residence etc. It likely is worth stating that adjusting for the factors included in the multivariable model had little influence on what was documented already (in the DHS report tables). AUTHORS We agree. We believe that reporting where the poor knowledge of HIV was concentrated can be beneficial for a resource constraint country like Myanmar. We hope this is fine. REVIEWER If the authors believe that the more strict definition of HIV knowledge (that requires respondents to correctly identify mosquitoes and food as NOT influencing risk of HIV) is in fact important, that it would be fair to say that an overall prevalence of 20.4% is extremely (abysmally?) low. If, however, they believe that in fact it is not crucial that the general population possesses the HIV knowledge specific to these questions, then it may be reasonable to describe this percentage in less stark terms: perhaps simply ‘low’ would suffice; I would avoid saying ‘relatively’ low, as this begs the question ‘relative to what’? the ideal? Other countries? AUTHORS Thank you. We have removed “relatively” in the manuscript. . LINE 206 of revised manuscript with track changes REVIEWER Absolute prevalence of comprehensive knowledge should be reported for each country compared to Myanmar (eg Nigeria, DRC and Uganda). AUTHORS Thank you. We revised it. LINE 209 of manuscript with track changes REVIEWER The adolescent figures can be placed in the context of the UNGASS goal to achieve comprehensive knowledge among 95% of adolescents. AUTHORS Thank you. We revised it. LINE 207 of manuscript with track changes REVIEWER The SES gradients (wealth and educational attainment) are striking, independent, and are not explained by observed variables included in this model. This likely deserves greater emphasis. Why single out “the poorest two quintiles” for having “poor comprehensive knowledge”… when only 41% possessed comprehensive knowledge even the highest wealth quintile? [hint: predictive margins may be useful here] AUTHORS Thank you for this comment. We have added predicted probabilities as Table 2. REVIEWER As above, it would be valuable to report a secondary outcome of the two HIV prevention methods, which results in a slightly higher prevalence 54% among women and 62% among men (p205 in the DHS). It would be valuable to know whether or not the associations appear similar when this more common outcome is used in place of the stricter “comprehensive knowledge” outcome is used (the PRRs will likely be somewhat smaller in magnitude if the denominator/baseline value is larger, though this can be taken into account in your interpretation). The manuscript might contrast HIV Knoweldge in the general population with that reported among key populations (ie from the respective IBBS among PWID and FSW). AUTHORS Thank you. We have included comprehensive knowledge among key population in Myanmar according to IBBS survey in the discussion. LINE 202 of manuscript with track changes REVIEWER Given the concentration of HIV risk among key populations – that in Myanmar include migrants, as noted in the more recent strategic plans of MOHS/NAP – the authors could more explicitly state the value of reporting HIV knowledge among the general public. One way to do this is to highlight the emphasis of NAP on PMTCT, and make explicit that the women in the present analysis are women of reproductive age. AUTHORS Thank you for this valuable comment. We included “Furthermore, low proportion of comprehensive knowledge of HIV/AIDS among reproductive aged women in general population is alarming for effective prevention of mother-to-child transmission of HIV.” LINE 214 of manuscript with track changes REVIEWER If a large number of children and adolescents do not attend school (attendance ratios are 83% for primary and only 60% among adolescents) then it is not clear how school-based HIV education would address the gap in HIV knowledge among this vulnerable group. AUTHORS Thank you. We revised the sentence as “School health education and peer intervention programsshould be strengthened to improve comprehensive knowledge among late adolescent.” LINE 225 of manuscript with track changes REVIEWER Addressing the massive SES gradients in HIV knowledge is an admirable goal, though it’s not at all clear what a “targeted” approach might look like, or whether it makes much sense given the low knowledge overall, and in every subgroup presented – though calculating the predictive margins might help to highlight whether it would be reasonable to omit certain subgroups from a ‘targeted’ campaign. AUTHORS Thank you for this interesting comment. We have included predicted probabilities as Table 2. REVIEWER Minor points Introduction The findings from Uganda belong in the discussion (along with DRC and Nigeria). AUTHORS Thank you. We have revised it in the manuscript. LINE 209 of manuscript with track changes REVIEWER Methods: the paragraph that begins “Comprehensive knowledge was considered…” should read “present” [drop as ‘yes’] AUTHORS Thank you. We have revised it in the manuscript. LINE 127 of manuscript with track changes REVIEWER Results: second paragraph -- repeating “17,622 respondents” is redundant AUTHORS Thank you. We have revised it. REVIEWER Figure 1 – I am not sure what is added by presenting the sampled clusters on a map, in the particular manuscript. Consider mapping the prevalence of the primary outcome of HIV knowledge. Since the crude prevalence of comprehensive knowledge by state/region is already provided in the DHS report (albeit separately for men and women, and in tabulated format); then the authors might present predicted probabilities ‘adjusted’ (standardized) for age, sex and nonurban residence… AUTHORS Thank you for your comment. We excluded the map figure and included the predicted probabilities by SES after adjusted model is presented in Table 2." } ] }, { "id": "79872", "date": "24 Feb 2021", "name": "Naohiro Yonemoto", "expertise": [ "Reviewer Expertise Epidemiology and 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\nThe study would be valuable, but I have some comments below.\nIn introduction, third paragraph was not clear. Why did you mention \"in Uganda\" ?\n\nDid you check validity and confidence as an composite measure of knowledge in \"Comprehensive knowledge\"? How to summarize them as composite score? This is a critical issue.\n\nHow to select socio-economic variables from original dateset? All?\n\nYou should more clearly describe the datasets.\n\nYou should describe data flow chart and align with STROBE statement.\n\nYou should discuss the difference of respondents by sex.\n\nHow to analyze them \"with weights\" in the model. You should more clearly describe them.\n\nWealth quin tile was not clear. You should more clearly describe them.\n\nHow to handle missing data in the analysis?\n\nYou mentioned use of \"robust variance\". Why did you use it and was used for 95%CIs?\n\nYou should more clearly describe variables of education and regions. How long educated? Where? Map?\n\nYou should more clearly discuss the gap between knowledge (by responded) and behaviors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6937", "date": "28 Jul 2021", "name": "Kyaw Lwin Show", "role": "Author Response", "response": "REVIEWER 2 Naohiro Yonemoto, Department of Public Health, Juntendo University School of medicine, Tokyo, Japan REVIEWER The study would be valuable, but I have some comments below. In introduction, third paragraph was not clear. Why did you mention \"in Uganda\" ? AUTHORS Thank you. We included Uganda as international example. We hope this is fine. REVIEWER Did you check validity and confidence as an composite measure of knowledge in \"Comprehensive knowledge\"? How to summarize them as composite score? This is a critical issue. AUTHORS Thank you. “Comprehensive knowledge” used as an outcome in this study was expressed in the DHS reports by many countries. It is the composite score and was considered as ‘present’ if a person (i) knew about condom use and that limiting sexual intercourse to one partner could prevent HIV and (ii) knew that a healthy looking person could have HIV and (iii) rejected the two most common local misconceptions about the transmission of HIV, which in Myanmar were that HIV could be transmitted through mosquito bites and that a person could get infected with HIV by sharing food with someone who has AIDS. It is stated in the paper LINE 127 of revised manuscript with track changes. REVIEWER How to select socio-economic variables from original dateset? All? AUTHORS Thank you. At the first place, we reviewed the dataset and based on our experience and available literatures, we selected SES variables to be included in our study. REVIEWER You should more clearly describe the datasets. AUTHORS Thank you. We analyzed publically available (https://dhsprogram.com/) secondary data which is the MDHS 15-16 dataset and was described briefly in LINE 35-49 of the manuscript. We did not describe the details because of nature of the brief report allowing 2,500 words and it was already presented in MDHS 15-16 report. REVIEWER You should describe data flow chart and align with STROBE statement. AUTHORS Thank you. As analyzed publically available secondary data which is the MDHS 15-16 dataset, the data flow is described in the MDHS 15-16 report (dhsprogram.com) and we decided not to repeat the same in our manuscript to save the space. Thank you for suggesting to check the quality of reporting with STROBE statement which was very useful. We recognized that the sampling procedure and sample size calculation were not specifically mentioned in our report since they were previously described in DHS report and we have indicated it in the manuscript. [LINE 113 of revised manuscript with track changes] REVIEWER You should discuss the difference of respondents by sex. AUTHORS Thank you. The difference of respondents by sex is happened because of sampling design by the DHS methodology. In the DHS report, it was mentioned as “All women age 15-49 who are either permanent residents of the selected households or visitors who stayed in the households the night before the survey were eligible to be interviewed. In half of the selected households (every second households), all men age 15-49 who were either residents or visitors who stayed in the households the night before the survey were eligible to be interviewed.” You may check the detail in the DHS report. (https://dhsprogram.com/) REVIEWER How to analyze them \"with weights\" in the model. You should more clearly describe them. AUTHORS Thank you. Because of non-proportional sample allocation by DHS methodology, weighting factors were added to the data file and calculated to be representative at the national level. This was also described in the MDHS 15-16 report and we did not repeat it to save the space. We hope this is fine. REVIEWER Wealth quintile was not clear. You should more clearly describe them. AUTHORS The wealth quantile was already classified as it is in the original dataset and we did not do any modification. In the DHS report, it was mentioned that the wealth index were derived from selected variables and you can see the detail in page number 9 of the MDHS report. REVIEWER How to handle missing data in the analysis? AUTHORS Thank you. Magnitude of missing data was minimal and hence, we did not include them in the model. However, we presented the number and proportion of missing data in the table. REVIEWER You mentioned use of \"robust variance\". Why did you use it and was used for 95%CIs? AUTHORS We used the robust option to obtain robust standard errors for the parameter estimates to control for mild violation of underlying assumptions. REVIEWER You should more clearly describe variables of education and regions. How long educated? Where? Map? AUTHORS Thank you. We have described education and regions as footnotes under the table 1. REVIEWER You should more clearly discuss the gap between knowledge (by responded) and behaviors. AUTHORS Thank you. We have added “Furthermore, access to HIV preventive equipment such as condoms and creating safe environment is critical” in the discussion. LINE 230 of revised manuscript with track changes" } ] } ]	1	https://f1000research.com/articles/9-5
https://f1000research.com/articles/10-663/v1	27 Jul 21	{ "type": "Brief Report", "title": "Efficacy of COVID-19 vaccination in individuals designated as clinically extremely vulnerable in Scotland", "authors": [ "Paul M McKeigue", "David A McAllister", "Jen Bishop", "Sharon Hutchinson", "Chris Robertson", "Nazir Lone", "Jim McMenamin", "David Goldberg", "Helen M Colhoun", "Paul M McKeigue", "David A McAllister", "Jen Bishop", "Sharon Hutchinson", "Chris Robertson", "Nazir Lone", "Jim McMenamin", "David Goldberg" ], "abstract": "Background: Although COVID-19 vaccines have been shown to have high efficacy in the general population, it has not been established whether this applies to vulnerable groups. The objective of this study was to estimate the efficacy of vaccination in reducing the risk of severe COVID-19 among those designated as clinically extremely vulnerable in Scotland. Methods: In a matched case-control design (REACT-SCOT), all 111295 cases of COVID-19 in Scotland diagnosed from 1 December 2020 to 16 March 2021 were matched for age, sex and primary care practice to 1093449 controls from the general population. This was linked to national data on vaccinations and those designated as clinically extremely vulnerable and thus eligible for shielding support. Severe COVID-19 was defined as cases with entry to critical care or fatal outcome. Rate ratios associated with vaccination within risk groups were estimated by conditional logistic regression. Results: The rate ratio for severe COVID-19 associated with vaccination at least 14 days before was 0.29 (95% CI 0.22 to 0.37) in those eligible for shielding, compared with 0.29 (95% CI 0.25 to 0.34) in those ineligible for shielding. The rate ratio for hospitalized or fatal COVID-19 was 0.39 (95% CI 0.33 to 0.46) in those eligible and 0.37 (95% CI 0.33 to 0.41) in those not eligible for shielding. Examined by specific shielding conditions, the rate ratio for hospitalized or fatal COVID-19 ranged from 0.33 (95% CI 0.21 to 0.51) in those with specific cancers to 0.74 (95% CI 0.36 to 1.51) in solid organ transplant recipients, and 0.53 (95% CI 0.33 to 0.84) in others on immunosuppressants (excluding solid organ transplant recipients). Conclusions: These results are reassuring with respect to efficacy in clinically vulnerable individuals including immunocompromised individuals, but studies in larger populations are needed to estimate efficacy in solid organ transplant recipients.", "keywords": [ "COVID-19", "record linkage", "case-control studies", "vaccination", "efficacy", "solid organ transplant", "immunosuppression", "severe respiratory disease", "cancer" ], "content": "Introduction\n\nA primary-care based observational study that included cases diagnosed up to 15 Feb 2021 in Scotland estimated maximal efficacy of a single vaccine dose to be 70% for ChAdOx1 nCoV-19 and 85% for BNT162b2 mRNA at 28 days post-vaccination.1 However policy on managing the epidemic depends critically upon the extent to which vaccination protects clinically vulnerable individuals, who account for most hospitalizations and deaths,2 against severe COVID-19. Whet levels of vaccine efficacy pertain among those designated as clinically extremely vulnerable to COVID-19 is unclear. In Scotland early in the epidemic Public Health Scotland established a register of all those with certain conditions or on certain drugs that were likely to lead to extreme vulnerability to COVID-19 was established based on electronic health care records and primary care physician reporting.3,4 This register was used to determine eligibility for the national shielding support programme. We previously reported on the risk of severe COVID-19 in this shielding eligibility cohort compared to the rest of the population.5 If vaccine efficacy were estimated to be lower in clinically extremely vulnerable individuals than in the rest of the population, additional measures to shield these individuals would be needed until overall transmission rates in the population are low. Lower vaccine efficacy is possible since many such persons are immunocompromised or on immunosuppressants. Therefore, the aim of this study was to examine the efficacy of COVID-19 vaccination in reducing the risk of severe COVID-19 among those designated as clinically extremely vulnerable in Scotland.\n\n\nMethods\n\nThis analysis is based on the REACT-SCOT matched case-control study, established at the beginning of the epidemic to investigate risk factors for severe COVID-19 in the population of Scotland.2 We used this study to take advantage of data linkages already established. The design has been described in detail previously.2 In brief, for every incident case of COVID-19 in the population ten controls matched for one-year age, sex and primary care practice and alive on the day of presentation of the case that they were matched to were selected using the Community Health Index database. COVID-19 cases are those with a positive nucleic acid test, or a hospital admission or death with COVID-19 ICD-10 codes. The REACT-SCOT case-control dataset is refreshed regularly and is linked to the vaccination database and to the regularly updated dataset of all individuals deemed eligible for the shielding programme.\n\nAs previously,2 to minimise ascertainment bias we pre-specified the primary outcome measure as severe COVID-19, defined as cases with entry to critical care within 28 days of presentation or fatal outcome (any death within 28 days of a positive test or any death for which COVID-19 was coded as underlying cause). As previously, cases and controls were classified as eligible or ineligible for shielding.5 For further analyses, the shielding category was subdivided as described previously into six categories: solid organ transplant, specific cancers, severe respiratory conditions, other rare conditions, on immunosuppressants, and additional conditions.5 Those ineligible for shielding were classified according to the presence of conditions designated by public health agencies as “moderate risk conditions” as described previously.2 This analysis is based on cases presenting from 1 December 2020 to 16 March 2021 (the latest linkage dataset available at the time of preparing this report).\n\nVaccination status in cases was coded as having had at least one dose of any vaccine at least 14 days before presentation date. This assigns cases (and their matched controls) who were vaccinated less than 14 days prior to the case date to the unexposed category. The numbers of cases and controls were too few to allow further subdivision of time since vaccination.\n\nThe effect of vaccination in each of the clinical vulnerability categories was estimated in a conditional logistic regression model (R function survival::clogit, package version_3.2-7, R version 3.6.3) fitted to the full dataset, specifying effects βR2,…,βRJ for the log rate ratio associated with risk categories 2 to J (βR1=0 for the reference category J=1), and nested effects βV1,…,βVJ for the log rate ratio associated with vaccination in each of the J risk categories. With this incidence density sampling design, the conditional odds ratio is the rate ratio. The efficacy of vaccination is 1 minus the rate ratio. The strata in the conditional logistic regression model are matched sets: typically one case and up to 10 controls, but where two cases with the same age, sex and GP practice present on the same day the matched set comprises two cases and up to 20 controls. When estimating the rate ratio within a risk group such as a shielding category, only cases and controls in that risk group are retained in the matched sets. Matched sets in which all are vaccinated or none are vaccinated do not contribute to the rate ratio. We emphasize that the unconditional odds ratios calculated from summary tables of the vaccination status of cases and controls in each risk group cannot be used to estimate rate ratios.6,7 The design controls not only for the matching factors of age, sex and general practice but also for calendar time. Because severe COVID-19 is strongly associated with care home residence and care home residents were a priority group for vaccination, the model includes care home residence as a covariate.\n\nSevere COVID-19 is strongly associated with recent hospital admission. This might confound the association of severe COVID-19 with vaccination if hospital admission causes vaccination appointments to be missed or alternatively if hospital admission increases the probability of being vaccinated by a given date. Therefore the analysis was repeated with adjustment for any hospital discharge from 2 to 8 weeks before presentation date, and for being an inpatient for at least 8 days before presentation date (the criterion for probable/definite hospital-acquired infection8). We also reported the vaccine efficacy for severe COVID-19 separating out those with conditions designated previously by Public Health Scotland as moderate risk from the remainder not eligible for shielding.\n\nTo allow inference on vaccine efficacy in smaller groups, the vaccine efficacy analysis was repeated with the case definition extended to include all cases that were hospitalized or fatal.\n\nThis study was performed within Public Health Scotland as part of its statutory duty to monitor and investigate public health problems. Under the UK Policy Framework for Health and Social Care Research set out by the NHS Health Research Authority, this does not fall within the definition of research and ethical review is not required. Individual consent is not required for Public Health Scotland staff to process personal data to perform specific tasks in the public interest that fall within its statutory role. The statutory basis for this is set out in Public Health Scotland’s privacy notice.\n\nA Data Protection Impact Assessment (DPIA) allows Public Health Scotland staff to link existing datasets. This study was approved under COVID-19 Rapid DPIA 20210023. Datasets were de-identified before analysis.\n\n\nResults\n\nOver the study period there were 863 severe cases among those eligible for shielding of which 731 were fatal and 3482 severe cases among those not eligible for shielding of which 2641 were fatal. 97% of severe cases had a positive nucleic acid test for SARS-CoV-2 and this proportion did not differ between those eligible and ineligible for shielding. Figure 1 shows the time course of daily severe cases in Scotland and vaccination among those eligible for shielding. The proportion vaccinated at least 14 days before is estimated from those sampled as controls who were eligible for shielding. From January to February 2020 the daily number of severe cases declined rapidly and the proportion vaccinated among those eligible for shielding rose from 2% to 87%. The association between vaccination and severe disease is thus confounded by calendar time. This confounding is controlled by the matching of cases and controls on calendar date but we reiterate that because of this the unconditional odds ratios calculated from Tables 1 and 2 cannot be used to estimate rate ratios.7 Information about vaccine efficacy in those eligible for shielding is contributed by matched sets of cases and controls in the narrow time window during which incidence of severe COVID-19 was high and at least some of those eligible for shielding had been vaccinated at least 14 days before. Up to 16 March 2021 the proportion who had received a second dose at least 14 days before the date of sampling was very low: 1069 (0.06%) of the 1831776 controls ineligible for shielding and 33 (0.05%) of the 65573 controls eligible for shielding.\n\n(a) Daily severe cases by risk category; (b) Controls eligible for shielding: proportion vaccinated at least 14 days before, by 7-day window of date on which they were sampled as controls.\n\nPresentation dates from 1 December 2020 to 16 March 2021.\n\nVaccine status coded as 1 if at least one dose at least 14 days before, 0 otherwise.\n\nPresentation dates from 1 December 2020 to 16 March 2021.\n\nVaccine status coded as 1 if at least one dose at least 14 days before, 0 otherwise.\n\nTable 1 shows the vaccination status of severe cases and their controls by risk stratum. Table 2 shows the same breakdown for hospitalised cases and controls. These tables illustrate that the data are very sparse for certain shielding groups especially solid organ transplant recipients.\n\nThe rate ratio for severe COVID-19 associated with vaccination at least 14 days before was 0.29 (95% CI 0.22 to 0.37) in those eligible for shielding, compared with 0.29 (95% CI 0.25 to 0.34) in those ineligible for shielding. When inpatient stay for at least 8 days before presentation and any hospital discharge from 15 to 56 days before presentation were included as covariates, the corresponding rate ratios were 0.40 (95% CI 0.30 to 0.53) in those eligible and 0.39 (95% CI 0.33 to 0.46) in those ineligible for shielding. Table 3 gives a breakdown of the rate ratios for severe COVID-19 associated with vaccination by shielding condition. Point estimates for the rate ratio associated with vaccination in risk groups were similar to the estimated rate ratio in those not eligible for shielding, with the exception of solid organ transplant recipients and others on immunosuppressants in whom the numbers of cases were small (Table 1) and confidence intervals were wide.\n\nConditional logistic regression model matched on age, sex, general practice and presentation date.\n\nVaccine effects nested within each level of risk group.\n\nCare home residence included as covariate.\n\nPresentation dates from 1 December 2020 to 16 March 2021.\n\nVaccine status coded as 1 if at least one dose at least 14 days before, 0 otherwise.\n\nWhen those ineligible for shielding were separated into those with moderate risk conditions and those with no risk condition the rate ratios for severe COVID-19 associated with vaccination were 0.26 (95% CI 0.22 to 0.30) and 0.37 (95% CI 0.29 to 0.47) respectively. Among people with diabetes who were ineligible for shielding (and thus in the “moderate risk conditions” category), the rate ratios for severe COVID-19 associated with vaccination were 0.09 (95% CI 0.01 to 0.70) and 0.16 (95% CI 0.11 to 0.22) in those with Type 1 and Type 2 diabetes respectively.\n\nFor a secondary analysis with larger numbers of cases, the case definition was broadened to include all hospitalized or fatal cases. The rate ratio for hospitalised or fatal COVID-19 was 0.39 (95% CI 0.33 to 0.46) in those eligible and 0.37 (95% CI 0.33 to 0.41) in those not eligible for shielding. Table 4 shows that there was evidence for efficacy in all groups except solid organ transplant recipients in whom even with this broader case definition the numbers of cases and controls were too small for efficacy to be estimated reliably.\n\nConditional logistic regression model matched on age, sex, general practice and presentation date.\n\nCare home residence included as covariate.\n\nPresentation dates from 1 December 2020 to 16 March 2021.\n\nVaccine status coded as 1 if at least one dose at least 14 days before, 0 otherwise.\n\n\nConclusions\n\nEfficacy of a single vaccination dose in protecting against severe COVID-19 was as high or higher in those eligible for shielding as in those without risk conditions. This is reassuring in the light of reports that vaccine-induced antibody conversion is impaired in clinically vulnerable groups such as those with haematologic malignancies9 and those on immunosuppressants for chronic inflammatory disease.10 However for solid organ transplant recipients – the group at highest risk among those eligible for shielding – the number of cases in the Scottish population is too small for the efficacy of vaccination to be estimated.\n\nStrengths of this analysis are the linkage to a pre-existing register of individuals designated as clinically extremely vulnerable, the ability to adjust for recent hospital exposure for which there was evidence of some confounding effect and the focus on severe COVID-19 rather than all test-positive cases. To ensure complete ascertainment of severe cases the case definition was pre-specified to include clinically diagnosed cases without a positive nucleic acid test; as only 3% of severe cases during the study period did not have a positive test, any diagnostic misclassification of these cases is unlikely to change the results appreciably. Limitations of our analysis are the small numbers with specific risk conditions, especially solid organ transplant recipients, and that the estimates of efficacy are effectively for first dose only as very few of those eligible for shielding had received a second dose of vaccine during the study period. Although data published by Public Health Scotland show that by 29 May 2021 90% of those eligible for shielding had received two vaccine doses, there have been too few cases of severe disease after February 2020 for an updated estimate of the efficacy of two doses in those eligible for shielding to be reliable. The estimates of vaccine efficacy that we report for the general population are similar to results reported from other countries but we cannot comment on generalizability with respect to the clinically extremely vulnerable, as studies of this in other countries have not been reported.\n\nThese estimates of efficacy of a first dose of vaccine over all time periods from 14 days post-vaccination were similar to those estimated at 14-20 days post-vaccination for the total population of Scotland.1 As we have previously shown that the shielding group overall have approximately six-fold higher risk of severe COVID-19 than those of the same age and sex without risk conditions,5 efficacy of at least 82% (a rate ratio no more than 0.18) with a single dose would be required to lower risk to the level of unvaccinated persons of the same age and sex without risk conditions.\n\nWhilst these results give reassurance that the vaccine is showing efficacy overall in people with extreme clinical vulnerability to COVID-19, such persons should also be advised that they remain at increased risk after a single dose (about twofold compared to those of the same age and sex without risk conditions, given the vaccine-associated rate ratio of about 0.3), and should remain vigilant until they have received their second dose and background transmission rates in their locale are low. A study in a larger population is needed to estimate efficacy in solid organ transplant recipients. This would lay the basis for advising people in this group about their ongoing risk level, for whether to recommend a third dose of vaccine to improve immunogenicity,11 and for assessing the need for development of alternatives to vaccines such as synthetic antibodies.\n\n\nDeclarations\n\nThe component datasets used in this study are available via the COVID-19 Research Database hosted by Public Health Scotland’s electronic Data Research and Innovation Service (eDRIS). A guidance document for applicants is available on the eDRIS website. There are no restrictions on eligibility to apply.\n\nAnalysis code available from: https://github.com/pmckeigue/covid-scotland_public/releases/tag/beta\n\nArchived analysis code as at time of publication: https://doi.org/10.5281/zenodo.5082340\n\nLicense: OSI", "appendix": "Acknowledgements\n\nWe thank all staff in critical care units who submitted data to the SICSAG database, the Scottish Morbidity Record Data Team, the staff of the National Register of Scotland, the Public Health Scotland Terminology Services, the HPS COVID-19 Laboratory & Testing cell and the NHS Scotland Diagnostic Virology Laboratories.\n\n\nReferences\n\nVasileiou E, Simpson CR, Shi T, et al.: Interim findings from first-dose mass COVID-19 vaccination roll-out and COVID-19 hospital admissions in Scotland: A national prospective cohort study. Lancet (London, England). 2021 May; 397(10285): 1646–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKeigue PM, Weir A, Bishop J, et al.: Rapid Epidemiological Analysis of Comorbidities and Treatments as risk factors for COVID-19 in Scotland (REACT-SCOT): A population-based case-control study. PLoS Med. 2020 Oct; 17(10): e1003374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHealth Protection Scotland: Search criteria for highest risk patients for shielding. Public Health Scotland. 2020 May.\n\nKanani N, Waller E: Caring for people at highest risk during COVID-19 incident.NHS England; 2020 Mar.\n\nMcKeigue PM, McAllister D, Caldwell D, et al.: Relation of severe COVID-19 in Scotland to transmission-related factors and risk conditions eligible for shielding support: REACT-SCOT case-control study. BMC Medicine 2021 Jun; 19:149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreslow NE, Day NE, Halvorsen KT, et al.: Estimation of multiple relative risk functions in matched case-control studies. Am J Epidemiol. 1978 Oct; 108(4): 299–307. PubMed Abstract \| Publisher Full Text\n\nGreenland S, Thomas DC: On the need for the rare disease assumption in case-control studies. Am J Epidemiol. 1982 Sep; 116(3): 547–53. PubMed Abstract \| Publisher Full Text\n\nEuropean Centre for Disease Prevention and Control: Surveillance definitions for COVID-19. European Centre for Disease Prevention and Control.2020. Reference Source\n\nMonin-Aldama L, Laing AG, Muñoz-Ruiz M, et al.: Interim results of the safety and immune-efficacy of 1 versus 2 doses of COVID-19 vaccine BNT162b2 for cancer patients in the context of the UK vaccine priority guidelines. medRxiv. 2021 Mar; 2021.03.17.21253131. Publisher Full Text\n\nDeepak P, Kim W, Paley MA, et al.: Glucocorticoids and B Cell Depleting Agents Substantially Impair Immunogenicity of mRNA Vaccines to SARS-CoV-2. medRxiv. 2021 Apr; 2021.04.05.21254656. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamar N, Abravanel F, Marion O, et al.: Three Doses of an mRNA Covid-19 Vaccine in Solid-Organ Transplant Recipients. N Engl J Med. 2021 Jun; 0(0): null. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "90959", "date": "22 Sep 2021", "name": "Andreas Martin Lisewski", "expertise": [ "Reviewer Expertise computational life sciences", "infectious diseases" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMcKeigue and colleagues report from a controlled observational study on COVID-19 (mRNA and adenoviral vector) vaccine efficacy (VE) among the clinically vulnerable population in Scotland. These individuals were identified at increased risk for severe COVID-19 and thus were specifically eligible for shielding support through vaccination. The study was conducted between 1 December 2020 and 16 March 2021 and severe COVID-19 was defined \"as cases with critical care or fatal outcome.\" It can thus be viewed as a continuation of earlier reports in a similar context (Vasileiou et al., 20211)\nWhile the authors' reasoning that has lead to the present study seems quite clear, the underlying COVID-19 epidemiology data from \"Public Health Scotland\" (PHS) for the above time period appears to be problematic.\nSpecifically, the “PHS COVID-19 Statistical Report, as of 21 June 2021” [2, p 28], reports mortality data that is directly relevant to the study by McKeigue et al: the number of “overall deaths that have occurred within 28 days of a COVID-19 vaccination” between 8 December 2020 and 11 June 2021, where a total of 5522 people died within 28 days of receiving a COVID-19 vaccine in Scotland (number of days between vaccine and death is 0-27, where 0 is the day of vaccination, all age groups). In contrast, by comparing the 2015-19 death rate, 8718 deaths would have been expected among the vaccinated population within 28 days of receiving their COVID-19 vaccination leading to mortality rate ratio of 0.59 (after 1 dose) and 0.66 (after 2 doses). When taking the 2020-21 death rate as reference [3], the overall reduction in mortality following COVID-19 vaccinations is even more striking because between Dec 2020 and June 2021 the number of deaths in Scotland was on average ~6% higher than in the corresponding 2015-19 months. Thus, following COVID-19 vaccination, we are facing an almost ~50% reduction in mortality across all causes of death during the study's relevant time period (Note 1: The remarks on page 29 of the PHS report do not explain this reduction. Note 2: During the same period, deaths involving COVID-19 [4] amounted to only about ~10% of the total deaths in Scotland.)\nFrom these PHS data, it would be therefore possible to infer a considerable efficacy of COVID-19 vaccines against all causes of death, including COVID-19. Clearly, such a conclusion would be epidemiologically unacceptable.\nBecause the observational study of McKeigue et al. is apparently based on these PHS data (e.g., compare Fig. 1a in McKeigue et al. with Fig. 7 in [2]), I ask the authors to clarify the above anomaly in mortality associated with COVID-19 vaccinations as well as its relationship to their own results. Without such clarification their reported COVID-19 VE among vulnerable groups cannot be fully trusted. I even predict that a complete explanation of the above anomaly would be of considerable public interest.\nIn my opinion, this important point first needs to be comprehensively addressed before reviewing any further details of their manuscript.\nReferences:\n[1] https://doi.org/10.1016/S0140-6736(21)00677-2\n[2] https://publichealthscotland.scot/publications/covid-19-statistical-report/covid-19-statistical-report-23-june-2021/\n[3] https://www.nrscotland.gov.uk/files//statistics/weekly-monthly-births-deaths-data/2021/aug/monthly-august-21-tab3.xlsx\n[4] https://www.nrscotland.gov.uk/files//statistics/covid19/covid-deaths-21-data-week-36.xlsx\n\nIs the work 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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7284", "date": "07 Oct 2021", "name": "Paul McKeigue", "role": "Author Response", "response": "The reviewer does not comment directly on our paper but rather queries whether the underlying dataset is correct. His concerns are based on a routine surveillance report from Public Health Scotland, not written by us, which reported a rate ratio of 0.59 for all-cause mortality in the first 28 days after first vaccination dose. As the reviewer notes, this is unlikely to be a causal effect of vaccination. The report from Public Health Scotland noted that this could be explained by a number of factors but did not elaborate further. A plausible explanation for lower total mortality shortly after vaccination in vaccine recipients is allocation bias. Specifically, people who are terminally ill and likely to die in the next few weeks are less likely to attend when they are sent an appointment for vaccination. Thus mortality in the first few weeks after vaccination will be lower than the average for people of the same age and sex. On this basis we would predict that mortality from cancer and other conditions that cause death after prolonged illness would be especially low in the first few weeks after vaccination, but mortality from causes where deaths are often sudden (circulatory disease, external causes) would be reduced less. Further comments can be found here." } ] }, { "id": "120320", "date": "14 Feb 2022", "name": "Tauqeer Hussain Mallhi", "expertise": [ "Reviewer Expertise Infectious Diseases", "Pharmaceutical Care", "Nephrology", "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\nThank you for the invitation. In this study, McKeigue and colleagues evaluated the efficacy of vaccines in an extremely vulnerable population. This study is conducted with a good design, the research question is quite reasonable and hypothesis testing is also up to the mark. However, I have a few additions, if the authors could incorporate them in the revised version:\nThe authors have to define a few terms in the methodology section under the heading of \"operational definitions\", these include a definition of a vulnerable population, definitions of vaccine efficacy (prime outcomes of the study), and definition of vaccine status.\n\nTo my knowledge about statistics, the authors used appropriate and relevant tests to make an inferential comparison. However, I suggest putting the information on how variables opted for logistic regression.\n\nPlease mention the total number of participants in the abstract section too.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-663
https://f1000research.com/articles/9-1449/v1	11 Dec 20	{ "type": "Research Article", "title": "Classification of small ruminant lentivirus subtype A2, subgroups 1 and 2 based on whole genome comparisons and complex recombination patterns", "authors": [ "Aaron M. Dickey", "Timothy P. L. Smith", "Michael L. Clawson", "Michael P. Heaton", "Aspen M. Workman", "Timothy P. L. Smith", "Michael L. Clawson", "Michael P. Heaton" ], "abstract": "Background: Small ruminant lentiviruses (SRLVs) cause a multisystemic chronic wasting disease in sheep across much of the world. SRLV subtype A2 is prevalent in North America and further classified into multiple subgroups based on variation in the group antigens gene (gag) and envelope (env) genes. In sheep, the ovine transmembrane protein 154 (TMEM154) gene is associated with SRLV susceptibility. Ewes with at least one copy of TMEM154 encoding a full-length protein with glutamate at position 35 (E35; haplotypes 2 and 3), are highly susceptible to SRLV infection while ewes with any combination of TMEM154 haplotypes which encodes lysine (K35; haplotype 1), or truncated proteins (haplotypes 4 and 6) are several times less so. A2 subgroups 1 and 2 are associated with host TMEM154 genotypes; subgroup 1 with the K35/K35 genotype and subgroup 2 with the E35/E35 genotype. Methods: The goals of this study were to analyze sequence variation within and among SRLV subtype A2 subgroups 1 and 2 and to identify genome-scale recombination patterns. This was done using full-length assemblies of virus samples. Results: Consensus viral genomes were assembled for 23 infected sheep, including animals of assorted TMEM154 genotypes comprised of haplotypes 1, 2, or 3. Viral genome analysis identified viral subgroups 1 and 2 among the samples, and revealed additional sub-structure within subgroup 2 based on models predicting complex patterns of recombination between the two subgroups in several genomes. Animals with evidence of dual subgroup infection also possessed the most diverse quasi-species and the most highly recombined genomes. Conclusions: The viral subgroup framework developed to classify SRLV consensus genomes along a continuum of recombination suggests that animals with the TMEM154 E35/K35 genotype may represent a reservoir for producing viral genomes representing recombination between A2 subgroups 1 and 2.", "keywords": [ "Small ruminant lentivirus", "recombination", "quasispecies", "ovine progressive pneumonia virus" ], "content": "Introduction\n\nSmall ruminant lentiviruses (SRLV) are a genetically diverse group of lentiviruses belonging to the family Retroviridae. SRLV infect sheep, goats, and wild ruminants worldwide causing a lifelong persistent infection clinically characterized by wasting, interstitial pneumonia with labored breathing, indurative mastitis, arthritis, and/or more rarely, encephalitis (Blacklaws, 2012). Disease progression is typically slow, and the genetic background of both the host and virus influence the clinical course and outcome of infection (Heaton et al., 2012; Sider et al., 2013).\n\nThe SRLV genome consists of two identical positive-sense single-stranded RNA subunits approximately 9 kb in length. The viral genome, which is integrated into host cells in the form of a provirus, contains three structural genes (gag, pol, and env) and three regulatory genes (tat, vif, and rev) flanked by non-coding long terminal repeat regions (LTRs). Phylogenetic analysis based on partial group antigens gene (gag) and polymerase (pol) gene sequences divides these viruses into five major genotype groups, A-E, which are further divided into different subtypes (Shah et al., 2004). The groups differ by 25–37% and the subtypes differ by 15–27% in nucleotide composition at these genomic loci (Ramírez et al., 2013). Genotypes A and B are distributed worldwide, while genotypes C-E are geographically restricted (Gjerset et al., 2006; Grego et al., 2009; Shah et al., 2004).\n\nThe comprehensive set of haplotypes from the same viral species in a single host is known as a quasispecies (Eigen et al., 1988). The viral quasispecies arises from the interplay of three evolutionary factors throughout the duration of a chronic infection. These three factors are mutation, recombination, and selection. Point mutations and small indels are introduced into the SRLV viral genome due to its low fidelity, error prone reverse transcriptase enzyme. Selection can be driven by the host immune system and antiviral medications. Work with related lentiviruses has revealed selection pressure on mutants can produce variants diverging by up to 5% from the founder strain in a few years, though this rate does not remain constant over time (Lee et al., 2008; Shankarappa et al., 1999). Co-infection (simultaneous infection) or superinfection (sequential infection) can lead to more dramatic genetic changes through recombination, but these two types of dual infection are difficult to distinguish in the absence of a detailed infection history. Regardless of the timing, when diverse viral subtypes infect the same host cell, the reverse transcriptase readily switches between viral genomes, with estimates of three to nine recombination events per replication cycle (Jetzt et al., 2000). This process allows rapid emergence of new viral strains that may exhibit novel phenotypes (reviewed in Ramírez et al., 2013). In vivo recombination has been documented between genotypes A and B and among genotype A and B subtypes (Andrésdóttir, 2003; Fras et al., 2013; Pisoni et al., 2007; Ramírez et al., 2011; Sider et al., 2013). However, recombination in SRLVs has not previously been characterized at the whole-genome level.\n\nThe virus-host interaction is a continuous co-evolutionary process. In sheep, genetic variation in the host transmembrane protein 154 (TMEM154) gene associates with SRLV susceptibility (Heaton et al., 2012; Leymaster et al., 2013; Leymaster et al., 2015; Molaee et al., 2018; Molaee et al., 2019; Yaman et al., 2019). Twelve TMEM154 haplotypes have been identified (Heaton et al., 2012), and ewes homozygous for haplotype 1, which encodes a lysine at position 35 (K35), had a decreased risk of SRLV infection compared to those with one or two copies of haplotype 2 or 3 (ancestral), both of which encode a glutamate at position 35 (E35). Two distinct SLRV A2 subgroups have been identified that infected sheep in association with their TMEM154 E35K genotypes and specific diplotypes (Sider et al., 2013). SLRV A2 subgroup 1 viruses were significantly more likely to infect sheep with either TMEM154 diplotypes 1,1 or 1,4. TMEM154 haplotype 4 contains a rare frameshift mutation (A4Δ53) and does not produce mRNA encoding functional amino acids downstream of amino acid position 4 of the precursor protein. Consequently, subgroup 1 associated with hemizygous or homozygous K35 genotypes. SLRV A2 subgroup 2 viruses were more likely to infect sheep with one or two copies of either haplotype 2 or 3, and that could also have one copy of haplotype 4 (Sider et al., 2013). Consequently, subgroup 2 viruses associated with hemizygous or homozygous E35 genotypes. While it has been proposed that TMEM154 E35K hemizygosity or homozygosity could be a factor in SRLV A2 subgroup associations, the biology remains obscured.\n\nSRLV A2 subgroups 1 and 2, and their associations with TMEM154 E35K are not well understood. The subgroups were previously defined by sequence variation in a partial region of the gag and the transmembrane region of the envelope gene (env), which were not thought to have critical roles in potential SRLV TMEM154 interactions (Sider et al., 2013). Due to relatively short sequenced regions of SRLV A2 subgroups 1 and 2 genomes and because of extensive recombination detected within the sequences, the cutoff between groups 1 and 2 was not clear (Sider et al., 2013). In this study, we obtained full-length consensus SRLV genomes from a cross-section of sheep belonging to the flock which was part of the original TMEM154 association studies (Heaton et al., 2012; Heaton et al., 2013; Leymaster et al., 2013; Leymaster et al., 2015; Sider et al., 2013). These sheep had all been genotyped as containing haplotypes 1, 2, or 3. Ovine TMEM154 haplotypes 4-12 were not represented in this study. The goals of this study were to 1) obtain full-length consensus genomes for members of SLRV A2 subgroups 1 and 2; 2) identify subgroup 1 and 2 specific variants, while accounting for recombination, and use these variants to estimate levels of intra-host sequence variation; 3) search for subgroup-specific functional viral variants relative to host TMEM154 E35K genotypes.\n\n\nMethods\n\nAll animal procedures were reviewed and approved by the United States Department of Agriculture (USDA), Agricultural Research Service (ARS), U.S. Meat Animal Research Center (USMARC) Animal Care and Use Committee prior to their implementation (Experiment Number 96). The source flock’s history of disease surveillance is also relevant when requesting reference samples described in any report. Since first stocking sheep in 1966, USMAC has not had a known case of scrapie. Until 2002, surveillance consisted of monitoring sheep for possible signs of scrapie and submitting brain samples to the USDA Animal and Plant Health Inspection Service (APHIS) National Veterinary Services Laboratory in Ames, IA for testing. All tests have been negative. Since April 2002, USMARC has voluntarily participated in the APHIS Scrapie Flock Certification Program, is in compliance with the National Scrapie Eradication Program, and is certified as scrapie-free. With regards to other transmissible diseases, it is recognized that the USMARC flock of 2000 to 4000 breeding ewes is located in a bluetongue medium incidence area and is known to have some prevalence of contagious ecthyma (sore mouth), foot rot, paratuberculosis (Johne’s disease), ovine progressive pneumonia (visna-maedi), and pseudotuberculosis caseous lymphadenitis.\n\nLungs from 22 sheep at the US Meat Animal Research Center in Nebraska that were a part of the original study for association of A2 subgroups 1 and 2 with TMEM154 haplotypes (Heaton et al., 2012; Sider et al., 2013) were used in this study (Table 1). These sheep were all genotyped as containing haplotypes 1, 2, or 3. SRLV seropositive sheep were originally diagnosed with clinical ovine progressive pneumonia (OPP) by gross morphology and histopathology of both lung and mediastinal lymph node. In addition, colostrum from one seropositive ewe (201373037) showing no clinical signs of disease was used in this study (Table 1).\n\nNo long reads: coverage includes only short reads, 10 to 26 nucleotides not fully resolved on 5' ends.\n\nThe population to which a consensus genome was assigned in the subgroup 1 vs 2a vs 2b recombination model (Figure 3A). r/d: recombinant/dual infections.\n\n*The short read + long read consensuses reported here are identical (200303013) and slightly different (199906011) from the long-read consensuses reported by Workman et al., 2017 (KY358787 and KY358788 respectively). See results.\n\nLung samples were homogenized using a gentleMACS dissociator (Miltenyl Biotec; San Diego, CA) in minimal essential medium (Gibco, Thermo Fisher Scientific, Waltham, MA). Homogenates were then subjected to two freeze/thaw cycles to further ensure cell lysis. Homogenates were clarified by centrifugation followed by sequential filtration through 0.45 and 0.2-µm syringe filters to remove cellular debris. Clarified samples (250 uL) were treated with 20 U RNase One (Promega, Madison, WI) and 30 U Turbo DNase (Ambion, Austin, TX) in 1x DNase buffer (Ambion) at 37°C for 90 minutes to degrade unprotected host and environmental nucleic acids. To ensure continuous DNase activity, 10 U of DNase was added to the sample every 30 minutes during the 90-minute incubation. Remaining nucleic acids were isolated using Trizol LS (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. A final DNase treatment was performed to remove final traces of DNA from the RNA preparation.\n\nColostrum (approximately 4 mL) was manually collected from a SRLV seropositive ewe within the first 24 hours after giving birth. Colostrum was diluted 1:2 with cold phosphate-buffered saline and centrifuged at 800 x g for 15 minutes at 4°C. The cream layer was skimmed from the top and 250 µL of milk was treated with nucleases and RNA was extracted as described above.\n\nRNA libraries were prepared as previously described (Workman et al., 2015; Workman et al., 2017; Workman et al., 2018). Briefly, 100 ng of total RNA were used as input material for Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA). Libraries were prepared as specified by the manufacturer’s protocol without the initial step of poly-A selection on oligo-dT beads. Libraries were sequenced on an Illumina MiSeq instrument with a 600-cycle kit (v3) to generate 2 × 300 bp paired-end reads. Index adapters were removed from raw sequence reads using cutadapt 1.9.1 (Martin, 2011) or BBDuk 35.82 (Brian Bushnell within Geneious 11.1.4 (Kearse et al., 2012) (Biomatters, Auckland, New Zealand) and trimmed reads were screened against the UniVec_Core database to remove contaminating vector sequences. Overlapping paired-end reads were merged using BBMerge 8.9 (Bushnell within Geneious).\n\nThe remaining RNA was used to generate long-read sequencing libraries according to a modified RNA Iso-Seq with poly(A) tails added to the 3’ ends to allow cDNA synthesis of subgenomic fragments. The resulting cDNA was amplified, size fractionated and the largest fragments were used to make SMRTbell templates, which were sequenced on a Pacific Biosciences RSII instrument. SMRT Analysis was used to generate error corrected circular consensus sequences (CCS) from the raw reads and adapters and poly(A) tails were removed with BBDuk.\n\nReads greater than 1,000 nucleotides in length were assembled de novo with the Geneious assembler. All reads were then mapped to the de novo assembly, and the resulting consensus sequence was reported. Four strains lacked good quality long-read sequence data (Table 1) so the MiSeq short reads were assembled using template-assisted assembly in Geneious following Workman et al. (2018) with accessions KY358787 and KY358788, respectively, used as subgroup 1 and 2 references. These two reference strains were included in this study.\n\nTo calculate total genome coverage for each sample, merged and unmerged paired-end reads plus long-read CCS’s were jointly mapped to the consensus genome using the Geneious mapper with 40% maximum allowable mismatches, a word length of 24, an index word length of 14, 10% allowable gaps and a maximum gap size of 50.\n\nUp to 26 nucleotides on the 5’ ends did not fully resolve in genomes with only short-read sequencing available (Table 1). Genomes were manually annotated based on alignments with full-length SRLV genomes available in GenBank.\n\nTo augment the newly reported genome sequences, full-length SLRV and SLRV-like genomes were also downloaded from GenBank using the following query of the Nucleotide database on October 11, 2019: txid11660[ORGN] OR txid2169971[ORGN] OR txid11653[ORGN] OR txid11663[ORGN] AND (\"8000\"[SLEN] : \"12000\"[SLEN]). 79 unique genomes were aligned using MUSCLE 3.8.425 (Edgar, 2004 in Geneious 11.1.5) and a neighbor net phylogenetic network was built using default settings in Splitstree5 (Huson & Bryant, 2006).\n\nPopulation assignment of each genome generated in this study was performed in FineStructure version 4 and its companion program, ChromoPainter version 1 (Lawson et al., 2012). To generate the alignment used in FineStructure, MUSCLE was used followed by manual refinement. Gaps in the alignment were converted to presence/absence characters as-is with simple gap patterns reduced to a single character regardless of size. The recombination rate map used in FineStructure and Chromopainter was estimated using the LDhat 2.2a interval program (McVean et al., 2004). For LDhat, pre-computed likelihoods were generated using a population-scaled per-site mutation rate inferred from the data (0.07587), a grid size of 101 and a maximum population-scaled whole-genome recombination rate of 100. The variable rate estimation was run for 10 million iterations with the first half discarded as burn-in and a block penalty of 20. To avoid alignment edge inaccuracies, the final 10% of SNPs from the 3’ terminus were placed preceding the 5’ terminus and the first 10% of SNPs from the 5’ terminus were placed following the 3’ terminus, essentially simulating circular genomes. The recombination rate point estimates at these simulated edges were removed. The outputs of LDHat were population-scaled recombination rates (p), which relate to the biochemical recombination rate (r) according to the formula p=2Ner where Ne is the effective population size. Ne is difficult to estimate. Estimates for HIV, a related lentivirus which also produces chronic infections, vary by several orders of magnitude (Pennings et al., 2014). Computational estimates of Ne in viruses also require time-series sampling data (Rousseau et al., 2017). Thus, Ne was not estimated for this study and the Chromopainter recombination outputs were interpreted as being scaled by 2Ne. The FineStructure analysis was run using the linkage model, the variable recombination rate map estimated as described above, and specifications for haploid genomes. ChromoPainter detected shared ancestry by reconstructing each genome as a probabilistic mosaic of ‘chunks’ derived via recombination from all other input genomes (termed ‘all vs all painting’) and FineStructure assigned the genomes to populations based on the quantity and lengths of these shared genomic chunks. The following settings were changed from the default in FineStructure and/or ChromoPainter to ensure convergence of estimated parameters: ChromoPainter chunks-per-region parameter k=38, ChromoPainter samples s=10, Markov Chain Monte Carlo (MCMC) iterations=1e6 FineStructure tree finding maximization steps=1e6 and FineStructure independent MCMC runs=10. ChromoPainter iterations i=100 were run for estimating the global switch rate parameter and the global mutation rate. In FineStructure, Strain 201373037 was excluded from the estimation of the global switch rate parameter since it trended toward 0 and stalled the program. This indicated very closely related samples in the dataset (G. Hellenthal, personal communication). Inconsistency in assignment of individuals to populations was resolved by assigning all ambiguously assigned individuals to the largest of the potential populations.\n\nTo model subgroup-specific recombination, ChromoPainter version 2 was run in ‘donor-mode’ using the population assignments, global switch-rate parameter (0.168355), and population specific mutation rates output from the FineStructure analysis. These models were run for 500 iterations to ensure convergence of copy probability. In contrast to all vs all painting, donor specific painting assigns genomic chunks to recipient genomes based on donor populations comprised of multiple genomes. To increase genome-wide assignment probability of subgroup specific SNPs, consensus genomes with evidence of large recombination blocks were iteratively removed from each subgroup pool of donor genomes if average copy probability was increasing. This was done to eliminate the most obvious recombinants from the pools while retaining enough donor genomes to optimize the recombination model. The output donor subgroup-assignments for each SNP were used to identify subgroup-specific SNPs while accounting for recombination. Recombination rates have been estimated for several RNA retroviruses, and most estimates are in the range of 10-3 to 10-5 (Tromas et al., 2014). Thus, we also ran the diagnostic SNP identification model using a range of fixed recombination rates (10-3 to 10-8 Morgans-per-base pair) to confirm that diagnostic SNP count did not change when varying input recombination rate by several orders of magnitude.\n\nThe subgroup-specific SNP content was quantified for each strain by extracting intra-host SNPs meeting default statistical restrictions (Maximum Variant P-value of 10-6, Minimum Strand-Bias P-value of 10-5 when exceeding 65% bias) relative to their final alignment in Geneious 11.1.4. The percentage of subgroup 1, subgroup 2, and ‘other’ SNPs at each diagnostic locus was calculated for each consensus genome. Subgroup partial dual infections were inferred as contiguous or nearly contiguous SNP blocks bearing both subgroup diagnostic alleles. For visualization relative to the consensus genome, these inferred partial dual infections were limited to genome blocks or scaffolds ≥50 nucleotides long where variants diagnostic for both subgroups co-occurred at a frequency of ≥5% and ≥2 reads. The 5% threshold was chosen as it was a conservative value accounting for sequencing error and mis-mapping when calling quasispecies SNPs. Multiple putative dual infection blocks were extended or scaffolded together when separated by <50 nucleotides and 1 diagnostic SNP. We characterize these as being caused by dual infection with unknown underlying viral haplotypes containing SNPs diagnostic to both subgroups at these regions as this is the most parsimonious explanation. However, quasispeciation in the absence of dual infection is an increasingly possible explanation as the numbers of adjacent subgroup diagnostic SNPs in the characterized regions decrease.\n\nOnce subgroup-specific SNPs were identified in the context of recombination, intra-host amino acid variation (functional quasispecies) at the subgroup diagnostic loci were identified by extracting variants from the alignments in Geneious 11.1.5 using the same statistical criteria applied to nucleotides and occurring at a frequency of ≥5%. Highly variable domains in gag and env previously shown to be important were analyzed in the context of subgroup assignment and host TMEM154 diplotype. Additionally, SignalP-5.0 (Nielsen, 2017) was used to predict the env signal peptide cleavage site.\n\n\nResults\n\nCoverage of the 23 genomes ranged from 52- to 2661-fold (Table 1). Complete or near-complete genomes ranged from 9164 to 9215 nucleotides in length. The combined short read and long read consensus genome of strain 199906011 was slightly different from the long read only consensus (KY358788). The sites with differences had a high frequency of the minor allele in the quasispecies in the long read only consensus and so switched the identity of the minor allele in the combined short read and long read consensus. The combined short read and long read consensus of strain 200303013 was identical to the long read only consensus (KY358787). A phylogenetic network using full-length SRLV genomes was dominated by groups A and B (Figure 1). Subtype A2 strains from the United States of America occupied a distinct cluster within the network. Additional clusters on the tree were generally represented by a single geographic region, with Italy representing the highest number of unique clusters. Recombination was evident in several clusters of the network including subtype A2. FineStructure analysis of the 23 genomes from this study identified six distinct populations across the consensus genomes based on all-vs-all genome painting (Figure 2). FineStructure identified two distinct subgroup 2 populations identified in Figure 2 as subgroup 2a and subgroup 2b. Subgroup 2a is intermediate between subgroup1 and subgroup2b on principal component 1 (Figure 2).\n\nColors correspond to genomes assigned to subgroup specific pools of donor genomes in recombination analysis (Table 1).\n\nThe first three principal components account respectively for 40, 18 and 11 percent of the variance in the data.\n\nThe recombination models utilized were (A) subgroup1 vs subgroup 2a vs subgroup 2b and (B) subgroup 1 vs subgroup 2b. The black boxes highlight large subgroup 1 recombination blocks in subgroup 2a genomes.\n\nDue to the large number of FineStructure identified populations, several recombination models were run in ChromopainterV2. Five of the six identified populations had >1 consensus genome. The model with five potential populations of donor genomes indicated that the three most frequent populations (subgroup 1, subgroup 2a and subgroup 2b) accounted for >88% of the SNPs and were the majority donors to 22 genomes (Extended data, Supplementary Figure 1 (Dickey & Workman, 2020b)). The model was run with these three donor populations only (Figure 3A). Due to the relative location of the four populations along principal component 1 (Figure 2), the model was also run with subgroup 2b and subgroup 1 as the only two donor populations (Figure 3B). This showed possible complex recombination blocks between subgroup 1 and subgroup 2b in subgroup 2a genomes (Figure 3B). Subgroup 2a genomes also showed intermediate average pairwise percent divergences between subgroups 1 and 2b (Table 3). All models showed many predicted recombination blocks spanning the consensus genomes (Table 2, Figure 3 and Extended data, Supplementary Figure 1 (Dickey & Workman, 2020b)). Finally, a recombination model was run with only 2 donor populations, subgroup 1 and subgroup 2 (as 2a+2b) (Figure 4). This was done to identify and extract subgroup specific SNPs while accounting for recombination. Viral strains 200050064 and 199916128 were removed from the subgroup 1 and 2 donor pools respectively based on having the highest proportion of inter-subgroup recombination (Table 2, Figure 3) and this improved the subgroup 1 vs subgroup 2 recombination model. Further removal of genomes as potential donors did not improve the model. The average number of alternate subgroup recombination blocks (Chromopainter’s chunkcount) was 2-fold higher in subgroup 2 genomes than subgroup 1 (Table 2). Chromopainter’s chunklengths parameter averaged 3-fold higher in subgroup 2 and predicted population specific mutation rate averaged 3-fold higher in subgroup 2 consensus genomes than subgroup 1 (Table 2).\n\nChromopainter’s ‘chunkcounts’ parameter is defined as the number of genomic chunks from a population of donor genomes, assigned to the recipient genome via recombination. ‘Chunklengths’ are the combined lengths (in centimorgans X 2Ne) of these chunks. Donor specific mutation rate is the amount of mismatching across the recipient chunklengths divided by the number of loci. Donor status is the population of donor genomes to which a consensus genome was assigned in the final subgroup 1 vs subgroup 2 recombination model (Figure 4). To improve recombination models, genomes with high inter-subgroup recombination were iteratively removed from the populations if model quality (as judged by average copy probability) was increasing.\n\nChromopainterV2 mutationprobs parameter.\n\nThe recombination model utilized was subgroup1 vs subgroup 2.\n\nSubgroup diagnostic SNP inference accounting for recombination between subgroup 1 and subgroup 2 (as FineStructure population 2a+2b) resulted in 413 diagnostic SNPs (Extended data, Supplementary Table 1 (Dickey & Workman, 2020a)). The frequency of alternate subgroup diagnostic alleles was 3-fold higher in subgroup 2 consensus genomes than subgroup 1 (Table 4).\n\nDonor status is the population of donor genomes to which a consensus genome was assigned in the final subgroup 1 vs subgroup 2 recombination model (Figure 4).\n\nIntra-host variation at subgroup-specific SNPs was analyzed to infer the presence of dual infections. The parameters specified for predicting subgroup dual infections resulted in 73 genomic regions indicative of dual infection across nine consensus genomes (range: 1–14, average: 8.2), averaging 261.6 nucleotides in length (range: 55–1482) and comprising 2%–45% of the genome (Extended data, Supplementary Table 2 (Dickey & Workman, 2020a), Figure 5).\n\nThe background is the subgroup 1 vs subgroup 2 recombination model (Figure 4). Genomic regions indicative of dual infection contained both subgroup diagnostic alleles at a frequency ≥5% for at least 2 consecutive diagnostic SNPs and 50 nucleotides.\n\nOf the 413 subgroup diagnostic SNPs identified, 106 were non-synonymous (Extended data, Supplementary Tables 1 and 3 (Dickey & Workman, 2020a)). A2 subgroup 1 and 2 specific variants were identified in all viral genes with frequencies ranging from 2.2 to 4.3%. Sequence analysis of the immunodominant epitope in the gag gene revealed two adjacent SNPs that resulted in a single amino acid change distinguishing subgroups 1 and 2 (Extended data, Supplementary Table 3 (Dickey & Workman, 2020a)). Analysis of the env gene variable regions V1-V5 (Valas et al., 2000) found no subgroup specific SNP in variable regions V1 and V2, five subgroup specific variants each in V3 and V4, and one in V5 (Extended data, Supplementary Table 3 (Dickey & Workman, 2020a)). Six subgroup defining variants were identified in the predicted env signal peptide.\n\n\nDiscussion\n\nThis study provides full-length or near-full-length consensus genomes from 21 new SRLV subtype A2 strains used in determining the viral subgroup association with TMEM154 E35K genotypes (Heaton et al., 2012; Sider et al., 2013) in addition to the two subgroup representative strains from Workman et al., 2017. These genomes were analyzed for recombination and population structure using a chromosome ‘painting’ model. Several genomes showed complex recombination patterns. Furthermore, this model was used to identify 413 subgroup-specific SNPs while accounting for recombination. This information was used to quantify intra-host genetic diversity at diagnostic SNPs and estimated nine animals were dually infected with viral recombinants such that they have diagnostic SNPs from both virus subgroups for portions of their genome. Lastly, we analyzed important functional domains in the virus genome in the context of virus subgroup and host TMEM154 diplotypes focusing only on haplotypes 1, 2 and 3.\n\nThe SRLV phylogenetic network contained subtype A2 as a distinct cluster (Figure 1). Several genomes in this cluster are connected by many nodes indicating inter-subgroup recombination. The FineStructure analysis identified two distinct subgroup 2 ‘populations’ of consensus genomes. These have been provisionally designated subgroup 2a and subgroup 2b. Subgroup 2a is intermediate between subgroup 1 and subgroup 2b both in terms of position along principal component 1 (Figure 2) and in terms of genetic distance (Table 3). The subgroup 2 genome reported by Workman et al., 2017 (viral strain 199906011, GenBank KY358788.1) belongs to subgroup 2a (Table 1). This is a case where it is difficult to distinguish, with certainty, the recombinant from the second viral donor, however there is some evidence that subgroup 2b may be a ‘purer’ representative of subgroup 2. Even when subgroup 2a genomes were included among the subgroup 2 donors, the only consensus genomes that resolved unambiguously as subgroup 2 were the four subgroup 2b strains (Table 2). In contrast, subgroup 1 was more clearly delimited by the recombination model. Of the nine subgroup 1 donor genomes, only 200103342 did not resolve unambiguously as subgroup 1 (Table 2).\n\nSubgroup 2 was more genetically diverse than subgroup 1 based on its higher mutation rates and increased recombination (Figure 4, Table 2). Subgroup 2 also had higher intra-host genetic diversity based on the dual infection analysis (Table 4). The consensus genomes with the highest intra-host diversity (Figure 5, Table 4 and Extended data, Supplementary Table 2 (Dickey & Workman, 2020a)) and highest recombination block count (Figure 4, Table 2) did not conform to a good concept of ‘population’ (Extended data, Supplementary Figure 1 (Dickey & Workman, 2020b)) despite being identified as populations by FineStructure analysis (Figure 2). But these genomes could be parsimoniously modeled as complex recombinants of subgroups 1 and 2 (Figure 4). We argue that more than one representative genome is required to properly distinguish subgroups from recombinant forms of subgroups 1 and 2. The subgroup 1 vs subgroup 2 recombination model used to identify diagnostic SNPs was informed by both recombination rate variation across the genome as well as differing mutation rates between subgroups. Models run with a constant recombination rate across the genome identified a slightly higher number (421) of diagnostic SNPs (see Methods). The dual infection analysis identified multiple genomic regions featuring modest frequencies of both subgroup diagnostic alleles (Extended data, Supplementary Table 2 (Dickey & Workman, 2020a)). These were identified in the most recombinant consensus genomes (Figure 4). Because the dual infection regions did not span the entire genome, the underlying haplotypes were most likely recombinant as opposed to ‘pure’ subgroup sequences.\n\nThe relatively small number of strains characterized in this study and a paucity of geographic variability biased our results and limited our ability to make recombination-based inference. While the subset of samples from the original TMEM154/A2 association studies chosen was a good starting place for modeling recombination, the addition of larger numbers of geographically diverse SRLV A2 genomes should improve the recombination model(s) substantially due to a larger pool of potential recombinant and parental genomes (Yahara et al., 2019). There are presently 79 unique full length SRLV genomes available with more than half of these published since 2019 (Colitti et al., 2019; present work) so the time has probably come to recharacterize SRLV diversity at the whole-genome level, expanding the current classification beyond partial gag/pol sequence. A revised classification system will better facilitate outbreak tracing and identification of recombinants circulating beyond their local flocks. Such circulating recombinant forms (CRFs) have been extensively characterized for HIV (Carr et al., 1999; Leitner et al., 2005) providing a possible model and framework for the SRLV research community to adapt. However, the current CRF framework for HIV utilizes consensus genomes so an accounting of the underlying haplotypes (quasispecies) contributing to these consensuses would benefit the genomic characterization of both lentiviruses.\n\nFras et al., 2013 have suggested that dual infection of small ruminant lentiviruses may be common, understudied, and underreported. Our results and those of Sider et al., 2013 confirm that dual infection is common though none of our samples showed evidence of having been dually infected by pure subgroup 1 and 2 representatives. Hopefully, declining sequencing costs and increased availability of whole genome sequencing will foster greater reporting of this phenomenon. Our results also conform to those of Sider et al., 2013 including the two subgroups identified and the existence of recombination. These results extend those of Sider et al., 2013 from partial gag/env to the complete genome while accounting for recombination. Recombination is also clearly delimited by the ChromoPainter models. The largest recombination blocks were also predicted by the software program, RDP (Martin et al., 2015) (data not shown), which also identified the largest 1+2b=2a recombination block spanning the middle portion of env (Figure 3B, individuals p and q). While our results extend the existence of two subgroups across the entire SRLV genome, subgroup 2 has additional population sub-structuring (Figure 2, Figure 3B). Subgroup 2a may represent a somewhat stable locally circulating recombinant of subgroup 1 and 2b (Figure 3). The genomes identified as 2a were found exclusively in TMEM154 2,2 and 2,3 diplotype animals (Table 1). Additionally, most strains with genomic regions indicative of dual infection were from TMEM154 susceptible 1,2 and 1,3 heterozygotes, i.e. animals with both an E and K at position 35. This suggests that animals that are E35K heterozygous due to TMEM154 1,2 and 1,3 diplotypes may facilitate recombination between subgroups 1 and 2.\n\nInterestingly, two subtype specific functional variants were found in a region of the env gene variable region 4 (V4) which was recently identified to contain “signature patterns” associated with different clinical status in sheep and goats (Mendez et al., 2020). This region of V4 also contains targets of neutralizing antibodies and is predicted to play a role in virus entry (Skraban et al., 1999). Multiple amino acid changes were also observed in the N-terminus of env. None of the amino acids were predicted to change the env signal peptide cleavage site; however, it would be interesting to know if the five subgroup-specific amino acids affect post-translational modifications such as cleavage timing, folding, or glycosylation, phenomena documented to affect HIV fitness (Asmal et al., 2011; Snapp et al., 2017; Upadhyay et al., 2018). As more genomes are sequenced, and we learn more about the function of TMEM154 in the context of the virus lifecycle, it will be interesting to see which, if any, of these viral sequences are biologically responsible for TMEM154 associations.\n\n\nData availability\n\nNCBI sequence accessions are provided in Table 1.\n\nFigshare: Supplemental Tables. https://doi.org/10.6084/m9.figshare.13109984 (Dickey & Workman, 2020a).\n\nThis project contains the following extended data:\n\nSupplemental Table 1. Four hundred and thirteen diagnostic SNPs distinguishing SRLV A2 subgroups.\n\nSupplemental Table 2: Seventy-three genomic regions indicative of dual subgroup infection among 9 SRLV A2 genomes.\n\nSupplemental Table 3: Within-host amino acid variability (functional viral quasispecies) at 106 subgroup diagnostic loci. Colors according to subgroup 1 vs subgroup 2 recombination model (Figure 4) can be seen in the downloaded file.\n\nFigshare: Supplemental Figure 1. https://doi.org/10.6084/m9.figshare.13109909 (Dickey & Workman, 2020b).\n\nThis file contains 23 SRLV subtype A2 genomes ‘painted’ with recipient genomic ‘chunks’ derived from populations of donor genomes. The five donor genome populations in the recombination model were determined using FineStructure.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author contributions\n\n\n\nConceptualization (AMW, MLC, MPH); Data Curation (AMW, AMD, TPLS); Formal Analysis (AMW, AMD); Investigation (AMW, TPLS); Methodology (AMW, AMD, TPLS); Project Administration (AMW); Resources (AMW, MPH, MLC, AMD, TPLS); Software (AMD); Visualization (AMD, AMW); Writing (AMD, AMW); Editing (AMW, MLC, MPH, AMD, TPLS).\n\n\nAcknowledgments\n\nSue Hauver, Brad Sharp, William Thompson, Jacky Carnahan, Kelsey McClure and Kristen Kuhn assisted with RNA library preparation and sequencing. Stephanie Schmidt provided secretarial support. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer.\n\n\nReferences\n\nAndrésdóttir V: Evidence for recombination in the envelope gene of maedi-visna virus. Virus Genes. 2003; 27(1): 5–9. PubMed Abstract \| Publisher Full Text\n\nAsmal M, Hellmann I, Liu W, et al.: A signature in HIV-1 envelope leader peptide associated with transition from acute to chronic infection impacts envelope processing and infectivity. PLoS One. 2011; 6(8): e23673. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlacklaws BA: Small ruminant lentiviruses: immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus. Comp Immunol Microbiol Infect Dis. 2012; 35(3): 259–269. PubMed Abstract \| Publisher Full Text\n\nCarr JK, Foley BT, Leitner T, et al.: Reference Sequences Representing the Principal Genetic Diversity of HIV-1 in the Pandemic. Human retroviruses and AIDS 1998. Los Alamos, New Mexico.: Los Alamos National Laboratory. 1999. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSkraban R, Matthíasodóttir S, Torsteinsdóttir S, et al.: Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype. J Virol. 1999; 73(10): 8064–8072. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSnapp EL, McCaul N, Quandte M, et al.: Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide. eLife. 2017; 6: e26067. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTromas N, Zwart MP, Poulain M, et al.: Estimation of the in vivo recombination rate for a plant RNA virus. J Gen Virol. 2014; 95(Pt 3): 724–732. PubMed Abstract \| Publisher Full Text\n\nUpadhyay C, Feyznezhad R, Yang W, et al.: Alterations of HIV-1 envelope phenotype and antibody-mediated neutralization by signal peptide mutations. PLoS Pathog. 2018; 14(1): e1006812. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValas S, Benoit C, Baudry D, et al.: Variability and immunogenicity of Caprine Arthritis-Encephalitis Virus Surface Glycoprotein. J Virol. 2000; 74(13): 6178–6185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorkman AM, Clawson ML, Heaton MP, et al.: First complete genome sequence of a genotype A2, subgroup 4 small ruminant lentivirus. Microbiol Resour Announc. 2018; 7(19): e01337–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorkman AM, Dickey AM, Heaton MP, et al.: Complete genome sequences of two genotype A2 small ruminant lentiviruses isolated from infected US Sheep. Genome Announc. 2017; 5(13): e00109–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorkman AM, Harhay GP, Heaton MP, et al.: Full-length coding sequences for 12 bovine viral diarrhea virus isolates from persistently infected cattle in a feedyard in kansas. Genome Announc. 2015; 3(3): e00487–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYahara K, Lehours P, Vale FF: Analysis of genetic recombination and the pan-genome of a highly recombinogenic bacteriophage species. Microb Genom. 2019; 5(8): e000282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYaman Y, Keleş M, Aymaz R, et al.: Association of TMEM154. variants with visna/maedi virus infection in Turkish sheep. Small Rum Res. 2019; 177: 61–67. Publisher Full Text" }	[ { "id": "77216", "date": "08 Feb 2021", "name": "Monika Olech", "expertise": [ "Reviewer Expertise Genetic and anitigenic characterization of Small Ruminant Lentiviruses (SRLV) in sheep", "goats and wildlife ruminants (molecular analysis", "phylogeny", "cross species infection", "genetic recombination)" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors focused mainly on the analysis of recombination of 23 SRLV strains belonging to subtype A2. In this paper a lot of different analyses were performed, but the results and figures are described very poorly. This makes the article very hard to read. For example on Fig. 1 I see only 15 isolates not 23. Lack is information on accesion numbers or isolate names of sequences from GenBank present on the tree. Please indicate to which genotypes they belong. In Figure 2, there are three images that should be labeled as A, B and C and their description should be more detailed (in figure and in results). In the third picture I see three populations not six. I don't understand why all 23 sequences were used in the recombination analysis. The authors write that only subgroubs 2a, 2b and 1 was used in the reombination models but some genomes not belonged to these subgroups (Fine structure Pop4, 5 and 6, Figure2). In the discussion the results of the study are repeated. There are no analyses regarding the occurrence of TMEM154E35K genotypes and susceptibility/resistance of sheep infected with and SRLV A2 subgroups 1 and 2. Did the research confirm previous studies described by Sider et al., 2013?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6923", "date": "26 Jul 2021", "name": "Aaron Dickey", "role": "Author Response", "response": "In this article, the authors focused mainly on the analysis of recombination of 23 SRLV strains belonging to subtype A2. In this paper a lot of different analyses were performed, but the results and figures are described very poorly. This makes the article very hard to read. For example on Fig. 1 I see only 15 isolates not 23. Response: Figure 1 is information dense, particularly the very similar subgroup 1 genomes denoted by the lavender-colored oval. However, we agree it is beneficial to list all genomes, so they are now listed in clockwise order in the new Supplemental Table 1. Lacking is information on accession numbers or isolate names of sequences from GenBank present on the tree. Response: An added benefit to the new supplemental table (see previous comment) is space to put the GenBank accession numbers of all strains in Figure 1. Genbank accession number, isolate name, county of origin, and virus genotype are now listed in Supplemental Table 1. Please indicate to which genotypes they belong. Response: This information is made clearer in the figure by curly brackets, additional annotations, and clarification in the legend. In Figure 2, there are three images that should be labeled as A, B and C and their description should be more detailed (in figure and in results). In the third picture I see three populations not six. Response: The figures sections have been relabeled A, B and C and additional details are provided in the results. We are not certain but think the reviewer may be referring to 3 clusters of strains, not six, since principal components 2 and 3 (PC2 and PC3) do not strongly discriminate subgroups 1 and 2b. We do not find this problematic since the first principal component strongly discriminates these two populations. I don't understand why all 23 sequences were used in the recombination analysis. The authors write that only subgroups 2a, 2b and 1 was used in the recombination models but some genomes not belonged to these subgroups (Fine structure Pop4, 5 and 6, Figure2). Response: Our initial recombination donor model had five donor populations and all 23 genomes as possible recipients (Supplemental Figure 1). fineSTRUCTURE population 6 was excluded from this analysis on the grounds that singletons could not comprise a population for this type of analysis. The result suggests fineSTRUCTURE populations 4 and 5 are probably not populations either, justifying our continued refinement of recombination models to those with fewer donor populations, which better characterize the subgroups for which there is sufficient evidence, and recombination patterns among them. Our explanation of this is that fineSTRUCTURE assigned ‘population’ status to groups of genomes that would be better described as complex recombinants of already defined subgroups, rather than true populations. Over-splitting of these types of datasets into too many ‘populations’ is a known problem. See: Falush D, van Dorp L, Lawson D, 2016 A tutorial on how (not) to over-interpret STRUCTURE/ADMIXTURE bar plots. bioRxiv 066431; 10.1101/066431. While, fewer potential donor genomes were used in the refined recombination models (Figures 3 and 4), all genomes were used as recipients, allowing us to characterize recombination. In the discussion the results of the study are repeated. Response: We believed this was beneficial to organizing points of discussion. There are no analyses regarding the occurrence of TMEM154E35K genotypes and susceptibility/resistance of sheep infected with and SRLV A2 subgroups 1 and 2. Did the research confirm previous studies described by Sider et al., 2013? Response: We generated complete genome sequences from a subset of genomes characterized in the Sider et al., 2013 paper. The goal was not to confirm or dispute the association, but rather, to generate complete genomes and develop a nucleotide polymorphism typing system for the two major subgroups while accounting for recombination. More complete SRLV sequences are required to identify which viral elements correlate with host adaptation." } ] }, { "id": "83037", "date": "28 Apr 2021", "name": "Carlos Loncoman", "expertise": [ "Reviewer Expertise Virology", "recombination", "herpesvirus" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 assembled full length viral genomes and then analyzed specific segments in order to identify recombination patterns by using chromosome painting model.\nGeneral comments:\nIt’s not clear in the introduction whether this chromosome painting model is used for recombination detection in any other virus? Perhaps any other RNA virus at all? I would highly appreciate if the authors could expand their introduction about this.\nI would suggest the authors to include RDP software in methods and at least 1 figure in the results to see where the recombination events take place within the genome and compare these results with the chromosome painting one. Currently, these results are as not shown so it’s not very clear whether these results are complementary.\nMy major concern with the recombination events that the authors describe in the manuscript are related with the differentiation between mutation and recombination. They mention in the introduction that RNA viruses have more mutations due to the low fidelity of the reverse transcriptase how did they differentiate these events from true recombination events?\nSpecific comments:\nAbstract - Methods – The goal. Could the author explain the method rather than the goal?\nIn the first paragraph would be good to read 1 or 2 lines about the importance or impact of this pathogen over sheep population not just the disease that cause the virus. Perhaps include incidence or any statistics about the disease that cause the virus. This would help to put this research in context.\nAs the genome contains more than one non-coding long terminal repeat regions. I wonder how did the authors manage to sequence that specific part as I know they could be problematic to determine sequence identity. Are these regions inverted?\nWhy the authors did not amplify the virus previous to sequencing? Is there any cell culture that could be used for this purposes? Could this help with the four strains that had low quality long read sequence?\nRDP software is mentioned in the discussion but not in the methods.\nThe split tree software has a tool to measure recombination called PHI test. Is there a reason why the authors did not include this in the manuscript?\nWhat does PC stands for in Figure 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? 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": "6924", "date": "26 Jul 2021", "name": "Aaron Dickey", "role": "Author Response", "response": "General comments: It’s not clear in the introduction whether this chromosome painting model is used for recombination detection in any other virus? Perhaps any other RNA virus at all? I would highly appreciate if the authors could expand their introduction about this. Response: The recombination modeling program utilized in these analyses has been previously used to analyze recombination in Helicobacter pylori prophages (Yahara et al., 2019) and Herpes Simplex Virus (Forni et al., 2020). This information has been added to the methods. The Yahara et al. reference may be most similar in initial approach. Ours may be the first use of ChromoPainter version 2 ‘donor mode’ to extract subgroup-diagnostic variants in viruses while accounting for recombination. I would suggest the authors to include RDP software in methods and at least 1 figure in the results to see where the recombination events take place within the genome and compare these results with the chromosome painting one. Currently, these results are as not shown so it’s not very clear whether these results are complementary. Response: We agree with the reviewer that our use of RDP belongs in the methods. We have attempted to do so in a way that does not distract from the primary focus of the manuscript, which was to identify subgroup-diagnostic variants, rather than recombination events. RDP was only used in this work to confirm a single putative event: the possible introgression of a large chunk of subgroup 1 sequence into a subgroup 2b background to create subgroup 2a (black box in Figure 3b). This event (approximate size, genomic position, and ChromoPainter assigned subgroup of RDP identified putative parental strains) was confirmed by RDP. This information has been added to the results and the details of the RDP event are provided in Extended data, Supplemental Table 2(Dickey & Workman, 2020a). My major concern with the recombination events that the authors describe in the manuscript are related with the differentiation between mutation and recombination. They mention in the introduction that RNA viruses have more mutations due to the low fidelity of the reverse transcriptase how did they differentiate these events from true recombination events? Response: All recombination analysis programs claim to account for mutation as this is a fundamental issue. ChromoPainter initializes at the mutation rate of Li and Stephens 2003, but we estimated it in our analyses by using the -iM flag according to program developer recommendations. Interestingly, ChromoPainter’s mutation rate estimates are 3-fold higher for subgroup 2 genomes compared to subgroup 1 (Table 2). Population specific mutation rate estimates were obtained by averaging the estimated rates from the fineSTRUCTURE run among members of the assigned population and incorporated into our increasingly refined recombination models using the -m flag. We have added this information and the Li and Stephens reference to the manuscript. Our subgroup-specific-colored blocks in figures 3-5 are best interpreted as regions of sequence assigned by the recombination model to those subgroups, not as events-per-se. We believe regions alternating back-and-forth between subgroups over short genomic spans (e.g. strains labeled k-r in figures 3-5), are sometimes best interpreted as having arisen from multiple recombination events over time. We have attempted to minimize the use of the term ‘event’ in the manuscript in service of this paradigm. Li N, Stephens M (2003) Modeling linkage disequilibrium and identifying recombination hotspots using single-nucleotide polymorphism data. Genetics 165: 2213–2233 Specific comments: Abstract - Methods – The goal. Could the author explain the method rather than the goal? Response: The methods section now reads: Methods: Sequence variation within and among full-length assemblies of SRLV subtype A2 subgroups 1 and 2 was analyzed to identify genome-scale recombination patterns. In the first paragraph would be good to read 1 or 2 lines about the importance or impact of this pathogen over sheep population not just the disease that cause the virus. Perhaps include incidence or any statistics about the disease that cause the virus. This would help to put this research in context. Response: This information has been added to the first paragraph of the introduction. As the genome contains more than one non-coding long terminal repeat regions. I wonder how did the authors manage to sequence that specific part as I know they could be problematic to determine sequence identity. Are these regions inverted? Response: We used PacBio long-read technology to help resolve the ends of the viral genome. We report some genomes as ‘near-complete’ because we did not obtain the complete LTR sequence for all genomes. Why did the authors not amplify the virus previous to sequencing? Is there any cell culture that could be used for this purposes? Could this help with the four strains that had low quality long read sequence? Response: It is true that the virus can be isolated in culture prior to sequencing; however, there were no commercially available cell lines for the isolation of SRLV at the time we initiated this project. While we could have developed appropriate cell lines, we chose not to isolate the virus first, as this can lead to the selection of a subpopulation of the infecting virus quasispecies. For these reasons, we sequenced the virus directly from the infected tissues. RDP software is mentioned in the discussion but not in the methods. Response: Please see the response to General comment 2. The split tree software has a tool to measure recombination called PHI test. Is there a reason why the authors did not include this in the manuscript? Response: SplitsTree5 does not have this analysis option, though we also remember it from SplitsTree4. We ran a PHI-test in RDP5 and now report it in the results. What does PC stand for in Figure 2? Response: Principal Component (PC), is now incorporated into the legend of Figure 2." } ] } ]	1	https://f1000research.com/articles/9-1449
https://f1000research.com/articles/10-384/v1	13 May 21	{ "type": "Research Article", "title": "Entry of antiepileptic drugs (valproate and lamotrigine) into the developing rat brain", "authors": [ "Samuel J. Toll", "Fiona Qiu", "Yifan Huang", "Mark D. Habgood", "Katarzyna M. Dziegielewska", "Shuai Nie", "Norman R. Saunders", "Samuel J. Toll", "Fiona Qiu", "Yifan Huang", "Mark D. Habgood", "Katarzyna M. Dziegielewska", "Shuai Nie" ], "abstract": "Background: Women with epilepsy face difficult choices whether to continue antiepileptic drug treatment during pregnancy, as uncontrolled seizures carry great risk to mother and fetus but continuing treatment may have adverse effects on baby’s development. This study aimed at evaluating antiepileptic drug entry into developing brain. Methods: Anaesthetised pregnant, non-pregnant adult females, postnatal and fetal rats were injected intraperitoneally with different doses, single or in combinations, of valproate and lamotrigine, all within clinical range. Injectate included 3H-labelled drug. After 30min, CSF, blood and brain samples were obtained; radioactivity was measured using liquid scintillation counting. Some animals were also exposed to valproate in feed throughout pregnancy and into neonatal period. Drug levels were measured by liquid chromatography coupled to mass spectrometry (LC-MS). Results are given as CSF or tissue/plasma% as index of drug entry. Results: Entry of valproate into brain and CSF was higher at E19 and P4 compared to adult but was not dose-dependent; placental transfer increased significantly at highest dose of 100mg/Kg. Lamotrigine entry into the brain was dose dependent only at E19. Chronic valproate treatment, or combination of valproate and lamotrigine had little effect on either drug entry, except for reduced valproate brain entry in adult brain with chronic treatment. Placental transfer decreased significantly after chronic valproate treatment. LC-MS measurement of valproate in adults confirmed that rat plasma values were within the clinical range and CSF/plasma and brain/plasma ratios for LC-MS and 3H-valproate were similar. Conclusion: Results suggest that entry of valproate may be higher in developing brain, the capacity of barrier mechanism is mostly unaffected by doses within the clinical range, with or without addition of lamotrigine. Chronic valproate exposure may result in upregulation in cellular mechanisms restricting its entry into the brain. Entry of lamotrigine was little different at different ages and was not dose dependent.", "keywords": [ "CSF", "blood-brain barrier", "choroid plexus", "efflux mechanisms", "epilepsy", "fetus", "neonate", "placenta." ], "content": "Abbreviations\n\nABC, ATP-binding cassette; CNS, central nervous system; CSF, cerebrospinal fluid; DPM, disintegrations per minute; E, embryonic (note that by longstanding convention all gestational ages in rodents are referred to as embryonic, but in this study E19 is a fetal stage); i.p., intraperitoneal; i.v., intravenous; LTG, lamotrigine; LC-MS, liquid chromatography coupled to mass spectrometry; P, postnatal; SD, standard deviation; μCi, micro Curie; VPA valproic acid.\n\n\nIntroduction\n\nEpilepsy is a neurological disorder characterised by seizures of varying types and severity ranging from absence, in which the patient becomes unresponsive to stimuli, to tonic–clonic or atonic seizures, where the individual suffers a loss of motor control (Thijs et al., 2019). Epilepsy affects people of all ages and sexes and it has been estimated that over 70 million people suffer from this disorder worldwide (Thijs et al., 2019).\n\nIn some cases of focal epilepsy, surgical removal of the provoking brain region can provide a cure (Rugg-Gunn et al., 2020; Thijs et al., 2019) but for many people epilepsy is a continuing condition requiring lifelong drug treatment. A particular clinical problem is that during pregnancy epileptic women need to continue their medication, otherwise the recurrence of seizures may adversely affect both their health and the health of their offspring (Tomson et al., 2019). Much is known about adverse effects of antiepileptic drugs, particularly in terms of the occurrence of congenital malformations when these drugs are taken early in pregnancy; less is known about the longer-term effects on brain development and behaviour in the offspring. Because of a lack of a regulatory framework drugs used in pregnancy are prescribed “off-label”. When advising patients clinicians therefore have to rely on experience and limited clinical and animal experimental reports aggregated into databases (e.g. Briggs et al., 2017). However, there is a general requirement (e.g. by the US Federal Drug Administration) that all new drugs before being used in any patients should be tested in animals (usually rodents) for possible teratogenic effects. Such tests have shown that a well-established antiepileptic drug, valproate causes a significant number of congenital abnormalities in animals (Jazayeri et al., 2020) but also in humans; Tomson et al. (2018) found that the risk for congenital malformation in children of mothers exposed to valproate was increased by 4–5-times especially at higher valproate doses, and particularly when used in the first trimester (see also Abou-Khalil, 2019; Vajda, 2012). The most common problems associated with gestational valproate use are cardiac malformations, hypospadias, renal defects, and neural tube defects, with higher doses increasing the risk of spina bifida (Jentink et al., 2010). Possible ill-effects of taking the drug later in pregnancy have been little studied but will be outlined later in the Discussion. Because of the high incidence of congenital malformations occurring in offspring of pregnant women taking valproate, some countries (e.g. France) have banned its use in pregnancy (Casassus, 2017). Other advisory bodies urge caution and advise avoiding or limiting the use of valproate in pregnancy (Australian Medicines Handbook, 2019; Briggs et al., 2017; The Royal Women’s Hospital Pregnancy and Breastfeeding Medicines Guide, 2020). The problem for clinicians and their patients is that valproate remains the most effective and in some cases, the only form of effective treatment for some forms of epilepsy (Tomson et al., 2015). Currently clinicians deal with this problem by using the lowest doses of valproate that will still suppress seizures. This is often achieved by combining a lower dose of valproate with a second antiepileptic drug, for example lamotrigine (Tomson et al., 2019). However, little is known about how these drugs interact in terms of entry into a fetus and its brain and still less about possible deleterious effects in the offspring. This paper describes an experimental approach using an established animal model (rat, Koehn et al., 2019; Koehn et al., 2020) aimed at shedding light on these problems. The starting point for such studies, as described here, is to determine the extent of age-related entry into the brain and CSF of valproate and lamotrigine used as mono- or combination therapies at doses which are within the clinical range.\n\n\nMethods\n\nThe Sprague–Dawley (RRID: RGD_728193) strain of Rattus norvegicus was used in this study. All animal experimentation was approved by the University of Melbourne Animal Ethics Committee (Ethics Permission AEC: 1714344.1) and conducted in compliance with Australian National Health and Medical Research Guidelines. All animals were assessed as healthy prior to commencement of experiments. All experiments were short term and conducted under anaesthesia. All efforts were made to ameliorate any suffering of animals. They were handled by experienced researchers in such a way as to minimise stress prior to being anaesthetised.\n\nSprague–Dawley rats were supplied by the Animal Resources Centre, WA, and the University of Melbourne Biological Research Facility. Animals were kept in a 12-hour light/dark cycle. They were provided with ad libitum access to food, consisting of dry pellets of a fixed formulation for rats (supplied by Specialty Feeds, Western Australia), with the exception of animals designated for chronic valproate experiments (see below). All animals had access to a continuous supply of water. Animals were housed in groups of 2–4 (adults) per cage (25cm×35cm×25cm on Breeders Choice paper bedding, made from 99% recycled paper; it is biodegradable with no added chemicals).\n\nThree ages were studied: fetuses from time-mated females (all primigravida) at embryonic day (E) 19, pups at postnatal day (P) 4, and adults. E19 is a stage of development when adequate volumes of blood and cerebrospinal fluid (CSF) can be obtained for analysis from fetal rats without pooling (Dziegielewska et al., 1981); also individual pups can be injected intraperitoneally while still inside the uterine horn and kept viable for periods of time (Koehn et al., 2019; Koehn et al., 2020). These ages represent developmental milestones that can be translated to stages of human brain development corresponding to the end of first trimester, late second trimester and adult (Clancy et al., 2013; Workman et al., 2013). They can also be compared with data from previous studies of other drugs carried out at these ages (Koehn et al., 2019; Koehn et al., 2020). All animals were injected with a drug dose of one of the following treatments: valproate, lamotrigine, or valproate and lamotrigine combined, with tracer amounts of 3H-labelled drug incorporated into the injectate (for details see Table 1). 3H-dextran was used to estimate residual blood amounts in brain tissue as described below. Animal numbers (Table 2) were based on previous experience of such experiments and were the minimum number required to detect a significant difference between groups at p<0.05. Animals were selected for treatment groups to ensure weights were statistically similar between direct comparisons. Where possible, equal numbers of male and female fetuses and postnatal animals were used. Animals were allocated to experiments by the Animal House staff, who had no knowledge of the particular experiments to be performed. The experimenters had no role in the selection of the animals, thus avoiding selection bias. Most experiments were carried out between 09.00 and 14.00 hours. Very few experiments were unsuccessful; details are given in the legend to Table 2.\n\nTime between injection and collection, method of administration (MoA) and number of animals used at each age (n = number of litters); i.p. (intraperitoneal), i.v. (intravenous). Animal losses (not recorded here): One E19 mother from blood loss during cannulation. Three P4 animals not used (2 dose errors, one died before end of experiment). In the chronic valproate feed experiments approximately half of the fetuses died in utero.\n\nDetails of the drugs and markers used are listed in Table 1, including doses. Sodium valproate (>98%) was administered in one of four doses: 10, 30, 100 or 200 mg/kg body weight. This covered the range of doses from below to above those used clinically in monotherapy or combined with lamotrigine (T O’Brien and F Vajda, personal communication). Sodium valproate was dissolved in sterile isotonic (0.9%) sodium chloride solution and 3H-valproate (for amount see Table 1) was added prior to injection.\n\nLamotrigine was administered in one of three doses: 6, 20 and 40 mg/kg body weight This covered the range of doses from below to above those used clinically in monotherapy or combined with valproate (T O’Brien, N Jones and F Vajda, personal communication). Lamotrigine was dissolved in 100% ethyl alcohol and 3H-lamotrigine was added prior to injection (Table 1). Four preparations were used for combination therapy experiments. These were intended to achieve blood concentrations in the rat equivalent to the range used in combination therapies clinically in humans (T O’Brien, N Jones and F Vajda, personal communication). For all combination experiments, two injections were used. The first injection always contained 30 mg/kg valproate dissolved in sterile saline. The second injection was one of either 2 mg/kg, 6 mg/kg, 12 mg/kg or 20 mg/kg of lamotrigine dissolved in 100% ethanol. Depending on the entry of which was to be estimated, either 3H-valproate was added to the first injection, or 3H-lamotrigine was added to the second injection.\n\nThe drugs were administered intraperitoneally (i.p.). 3H-dextran was administered intravenously for estimation of residual vascular space as described below.\n\nFor chronic experiments, female rats were fed for two weeks prior to mating on a feed premixed with 20 g/kg valproate (Specialty Feeds, Sigma Aldrich). This feed has been shown to achieve consistent blood concentrations of ~220 μmol/L (approaching human clinical range of 300–600 μmol/L) of valproate across a sustained period (Jazayeri et al., 2020). For successfully time-mated females, feeding continued up until the point of fetus or postnatal (P4) sample collection. At termination of the experiment drug entry was estimated as for acute experiments, i.e. animals then received an acute injection of 100 mg/kg valproate with 3H-valproate, the preparation of which is described below.\n\nAll time-mated pregnant females at E19, as well as postnatal animals used for residual vascular space estimations received an i.p. injection of 25% w/v urethane (1.0 mL/100 g body weight). Postnatal animals in valproate, lamotrigine and combination treatment groups were anaesthetised using inhaled isoflurane. The reason for the difference in anaesthesia was the duration of the experiment and/or route of sample collection, see below. Deep anaesthesia was ensured by unresponsiveness to the pinching of the toe and tail before surgery commenced.\n\nE19: Following deep anaesthesia, pregnant females at E19 were placed on a 39°C heating mat in a supine position. An endotracheal catheter was inserted to provide a clear airway. The femoral artery was cannulated to allow time-matched maternal blood samples to be taken at the time of each individual pup sampling (Koehn et al., 2019). Blood volume loss was approximately replaced with 0.9% sterile sodium chloride solution. Samples were collected from the first fetus 30 min following i.p. drug injection, continuing up to 2.5 hours post-injection, at approximately 10 min intervals. Time-matched blood samples were used to estimate placental transfer at the time of each fetal collection, according to Equation 3. Previous experience has shown that fetuses can be maintained in reasonable physiological condition for about 3-4h following induction of anaesthesia.\n\nP4 and adult animals: at both ages the animals underwent similar procedures. 30 min following the i.p. injection of drug, the animals were anaesthetised using inhaled isoflurane and the diaphragm was cut to terminate the experiment. Blood was drained from the right ventricle of the heart using a heparinised glass micropipette attached to PVC tubing for P4 animals or a heparinised syringe for adult animals.\n\nBlood, CSF, cortex, brainstem, and thymus samples were collected from fetuses, pups and adults. As ABC transporters are a key mechanism of limiting drug entry into the brain and the main such transporter appears to be P-glycoprotein, the thymus was included for comparison, as it is known to have limited expression of this efflux transporter (Valente et al., 2008, and see Discussion). At E19, at the end of each period of exposure to drug the experiment was terminated by exsanguination of the fetus by sampling blood from the right ventricle using heparinised glass micropipettes attached to PVC tubing (Habgood et al., 1993). Maternal blood samples were taken via the arterial cannula with a final sample taken from the right ventricle of the heart with a syringe. In all animals, CSF was taken from the cisterna magna, with careful suction through glass micropipettes attached to PVC tubing. CSF was briefly centrifuged and checked under a microscope for blood contamination after collection. As little as 0.2% of blood contamination can be detected in 15–20 μL (Habgood et al., 1992). The left and right cortices of the brain were taken following careful exposure of the lateral ventricles, to avoid contamination from the choroid plexus and CSF, followed by the brainstem. For comparison with postnatal animals, additional experiments were conducted following direct i.p. injection to the fetus (sample collected 30 min later) as described for postnatal animals.\n\nBlood samples were centrifuged at 5000 rpm for 5 mins to separate the plasma. Measured volumes (not exceeding 20 µL) of plasma, CSF, and injectate were transferred into scintillation tubes. 5 mL of scintillation fluid (Emulsifier-safe, PerkinElmer) was then added. Tissue samples were weighed and up to 50 mg were dissolved in 500 µL of Soluene 350 (PerkinElmer) overnight in a scintillation tube. Two drops of acetic acid were added to each tube to neutralise the alkaline Soluene 350, followed by 5 mL of scintillation fluid (Emulsifier Safe, PerkinElmer). Samples were placed on a liquid scintillation counter (Tri-Carb 4910 TR, PerkinElmer) for 5 mins per sample to count radioactivity disintegrations per minute (DPM). Blank samples taken from rats with no radioactivity were also counted with each run to establish background levels. These were always subtracted from the plasma, CSF or tissue sample counts.\n\nValproic acid and D6-valproic acid (Novachem, Aus) were dissolved in water at 5 µg/µL as stock. All stock solutions were stored at -20 ℃. The internal standard mixture containing 12 or 120 ng/µL D6-valproic acid in methanol was prepared from diluting stock solutions using methanol immediately before use. 10 µL of internal standard mixture and 80 µL methanol were added to 10 µL plasma, CSF or brain homogenate. Approximately 20 mg of brain tissue were minced and homogenised in a volume of deionized water (in µL) equal to twice the weight of the tissue using a glass homogeniser; 10 µL of the homogenate was used for each sample. After vortexing for 30 seconds, the sample was centrifuged at 14,000×g for 10 min and the top 50 µL was transferred to glass vial for liquid chromatography coupled to mass spectrometry (LC–MS) analysis using a Vanquish ultrahigh performance liquid chromatography (UHPLC) linked to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) operated at positive ion mode. Solvent A was 10 mM ammonium formate with 0.1% formic acid in water and solvent B was acetonitrile. 25 µL of each sample was injected into an RRHD Eclipse Plus C18 column (2.1×1000 mm, 1.8 µm; Agilent Technologies, Santa Clara, CA, USA) at 50°C at a flow rate of 350 μL/min for 1 min using 5% solvent B. During separation, the percentage of solvent B was increased from 5% to 40% in 4 min. Subsequently, the percentage of solvent B was increased to 80% in 0.5 min and then maintained at 99% for 2 min. Finally, the percentage of solvent B was decreased to 5% in 0.1 min and maintained for 2.4 min.\n\nAll MS experiments were performed using a heated electrospray ionization (HESI) source. The spray voltage was 3.0 kV in negative ionisation mode. The flow rates of sheath, auxiliary and sweep gases were 20 and 6 and 1 arbitrary unit(s), respectively. The ion transfer tube and vaporizer temperatures were maintained at 350°C and 400°C, respectively, and the S-Lens RF level was set at 50%. The full-scan MS-spectra were acquired in the Orbitrap at a mass resolving power of 120,000 (at m/z 200) across an m/z range of 100–1000 using quadrupole isolation, auto-gain control (AGC) target of 4E5, auto maximum injection time at both polarities. Targeted higher-energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS) scan of valproic acid at m/z 143.1072 and D6-valproic at m/z 149.1394 acid were performed with normalized collision energy (NCE) at 0%, isolation width of 4 Da, Orbitrap resolution at 15,000 (at m/z 200), maximum injection time of 22 milliseconds and AGC target of 2.5E5. Ion chromatogram peak area of ions in MS/MS scans at m/z 143.1072 from valproic acid and 149.1394 from D6-valproic acid at 6.6 min were extracted using Skyline 20.2 (RRID:SCR_014080) to calculate the concentration of drug in each sample. The linear response range of valproic acid in different sample types tested for the LC–MS/MS analysis is provided in the extended data (Toll et al., 2021).\n\nAll radioactivity counts were normalised to weight/volume and expressed as DPM/mg or μL of sample. The residual vascular space for each tissue sample was calculated (Equation 1). These values were then used to correct tissue counts with results expressed as tissue/ or fluid/plasma ratios % (Equation 2). Fetal and maternal blood samples at E19 were used to obtain placental transfer (Equation 3).\n\nEquation 1\n\nEquation 2\n\nEquation 3\n\nData from all experiments are expressed as mean±standard deviation (SD). Statistical differences across time-course experiments, dose experiments with greater than two doses, combination experiments with greater than two doses and age comparisons were obtained using a one-way ANOVA with Tukey’s multiple comparisons test. Differences between dose experiments with two treatments, combination experiments with two treatments, and comparisons between acute and chronic groups were obtained using an unpaired two-tailed t-test. All tests were completed using Prism (GraphPad Software Inc; RRID:SCR_002798) with p<0.05 accepted as statistically significant. All statistical analyses can be performed using JASP (RRID:SCR_015823), an open-source alternative.\n\n\nResults\n\nMost results are expressed as tissue or CSF to plasma concentration ratios (equations 1–3 above). This is an established way to represent the entry (permeability) of markers that cross cellular interfaces as this takes into account variability in marker concentrations in plasma due to variations in in vivo experimentation (Davson & Segal, 1996)\n\nResidual vascular space in the brain was estimated using 70 kDa dextran radiolabelled with 3H which was injected i.v. and left to circulate for only 5 min. The concentration ratio of the dextran in the brain tissue compared with plasma was used as an indicator of the residual blood space, as such a large molecule is not expected to leave the circulation in 5 min only (Habgood et al., 1993). The ratio of 3H-dextran into cortex and brainstem compared with plasma, illustrated in Figure 1A and B, showed no significant difference at any of the ages studied and was around 2–4%. In contrast, thymus residual vascular space appeared to be greater at younger ages (E19 and P4) than in the adults but samples were too limited in numbers (due to the necessity of pooling) for this to be tested formally (Figure 1C).\n\nA. Cortex/plasma, B. Brainstem/plasma and C. Thymus/plasma concentration ratios (%) of 3H-dextran in rats at E19, P4 and in non-pregnant female adults, collected 5 minutes after a single intravenous injection of radioactive tracer (3H-dextran). Each point represents the result from a single animal. Mean±standard deviation (SD). E19; n=6, except for thymus where a single sample was obtained by pooling (1 litter), P4; n=3–5, adult; n=2–3. Note different scale in C.\n\nAll results for brain and thymus drug permeability studies described subsequently have been corrected for residual vascular space. CSF was checked for blood contamination as described in the Methods. Four contaminated samples were discarded.\n\nTo establish if entry of valproate and lamotrigine into the brain and CSF changed depending on the duration of exposure to the drug, time course experiments were conducted at P4. Results are shown in Figure 2. Following a single i.p. injection, the entry of 3H-valproate into the CSF, cortex, brainstem, and thymus of P4 rats was similar between 15 min and 90 min, as was also the case for entry of 3H-lamotrigine between 30 min and 120 min (Figure 2); therefore, all subsequent acute experiments were performed 30 min following i.p. injection, as in previous similar experiments (Koehn et al., 2019).\n\nCSF/plasma, Cortex/plasma, Brainstem/plasma, and Thymus/plasma concentration ratios (%) of A. Valproate (single intraperitoneal injection of 100 mg/kg valproate with radioactive tracer 3H-valproate) and B. Lamotrigine (single intraperitoneal injection of 20 mg/kg of lamotrigine with 3H-lamotrigine). Samples were collected between 15 and 120 min after injection. Each point represents the result from a single animal.\n\nThe doses of valproate and lamotrigine and the ages at which they were studied are shown in Table 1. The entry of 3H-valproate into the CSF, cortex and brainstem following i.p. injection into the fetuses, postnatal animals or adults are shown in Figure 3\n\nCSF/plasma, Cortex/plasma and Brainstem/plasma concentration ratios (%) in E19 (A–C), P4 (D–F) and non-pregnant female adult (G–I) rats, collected 30 minutes after a single intraperitoneal injection of valproate with radioactive tracer (3H-valproate). Doses in mg/kg indicated on the x-axis. Each point represents the result from a single animal. Mean±SD; n=3-5. p<0.05, p<0.01, p<0.001, *p<0.0001. Note that some error bars are too small to be clearly visible.\n\nTransfer into the brain and CSF at E19 was not significantly different between the two doses tested (30 mg/kg and 100 mg/kg). This indicates that drug concentration in this range had little influence on the permeability of valproate across the blood–brain and CSF–brain barriers at E19. Actual values for CSF/plasma and tissue/plasma ratios are shown in extended data Table 3A.\n\nAt P4, the entry of 30 and 100 mg/kg valproate was appreciably less than at E19 and this was even more the case in the adult (Figure 3). However, there was no difference in entry of these two doses at P4 or in adults. At the higher doses (100 and 200 mg/kg) the CSF and brain/plasma ratios were significantly higher than for the lower doses (actual values shown in the extended data Table 3A), indicating a dose-dependent relationship of valproate entry in postnatal pups and in adults. In the adult the entry of the highest dose (200 mg/kg) was nevertheless still substantially below that of the lower doses at E19 (Figure 3).\n\n3H-lamotrigine entry into the CSF, cortex and brainstem at all three ages is illustrated in (Figure 4). The only significant differences were for CSF and cortex values at E19 when the entry following injection of 20 mg/kg was marginally less (p<0.05). In general, permeability across the blood–brain and blood–CSF barriers did not appear to be dose-dependent over the concentration range studied at P4 and in adults and only marginally so at E19. Actual values for CSF/plasma and tissue/plasma ratios are in the extended data Table 4A.\n\nCSF/plasma, Cortex/plasma and Brainstem/plasma ratios (%) in E19 (A–C), P4 (D–F) and non-pregnant female adult (G–I) rats, collected 30 minutes after a single intraperitoneal injection of lamotrigine with radioactive tracer (3H-lamotrigine). Doses in mg/kg indicated on the x-axis. Each point represents the result from a single animal. Mean±SD; n=3–12. p<0.05. Note that some error bars are too small to be clearly visible.\n\nTo illustrate the age dependence of drug entry into CSF and brain data from Figure 3 and Figure 4 have been replotted in Figure 5 for 100 mg/kg valproate and 20 mg/kg lamotrigine. For 3H-valproate, there was little difference between E19 and P4, but the adult values were substantially and significantly lower (p<0.01–0.001). For 20 mg/kg lamotrigine entry into CSF was significantly greater at P4 compared with E19 (p<0.01). There were also increases in brain entry between E19 and adults (p<0.05–0.01). Values of CSF/plasma and tissue/plasma are in the extended data Table 5A.\n\nCSF/plasma, Cortex/plasma and Brainstem/plasma concentration ratios (%) of valproate (A–C) or lamotrigine (D–F) at E19, P4 and in non-pregnant female adult rats collected 30 min after a single intraperitoneal injection of 100 mg/kg valproate or 20 mg/kg lamotrigine respectively. Each point represents the result from a single animal. Mean±SD. n=3–8. p<0.05, * p<0.01. Note that some error bars are too small to be clearly visible.\n\nValproate. In these experiments, entry of 3H-valproate in the presence of increasing doses of lamotrigine (0–6 mg/kg at E19 and in adults and up to 20 mg/kg at P4) was estimated (Figure 6). Even over the extended dose range at P4 there were no differences in CSF or brain/plasma ratios. At E19, there was a small but significant (p<0.05) decrease in the brainstem entry with the 6 mg/kg dose. In the adult there was a small but significant (p<0.05) increase in entry into CSF with the 6 mg/kg dose. Values for CSF/plasma and tissue/plasma ratios are in Supplementary Material Table 6A.\n\nLamotrigine. In the converse experiment P4 and adult animals were injected i.p. with 6 mg/kg lamotrigine with 3H-lamotrigine, alone or with 30 mg/kg valproate (Figure 7). The only significant difference (p<0.05) was at P4 in the cortex. Otherwise the addition of valproate to lamotrigine did not appear to influence the entry of lamotrigine. Values for CSF/plasma and tissue/plasma ratios are in the extended data Table 7A.\n\nAnimals assigned to the chronic group were treated with a feed containing 20 g/kg valproate (see Methods and Jazayeri et al., 2020) for at least 3 weeks for non-pregnant adults, and at least 2 weeks prior to mating followed by a further 19 days for E19 experiments and 25–26 days for P4 experiments. They were compared with animals fed chronically with food not containing valproate. Thus E19 pups received the drug exclusively via placental transfer and following birth P4 animals continued to receive valproate via breastmilk. All chronically fed animals and controls were then treated with the same protocol as the acutely treated animals (see Methods) that received an injection of 100 mg/kg valproate containing a radioactive 3H-valproate 30 min prior to sample collection.\n\nEntry of 3H-valproate into the cortex of E19 fetuses following chronic feeding of the mother was significantly lower (p<0.05) than in the fetuses of the acutely treated mother (Figure 8). At this age, there were no significant differences for CSF or brainstem for acutely and chronically treated animals (Figure 8). At P4, there were no significant differences for CSF or brain. In the adult there were small but statistically significant (p<0.05) differences for both brain regions in which ratios were slightly lower in the chronically fed animals. In adult animals the entry of valproate in those fed chronically with chow containing valproate or in those on drug free chow was substantially below that in the younger animals. Values for CSF/plasma and tissue/plasma ratios are in the extended data Table 8A.\n\nMeasurement of 3H-labelled valproate and lamotrigine in maternal and fetal plasma following maternal drug administration allowed an estimate of the level of protection provided by the placenta against drug entry from the maternal circulation into the fetus. At E19, pregnant dams were injected with either valproate or lamotrigine as described for acute experiments. Thirty minutes later samples began to be serially taken from individual fetuses at the times indicated in Figure 9A (valproate) and Figure 9C (lamotrigine) together with time-matched maternal blood samples. In one experiment, the rat was fed with food containing 100 mg/kg valproate from 2 weeks before pairing up to E19 (see Methods). Figures 9A and 9C also show the fetal/maternal ratios for the individual pups. Figures 9B and 9D show the aggregated data up to 110 min. For valproate, in fetuses that received only a single dose of 100 mg/kg valproate, the transfer of the drug expressed as fetal/maternal plasma ratio was significantly higher (p<0.001) than values obtained following either the 30 mg/kg single dose or the chronic 100 mg/kg dose. For lamotrigine there was no significant difference between the two doses administered.\n\nThymus tissue was collected in this study because it is known to contain little expression of the ABC efflux transporter P-glycoprotein, Abcb1 (Valente et al., 2008) which is the most studied of the efflux mechanisms that exclude or limit the entry of many drugs at brain barriers and for which there is some evidence that lamotrigine may be a substrate (Zhang et al., 2012, see below).\n\nOnly limited data are available for E19 because the small size of the tissue required pooling to have enough to measure the labelled drugs. However, they indicate that, as with brain and CSF, entry of valproate appears to have been higher at E19 than at later ages. At P4, there was very little entry of 3H-valproate following injection of either 30 or 100 mg/kg. However, with 100 mg/kg chronic treatment or acute 200 mg/kg dose, there was a substantial and significant (p<0.05–0.01) increase compared with the lower doses. For lamotrigine, there appeared to be no significant differences in the entry for the different doses and the entry at each age was similar (Figure 10). The thymus/plasma ratios for a single intraperitoneal injection of 100 mg/kg valproate or 20 mg/kg lamotrigine at E19, P4 and adult are shown in Figure 11. The entry of valproate at E19 may have been greater than later but only one pooled sample was available. There did not appear to be any age-related differences for lamotrigine. Combining doses of valproate and lamotrigine did not appear to have much effect on the entry of either drug (Figure 12). There were no differences for valproate entry following acute and chronic treatment (Figure 13).\n\nValproate concentrations were estimated by LC–MS in plasma, CSF and brain of adult and P4 rats (Table 3). Following a single dose of 30 mg/kg (within the clinical range in adult patients, Johannessen & Johannessen, 2003) the plasma levels in adult rats were 34±8 µg/mL and in P4 pups 67 ± 12 µg/mL. At a dose of 100 mg/kg, a dose that is above the usual clinical range, the estimated plasma levels in experimental animals at both ages were also above the clinical range (141±6 µg/mL at P4 and 166±28 µg/mL in adults, Table 3). In order to compare and validate the two methods (LC–MS and radioactivity counting), CSF/plasma and brain/ plasma ratios were compared and were not significantly different at either age (Figure 14).\n\nP4 and non-pregnant female adult rats. collected 30 minutes after a single intraperitoneal injection of 30mg/kg or 100mg/Kg valproate; measured by LC–MS. Mean± SD. n=3–4. Johannessen & Johannessen, (2003). Clinical dose range 20–60 mg/kg: The Australian Medicines Handbook, US Food and Drug Administration (2016).\n\n\nDiscussion\n\nThis study provides data on the role of barriers in the brain and placenta in modulating entry of valproate and lamotrigine into the developing brain, when used alone or in combination. The rat has been used at an embryonic stage of development (E19), a postnatal age (P4) and adult. Drug entry into the CSF, cortex, brainstem has been estimated using radiolabelled drugs and compared with the thymus as this organ is known to have limited expression of one of the main efflux transporters, P-glycoprotein (Valente et al., 2008) for which lamotrigine and some other anti-epileptic drugs are thought to be a substrate (Ambroziak et al., 2010; Baltes et al., 2007; Jentink et al., 2010; Potschka et al., 2002; Rizzi et al., 2002).\n\nThe blood–brain, blood–CSF and placental barriers could be expected to contribute to the overall protection of the developing brain in utero; however, once born, the pup’s brain would rely solely on the protection provided by its own barriers. It has been previously reported that this protection is developmentally regulated for some drugs such as digoxin, cimetidine and paracetamol (Koehn et al., 2019) but the degree of protection for valproate and lamotrigine is not known.\n\nClinical doses of valproate range from 20–60 mg/kg (Johannessen & Johannessen, 2003). We estimated valproate concentration in plasma, CSF and cortex of adult and P4 pups following an acute i.p. injection of either 30 mg/kg or 100 mg/kg. The dose of 30 mg/kg is well within the clinical range but 100 mg/kg is above. This was chosen on purpose to cover a wide range of drug doses as the route of administration we applied was different to that used in clinic (i.p. versus oral). Nevertheless, following a single dose of 30 mg/kg, concentrations of valproate measured by LC–MS in plasma from P4 and adult rats were within (adult) or close to (P4) the clinical range (Table 3). The higher dose of 100 mg/kg was above the recommended clinical concentration in patients’ plasma at both ages but clinical doses refer to valproate concentration at a steady level, not within 30 min of administration. The two methods (LC–MS and radioactivity counting) were also used for comparison to express results as ratios for CSF/plasma and brain/plasma in adults. These were very similar (Figure 14).\n\nEntry of valproate into the CSF and brain was lower in the older animals studied (Figure 3 and Figure 5). For a dose of 30 mg/kg (within the clinical range, Johannessen & Johannessen, 2003) and 100 mg/kg above the usual clinical range there was no difference in entry at any of the three ages studied. For the even higher dose of 200 mg/kg at P4 and adult, there was a small but significant (p<0.01–0.001) increase in entry of the drug. In the placenta, there was a substantial increase in fetal/maternal plasma ratio for the 100 mg/kg dose (Figure 9B and further discussion below). These findings suggest that even above the clinical dose range brain barrier mechanisms were able to deal with the increased dose of valproate, in spite of the lesser effectiveness of the placenta. The mechanisms that might be involved are discussed below. The greater entry of the highest dose tested provides support for the current clinical practice of reducing valproate dose at the later stages of pregnancy, in order to limit the risks of cognitive impacts (Thijs et al., 2019; Tomson et al., 2015; Tomson et al., 2019).\n\nFor lamotrigine at the doses studied, there was little difference in entry into CSF and brain at each dose (Figure 4). But unlike valproate and other dugs examined in similar studies (Koehn et al., 2019) the level of lamotrigine entry appeared to increase in CSF and brain with age (Figure 5). This suggests that the mechanisms protecting the brain against entry of lamotrigine are well established at E19 but appear less effective at older ages. Unlike valproate at the placental barrier, increasing the dose of lamotrigine did not affect the entry of the drug into the fetus (Figure 9D) suggesting that the placental protective mechanisms were effective in this dose range (see below for further discussion).\n\nPregnancy registries and clinical studies have reported small numbers of a wide range of abnormalities in offspring of mothers that took lamotrigine, but mainly when exposure was in early pregnancy (Briggs et al., 2017). It is not clear whether the higher than adult rate of lamotrigine entry into brain at E19 represents a risk to the fetus and offspring when lamotrigine exposure is later in pregnancy.\n\nIn experiments in which entry of 30 mg/kg 3H-valproate was measured in the absence or presence of different doses of lamotrigine (Figure 6) or the converse of measuring entry of 6 mg/kg lamotrigine in the absence or presence of 30 mg/kg valproate (Figure 7) there was little influence on drug entry by the combinations at the three ages studied. The dose combinations used were within that employed clinically (see Methods) which provides some reassurance that the current clinical practice of using lower doses of antiepileptic drugs in combination does not result in interference in brain entry between the drugs. This finding is thus relevant to the clinical practice of attempting to mitigate the ill effects of valproate by using lower doses combined with a second antiepileptic drug. Further studies could involve a wider range of doses and other antiepileptics with which valproate has been combined, particularly with longer term exposure to the drugs.\n\nAll animals received 30 mg/kg of valproate with radioactive tracer (3H-valproate) as a monotherapy, or in combination with different doses of lamotrigine. CSF/plasma, Cortex/plasma and Brainstem/plasma concentration ratios (%) in E19 (A–C), P4 (D–F) and non-pregnant female adult (G–I) rats of valproate, collected 30 min after intraperitoneal injections. Each point represents the result from a single animal. Mean±SD. n=3–10. p<0.05.\n\nNote that some error bars are too small to be clearly visible.\n\nAll animals received 6 mg/kg of lamotrigine with radioactive tracer (3H-lamotrigine) as a monotherapy, or in combination with 30 mg/kg of valproate. CSF/plasma, Cortex/plasma and Brainstem/plasma concentration ratios (%) in P4 (A–C) and non-pregnant female adult (D–F) rats of lamotrigine, collected 30 min after intraperitoneal injections. Each point represents the result from a single animal. Mean±SD. n=3–4. p<0.05.\n\nCSF/plasma, Cortex/plasma and Brainstem/plasma concentration ratios (%) in E19 (A–C), P4 (D–F) and non-pregnant female adult (G–I) rats of valproate, collected 30 min after intraperitoneal injections of 100 mg/kg valproate with radioactive tracer (3H-valproate). Dams of the chronic group were treated with a special feed containing valproate for at least two weeks prior to mating whereas dams of the acute group received a control feed. Non-pregnant female adult rats in the chronic group were treated with a special feed containing valproate for at least three weeks prior to experimentation. Each point represents the result from a single animal. Mean±SD. n=3–6. p<0.05. Note that some error bars are too small to be clearly visible.\n\nA. Time-course of fetal plasma/maternal plasma concentration ratios (%) of valproate collected 30–110 min after an intraperitoneal injection to the mother of valproate with radioactive tracer (3H-valproate). B. Results from A are mean±SD. C. Time-course of fetal plasma/maternal plasma concentration ratios (%) of lamotrigine collected 40–170 min after an intraperitoneal injection to the mother of lamotrigine with radioactive tracer (3H-lamotrigine). D Results from C are mean±SD. For B and D results were taken up to 110 min (box) to be comparable between experiments. Each point represents the result from a single animal; n=5–12. **p<0.001.\n\nThymus/plasma concentration ratios (%) of valproate (A–C) or lamotrigine (D–F) at E19, P4 and in non-pregnant female adult rats collected 30 minutes after a single intraperitoneal injection of either drug. Doses in mg/kg indicated on the x-axis. Each point represents the result from a single animal except for E19 thymus where a single sample was obtained by pooling (1 litter). Mean±SD; n=3–4. p<0.05, *p<0.01. Note that some error bars are too small to be clearly visible.\n\nThymus/plasma concentration ratios (%) of valproate (A) or lamotrigine (B) at E19, P4 and in non-pregnant female adult rats collected 30 minutes after a single intraperitoneal injection of 100 mg/kg valproate or 20 mg/kg lamotrigine respectively. Each point represents the result from a single animal except for E19 thymus where a single sample was obtained by pooling (1–2 litters). Mean±SD; n=3.\n\nThymus/plasma concentration ratios (%) of valproate at E19 (A), P4 (B) or non-pregnant female adult rats (C) collected 30 min after intraperitoneal injections of 30 mg/kg of valproate with radioactive tracer (3H-valproate) as a monotherapy, or in combination with different doses of lamotrigine. Thymus/plasma concentration ratios (%) of lamotrigine at P4 (D) or non-pregnant female adult rats (E) collected 30 min after intraperitoneal injections of 6 mg/kg of lamotrigine with radioactive tracer (3H-lamotrigine) as a monotherapy, or in combination with 30 mg/kg of valproate. Each point represents the result from a single animal except for E19 thymus where a single sample was obtained by pooling (1 litter). Mean±SD; n=3–4. p<0.05.\n\nThymus/plasma concentration ratios (%) of valproate at E19 (A), P4 (B) or non-pregnant female adult rats (C) collected 30 min after intraperitoneal injections of 100 mg/kg valproate with radioactive tracer (3H-valproate). Dams of the chronic group were treated with a special feed containing valproate for at least two weeks prior to mating whereas dams of the acute group received a control feed. Non-pregnant female adult rats in the chronic group were treated with a special feed containing valproate for at least three weeks prior to experimentation. Each point represents the result from a single animal except for E19 thymus where a single sample was obtained by pooling (1 litter). Mean±SD. n=3–4.\n\nA. CSF/plasma and B. Cortex/plasma concentration ratios (%) of valproate in non-pregnant female adults, collected 30 min after i.p. injection of 30 mg/kg or 100 mg/kg valproate with or without radioactive tracer (3H-valproate). LC–MS, liquid chromatography–mass spectrometry. Each point represents the result from a single animal. Mean±SD. n=3–4.\n\nThe chronic treatment used in the present study was that developed by Jazayeri et al. (2020) who showed that a feed containing 20 g/kg valproate resulted in blood levels in pregnant rats similar to those in pregnant mothers on treatment with this antiepileptic drug. At E19 there was a small but significant (p<0.05) decrease in cortical brain plasma ratios following injection of a dose of 100 mg/kg that included 3H-valproate compared with an acute injection (Figure 8). At P4, there were no differences in the ratios for brain cortex, brainstem or CSF (Figure 8). In adults, there was a significant decline (p<0.05) in ratio for both cortex and brainstem from about 10% to 6%, but no difference for CSF (Figure 8). These reductions in entry at E19 and in adults are small and may not be functionally significant. However, the decline in brain ratios at E19 and in the adults may reflect an upregulation in the mechanisms that limit entry of valproate into the brain, as considered below. The lack of such an effect at P4 may just reflect the variation in response to valproate that sometimes occurs in patients.\n\nFetal/maternal plasma ratios were used as an estimate of drug entry into the fetus and as an indication of the level of protection provided by the placenta. For valproate, in acute dose experiments, there was a significant increase from 38.2±4.4% with a dose of 30 mg/kg to 71.3±11.5% following a dose of 100 mg/kg (Figure 9B, p<0.0001). This suggests the possibility of a mechanism limiting entry of valproate across the placenta that can be saturated at higher doses. Following chronic treatment the fetal/maternal plasma ratio was reduced to the same as 30 mg/kg valproate, indicating the possibility of upregulation of efflux mechanisms. It is of potential clinical significance that entry into the fetus with chronic valproate treatment was reduced substantially (Figure 9B) with a much smaller effect on the clinically important entry of valproate into the mother’s brain (Figure 8).\n\nFor lamotrigine there was no significant difference in the fetal/maternal plasma ratios for the two doses tested (Figure 9D).\n\nThymus tissue was collected in this study because it has been found to contain limited expression of the ABC efflux transporter P-glycoprotein (Abcb1) a much-studied efflux mechanism that excludes or limits the entry of many drugs at brain barriers; there is some evidence that lamotrigine may be a substrate (Zhang et al., 2012 and see below). The residual vascular space was much larger in the thymus than in the brain regions sampled Figure 1) indicating a much larger vascularization of the tissue. This means that corrections applied for drug in blood in the tissue samples collected were much larger in the case of the thymus. At E19, the data for thymus are limited because measurement required pooling of tissue. However, the level of entry of valproate and lamotrigine appeared to be similar in thymus and brain and was not affected by increases in the drug doses (c.f. Figure 3, Figure 4, Figure 10).\n\nAt P4 for doses of 30 and 100 mg/kg the entry was substantially less than at E19 or in the adult but increased with a dose of 200 mg/kg valproate. The thymus thus appears to have effective mechanisms limiting entry of valproate in the postnatal period, a time when the development of the thymus has important biological functions (Miller, 2020). Entry of lamotrigine was not much different at the different ages suggesting that efflux mechanisms for the drug develop early.\n\nAddition of lamotrigine to valproate did not affect the entry of valproate into the thymus (Figure 12) whereas addition of valproate to lamotrigine increased the entry of lamotrigine from a ratio of 191.9±23.2% to 286.3±36.8% (p<0.05) at P4 but not in adults (Figure 12). Chronic treatment with valproate did not appear to affect its entry into the thymus (Figure 13).\n\nThe main mechanisms that limit drug entry into the brain are the ABC efflux transporters and a few SLC transporters that are bidirectional (Han et al., 2018; Roberts et al., 2008; Saidijam et al., 2018; Strazielle & Ghersi-Egea, 2015). In the case of valproate, the evidence is conflicting. This is partly due to a substantial use of in vitro systems some of which give different results depending on the cells used (Grewal et al., 2017; Zhang et al., 2012) or the species from which the cells were derived (Alms et al., 2014). In vivo studies may suffer from the limitation that generally effects of valproate (and other antiepileptics) on ABC transporter gene expression or changes in protein functionality were carried out using samples of whole brain. This does not allow distinguishing between effects on ABC transporters in cerebral endothelial cells from brain parenchymal cells. In a study in rabbits Scism et al. (2000) used probenecid, an organic anion transporter inhibitor and micro dialysis to measure drug concentrations in brain extracellular fluid of valproate administered intravenously and compared that with estimates of plasma and brain levels of valproate. Their measurements showed that probenecid increased intracellular brain tissue valproate 2.5 times but did not affect extracellular fluid/plasma concentration ratios taken as an estimate of blood–brain barrier transfer. Thus an additional factor to be taken account of in studies of barrier mechanism in relation to drug entry is the localisation of potential ABC or other efflux transporters. Wang et al. (2013) avoided this problem by studying the transport of a P-glycoprotein substrate in freshly isolated mouse cerebral capillaries. They reported that valproate increased the transfer of the specific P-glycoprotein fluorescent substrate NBD-CSA, suggesting that valproate itself interacts with P-glycoprotein. Weiss et al. (2003) have also shown that valproate, lamotrigine and verapamil increased entry of the fluorescent P-glycoprotein substrate calcein-AM into LLC-PK1 cells transfected with human MDR1 and into primary cultures of porcine brain capillary endothelial cells. However, this was only seen at high valproate and lamotrigine concentrations and to a much lower level (+10%) compared with the high-affinity P-glycoprotein substrate verapamil. In contrast, Moerman et al. (2011) found that in vivo in mice valproate did not interact with P-glycoprotein at therapeutic or higher doses. In support of this finding is the report that P-glycoprotein deficient Mdr1a/b(-/-) mice in the intrahippocampal kainate model of mesial temporal lobe epilepsy there we no significant differences in the anti-seizure efficacy of valproate and lamotrigine (Bankstahl et al., 2016).\n\nIn a comprehensive study using both in vitro and in vivo methods, Baltes et al. (2007) concluded that there is no evidence that valproate is a substrate for P-glycoprotein.\n\nThus on balance current evidence suggests that valproate is unlikely to be a P-glycoprotein substrate at the brain barriers.\n\nIn vitro studies using MDCKII cells transfected with human BCRP or mouse Bcrp did not find any evidence of valproate transport by BCRP (Cerveny et al., 2006; Römermann et al., 2015).\n\nIt is not clear what mechanism(s) limit entry of valproate across the placental barrier. Jinno et al. (2020) carried out a detailed study of 8 ATP and 10 SLC transporters at E13 and E20 using RTqPCR in pregnant rats. Abcb1a, 1b, Abcc2, Abcc4 were upregulated between these ages as were Slc7a5, Slc16a3, Slc22a3, Slc22a4, Slco2b1, Slco4a1. Effects of a single dose and treatment over 4 days with valproate were also studied. In response to multiple doses of valproate Mdr1a (P-glycoprotein Abab1a) and Mrp4 (Abcc4) were increased over 2-fold at E20, which may account for the decreased entry of valproate across the placenta observed in our experiments (Figure 9) in response to chronic treatment.\n\nIn the case of lamotrigine, there have been some conflicting reports, but the accumulating evidence from in vitro and in vivo studies is that lamotrigine is a low affinity substrate for both BCRP and P-glycoprotein. Zhang et al. (2012) summarize evidence from patient data, in vivo and in vitro experiments supporting this with additional recent human data available from Domjanović et al. (2018). In addition there has been a report of active transport of lamotrigine into brain via the OCT1 transporter (Dickens et al., 2012).\n\nPotschka et al. (2002) provided evidence that lamotrigine was a substrate for P-glycoprotein in vivo with increased entry of lamotrigine into rat brain extracellular fluid when a competing P-glycoprotein substrate (verapamil) was present. These authors used twin dialysis probes with guide catheters, implanting one probe into the left frontal cortex and the other into the right. Lamotrigine was administered to the animals i.p. and then verapamil infused via one of the guide catheters to one frontal cortex whilst the other side acted as a vehicle only control. The concentration of lamotrigine in the dialysate from the verapamil infused cortex was found to be approximately double that measured in the probe from the control hemisphere in the same animals.\n\nFor valproate, tissue/plasma ratios in E19 fetuses were not significantly affected by a large difference in doses used in the study (30 mg/kg compared to 100 mg/kg, Figure 3). However, the ratios were higher than at P4 and much higher than in the adult for the same doses (Figure 3 and Figure 5). Placental transfer at E19 considerably increased for 100 mg/kg dose compared to 30 mg/kg dose (Figure 9B). The lesser protection against the higher dose combined with the much higher tissue/plasma ratios suggests that fetal brain may be particularly susceptible to effects of valproate administered in late-gestation rats; a comparable stage in human brain development would be the first trimester (Clancy et al., 2013). At both P4 and in adults the CSF/plasma and brain/plasma ratios increased with increasing doses of between 10 mg/kg and 200 mg/kg (Figure 3). The ratios were approximately two-fold higher at P4 than in the adult. The progressive decline in ratios between E19 and adult suggest the development of brain barrier protective mechanisms against valproate. The nature of such mechanisms is unclear (see previous section).\n\nThe CSF/plasma and brain/plasma ratios for lamotrigine were similar at P4 and in adults and were not affected by increasing the dose (Figure 4). At E19 the ratios were significantly lower for a dose of 20 mg/kg in the CSF and cortex (Figure 4). This suggests that brain barrier protective mechanisms against lamotrigine are developed as early as E19 (equivalent to 13–14 weeks gestation in human, Clancy et al., 2013) but appear less effective at older ages (Figure 5). Current evidence suggests that the mechanisms involved could include the ABC efflux transporters P-glycoprotein and BCRP (see previous section).\n\nAdding 6 mg/kg lamotrigine to 30 mg/kg valproate (doses that are similar to the clinically used range, see Methods) at E19 and adult and up to 20 mg/kg at P4 (Figure 6) did not affect the CSF/plasma, brain/plasma or fetal/maternal plasma ratios of valproate.\n\nThis is an important validation of the current clinical approach to control seizures during pregnancy by reducing valproate doses by adding lamotrigine (Tomson et al., 2019). The increase in CSF/plasma ratio of valproate in the presence of 6 mg/kg lamotrigine in the adult and much smaller non-significant increase in the brain are not relevant to the antiepileptic drug status of the developing brain, but might indicate that the clinical dose of valproate for treatment of the mother could be reduced further.\n\nIn the converse experiment of adding 30 mg/kg valproate to 6 mg/kg lamotrigine in P4 and adult rats the ratios were not affected by the addition of valproate except for a small increase in the cortex but not brainstem at P4 (Figure 7); this further supports the clinical use of this drug combination in pregnant patients with epilepsy.\n\nResults from experiments in which female rats were fed a diet containing 20 g/kg valproate from before pairing and throughout pregnancy were compared with results from female rats fed on chow without the drug. At E19 there was a small but significant reduction in cortex/plasma ratio (Figure 8) and a substantial significant reduction in the fetal/maternal plasma ratio in the chronically treated animals from 70% to 35% (Figure 9B). For patients delivered at term this ratio was reported to be 120% (Bank et al., 2017). The difference might be due to the difference in gestation at which the measurements were made or reflect greater protection by the rat placenta.\n\nAt P4, the various ratios were unaffected by chronic treatment but in the adults the cortex and brainstem ratios were reduced in the chronically treated animals (Figure 8). These reductions in some ratios in the treated animals suggest that there may be a degree of upregulation of the mechanisms limiting valproate entry across barrier interfaces, as described previously for other drugs (Koehn et al., 2019 and see previous section). The relatively large decrease in fetal/maternal plasma ratio for valproate in chronically treated pregnant animals is of clinical significance as it suggests an increased degree of fetal protection with only a smaller effect on valproate entry in the maternal brain, which is important for treatment of the mother. Löscher (2007) has provided a valuable comparison in rats and humans of the pharmacokinetics of antiepileptic drugs including valproate and lamotrigine. The results from LC–MS measurement of valproate gave values in plasma in adult rats that were within the clinical range for a dose of 30 mg/kg, a dose that is with the range used in patients. This confirms that doses to be used can be based on body weight for such animal experiments. For this dose at P4, the plasma level was above the normal clinical upper limit (Table 3); this is hardly surprising given likely differences in drug metabolism and plasma protein binding in immature animals. It does suggest that in future experiments in developing animals it may be appropriate to scale down the amount administered.\n\nThis study was carried out during two periods of lockdown in Melbourne due to the Covid-19 pandemic. In the first period from Easter to mid-June, our laboratories were closed and the animal house ran down its breeding colonies because of uncertainty about how long the lockdown would continue. We were allowed to re-open our laboratories in mid-June under considerable restrictions on the number of people allowed in the lab and the periods they could be there for. These restrictions continued throughout the lockdown (late July to early November) and were still current into 2021. The staff restrictions and limited supply of pregnant and postnatal animals made it difficult to plan adequate numbers of experiments. However, it has been possible to carry out a comprehensive range of experiments on valproate and lamotrigine alone and in combination as well as some limited chronic experiments using valproate-containing feed. The results are internally consistent and show clear developmentally related patterns and no results have previously been published in which drug levels have been examined in fetal and postnatal blood and CSF. Such measurements are essential for determining the relative quantitative roles of the placental, blood-brain and blood-CSF barrier interfaces for antiepileptic drugs. It was only possible to study one dose of one of the drugs (valproate) in chronic experiments in which valproate was incorporated into the feed. It took several months to obtain the feed and the experiments took 5–6 weeks to conduct and only when suitable animals were available. However, this feeding regime has been shown previously to achieve clinically appropriate blood levels of the drug (Jazayeri et al., 2020). We have used radiolabelled drugs for most of the studies as liquid scintillation is a highly sensitive method that was able to detect measurable amounts of labelled drug in the limited volumes of fetal CSF and plasma available. However, these results have been validated by some estimations of valproate using LC–MS.\n\nIt is, of course, important to be cautious about extrapolating results from animal studies to humans. However, we are dealing with fundamental biological mechanisms so that, although there may be differences in detail, it is reasonably likely that the overall pattern of results will be significant for the human condition. There is a wealth of information which allows brain development to be compared in many species but particularly humans and rodents (Clancy et al., 2013; Workman et al., 2013). Also, the placentas of these species are classified as haemochorial and although there are some structural differences they are much more similar than for example the much “tighter” epitheliochorial multicotyledonary placenta of the sheep (Schneider, 1991) a species that has been used for many valuable developmental studies but are born at an advanced stage of brain development compared with humans (Clancy et al., 2013).\n\n\nConclusions and significance of the study\n\nStudies described here show that in the late gestational rat the placenta provides a significant impediment to transfer of the antiepileptic drugs valproate and lamotrigine, from the mother to the fetus but the degree of protection was less at higher doses of the drugs. In chronic experiments with valproate delivered via the feed there was evidence of a substantial upregulation in placental protection after a prolonged exposure to the drug. At the blood–brain and blood–CSF barriers, the results show that entry of the drugs at E19 is high but declines with age, indicating the development of protective mechanisms at these interfaces. In experiments using different combinations of doses of valproate and lamotrigine there was generally no difference in drug entry when compared to entry of either drug alone. This provides support for the current clinical practice of using lamotrigine to reduce the amount of valproate required to control seizure where valproate in some patients is the only effective antiepileptic drug; this limits the potential for this drug to cause congenital malformations. The finding of higher entry of valproate and possibly other AEDs still to be investigated early in brain development. The decline as the brain matures, provides a basis for future studies of the mechanisms involved and for studies of potential deleterious effects on brain development and behaviour in the offspring of pregnant females treated with AEDs.\n\n\nData availability\n\nFigshare: Underlying data for ‘Entry of antiepileptic drugs (valproate and lamotrigine) into the developing rat brain’. https://doi.org/10.26188/14374379 (Toll et al., 2021)\n\nThe project contains the following underlying data:\n\nRaw data for liquid scintillation experiments:\n\n• Lamotrigine adult: CPM, DPM and weight for all samples in adult lamotrigine experiments\n\n• Lamotrigine P4: As above\n\n• Lamotrigine E19: As above\n\n• Valproate adult : As above\n\n• Valproate P4: As above\n\n• Valproate E19: As above\n\nRaw data for LC-MS:\n\n• 210319: Raw data files for measurements on 19th March 2021\n\n• 210330: Raw data files for measurements on 30th March 2021\n\n• 210402: Raw data files for measurements on 2nd April 2021\n\n• LCMS peakarea: Peak area & concentration calculation for all samples\n\n• LCMS Skyline template: Template used to extract peak area from raw data in Skyline\n\nFigshare: Extended data for ‘Entry of antiepileptic drugs (valproate and lamotrigine) into the developing rat brain’. https://doi.org/10.26188/14374379 (Toll et al., 2021)\n\nThis project contains the following extended data:\n\n• Tables of numerical values for each figure\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAbou-Khalil BW: Update on antiepileptic drugs 2019. 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PubMed Abstract \| Publisher Full Text\n\nBank AM, Stowe ZN, Newport DJ, et al.: Placental passage of antiepileptic drugs at delivery and neonatal outcomes. Epilepsia. 2017; 58(5): e82–e86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBankstahl M, Klein S, Römermann K, et al.: Knockout of P-glycoprotein does not alter antiepileptic drug efficacy in the intrahippocampal kainate model of mesial temporal lobe epilepsy in mice. Neuropharmacology. 2016; 109: 183–195. PubMed Abstract \| Publisher Full Text\n\nBriggs GG, Freeman RK, Towers CV, et al.: Drugs in Pregnancy and Lactation: A Reference Guide to Fetal and Neonatal Risk. 11th Ed. Lippincott Williams & Wilkins (LWW). 2017. Reference Source\n\nCasassus B: France bans sodium valproate use in case of pregnancy. Lancet. 2017; 390(10091): 217. PubMed Abstract \| Publisher Full Text\n\nCerveny L, Pavek P, Malakova J, et al.: Lack of interactions between breast cancer resistance protein (bcrp/abcg2) and selected antiepileptic agents. Epilepsia. 2006; 47(3): 461–468. PubMed Abstract \| Publisher Full Text\n\nClancy B, Charvet CJ, Darlington RB, et al.: Extrapolating brain development from experimental species to humans. 2013. Reference Source\n\nDavson H, Segal M: Physiology of the CSF and blood-brain barriers. CRC press, Boca Raton, 1996; 36. Reference Source\n\nDickens D, Owen A, Alfirevic A, et al.: Lamotrigine is a substrate for OCT1 in brain endothelial cells. Biochem Pharmacol. 2012; 83(6): 805–814. PubMed Abstract \| Publisher Full Text\n\nDomjanović IK, Lovrić M, Trkulja V, et al.: Interaction between ABCG2 421C>A polymorphism and valproate in their effects on steady-state disposition of lamotrigine in adults with epilepsy. Br J Clin Pharmacol. 2018; 84(9): 2106–2119. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan LW, Gao C, Mao Q: An update on expression and function of P-gp/ABCB1 and BCRP/ABCG2 in the placenta and fetus. Expert Opin Drug Metab Toxicol. 2018; 14(8): 817–829. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJazayeri D, Braine E, McDonald S, et al.: A rat model of valproate teratogenicity from chronic oral treatment during pregnancy. Epilepsia. 2020; 61(6): 1291–1300. PubMed Abstract \| Publisher Full Text\n\nJentink J, Loane MA, Dolk H, et al.: Valproic acid monotherapy in pregnancy and major congenital malformations. N Engl J Med. 2010; 362(23): 2185–2193. PubMed Abstract \| Publisher Full Text\n\nJinno N, Furugen A, Kurosawa Y, et al.: Effects of single and repetitive valproic acid administration on the gene expression of placental transporters in pregnant rats: An analysis by gestational period. Reprod Toxicol. 2020; 96: 47–56. PubMed Abstract \| Publisher Full Text\n\nJohannessen CU, Johannessen SI: Valproate: Past, Present, and Future. CNS Drug Rev. 2003; 9(2): 199–216. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoehn L, Habgood M, Huang Y, et al.: Determinants of drug entry into the developing brain [version 1; peer review: 3 approved]. F1000Res. 2019; 8: 1372. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoehn LM, Huang Y, Habgood MD, et al.: Effects of paracetamol (acetaminophen) on gene expression and permeability properties of the rat placenta and fetal brain [version 2; peer review: 2 approved]. F1000Res. 2020; 9: 573. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLöscher W: The pharmacokinetics of antiepileptic drugs in rats: consequences for maintaining effective drug levels during prolonged drug administration in rat models of epilepsy. Epilepsia. 2007; 48(7): 1245–1258. PubMed Abstract \| Publisher Full Text\n\nMiller JFAP: The function of the thymus and its impact on modern medicine. Science. 2020; 369(6503): eaba2429. PubMed Abstract \| Publisher Full Text\n\nMoerman L, Wyffels L, Slaets D, et al.: Antiepileptic drugs modulate P-glycoproteins in the brain: a mice study with (11)C-desmethylloperamide. Epilepsy Res. 2011; 94(1–2): 18–25. PubMed Abstract \| Publisher Full Text\n\nPotschka H, Fedrowitz M, Löscher W: P-Glycoprotein-mediated efflux of phenobarbital, lamotrigine, and felbamate at the blood-brain barrier: evidence from microdialysis experiments in rats. Neurosci Lett. 2002; 327(3): 173–176. PubMed Abstract \| Publisher Full Text\n\nRizzi M, Caccia S, Guiso G, et al.: Limbic seizures induce P-glycoprotein in rodent brain: functional implications for pharmacoresistance. J Neurosci. 2002; 22(14): 5833–5839. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoberts LM, Black DS, Raman C, et al.: Subcellular localization of transporters along the rat blood-brain barrier and blood-cerebral-spinal fluid barrier by in vivo biotinylation. Neuroscience. 2008; 155(2): 423–38. PubMed Abstract \| Publisher Full Text\n\nRömermann K, Helmer R, Löscher W: The antiepileptic drug lamotrigine is a substrate of mouse and human breast cancer resistance protein (ABCG2). Neuropharmacology. 2015; 93: 7–14. PubMed Abstract \| Publisher Full Text\n\nRugg-Gunn F, Miserocchi A, McEvoy A: Epilepsy surgery. Pract Neurol. 2020; 20(1): 4–14. PubMed Abstract \| Publisher Full Text\n\nSaidijam M, Karimi Dermani F, Sohrabi S, et al.: Efflux proteins at the blood-brain barrier: review and bioinformatics analysis. Xenobiotica. 2018; 48(5): 506–532. PubMed Abstract \| Publisher Full Text\n\nSchneider H: Placental transport function. Reprod Fertil Dev. 1991; 3(4): 345–353. PubMed Abstract \| Publisher Full Text\n\nScism JL, Powers KM, Artru AA, et al.: Probenecid-inhibitable efflux transport of valproic acid in the brain parenchymal cells of rabbits: a microdialysis study. Brain Res. 2000; 884(1--2): 77–86. PubMed Abstract \| Publisher Full Text\n\nStrazielle N, Ghersi-Egea JF: Efflux transporters in blood-brain interfaces of the developing brain. Front Neurosci. 2015; 9: 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Royal Women’s Hospital: Pregnancy and Breastfeeding medicines Guide. 2020. Reference Source\n\nThijs RD, Surges R, O'Brien TJ, et al.: Epilepsy in adults. Lancet. 2019; 393(10172): 689–701. PubMed Abstract \| Publisher Full Text\n\nToll SJ, Qiu F, Huang Y, et al.: Underlying and extended data for ‘Entry of antiepileptic drugs (valproate and lamotrigine) into the developing rat brain’. 2021. http://www.doi.org/10.26188/14374379\n\nTomson T, Marson A, Boon P, et al.: Valproate in the treatment of epilepsy in girls and women of childbearing potential. Epilepsia. 2015; 56(7): 1006–19. PubMed Abstract \| Publisher Full Text\n\nTomson T, Battino D, Bonizzoni E, et al.: Comparative risk of major congenital malformations with eight different antiepileptic drugs: a prospective cohort study of the EURAP registry. Lancet Neurol. 2018; 17(6): 530–538. PubMed Abstract \| Publisher Full Text\n\nTomson T, Battino D, Bromley R, et al.: Management of epilepsy in pregnancy: a report from the International League Against Epilepsy Task Force on Women and Pregnancy. Epileptic Disord. 2019; 21(6): 497–517. PubMed Abstract\n\nUS Food and Drug Administration. 2016.\n\nVajda F: Dose issues in antiepileptic therapy. J Clin Neurosci. 2012; 19(11): 1475–1477. PubMed Abstract \| Publisher Full Text\n\nValente RC, Capella LS, Nascimento CR, et al.: ABCB1 (P-glycoprotein) but not ABCC1 (MRP1) is downregulated in peripheral blood mononuclear cells of spontaneously hypertensive rats. Pflugers Arch. 2008; 456(2): 359–368. PubMed Abstract \| Publisher Full Text\n\nWang X, Cabrera RM, Li Y, et al.: Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice. FASEB J. 2013; 27(3): 1167–1175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeiss J, Kerpen CJ, Lindenmaier H, et al.: Interaction of Antiepileptic Drugs with Human P-Glycoprotein in Vitro. J Pharmacol Exp Ther. 2003; 307(1): 262–267. PubMed Abstract \| Publisher Full Text\n\nWorkman AD, Charvet CJ, Clancy B, et al.: Modeling transformations of neurodevelopmental sequences across mammalian species. J Neurosci. 2013; 33(17): 7368–7383. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang C, Kwan P, Zuo Z, et al.: The transport of antiepileptic drugs by P-glycoprotein. Adv Drug Deliv Rev. 2012; 64(10): 930–942. PubMed Abstract \| Publisher Full Text" }	[ { "id": "85603", "date": "07 Jun 2021", "name": "Alan Leviton", "expertise": [ "Reviewer Expertise neonatal neurology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study of rats was designed to evaluate the entry of two antiepileptic drugs' (valproate and lamotrigine) entry into developing brain. The antiepileptic drugs were injected into the peritoneal cavity of pregnant rats and the offspring were assessed at embryonic day (E) 19, postnatal day (P) 4, and when adults. Based on their findings, the authors conclude that “the placenta provides a significant impediment to transfer of the antiepileptic drugs valproate and lamotrigine from the mother to the fetus.”\nThis is a thorough evaluation of the very drugs the fetuses of epileptic women are likely to be exposed to, and provides clinicians with the confidence they need to assure their patients that their anti-epileptic drugs are unlikely to damage their baby's brain.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6843", "date": "02 Jul 2021", "name": "Norman Saunders", "role": "Author Response", "response": "We should like to thank Dr Leviton for his review of our paper. We appreciate his comment that the study was a thorough evaluation and likely to be of value to clinicians in advising their patients who are required to take antiepileptic drugs during pregnancy." } ] }, { "id": "86533", "date": "15 Jun 2021", "name": "Neil Dani", "expertise": [ "Reviewer Expertise Developmental neuroscience", "neurobiology", "blood-CSF barrier." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 evaluate antiepileptic drug entry into the developing brain in fetal rat models. They test entry of valproate and lamotrigine into the fetus by LC-MS and report higher valproate entry in the developing brain. Chronic valproate exposure was found to restrict entry into the fetal brain. Further, lamotrigine showed milder differences with age and dose dependence. Finally, no significant interactions were seen in the combination therapy models. Overall, the study is informative and provides dosing guidelines for future experiments that investigate anti-epileptic drug effects on fetal brain development.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6844", "date": "02 Jul 2021", "name": "Norman Saunders", "role": "Author Response", "response": "We should like to thank Dr Dani for his review of our paper and his comment that our study will provide guidelines for future experiments. In fact, we are already embarking on these." } ] }, { "id": "86365", "date": "24 Jun 2021", "name": "Stefan Liebner", "expertise": [ "Reviewer Expertise Vascular biology", "blood-brain barrier research", "brain and vascular development", "cell-cell communication and signaling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nToll et al. have evaluated in the present manuscript the entry of anti-epileptic drugs into the developing rat brain. Specifically, they have explored the entry of valproate, known as the most effective treatment for maternal epilepsy, together with the accessory drug lamotrigine. Considering the fact that valproate is mostly combined with a second anti-epileptic drug since its high doses lead to congenital abnormalities, Toll et al. have studied the entry of the drugs valproate and lamotrigine when applied alone and in combination. Sprague-Dawley rats were used at three stages, including embryonic day (E)19, pups at postnatal (P) day 4, and adults since these ages represent developmental milestones that can be translated to stages of human brain development, corresponding to the end often first trimester, late second trimester and adult. Moreover, the authors compared acute versus chronic treatment of pregnant rat with valproate. The entry of valproate into brain and CSF was identified to be higher at E19 and P4 compared to adult stage. The entry into the brain was not observed to be dose-dependent, whereas the transfer of valproate across the placenta increased with its dose and was highest at 100mg/kg.\nInstead, lamotrigine did not show significant differences regarding its permeability between the three stages. Nor did the combination treatment of valproate with lamotrigine show significant differences in brain entry of the drugs. Chronic treatment of pregnant rats with valproate resulted in reduced brain entry of valproate as well as in decreased placental transfer.\nThe presented data in this manuscript are highly relevant and interesting to the field of clinical epilepsy research. The work may have impact on the treatment of pregnant women suffering from epilepsy.\nHowever, to augment the scientific merit of the manuscript, some issues require the author's attention:\nMajor points:\nThe major points of critique on the manuscript by Toll et al. are listed below:\nFigure 3E shows that there is a significant difference in the entry of 30 and 100 mg/kg valproate into cortex at P4. However, the authors claim on Page 13, in Paragraph 2: \"For a dose of 30 mg/kg (within the clinical range, Johannessen & Johannessen, 2003) and 100 mg/kg above the usual clinical range there was no difference in entry at any of the three ages studied.\" The authors should comment on this discrepancy.\n\nFigure 1 shows the residual vascular space in the organs cortex, brainstem, and thymus. The authors report that each point represents the result from a single animal. Assuming that one animal was used to collect these three organs, why is the number of animals different for the different organs? For example the figure shows in A that n=3 animals were used to analyze the residual vascular space in the adult cortex, while the number of animals for the analysis of the brainstem is only n=2.\n\nThere is a high variation in the n numbers between different groups and/or different time points. Moreover, in several cases, the n number is not high enough to perform statistical analysis. For instance, in Figure 4E, one of the three animals might be outlier, suggesting that more replicates are needed. The authors should comment on this.\n\nThe authors have evaluated the entry of 3H-valproate into the cortex of E19 fetuses following chronic feeding of the mother. Considering the idea of combining valproate with lamotrigine, additional studies in which the entry of the drugs when combined in a chronic treatment paradigm would be complementary.\n\nIt is not very clear in the methods part whether the animals in the chronic group received 100 mg/kg i.p. valproate injection or not. If they did, isn’t it a combination of chronic+acute instead of just a chronic phase? If they did not, isn’t this time frame following the i.p. injection too short to evaluate a chronic phase outcome?\n\nAs it is written in the figure legend and on Page 12, Paragraph 6, Figures 9A and 9C show results from only one animal for each time point which does not seem to be sufficient to draw a conclusion. Moreover, Figures 9B and 9C show aggregated data along the time frame following the injection. A setup in which enough number of replicates were used for each time point and in which the aggregated data is from those replicates for each time point would be better to understand the dose and time-dependent entry of the drug into the fetus.\n\nThe authors write in the thymus permeability section: \"At P4 for doses of 30 and 100 mg/kg the entry was substantially less than at E19 or in the adult but increased with a dose of 200 mg/kg valproate.\" However, Figures 10B and C show that valproate entry to the thymus at 100 mg/kg dose is higher in P4 compared to the adult. The authors should clarify this.\n\nConsidering the increase in the entry of lamotrigine to CSF and brain with age, it is suggested in the manuscript that mechanisms protecting the brain against entry of lamotrigine are well established at E19 but appear less effective at older ages. This sounds like an assertive explanation for the observed phenomena with statistically not satisfying numbers of replicates. Given that lamotrigine potentially is a substrate of P-glycoprotein, the authors should comment on the regulation of the ABC transporter between E19 and postnatal stages up to adulthood.\n\nThe \"Limitations of the study\" part does not add anything to the scientific quality of the article. These limitations might be valid for any research group during the pandemic. The reviewer can only judge if the presented data are valid and if experimental and statistical rigidity were applied. Why and how it came about, and their ability to present solid data, cannot be judged by the reviewer.\n\nMinor points:\nPage 1, Results section: The sentence \"Entry of valproate into brain and CSF was higher at E19 and P4 compared to adult but was not dose-dependent;..\" could be written in a different way to make it clear that valproate entry is dose dependent but only at E19 this is not the case.\n\nPage 3, line 69: Check the format of the reference \"(rat, Koehn et al., 2019; Koehn et al., 2020)\". Was the word \"rat\" supposed to be in the reference?\n\nPage 7: \"The ratio of 3H-dextran into cortex and brainstem compared with plasma, illustrated in Figure 1A and B, showed no significant difference at any of the ages studied and was around 2–4%.\" Was it supposed to be \"entry into\" instead of just \"into\"?\n\nFigure 9C: Why the 20mg/kg case lasts only until app. 95 min? It is written on Page 12 \"Figures 9B and 9D show the aggregated data up to 110 min.\" However, the 20mg/kg does not reach that time point.\n\nFigure 10: Why is \"Thymus\" written on top of some graphs while on top of others not? This \"subtitle\" is not needed anyway, since there is already main title of the figure.\n\nOverall the manuscript by Toll et al. provides interesting and novel data on the treatment of pregnant rats with the anti-epileptic drugs valproate and lamotrigine.\nNevertheless, in its present form, the manuscript requires some additional work to augment its scientific merit. Hence the reviewer recommends the manuscript for indexing after major revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "6930", "date": "26 Jul 2021", "name": "Norman Saunders", "role": "Author Response", "response": "We thank Drs Liebner and Fidan for their detailed appraisal of our paper. We list our responses below. Where appropriate changes in the ms have been tracked. Major points: The major points of critique on the manuscript by Toll et al. are listed below: Figure 3E shows that there is a significant difference in the entry of 30 and 100 mg/kg valproate into cortex at P4. However, the authors claim on Page 13, in Paragraph 2: \"For a dose of 30 mg/kg (within the clinical range, Johannessen & Johannessen, 2003) and 100 mg/kg above the usual clinical range there was no difference in entry at any of the three ages studied.\" The authors should comment on this discrepancy. We thank the reviewers for drawing out attention to this discrepancy which has now been corrected. “For a dose of 30 mg/kg (within the clinical range, Johannessen & Johannessen, 2003) and 100 mg/kg (above the usual clinical range) there was no difference in entry into CSF at any of the three ages studied; in cortex and brainstem the entry of valproate was higher at P4 with the larger clinical dose, but not at the other ages (Figure 3).” Figure 1 shows the residual vascular space in the organs, cortex, brainstem, and thymus. The authors report that each point represents the result from a single animal. Assuming that one animal was used to collect these three organs, why is the number of animals different for the different organs? For example the figure shows in A that n=3 animals were used to analyze the residual vascular space in the adult cortex, while the number of animals for the analysis of the brainstem is only n=2. We initially only took cortex but later on decided to investigate brainstem as well so the first adult used for dextran blood space didn’t have a brainstem value. There is a high variation in the n numbers between different groups and/or different time points. Moreover, in several cases, the n number is not high enough to perform statistical analysis. For instance, in Figure 4E, one of the three animals might be outlier, suggesting that more replicates are needed. The authors should comment on this. We do comment on this in “Limitations of the Methods” and we agree, that more experiments would have been desirable. The authors have evaluated the entry of 3H-valproate into the cortex of E19 fetuses following chronic feeding of the mother. Considering the idea of combining valproate with lamotrigine, additional studies in which the entry of the drugs when combined in a chronic treatment paradigm would be complementary. We agree with this point and these experiments are planned. It takes considerable time to prepare feeds, check if animals consume enough to obtain a clinical range of the drug in plasma and make sure they are willing to mate when on this treatment. It is not very clear in the methods part whether the animals in the chronic group received 100 mg/kg i.p. valproate injection or not. If they did, isn’t it a combination of chronic+acute instead of just a chronic phase? If they did not, isn’t this time frame following the i.p. injection too short to evaluate a chronic phase outcome? All animals with chronic feeding received a final injection with radiolabelled valproate (described on page 6). The final acute injection is used to measure permeability to find out whether chronic exposure alters this aspect of the barrier mechanisms and therefore drug entry (see Koehn et al, 2019). As it is written in the figure legend and on Page 12, Paragraph 6, Figures 9A and 9C show results from only one animal for each time point which does not seem to be sufficient to draw a conclusion. Moreover, Figures 9B and 9C show aggregated data along the time frame following the injection. A setup in which enough number of replicates were used for each time point and in which the aggregated data is from those replicates for each time point would be better to understand the dose and time-dependent entry of the drug into the fetus. Each point is one fetus but the whole line graph shows data for that litter. Fetuses are taken one after the next therefore there will always be a time lag between littermates. The authors write in the thymus permeability section: \"At P4 for doses of 30 and 100 mg/kg the entry was substantially less than at E19 or in the adult but increased with a dose of 200 mg/kg valproate.\" However, Figures 10B and C show that valproate entry to the thymus at 100 mg/kg dose is higher in P4 compared to the adult. The authors should clarify this. We thank the reviewers for drawing our attention to this error which has been corrected. “At P4 for the dose of 30 mg/kg the entry was substantially less than at E19 or in the adult but increased with a dose of 200 mg/kg valproate. At 100 mg/kg the entry declined with age.” Considering the increase in the entry of lamotrigine to CSF and brain with age, it is suggested in the manuscript that mechanisms protecting the brain against entry of lamotrigine are well established at E19 but appear less effective at older ages. This sounds like an assertive explanation for the observed phenomena with statistically not satisfying numbers of replicates. Given that lamotrigine potentially is a substrate of P-glycoprotein, the authors should comment on the regulation of the ABC transporter between E19 and postnatal stages up to adulthood. As the reviewers comment lamotrigine is at best a potential substrate for P-glycoprotein. We have modified the statement in the text. “One possible explanation for the observed age-related differences in the entry of LTG and VPA into the brain could be the time frame of when responsible transporters become functional (see Discussion in Koehn et al, 2019).” The \"Limitations of the study\" part does not add anything to the scientific quality of the article. These limitations might be valid for any research group during the pandemic. The reviewer can only judge if the presented data are valid and if experimental and statistical rigidity were applied. Why and how it came about, and their ability to present solid data, cannot be judged by the reviewer. We disagree and note that two other Reviewers have not commented upon this aspect of the paper. We have nevertheless restricted the comment on the impact of the pandemic to a single sentence. We feel strongly that all papers should carry a section detailing limitations. If more authors were willing to acknowledge the experimental limitations of their studies it might lead to an improvement in standards of research and help readers assess validity of results (e.g. Begley & Ioannidis, 2015, Circ Res. 2015;116:116-126. DOI: 10.1161/CIRCRESAHA.114.303819.). Minor points: Page 1, Results section: The sentence \"Entry of valproate into brain and CSF was higher at E19 and P4 compared to adult but was not dose-dependent;..\" could be written in a different way to make it clear that valproate entry is dose dependent but only at E19 this is not the case. Revised to read “Entry of valproate into brain and CSF was higher at E19 and P4 compared to adult and was dose-dependent, except at E19.\" Page 3, line 69: Check the format of the reference \"(rat, Koehn et al., 2019; Koehn et al., 2020)\". Was the word \"rat\" supposed to be in the reference? Can remove? This was to indicate that the animal model was rat. Text modified to indicate this. Page 7: \"The ratio of 3H-dextran into cortex and brainstem compared with plasma, illustrated in Figure 1A and B, showed no significant difference at any of the ages studied and was around 2–4%.\" Was it supposed to be \"entry into\" instead of just \"into\"? Detectable dextran does not enter the brain in 5 minutes. The wording has been changed to “in brain tissue” to clarify this. Figure 9C: Why the 20mg/kg case lasts only until app. 95 min? It is written on Page 12 \"Figures 9B and 9D show the aggregated data up to 110 min.\" However, the 20mg/kg does not reach that time point. Usually fetuses are collected at 10 minutes intervals, the particular dam used for the 20mg/kg experiment had 5 pups. We limit the time to present results from all experiments to 110 min to allow longer experiments (more fetuses) to be comparable with shorter experiments. Figure 10: Why is \"Thymus\" written on top of some graphs while on top of others not? This \"subtitle\" is not needed anyway, since there is already main title of the figure. Thank you for drawing our attention to this anomaly. We have asked for “Thymus” to be removed from Figure 10 B and C and Figure 12 B." } ] } ]	1	https://f1000research.com/articles/10-384
https://f1000research.com/articles/10-641/v1	23 Jul 21	{ "type": "Opinion Article", "title": "The imperative to find the courage to redesign the biomedical research enterprise", "authors": [ "Bibiana Bielekova", "Shannon Brownlee", "Shannon Brownlee" ], "abstract": "Medical research aims to improve health for everyone. While its advances are undeniable, the pace and cost of the progress are not optimal. For example, independent analyses concluded that at least half of the published biomedical research findings are irreproducible, and most scientific papers are never read or cited. This paper examines biomedical research holistically, as a system of incentives that shape the behavior of scientists, administrators, publishers, and funders, and are detrimental to medical progress. We identify opportunities to change and improve those incentives by altering the way research output is disseminated and evaluated, and recommend transparent, data-driven measures of methodological rigor, reproducibility, and societal value of scientific discoveries. Embracing these opportunities would maximize our investments in biomedical research and optimize its value to human health, while simultaneously increasing freedom, creativity, and satisfaction of the scientific workforce.", "keywords": [ "research reproducibility", "science administration", "scientific publication system", "incentive structure in science" ], "content": "Introduction\n\nAt least half of the human and financial resources currently devoted to the biomedical academic research generates findings that may not enhance collective knowledge or are unlikely to improve health. Every independent attempt to reproduce published biomedical scientific studies found stunning irreproducibility rates ranging from 51 to 89%.1–7 In a survey of more than 1500 researchers, 90% of respondents believe there is a reproducibility crisis in biomedical sciences.8\n\nCurrent solutions to this crisis focus on encouraging better training of scientists in the methods of reproducible research and transparent reporting of methodological details.9,10 While these efforts target important aspects of the problem, they fail to address its roots.\n\nThis paper treats the research enterprise as a system and examines how that system shapes the behavior of scientists and administrators. It asks whether the current design of publicly funded biomedical research maximizes its societal value and considers modifications to the organizational structure to enhance research reproducibility and its relevance for human health.\n\n\nWhat is the desired outcome or mission of biomedical research?\n\nThe mission of the US National Institutes of Health (NIH), the largest publicly funded biomedical research enterprise in the world, is to “Uncover new knowledge that will lead to better health for everyone.” One could assume that “uncovering new knowledge” should be optimized in terms of speed and value to improving health. To “lead to better health for everyone,” the knowledge must be both reproducible, which ensures that we can trust the results and build upon them, and relevant to human health.11 To achieve optimal speed and value in advancing the NIH’s mission, a system must be organized around the pursuit of these implicit goals, which presently it is not. One way to see this is to explore the differences between the current biomedical research enterprise, and uniquely successful past public projects such as the Manhattan Project,12 and the Apollo Mission.13\n\nThe Manhattan and Apollo projects were entirely mission-driven with defined, concrete end-products. Accomplishment of their missions, in the expected timeframe, was the definition of success for all actors from funders to administrators and scientists (Figure 1A). Every expense, every human resource decision, every regulation imposed was judged based on its relevance to the mission: will it bring us closer or further away from the goal? Thus, the administrators and funders of successful organizations facilitate innovation by dismantling barriers and producing environments conducive to creativity and productivity.\n\n\n\nA. The successful institutions or temporary endeavors such as the Manhattan or NASA’s Apollo projects are strictly mission-driven: all players (i.e., funders, administrators and scientists) are awarded based on achieving defined organizational goals. This leads to synergy between players, creating an environment that seeks and values creativity, diversity, cooperation, and shared responsibility.\n\nB. In most current, publicly funded biomedical research institutions, the mission exerts only a minor influence on daily decisions. Goals and award structures are largely separate for different actors. This creates inefficiencies, administrative burdens, and wastes resources.\n\nFor example, at the inception of the Apollo project, the paucity of skilled aeronautic engineers was solved by the administrators flying to Canada, recruiting Canadian engineers and facilitating their integration to NASA teams.13 There was a concerted effort to foster collaboration within the endeavor, challenging the current belief that internal competition generates efficient organizations.14–18 Analogously, funders of productive organizations either directly lead the effort or facilitate and monitor accomplishment of the mission, consistent with their (often financial) interest in the outcome.\n\nIn stark contrast, while the current publicly funded biomedical research institutions defined the broad aims (to improve health), there are few concrete steps or timelines, and these exert only minor influence on daily decisions by researchers, administrators, and funders. Although researchers need freedom to pursue novel hypotheses, as these often lead to important discoveries, the award structures must unite the various actors within the system behind the organizational mission. Disturbingly, this is currently not true, as the goals and awards are often completely disconnected from the mission (Figure 1B).\n\nFor example, the NIH intramural research program has close to 100 different electronic systems. Most fulfill necessary administrative or regulatory needs, but generally disregard the burdens their mandated use imposes on researchers. Because these systems largely do not communicate with each other, researchers must repeatedly input identical data in different formats and at different time-intervals mandated by each electronic system. Thus, medical providers and researchers with advanced degrees perform repetitive tasks that can be completed much faster and without errors by the computers. Even worse, the lack of harmonization creates an almost comical level of complexity, where the researchers must “remember” that the same event, for example a side effect of a medication, is considered “serious” by one regulatory system and must be imputed immediately, “expected” by another system and should be imputed as aggregate yearly data, and “serious but expected” by the third system. These arbitrary complexities cause mistakes, which must be found and reported, creating a vicious cycle of useless paperwork.\n\nOne reason for such inefficiencies is the fact that administrators are judged not on the output of the scientists in their division, much less the reproducibility of the work coming out of their labs, but rather on the administrator’s skill in accomplishing administrative tasks and, often, their ability to raise money for the institution. No administrator is punished for being unwilling to remove an impediment to efficient scientific production, such as the NIH’s byzantine electronic systems. On the contrary, the disincentives of potential failure and the effort needed for transformative leadership eventually stifles even the most mission-driven administrator.\n\n\nExamining incentive structure for scientists\n\nSimilarly, the research teams, the “engines” discovering new knowledge, also operate within a reward structure which is not conducive to producing the most relevant and reproducible research possible.\n\nResearch teams typically consist of three to 30 people led by a single principal investigator (PI). Up to 80% of the team’s workforce are trainees. Their inevitable turnover impedes the continuity of long-term projects, such as clinical trials. The peak of a career trajectory for an academic researcher is to attain a (scarce) tenured PI position with an independent laboratory and budget. The currency of their realm is publications: how many articles are published; the prestige of the journals in which the articles are published; and the fame these articles generate.\n\nAstonishingly, research reproducibility and its value to health are almost never considered among hiring, promotion and tenure criteria.19 There are few penalties for scientists who routinely publish irreproducible results except in cases of outright fraud or research misconduct. This gross, de-facto misalignment of the researcher’s incentives causes the irreproducibility crisis.\n\n\nIncremental changes versus system re-design?\n\nNumerous authors and organizations have suggested or implemented incremental enhancements of research enterprise. Partially successful fixes, such as pre-print services, and journals that embrace open reviews and/or implement publication checklists that provide transparency in disclosing methodological details, are steps in the right direction, but thus far have failed to make a significant dent in irreproducibility. These incremental solutions often cannot become permanent in the face of existing incentives, as was the case of the PubMed Commons post-publication open peer review,20 which was discontinued because its use offered no advantage to scientists’ careers.\n\nChange is difficult. Even people deeply dissatisfied with the status quo fear its disruptive consequences. Furthermore, redesigning the publicly funded biomedical research enterprise is a daunting task, as no single entity oversees research in its entirety. Nevertheless, NIH is the leading force in the US, and as demonstrated in the coronavirus disease 2019 (COVID-19) pandemic, is best positioned to establish fruitful collaboration among funding agencies, academic institutions, and private entities to lead transformative initiatives.\n\n\nReforming the scientific enterprise into a self-regulating system\n\nA mission-driven approach to scientific reform includes the following desired outcomes:\n\n1. Incentivize reproducible, impactful research that is efficiently performed and rapidly disseminated.\n\n2. Turn scientists into collaborators by rewarding sharing of resources, dynamic data re-analyses, and transparent criticism and correction of the methods, results, and interpretations.\n\n3. Re-envision scientific teams by utilizing individuals’ strengths, by fairly and transparently distributing benefits from scientific accomplishments to team members, and by facilitating dynamic collaborations.\n\nTo attain these outcomes, the greatest opportunity is leveraging technological advances that can integrate the existing system of cataloguing scientific publications (e.g. PubMed) with objective, data-driven assessment of scientific papers’ technical quality, generalizability, and resulting societal value. We call such a dynamic data integration the “biomedical research network” (BRN, Figure 2). This free, transparently curated resource would transform the way in which scientists (and their institutions) are judged and rewarded, by their funders and the public, thereby affecting all outcomes listed above.\n\nA. The BRN represents a massive multi-dimensional network of all biomedical science studies. Electronic navigation allows to zoom in a specific branch of scientific investigation, exemplified by IL17 subnetwork (B). The IL17 branch is initiated by the discovery/sequencing of the IL17 gene in 1993. There are many different new branches of investigation related to IL17, but the branch that links IL17 to autoimmunity is the most successful. Although IL17 has been associated with many different animal models of autoimmunity and with more human diseases, only three subdivisions are highlighted due to space constraints: IL17 and ankylosing spondylitis (AS), IL17 and psoriasis, and IL17 and multiple sclerosis (MS). Of these, the IL17 and AS, and IL17 and psoriasis branches are highly successful, with proven therapeutic efficacy of many direct and indirect IL17 inhibitors. Due to this commercial and public health impact, the publications forming branches highlighted in green color accumulate positive Societal Impact (SI) scores, whereas the IL17 and MS branch thus far had no commercial success; therefore, its publications do not partake in positive SI scores allocation. The size of the circles of the defining papers (network “hubs” exemplified by publication of the discovery of IL17, 1st publication that associated IL17 with autoimmunity, 1st publication that associated IL17 with human psoriasis etc.) is proportional to the assigned SI scores: discovery of IL17 has the highest Scientific Impact subscore; whereas positive clinical trials of IL17-blocking agents in AS and psoriasis have a higher Commercial impact subscore of SI scores. The Studies #1-3 are described in detail in the text of the article, where the studies #2-3 attempted to reproduce findings of the high-impact Study#1 but failed. The font color in the Integrated knowledge diagram is proportional to the MR scores of the three publications.\n\n\nBRN and its metrics\n\nWe envision three ways to measure contributions to biomedical research objectively: a methodological rigor (MR) score; a reproducibility (R) score; and a societal impact (SI) score. Every publication would be assessed by each of these metrics.\n\nThe first metric, the MR score, is the most critical and the easiest to implement. It would be based on established attributes of reproducible research, such as blinding, power calculation, randomization, use of controls, validation of results in an independent cohort, availability of raw data and analysis codes.4,21,22 Though the quickest way forward is to assign an MR score to a paper based on a self-reported checklist by the author(s), this may generate bias and cannot be applied to already-published papers. Thus, at the BRN’s inception, reviewers would need to validate the self-reported MR score against the Methodology section of the paper. The latter can be accomplished through an open review system, associated e.g., with free/public pre-print service such as medRxiv. MedRxiv is run by a consortium of two academic institutions and the BMJ, and funded by a private foundation, the Chan Zuckerberg Initiative. MedRxiv has proven to be an invaluable aggregator of science associated with the COVID-19 pandemic. Reviewers’ curated MR scores would serve as the dataset for the development, optimization, and validation of an automated, word-recognition based algorithm, which assigns MR score by scanning the Methods section of scientific papers.\n\nScoring of all publications can produce an MR score for individual scientists, averaged across their publications. This would incentivize all co-authors to support methodological rigor for every experiment, effectively self-regulating high-quality experimental design. The MR score would also help non-experts to judge the technical rigor of the presented work.\n\nScientists may have two objections: one, the retrospective grading may not accurately reflect the quality of the original experimental design, because in the past, journals restricted word count for the Methods section. This is remediated by standardizing an individual paper’s MR score to the yearly averages, a solution used in standardized testing. Two, the authors may disagree with an assigned score. True mistakes, if validated in an open review, would improve the automated algorithm, possibly through public competition(s) similar to the Netflix prize.\n\nThe evolution of MR scores on a population level would objectively measure the success of the BRN strategy to combat irreproducibility. Improvements in a scientist’s standardized MR scores could represent professional growth. Additional training initiatives intended to improve methodological deficiencies and currently imposed on all scientists, could be more effectively targeted to the scientists with consistently low MR scores.\n\nUnless we demand pre-registration of all experimental designs akin to pre-registration of clinical trials in www.clinicaltrials.gov, the MR score will depend on what scientists choose to disclose. Some fear that pre-registration of all experiments will increase administrative burden23 and stifle innovation. Others believe that a lack of preregistration substitutes scientific predictions by less credible “postdictions”,24 exacerbating irreproducibility. A solution that integrates both views is to objectively assess reproducibility of results, or in broad understanding, the “scientific truth”25 of each study, post-publication.\n\nUsing language recognition, the BRN would assemble all related publications to a sub-network (Figure 2B), where the publication describing the crucial discovery that spurred a subsequent line of investigation would represent the main hub located at the center of the subnetwork. For example, the discovery of cytokine IL17 would sit at the hub of a subnetwork, and subsequent papers describing its role in autoimmune diseases would be one of the most successful “branches” of this IL17 subnetwork. Each new, related publication would be placed in the periphery of such a subnetwork, while minimizing the network “distance” between closely related research papers.\n\nTo illustrate how the BRN associates related articles and uses their MR score to assign a Reproducibility, or R score, consider three related articles that investigate the role of IL17F in predicting therapeutic response to the drug interferon beta (IFNb) for treating multiple sclerosis (MS). Study #126 was published in the high impact journal, Nature Medicine. By using only 26 MS patients and not employing methods to prevent bias, this study expands observations in mouse models to humans, concluding that high blood levels of IL17F predict poor response to IFNb. Two validation studies (Studies #227 and #328) that collectively (and blindly) analyzed samples from 357 randomly selected MS patients demonstrated unequivocally that serum levels of IL17F do not predict IFNb response in MS. In contrast to PubMed, which does not automatically associate related studies, the BRN algorithm places the validation studies in proximity of the Study #1 (i.e., on the same branch). Additionally, the BRN automatically integrates knowledge (Figure 2B) from related studies: because Studies #2 and #3 have high MR scores and validate each other, both receive high R scores. In contrast, Study #1 receives a low R score because it has a low MR score and was, in fact, not reproducible. To compute the R score, the algorithm compares the results of the two congruent studies “weighted” by their MR scores against the MR score of the conflicting study, to assign the probability that the specific result is true: the probability that serum IL17F levels predict therapeutic response to IFNb based on this integrated knowledge is very low – let’s say 0.1%; therefore, the Study#1 receives an R score of 0.001, whereas Studies#2 and #3 receive R scores of 0.999.\n\nThe goal is to employ technology to curate knowledge, while rewarding scientists for greater rigor. Because experimental rigor positively correlates with reproducibility,21 receiving consistently low R scores for consistently high MR scores will question accuracy of self-reported experimental designs. Such discrepancies would become exceedingly rare, as the mere understanding that objective curation will identify contradictions would naturally limit any temptation to cheat.\n\nThe most obvious objection against implementing the R score is its potential inaccuracy. Like every machine-learning algorithm, generated R scores will not be 100% accurate at its first iteration, but access to large, curated datasets (provided by author’s contestation and human reviewers’ adjudication of R scores), will allow data-driven optimizations. Ultimately, this will generate algorithm(s) that consistently outperform humans.29\n\nInnovation and relevance of scientific discoveries to health are essential attributes of the NIH mission. Indeed, science can be well-designed, reproducible, but still irrelevant.11 To assure that biomedical science is innovative and generates societal value, we must learn to quantify its worth objectively.\n\nThe impact of a discovery on science itself can be measured within the BRN as a growth of a new branch of scientific explorations. For example, basic scientific discoveries, such as the reprogramming of somatic cells to induced pluripotent stem cells (iPS;30) or CRISPR-CAS9 genetic editing both established new branches of science.31 Thus, their scientific impact is enormous. To assess the public health and commercial impacts of scientific discoveries, the BRN must link to databases of patents, newly approved drugs, medical products, and new technologies. This will trace the origin of societal and public health advances back to individual scientists in order to develop the integrated SI scores. The goal of the SI score is to objectively measure the impact of individual discoveries and cumulative impact of individual scientists or even scientific institutions, which can be used to reward institutional leaders. Current attempts to assign value to a publication or a researcher, such as NIH’s Relative Citation Ratio are insufficient, because they rely on the number of accrued citations. In our concrete example, the irreproducible publication in the prestigious journal is easily found in internet searches and continues to accrue citations years after its findings were unequivocally refuted: Study #126 accrued (as of May 2021) 590 citations, while the two congruent validation studies, published in less prestigious journals, accrued only 114 citations in total. This example is hardly an exception: a major effort to reproduce important scientific discoveries found non-reproduced articles published in higher impact journals and were accruing more citations than reproducible articles.3\n\nUndoubtedly, the SI score is the most difficult metric to derive. The challenge is to assign the accurate proportion of any given publication’s SI score to individual discoveries, as no discovery is made in isolation (exemplified in CRISPR technology, see the Broad Institute’s CRISPR Timeline). In other words, as a pie grows (i.e., measured by the growth of subsequent publications and their societal impact), the number of scientists credited for the share of a pie also expands proportionally to the calculated impact of their discoveries. The SI score will likely receive the greatest push-back, as scientists have limited influence over commercialization of their discoveries and comparing societal impact of discoveries in different scientific fields may seem unsurmountable. But should we shrink from the enormity of this task and accept that the opinion of a few “experts” who nominate and select scientists for prestigious awards, is the best we can ever do?\n\n\nLinking the BRN with the reform of the scientific publication system\n\nThe overarching goal of the BRN is to align research incentives to promote scientific breakthroughs with meaningful societal impacts. Current emphasis on publications, detached from their scientific rigor, reproducibility, and societal impact has been detrimental: thousands of scientists publish a paper every five days,32 most of which are unread and never cited (see33 and the NIH’s iCite database). No human being can keep up with this exponential growth of publications, even in his/her research field. Without transparent algorithms that curate this publication excess, work may be unnecessarily duplicative and findings with potential societal value may be difficult to find in search engines, slowing medical progress.\n\nAdditionally, publications must become the means, while transformative advances in basic sciences and medical cures are the ends. Therefore, although the BRN can be developed in parallel with the existing publication and dissemination system, reforming that system offers major advantages. First, the current unacceptably long and pointlessly expensive publication process stifles innovation. The public may be unaware that most scientific articles take years to publish, with research teams reformatting rejected papers to conform to requirements of different journals and paying thousands of USD per publication, even if these are no longer printed and only published electronically. This causes frustration, and wastes resources that should be dedicated to new discoveries. Making the BRN a universal, immediate and free dissemination system for biomedical sciences (e.g., by merging PubMed with current pre-print services such as bioRxiv and medRxiv) would avoid wasted time, labor and money.\n\nSecond, a single-blinded review system is morally indefensible. It empowers reviewers to determine the fate of the publication without accepting public responsibility (or reward) for the review. This system institutionalizes bias by limiting scientific contributions from women and minorities, and has shown itself to be incapable of weeding out irreproducible science.3\n\nOpen peer review is often disparaged on the assumption that few scientists would choose to participate, fearing retaliation for a critical review. If this is true, the scientific culture is broken. Engaging in discourse on validity and interpretation of scientific discoveries is a precondition to scientific progress. Providing constructive criticism of presented work and identifying ways for its improvement, while respecting the dignity of the scientists who produced it, underlies scientific maturity. This is very different from what can occur now, the writing of anonymous reviews containing publicly indefensible arguments and refusing to take personal responsibility for their accuracy.\n\nThe validity of science is the responsibility of all scientists. The irreproducibility crisis reflects on all of us, whether we adhere to sound or poor experimental design. We are the ones who form and embrace scientific culture and the only ones who can reform it.\n\nIf we want to achieve sustainable open peer review,34 we must reward scientists for their willingness to provide critical, creative thinking. Let’s return to the example described in the legend of Figure 2 and consider three hypothetical scenarios: in scenario #1, Study #1 is submitted to the BRN and a reviewer alerts the authors to the methodological flaw through open review. The authors acknowledge their mistake and amend or retract the problematic experiment in their paper. Because there were no negative societal consequences, the authors’ MR score would go down slightly for submitting a flawed experiment, and the reviewer would accrue positive RI score for identifying the paper’s shortcoming and triggering a correction.\n\nIn scenario #2, the authors submit a flawed experiment but choose to ignore the raised criticism. With the critical review in the public domain, an economical, well-designed (i.e., of high MR score) independent validation study is performed and fails to reproduce original finding. Now, the authors accrue not only a low MR score but also a low R score and negative SI score, equivalent to the value of wasted resources.\n\nFinally, in scenario #3, which in many ways reflects the status quo, all reviewers fear the consequences of posting a negative review of the experiment coming from an influential scientist. The reported findings appear to be so important that a pharma company decides to perform a new clinical trial based on the published data. After considerable expenditures, the trial unequivocally demonstrates the irreproducibility of the original publication. The authors would accumulate not only low MR and R scores, but also highly negative SI score due to accumulated costs of wasted resources. Everybody loses. Through transparent, responsible review we can correct mistakes while they are still small, help each other grow as scientists and maximize the value of our discoveries.\n\nScientists should freely publish their experiments, and any reader should be able to transparently (and succinctly) critique them, accepting full responsibility for the review. Reviewers with habitually frivolous criticism not supported by data or scientific understanding would themselves accrue low cumulative RI scores, prompting them to change this behavior. By contrast, critiques from scientists with high cumulative RI scores would impel the authors to carefully consider the reviewers’ arguments and perform additional experiments to ensure the validity of findings.\n\nThe BRN would automatically limit irreproducible science if career rewards depended on high MR, R, and SI metrics. If performing bad science were detrimental to one’s career, why would anybody conduct much less try to publish flawed experiments? And, if scientists read papers anyway (which they must do) and rating them were a simple task that promoted their careers, why would they not review?\n\nFinally, an open review process would level the playing field of scientific education by allowing all trainees and even journalists to learn critical thinking from the greatest minds. Everybody wins.\n\nWe also wholeheartedly embrace the concept of dynamic publications.35 Publications should be transparently modified (with the modification history recorded in the BRN) based on new experiments or evolved knowledge. Such modifications may enhance clarity, adjust conclusions, and add, improve, or retract experiments in response to open review. Furthermore, scientists who were not original authors should be able to attach results of subsequent experiments to an existing publication, rather than publishing independent papers. For example, an investigator publishes an intriguing finding that lacks an independent validation cohort. Another scientist has such a cohort and validates the published findings. Rather than submitting this as a new publication (and thus needlessly duplicating information already contained in the introduction and discussion of the original publication), this independent lab could simply submit the results of the validation experiment and its related methods to the BRN, and link it to the previous paper. If the results subsequently translate into societal value, all contributing scientists will share the benefit proportional to their contribution. This way, many scientists can efficiently strengthen or refute existing publications, solving the current problem of (not) publishing negative data36 and replication studies, which are essential for assessing the generalizability of discoveries.37,38\n\nRelated to the publication process is the issue of depositing full datasets into the public domain, not only to verify published results but also for the datasets to be reused to test new hypotheses. The societal benefit of sharing raw data is widely recognized39,40 but practically left at the discretion of the scientists. For example, the NIH encourages data sharing, and many journals request that the authors include a statement on data availability, such as: “Anonymized data not published within this article will be made available by request from any qualified investigator.” Such requests are not always granted and the declarations in the publication cannot be enforced.41\n\nThe reason for the lack of data sharing, especially from well-designed clinical studies, is that these datasets remain valuable; they allow scientists to ask new questions and publish new papers—the same value society loses if the data are not shared. Because different researchers ask different questions, some essential analyses may never be performed. Acknowledging truth on both sides, the solution is to incentivize data-sharing by assigning SI score to the investigators who generated valuable datasets whenever their reuse led to new societal benefits. If one team published raw data after spending 10 years generating an extremely well-characterized longitudinal cohort, and a second team re-analyzes these data and gains valuable insights, then both teams should share the resulting SI score for such invention, proportional to their efforts and intellectual contributions. Everybody wins, especially patients.\n\nWould the BRN destroy the current publication industry? Not necessarily. First, the BRN may be based on public-private partnership and employ current publication industry experts. Second, the BRN would provide new job opportunities, such as science writers analyzing aggregate knowledge to identify new trends and knowledge gaps, or to promote commercialization of discoveries by guiding investment funds. These would allow the current scientific publication workforce to directly advance science and health.\n\n\nRe-envisioning scientific teams by mapping individuals’ strengths, by fairly and transparently distributing benefits from scientific accomplishments and by facilitating dynamic collaborations\n\nSuccessful research requires a diverse workforce with complementary skills, as again highlighted by the Manhattan and Apollo projects. When people do their job expertly, they become invaluable.\n\nCurrent co-authorship criteria on scientific publications favor intellectual over manual or technical contributions. But what prevents us from fairly crediting the work of all team-members? The current acknowledgement style is needlessly vague: e.g., writing that persons #1-3 contributed to data analysis does not give justice to the reality if person #1 performed 90%, person #2, 9% and person #3, 1% of the work. The BRN could measure scientific contributions comprehensively, providing person-specific aggregate data, essential for junior scientists and support staff. This level of transparency and granularity, if linked to reward structures (recruitment, promotion, salary) would inspire the scientific workforce to assume more responsibilities, enhance their skillset and increase productivity. This productivity and cumulative societal value of institutional discoveries measured by the BRN metrics could re-align incentive structures of administrators and empower funders to make more impactful funding decisions.\n\n\nConclusion\n\nBiomedical data has become unmanageably vast, and much of it is not contributing to new knowledge or better health, which are the stated goals of the NIH and undoubtedly of many if not most biomedical researchers. We need advanced computer algorithms to help us make sense of all that data, as well as a new set of incentives to encourage all researchers to produce replicable results. Everything described in this article is technologically feasible. The COVID-19 pandemic demonstrated that medical innovation can be both fast and transformative, as long as leaders are willing to dismantle administrative barriers and foster an environment conducive to creativity and collaboration. Courageous leadership in science administration can propel us to a new era of accelerated biomedical advances, that will achieve the mission of the NIH: to improve health for everyone.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgments\n\nThe authors express gratitude to Drs. Gary Birnbaum and Alison Wichman for their thoughtful suggestions and Brian Brown, NIH Library Editing Service, for reviewing the manuscript. 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PubMed Abstract \| Publisher Full Text\n\nAxtell RC, et al.: T helper type 1 and 17 cells determine efficacy of interferon-beta in multiple sclerosis and experimental encephalomyelitis. Nat Med. 2010; 16(4): 406–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHartung HP, et al.: Interleukin 17F level and interferon beta response in patients with multiple sclerosis. JAMA Neurol. 2013; 70(8): 1017–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBushnell SE, et al.: Serum IL-17F does not predict poor response to IM IFNbeta-1a in relapsing-remitting MS. Neurology. 2012; 79(6): 531–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilver D, et al.: A general reinforcement learning algorithm that masters chess, shogi, and Go through self-play. Science. 2018; 362(6419): 1140–4. PubMed Abstract \| Publisher Full Text\n\nTakahashi K, et al.: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5): 861–72. PubMed Abstract \| Publisher Full Text\n\nScudellari M: How iPS cells changed the world. Nature. 2016; 534(7607): 310–2. PubMed Abstract \| Publisher Full Text\n\nIoannidis JPA, Klavans R, Boyack KW: Thousands of scientists publish a paper every five days. Nature. 2018; 561(7722): 167–9. PubMed Abstract \| Publisher Full Text\n\nFranca TFA, Monserrat JM: To Read More Papers, or to Read Papers Better? A Crucial Point for the Reproducibility Crisis. Bioessays. 2019; 41(1): e1800206. PubMed Abstract \| Publisher Full Text\n\nFlier JS: Irreproducibility of published bioscience research: Diagnosis, pathogenesis and therapy. Mol Metab. 2017; 6(1): 2–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIoannidis JP, Tatsioni A, Karassa FB: Published articles should not be dead and buried: introducing research updates. Eur J Clin Invest. 2010; 40(9): 767–9. PubMed Abstract \| Publisher Full Text\n\nIoannidis JP: Journals should publish all “null” results and should sparingly publish “positive” results. Cancer Epidemiol Biomarkers Prev. 2006; 15(1): 186. PubMed Abstract \| Publisher Full Text\n\nIoannidis JPA: Why replication has more scientific value than original discovery. Behav Brain Sci. 2018; 41: e137. PubMed Abstract \| Publisher Full Text\n\nIoannidis JP: Translational research may be most successful when it fails. Hastings Cent Rep. 2015; 45(2): 39–40. PubMed Abstract \| Publisher Full Text\n\nTaichman DB, et al.: Sharing Clinical Trial Data–A Proposal from the International Committee of Medical Journal Editors. N Engl J Med. 2016; 374(4): 384–6. PubMed Abstract \| Publisher Full Text\n\nMcCarthy M: Sharing clinical trial data should become the “expected norm,” US panel says. BMJ. 2015; 350: h240. PubMed Abstract \| Publisher Full Text\n\nMiyakawa T: No raw data, no science: another possible source of the reproducibility crisis. Mol Brain. 2020; 13(1): 24. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "90258", "date": "27 Aug 2021", "name": "Paul Glasziou", "expertise": [ "Reviewer Expertise Clinical Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and valuable article which looks at the current problems in the health and medical research systematic such as (lack of) reproducibility, accessibility, and relevance. Importantly, the paper considers the research enterprise as a system - of researchers, funders, publishers etc., with varying incentives and motivations - and examines potential solutions at this systems level. This is neatly illustrated by Figure 1 showing the poor alignment of the various elements and actors within the research system. There are the seeds of many good ideas within this paper. However considerable meta-research would be necessary before implementing the metrics and solutions proposed - for the detailed development and testing.\n\nI had a few suggestions to consider for changes:\nAlongside the Manhattan project an Apollo mission examples, given the current pandemic it would be useful to mention the CEPI vaccine collaboration - Coalition for Epidemic Preparedness Innovations (CEPI) which was launched at Davos 2017 and without which we would not have the vaccines we now have - CEPI\n\nThe proposed solutions rightly focus on aligning incentives with the need for impactful research requiring collaborations, and well-functioning teams. In \"COM-B\" terms (Capability + Opportunity + Motivation = Behaviour) the authors' focus is particularly on Motivation. However, it would be helpful to consider some of the Capability and Opportunity elements, such as skills, training, infrastructure, tools, access, etc.\n\nThe authors also proposes three new measures: methodological rigour score, a reproducibility score, and a societal impact score - again aimed at incentives/motivation. This is worthwhile, but a complex task and some suggestions on who and how the needed meta-research and experimental studies could be done would be helpful.\n\nThere are a number of important initiatives already underway which are aligned with the ideas presented here and it would be useful to set those out in a table, with examples such as:\n\nDORA: The Declaration on Research Assessment (DORA) which set out principles addressing the need to improve the ways in which researchers and the outputs of scholarly research are evaluated.\n\nThe Hong Kong principles: which provide a more detailed set of criteria for assessing researchers for promotion and tenure.\n\nThe EQUATOR Network centres - for Enhancing the QUAlity and Transparency Of health Research which develop and disseminate the reporting guidelines (CONSORT, PRISMA, etc).\n\nEVIR forum - a forum of over 40 research founders founded in 2017 to help health-related research funders increase the value of their research.\n\nThe evidence based research network - which aims to reduce waste in research by promoting: (a) No new studies without prior systematic review of existing evidence, (b) Efficient production, updating and dissemination of systematic reviews.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "101577", "date": "15 Dec 2021", "name": "Andrew Lee Hufton", "expertise": [ "Reviewer Expertise Genetics & computational biology", "scientific publishing", "data sharing", "open science" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review the Opinion Article by Drs. Bielekova and Brownlee, entitled \"The imperative to find the courage to redesign the biomedical research enterprise\".\nIn this piece, the authors argue that the incentive structure of modern biomedical research does not align with its supposed mission, and propose a series of changes that could reform this incentive structure in a way that they believe will \"maximize our investments in biomedical research and optimize its value to human health\".\nThe authors build their argument on the now quite mature literature documenting the so-called \"reproducibility crisis\". I tend to agree with the authors that this is a serious and urgent issue, and indeed many of the reforms proposed by the authors strike me as sensible. Despite this, however, I find a number of elements of the authors' argument hard to follow or poorly supported.\nThe authors begin by using the NIH as their dominant institutional example, and compare it to the Apollo Mission and the Manhattan Project, which they present as two enterprises with clearer, mission-driven management. I find this comparison, at minimum, unhelpful. First, let's be clear that the mission of one of these projects was the production of an instrument of mass death, which makes for a poor comparison to the NIH. That aside, both were much more defined projects with clear end-points, making any comparison to the NIH hard for me to really engage with. I think, in any analysis, the NIH and the research it has funded would be found to have had a far more positive impact on humanity than either of these projects.\n\nI would prefer to see a more informed comparison between the NIH and other organizations of similar scale or purpose. Can the authors support their argument that the NIH's system of organization is non-optimal by providing more relevant examples of organizations that have been able to more efficiently advance human health research?\nThe authors then propose a framework of radical changes to the way that scientific publications are published, assessed, and to how they are used to evaluate research programs and researchers. Before I address the authors' specific proposal, I would like to note that at this point I have lost the connection to the authors' original argument that the NIH's organizational system should be more mission-driven. The changes being proposed here reach far beyond the NIH's scope and its leverage to implement.\nCentral to the authors' proposal is a network based, multi-criteria method for assessing the value of scientific publications, which they call the BRN for \"biomedical research network\". I like some of the ideas embedded in this concept, but as a whole I remain far from convinced that this is technically feasible or that the ratings of a such system would better align with the NIH's mission. I appreciate that this is an opinion paper, but the BRN proposal being put forward is relatively specific, and I think should be supported with some evidence of feasibility for a scholarly work of this kind.\nI question whether reviewers could reliably quantify \"methodological rigor\", in a manner that would be meaningful across topics and disciplines. I am also not convinced that an automated system could identify related publications with sufficient semantic rigor to allow any calculations of reproducibility, in the manner proposed by the authors. Similarly, how would one rate social or commercial impact objectively in the short term, when these kinds of impacts often take years or even decades to mature?\nThese concerns are not trivial in my mind, since we have already seen the harm that poorly designed metrics have done to the scientific enterprise. I share the authors' concern about the reproducibility crisis, but I think there are very legitimate concerns in the scientific community that a rush to create new reproducibility metrics, could end up causing further harm, for example, by de-incentivizing cutting edge or interdisciplinary research where standards are still evolving and sparser research networks means that reproducibility is harder to assess.\nIn the rest of the paper, the authors make further recommendations, endorsing rating reviewers, open peer-review, dynamic publications, and data sharing, among other ideas. These are sensible recommendations, although I feel the authors do not manage to relate all of them directly back to their idea that biomedical science organizations should be more mission-driven.\nOverall, I am sympathetic to many of the authors' ideas and the overall sense of urgency that pervades the piece, but I am not convinced at this stage that the authors have made a coherent argument for how biomedical research could be reformed.\nMy advice, were the authors to consider developing this further, would be to think more critically about how the NIH or a similar organization could specifically enact change, using the tools and leverage available to it, and expounding more on how such an organization could concretely use mission-driven assessments to improve its outputs. As a reader, I personally would like to see this supported with more relevant organizational management comparisons, and some consideration of the costs of the proposed reforms relative to current organizational systems.\n\nThe BRN itself, while interesting, feels too underdeveloped at this stage. For such a proposal, I would like to see at least some evidence of feasibility, including numerical rankings and networking-building with actual papers.\nFinally, I would like to note that this paper at present is generally well written, after setting aside my more conceptual criticisms above.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No", "responses": [] } ]	1	https://f1000research.com/articles/10-641
https://f1000research.com/articles/10-634/v1	22 Jul 21	{ "type": "Research Article", "title": "Prevalence and predictors of multimorbidity among immigrant Asian Indian women residing in Sydney Australia: A cross-sectional study", "authors": [ "Cathy O'Callaghan", "Uday Yadav", "Sudha Natarajan", "Saroja Srinivasan", "Ritin Fernandez", "Uday Yadav", "Sudha Natarajan", "Saroja Srinivasan", "Ritin Fernandez" ], "abstract": "Abstract Background: There has been a rise in multimorbidity as people age and technology advances which is challenging for health systems. Multimorbidity prevalence varies globally due to various biological and social risk factors which can be accentuated or mitigated for populations in migration. This study investigated the prevalence and predictors of multimorbidity amongst a group of migrant Asian Indian women living in Australia. Methods: A cross-sectional descriptive study design using convenience sampling investigated the multimorbidity risk factors among first generation migrant Asian Indian women in Australia. This study was part of a larger study titled “Measuring Acculturation and Psychological Health of Senior Indian Women Living in Australia” that was conducted in Sydney, Australia. Data were collected using validated instruments as well as investigator developed questions. Women completed questionnaire surveys either by themselves or through the assistance of bilingual coordinators as English was not their first language. Results: 26% of the participants had one chronic condition and 74% had multimorbidities. The prevalence of individual conditions included cardiovascular disease 67.0%, osteoarthritis 57.6%, depression 37.4%, diabetes 31.5%, chronic respiratory conditions 10.8%, cancer 4.9% and nephrological problems 1.47%. In the unadjusted model, factors such as increasing age, education level, employment status, living arrangements, low physical activity, and elements of acculturative stress were significantly associated with multimorbidity. Multi-variable analysis identified the acculturative stress factor of threat to ethnic identity as a predictor of multimorbidity. Conclusion: Identifying the key determinants of multimorbidity in older adults from a migrant community with pre-existing risk factors can assist with the development of culturally appropriate strategies to identify people at risk of health conditions and to mitigate the health effects of acculturative stress.", "keywords": [ "Acculturation", "Asian Indian", "immigrant", "multimorbidity", "women" ], "content": "Introduction\n\nAdvances in technology and increased awareness and treatment of diseases has enhanced the longevity of the world population, in particular women.1 Currently 70% of those aged 90 or older and 60 % of those aged 70 or older are women.2 Living longer is associated with an increased susceptibility to multimorbidity - the simultaneous occurrence of two or more chronic diseases.3 Global estimates of multimorbidity for people aged 65 years and older range from 12.9% to 95.1%,4–6 with 51% multimorbidity in the Australian population.7 The large prevalence variance indicates methodological differences in measurement globally.\n\nNumerous studies have demonstrated that the prevalence of multimorbidity is higher in women compared to men.4,7–11 This difference might be related to biological, sociocultural, environmental, or economic factors. In particular, elderly women of lower socio-economic status have been reported to have the highest risk for multimorbidity.4 Research has seen a protective effect of estrogen on the development of certain chronic conditions such as heart disease, however the hormonal change post-menopause removes this protective effect and the risk increases significantly.12 Nevertheless, as these factors vary globally, their associations with multimorbidity may differ across populations.\n\nMultimorbidity has a significant impact on patients, their families, the health care system and society in general.13 The evidence suggests a higher risk of mortality among those with multimorbidity.14,15 Similarly, the presence of multimorbidity increases the risk of hospitalizations,16–18 and causes a decrease in physical functioning19 and overall quality of life.7,20–22 In addition, multimorbidity increases polypharmacy23–25 and psychological distress25–28 resulting in substantial economic burden for health systems.14 Among those who are employed, multimorbidity is also associated with lower work productivity7 including a higher number of sick leave days and early retirement.27,29 For those unemployed, the odds of obtaining employment are lowered. All these factors place an immense burden on society.\n\nThe magnitude of multimorbidity and the rise of noncommunicative diseases is rapidly increasing in developing countries. A systematic review of multimorbidity research in South Asia demonstrates that prevalence levels are up to 83%.28 The number of chronic conditions reported for people in South Asia ranges from 7 to 22,28 with the most common chronic conditions being hypertension, arthritis, diabetes, cardiac problems and skin diseases.28 Fewer patients have reported the presence of stroke, cancer, renal disease, and depression.30\n\nPopulations of South Asian background have been identified of having increased risk of cardiovascular disease and other chronic conditions,31,32 which could contribute to multimorbidity. The determinants of chronic diseases include a range of biological and societal factors which can persist when people from South Asia migrate overseas.33,34 In the United States, up to 32.4 % of Asian Indians had a combination of high cholesterol and hypertension which are risk factors for diabetes and coronary heart disease.35 In a study undertaken among migrant Asian Indian women in Australia, over a third experienced obesity, hypertension and diabetes which was higher than the national average of hypertension incidence.36\n\nThere is growing research about the effects of migration and acculturative stress on migrant populations, which can also have significant effect on those with pre-existing chronic health conditions. Acculturation is the process when individuals and groups from different cultures meet and adopt the attitudes, beliefs and behaviours of another culture.37 This process can have positive or negative effects on the psychological and physical health of migrants.38 Theories of the effect of acculturation on health have moved from one dimensional understandings to more sophisticated multidimensional models that outline the effects of various degrees of acquiring host culture and/or retaining heritage culture, and the development of acculturative stress associated with some of these variations.39 There have been reports that the longer that migrants from low-middle income countries live in high income countries and adopt unhealthy behaviours, the more weight they can gain, but there are numerous factors affecting these outcomes including the negative effect of acculturative stress.37 Migrant older women are seen to be more vulnerable to experiencing cultural change or acculturative stress when adjusting to a new society, as they can face more social isolation, changes in socioeconomic status and language barriers.36,40\n\nThere have been limited studies which have examined the effect of acculturative stress on chronic health conditions in Indian migrant communities. Indian migrants who settle in Australia may face numerous difficulties as they settle in a new culture which is vastly different from their own.41 Acculturative stress and depression has been linked to having a negative effect on cardiovascular health and other chronic conditions with Asian Indian migrants in the United Kingdom.33\n\nIn Australia, the Asian Indian born migrant population is the fastest growing minority group, and at the time of this study, there were an estimated 400,000 Indian born migrants living in Australia.42 However, there is limited information on multimorbidity prevalence particularly among South Asian women residing in Australia. Therefore, this study aimed to investigate the prevalence and predictors of multimorbidity among migrant Asian Indian women in Australia. Identifying the key determinants of multimorbidity can assist in the development of culturally appropriate strategies to prevent and manage chronic morbidities.\n\n\nMethods\n\nA cross-sectional descriptive design using convenience sampling was conducted among migrant Asian Indian women in Australia. Ethical approval was obtained from the Local health district Human Research Ethics Committee. This study was part of a larger study titled “Measuring Acculturation and Psychological Health of Senior Indian Women Living in Australia” that was conducted in a metropolitan Local Health District (LHD) in Sydney, Australia during 2013. Non-probability sampling was used to recruit Asian Indian women who were members of the Resourceful Australian and Indian Network (RAIN). RAIN is a community organisation that was established in 2006 to provide support to migrants originally from the Indian subcontinent and at the time had a membership of 300.\n\nA detailed description of the recruitment strategy has been presented elsewhere.43 In brief, women were included in the study if they are aged between 50-100 years, spoke English, Hindi, Punjabi, Marathi, Gujarati and/or Tamil and were willing to complete a questionnaire. Women were informed about the project through an announcement being made and a flyer being distributed at a RAIN meeting. Women who expressed an interest were given a subject information sheet and asked if they preferred to self-administer a questionnaire survey in English or required the assistance of one of the bilingual coordinators who spoke their language and could assist them to undertake the surveys (see questionnaire https://doi.org/10.5061/dryad.3r2280gfz).\n\nThe self- reported questionnaire was developed following intensive discussions with RAIN staff and multicultural health staff of the LHD along with a comprehensive literature review. The questionnaire consisted of items relating to demographic details (age, gender, country of birth, level of education, living arrangements, length of stay in Australia), smoking status, anthropometrics (height, weight, and waist circumference measured at the RAIN centre), and participation in physical activity (assessed using the Active Australia Survey (AAS)44 with correlation coefficients: 0.71 to 0.86; and Spearman’s Rho: 0.54 to 0.7745). Acculturative stress was measured using the Multi-dimensional Acculturative Stress Scale (MASS)46 which consisted of 24 items in five subscales. These sub-scales measured discrimination, threat to ethnic identity, lack of opportunities for occupational/financial mobility, homesickness, and language barriers. The items were rated on a four-point scale with 1 = disagree, 2 = to some extent disagree, 3 = to some extent agree, 4 = agree. Lower scores indicated a lower level of acculturative stress and higher scores showed higher levels of acculturative stress. The 24-items MASS had a coefficient alpha of .89 and the discrimination, threat to ethnic identity, lack of opportunities for occupational and financial mobility, homesickness and language barriers subscales had alphas of .82, .77, .87, .56, and .64, respectively. Depression was assessed using the PHQ 12 questionnaire developed for Asian Indians47 which had an internal consistency scale of 0.88.\n\nThe presence of multimorbidity was measured through questions about prevalence of non-communicable chronic conditions (diabetes, high blood pressure, high cholesterol, cardiovascular disease, chronic respiratory conditions, osteoarthritis, depression, nephrological problems and cancer). Participants gave self-reported information which was verified through the assistance of the bilingual coordinators and through survey questions asking if they were taking any medications for the treatment of the same. Table 1 presents the definitions of the individual chronic conditions used in this study.\n\n\n\n• Self-report of “heart problems” or taking medication for ‘heart condition (eg. Digoxin)’\n\n• Self-report of ‘high blood pressure' or taking medications ‘to control blood pressure’\n\n• Self-report of ‘high blood cholesterol’ or taking medications to ‘control blood cholesterol’.\n\n• Self-report of ‘stroke/Cerebro-vascular accident’ or ‘taking medications to prevent blood clots (eg. Aspirin)’\n\nAll analyses were completed using SPSS Version 25. Descriptive analyses (frequencies, percentages, means and standard deviations) were undertaken to assess the characteristics of the participants and associated outcomes. Multimorbidity was defined as the presence of more than one of the seven conditions in the same individual. Multimorbidity was then dichotomized as present or absent for assessing its relationship with the independent variables. Each of the individual conditions were coded as a binary response (0 = absent, 1 = present). The cumulative effect of the seven conditions (range 0 = absence of all seven conditions to 7 = all seven conditions present) were further dichotomized into presence or absence of multimorbidity, where multimorbidity was defined as the presence of two or more conditions (cumulative score of ≥2).\n\nWe assessed multicollinearity of covariates using Variance Inflation Factors (VIFs). The VIFs for all covariates that were included in the logistic regression analysis were less than 2.0. Associations between the covariates and multimorbidity were checked using a Chi-square test. All the co-variates with p-value ≤ 0.05 with the dependent variables in univariate analysis were considered in the multivariable analysis.\n\nEthical approval was obtained from the Local health district Human Research Ethics Committee (reference no HREC 12/282).\n\nConsent for publication: Written informed consent to publish aggregate details of participants was obtained from the participants as part of the consent process.\n\n\nResults\n\nWe included 203 immigrant women in the study, the majority of which were born in India, while a third came from other countries in the Indian diaspora (Kenya, South Africa, Fiji, East Africa, Malaysia, Pakistan, Sri Lanka). The mean age of the study participants was 66.11 (±9.59) years, with 33% in the 50-60 and 61-70 years age groups, and 34% in the 70 + years age group. Most ascribed to the Hindu religion. Around half of the women reported to be married or living with their partner (56.7%) at the time of the survey. Of the total, only 24.1% recorded themselves as employed. In terms of education, 18.7% had received primary education, 39.4% secondary and 41.9% had gained a higher education. The length of stay in Australia ranged from < 1month to 42 years (mean 18 years). Of the surveyed participants, 28.1% had a history of alcohol consumption, 1% were smokers and 37.4% were physically active. A large proportion of women indicated use of both Indian and English languages with the majority speaking both Hindi and English. Fewer than half of the women spoke Gujarati.\n\nOverall, 73.9% of the study participants had multiple chronic conditions and 26.1% were found to have one chronic condition. The prevalence of diabetes, cardiovascular disease, chronic respiratory conditions, osteoarthritic, depression, nephrological problems and cancer was 31.5%, 67.0%, 10.8%, 57.6%, 37.4%, 1.47%, and 4.9%, respectively (Table 2).\n\nIn our study sample, we observed significant differences between age group, education level, employment status, living arrangements, physical activity, the acculturative stress of threat to ethnic identity and homesickness, and the presence of multiple morbidities (Table 3). The multi-variable analysis identified threat to ethnic identity as a predictor for multimorbidity among the Immigrant Asian Indian women (Table 4).\n\n\nDiscussion\n\nThe aim of this study was to investigate the prevalence and predictors of multimorbidity amongst a group of immigrant Asian Indian women residing in Sydney, Australia. This is the first study that presents self-reported multimorbidity and its associates among this group. Identifying the key determinants of multimorbidity can assist with the development of culturally appropriate strategies that can identify people at risk of health conditions and recognise the impact of migration and other risk factors on their health.\n\nThe study results demonstrate that the prevalence of multimorbidity was 74%. This result is much higher compared to the prevalence worldwide,3 in South Asia26 and in the Australian population.48 There was a high prevalence of the combined chronic conditions of cardiovascular disease 67.0% and osteoarthritis 57.6%, a combination which has been recorded in other studies.3 The high prevalence of cardiovascular disease among Asian Indians has been reported in the literature.32 Among this sample group, the high prevalence could be due to the consumption of a carbohydrate dense diet and lack of physical activity when living in a new country.33,49–51\n\nA potential cause for higher prevalence of multimorbidity could be because the women were members of the RAIN group which provides social support to women, and as a result the women felt more confident to share their health conditions. In addition, the data were collected using self-reports which may have reduced accuracy,48,52 and that women may have a higher tendency to share their conditions in self-reports.47,53\n\nOther influences for this excess multimorbidity in women could be higher exposure to biological and sociocultural risk factors for non-communicable diseases or gender inequality in access to healthcare.4,9,10,32,36,37,41 Results from this study demonstrated that older women had a higher prevalence of multimorbidity which is consistent with the literature.4,7 The increase in life expectancy means that older adults experience a higher incidence of aging conditions such as diabetes, CVD and osteoarthritis.3\n\nThere was a significant correlation between multimorbidity and the factors of increasing age, low education, being unemployed, living alone, lack of physical activity, threat to ethnic identity and homesickness. Women with higher education levels had a lower prevalence of multimorbidity. It could be postulated that women with higher educations have higher incomes, access to better quality food, and access to medications thus leading to improved quality of life and lower prevalence of multimorbidity. Research has also indicated the effects of lower socio-economic status on multimorbidity.7,54\n\nThree quarters of the women who did not have multimorbidity were married or living with a partner. It has been well established that a partner is form of social support and that the presence of a personal relationship leads to better health outcomes55 particularly among older adults. The absence of a partner could indicate a lower level of support particularly in attending medical appointments and obtaining medications for health conditions. What is known is that migrants may struggle in relation to chronic conditions as patient self-management may be an unfamiliar concept63,33; especially for older migrants, and women who are living alone.64 For the Indian women, living alone could also be an indication of widowhood and possible cultural stigma.43,50 For Indian women who have been widowed, traditional Hindu expectations and taboos may contribute to social isolation.56\n\nPhysical activity continues to remain the corner stone of overall health and wellbeing. The risks of being physically inactive in relation to multimorbidity have been reported.7,57–60 Numerous studies have reported that participation in physical activity reduces the risk of diabetes, blood pressure, cardiovascular disease and arthritis.61,62 It is no wonder in our study that there was a low prevalence of multimorbidity among those who participated in physical activity. The link between acculturative stress and increased settlement time on obesity suggests that health interventions should target first generation migrants to promote retention of their original healthy behaviours.37\n\nThe results also demonstrated an association of multimorbidity and the acculturative stress factor of homesickness. This acculturation stress is intensified by the existence of cardiovascular disease and frequency of modifiable risk factors49 especially for womenAdd gap between footnote numbers.63,64\n\nThe multi-variable analysis identified threat to ethnic identity as a predictor for multimorbidity. This factor is one of the components taken from the MASS acculturation scale survey and includes the indicators of feeling sad when not seeing cultural roots in this society; losing Indian identity; feeling neither Indian nor Australian; feeling divided between India and Australia; and worrying that the next generation will become very broad minded.46 The impact of cultural displacement and acculturative stress on health64,65 and mental health66 has been noted in research. Feelings of being divided between the host and native country can contribute to being marginalised in the acculturation model, a psychological state which produces the most stress.39 This confirms the other reported results of this research stating that the senior Indian women experienced feelings of cultural stigma and thus social isolation.43\n\nAlthough non-modifiable risk factors such as age play a significant role in the prevalence of multimorbidity, modifiable risk factors can be addressed. This includes considering the impact of adjusting to a new culture, loss in networks, family support, lifestyle/physical activity, and lack of employment on health.67 Public health intervention strategies and community support need to be tailored to the cultural background of the women and recognise the impact of acculturative stress and homesickness to ensure healthy and active ageing.51\n\nCommunity focused strategies to address multiple chronic conditions should take an integrated approach including increasing the knowledge and skills of the older Indian population in preventing and managing cardiovascular disease, diabetes, depression, and arthritis; facilitate interventions targeting emotional health and wellbeing to improve self-confidence; and promoting culturally and linguistically relevant health resources to the community.\n\nHealth practitioners should be informed about the psychosocial and cultural issues amongst older Indian women; develop partnerships between multicultural health service providers for ongoing dialogue of specific community health needs; develop multidisciplinary management packages and care plans for chronic multimorbid conditions; and tailor self-management programs and coaching programs to the patient’s cultural and linguistic background and level of health literacy.68\n\nReducing acculturative stress could be addressed through linking and referring vulnerable individuals to existing Indian and culturally appropriate social support services. Acculturation studies have indicated integration to Australian society while also maintaining strong connections with cultural traditions incur the best outcomes for psychological wellbeing.39,69\n\nSome of the strengths of this study include data collection in a variety of languages of the participants by trained data collectors. In addition, the use of validated questionnaires added to the robustness of the study. Despite the evidence presented, some of the limitations inherent in undertaking such a study need to be acknowledged. Firstly, the sample comprised of only 203 women from a single community organisation. Hence further research using larger samples from the wider Indian diaspora in Australia is needed. The data collected was self-reported data, hence participants may have overestimated the severity of their illnesses. It is worth mentioning that this information is related to data collected approximately 10 years ago at a subnational level.\n\nFurther research is needed to address the multidimensional factors contributing to multimorbidity especially for older adult migrants to prevent poor health outcomes. The effect of acculturative stress on multimorbidity is an area that should be further researched. While this study examined a range of factors, the effects of having a low English proficiency for migrants should be further examined.\n\n\nConclusion\n\nRisk factors contributing to increased multimorbidity is a growing area of research which will assist in containing its human and economic costs. Although the evidence base for the link between acculturative stress and multimorbidity needs to be further developed and much more research is still needed beyond just self-reports, current best practice should focus on the culturally responsive prevention and management program for chronic multimorbidity and older migrants who may have pre-existing risk factors.\n\n\nAuthors’ contribution\n\nConceived and designed the study: COC, UNY, RF, SN and SS. Performed field work: SN, SS, RF and COC. Analysed data in the present study: COC, UNY and RF. Initial draft preparation: UNY, RF and COC. All authors read and approved the final manuscript.", "appendix": "Acknowledgements\n\nThe principal researchers were Professor Ritin Fernandez, Dr Sudha Natarajan, Dr Gunu Naker and Dr Saroja Srinivasan, Dr Cathy O’Callaghan and Ms Patty Loukas. The Project Steering Committee included representatives from Gymea Community Aid; Mental Health Service, St George Hospital; Diversity Health Coordinators at St George and Sutherland Hospitals; and The Resourceful Australian Indian Network (RAIN). The authors would like to thank the women who participated in this project and the Bilingual Coordinators (Mrs Kapila, Ms Hunt, Mr Amin, Mr Dhananjay, Mrs Loganayaki, Mrs Hirani, Mrs Powar, Dr Srinivasan and Mrs Pandit) who played an important role in collecting data.\n\n\nData availability\n\nFree access to de-identified individual data is restricted for ethical reasons as i) doing so may potentially identify the psychological and physical health details of individuals from a minority population; and ii) the investigators have not sought approval of the patients to share the raw data and have informed them that data will be published in aggregate. The South Eastern Sydney Local Health District’s Human Ethics Committee have approved these prohibitions (reference no HREC 12/282, SESLHD-RSO@health.nsw.gov.au).\n\nDryad: Measuring Acculturation and Health of Indian Women Living in Australia (MAHILA) study questionnaire. https://doi.org/10.5061/dryad.3r2280gfz.70\n\nThis project contains the following extended data.\n\n• MAHILA_questionnaire.pdf (a copy of the questionnaire given to participants).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nLunenfeld B, Stratton P: The clinical consequences of an ageing world and preventive strategies. Best Pract Res Clin Obstet Gynaecol. 2013; 27(5): 643–59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: 10 facts on health inequities and their causes. Im Internet, 2011. 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BMJ Open. 2015; 5(10): e007235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSum G, Ishida M, Koh GC-H, et al.: Implications of multimorbidity on healthcare utilisation and work productivity by socioeconomic groups: Cross-sectional analyses of Australia and Japan. PLoS One. 2020; 15(4): e0232281. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPati S, Hussain MA, Swain S, et al.: Development and validation of a questionnaire to assess multimorbidity in primary care: an Indian experience. Biomed Res. Int. 2016; 2016. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel V, Chatterji S, Chisholm D, et al.: Chronic diseases and injuriesi n India. Lancet. 2011; 377: 413–28. Publisher Full Text\n\nMahajan D, Bermingham M: Risk factors for coronary heart disease in two similar Indian population groups, one residing in India, and the other in Sydney, Australia. Eur J Clin Nutr. 2004; 58(5): 751–60. 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PubMed Abstract \| Publisher Full Text\n\nMinas H, Kakuma R, Too L, et al.: Mental health research and evaluation in multicultural Australia: Developing a culture of inclusion. BioMed Central. 2012. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAustralian Institute of Health and Welfare: Women and heart disease: cardiovascular profile of women in Australia.Canberra: 2010.\n\nHarris M, Harris-Roxas B, AW K: Care of patients with chronic disease: achievements in Australia over the past decade. Med J Aust. 2018; 209(2): 55–7. PubMed Abstract \| Publisher Full Text\n\nSam D, Berry J: Acculturation when individuals and groups of different cultural backgrounds meet. Perspect Psychol Sci. 2010; 5(4): 472–81. PubMed Abstract \| Publisher Full Text\n\nO'Callaghan C, et al.: Measuring Acculturation and Health of Indian Women Living in Australia (MAHILA) study questionnaire. Dryad, Dataset. 2021. Publisher Full Text" }	[ { "id": "284965", "date": "05 Jul 2024", "name": "Carol El-Hayek", "expertise": [ "Reviewer Expertise Epidemiology and public health research and evaluation", "with a focus on priority populations" ], "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 chance to review this manuscript. It presents a cross-sectional analysis of survey data to quantify the prevalence and identify predictors of multimorbidity among migrant Asian Indian women in Sydney, Australia. The study addresses a timely issue among an important demographic within the Australian context, however I believe this manuscript needs to present a stronger narrative and a more committed interpretation of the results and revise the potentially stigmatising language throughout. Please see my comments and suggestions below.\nMajor revisions:\nThe Introduction highlights factors contributing to multimorbidity although it reads like a broad list which can make it difficult to follow the narrative and understand the connection between the points being made, including the justification for the population studied. The Introduction needs a more logical flow and needs to more strongly justify why the study is specifically necessary for migrant Asian Indian women in Australia. The data presented in this paper originate from a previous study. Were there particular findings from that study, for example, or other studies that warranted this investigation in this demographic?\n\nThe Methods section needs to provide more detail of the data and statistical methods used in the study. Some examples below.\nDespite including “This study was part of a larger study titled … that was conducted in a metropolitan Local Health District (LHD) in Sydney, Australia during 2013” it is not necessarily clear that this study is an analysis of data collected in 2013. Many of the sample characteristics, described in the first paragraph of Results, are not included in the univariate analysis presented in Table 3. Information is missing on response rates, whether responses were excluded based on completeness, consistency, or any other criteria and how incomplete or unverifiable responses were handled. The exact logistic regression model used is not specified, whether it was binary or multinomial, stepwise selection or full model. This is needed to interpret Table 4.\n\nThe comparison of prevalence in the Discussion is unclear. The Australian article cited shows multimorbidity ranged between 64% and 88% depending on the conditions and therefore the statement “This result is much higher compared to the prevalence … in the Australian population” appears questionable. Also, were results presented by gender or migrant status to make a reasonable comparison? If not, this is worthy of discussion.\n\nThe Discussion provides an overview of the predictors of multimorbidity among Asian Indian women in Sydney found in this study, however, it tends to list and address points superficially, leaving the reader to connect the dots and look up the citations to better understand the implications. It would be great to see a deeper dive into the key findings, with more detail on how they connect and what they mean. Additionally, many links made between the findings in this study and possible explanations are not well supported, sometimes over simplified and often generalised. Some examples are appended below.\n\nHow is acculturative stress linked to multimorbidity? This is the main finding and needs to be addressed directly in a way that provides readers with an understanding of the possible mechanisms. What do the data presented in this study contribute to our understanding or to this population? The article cited to support the statement “It has been well established that a partner is [a] form of social support and that the presence of a personal relationship leads to better health outcomes, particularly among older adults” appears to be a U.S perspective on the link to “thriving” and makes no mention of migrant women of any background (I was only able to access the abstract). Hence its relevance here is questionable. If it is “well established” as authors state, more relevant articles should be used to support the statement. My concern is that multicultural women’s health research does not necessarily show that women with partners are better supported. “The link between acculturative stress and increased settlement time on obesity suggests that health interventions should target first generation migrants to promote retention of their original healthy behaviours” appears to repeat the conclusion of the article cited rather than provide an insight into how the link was made in this study. Also, the length of time did not appear to be included in the univariate or multivariate analyses of this study.\n\nWhile limitations are acknowledged they should be more explicitly discussed in terms of how they might impact the findings (including the generalisability to the broader population of Asian Indian women in Australia) and link to gaps in the research and how these limitations could be addressed in future work.\n\nThe section “Addressing risk factors” and parts of the Conclusion are beyond the scope of the research conducted in this study. Making recommendations and referring to “current best practice” interventions should be the result of co-design with community or a comprehensive review of health promotion evaluation research, which is not the objective of this study.\n\nFinally, a note on language. It is important to avoid attributing negative behaviours or outcomes specifically to migrants that can potentially cause stigmatisation and related harms to communities. Instead focus on systemic and environmental factors. There are many examples in this manuscript of where stigmatising statements require revision. One is “There have been reports that the longer that migrants from low-middle income countries live in high income countries and adopt unhealthy behaviours, the more weight they can gain…” This can be seen as attributing unhealthy behaviours and weight gain to migrants. A possible revision might be \"Some studies suggest that prolonged residence in high-income countries can lead to lifestyle changes and weight gain among migrants from low-middle income countries...\" which acknowledges factors beyond the individual. Another is use of the term “excess multimorbidity” which can be seen as negative whereas referring to the prevalence estimate of multimorbidity in this sample population is more neutral.\n\nMinor revisions\nThe last sentence of the third paragraph needs a reference. The manuscript alternates between past and current tense. The Introduction mentions that there are 400,000 Indian born migrants living in Australia. This needs some context for readers who may not know the significance of this number. Methods state “The items were rated on a four-point scale with 1 = disagree, 2 = to some extent disagree, 3 = to some extent agree, 4 = agree. Lower scores indicated a lower level of acculturative stress and higher scores showed higher levels of acculturative stress.” What was the cut-off for acculturative stress =0 vs acculturative stress =1? If a chi-squared test was used to analyse the association between multimorbidity and various factors, it is more accurate to describe the results in terms of associations rather than correlations. Marital status is a point in the Discussion however the data are not shown. Consider a table describing participant socio/demographic characteristics. Given the Discussion regarding socioeconomic status, the Methods could indicate the survey questions that were used to define a participant’s socioeconomic status. The trained data collectors are one of the strengths mentioned in the discussion and no other information about them is specified (such as bilingual or bicultural) or supporting literature citing this as a strength. The statement “women were members of the RAIN group which provides social support to women” is discussed in the context of the reliability of self-report but not on possible selection bias and representativeness. There is no mention of the low alpha coefficient of homesickness and what that might mean for interpretation of findings. Reference number 50 requires more information. It is unclear what the material being cited is and where it can be located. A google search did not help.\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?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-634
https://f1000research.com/articles/10-633/v1	22 Jul 21	{ "type": "Research Article", "title": "Regulatory landscapes of specific miRNAs are conserved between cell lines and primary tumors", "authors": [ "Hanwen Zhu", "Boting Ning", "Hanwen Zhu" ], "abstract": "Background: MicroRNAs are essential gene expression regulators and play important roles in various biological processes, such as cancer. They have shown great translational promise as either diagnostic biomarkers or therapeutic targets. While the similarities between transcriptomic profiles from The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia have been thoroughly studied before, less is known on the microRNA side. This project aims to provide critical biological knowledge on the extent of consensus microRNA expression and regulation between cell line models and primary human tumors.\n\nMethod: First, we examined the similarity of miRNA expression profiles between CCLE cell lines and TCGA tumor samples for each cancer type. Next, we compared the expression of miRNAs associating the hallmarks of cancer pathways. Finally, we constructed miRNA-mRNA regulatory network for each cancer type and evaluated whether the regulatory role of each miRNA is conserved between cell lines and tumor samples.\n\nResults: Our results indicate that, similar to gene expression, how well cancer cell line microRNA expression would capture the transcriptomic profile of human cancer tissues is greatly affected by the tumor type and purity. The cell-type composition for a cancer type also affects how accurately cancer cell lines could reflect the miRNA expression in tumor tissues. Furthermore, through network analysis, we show that certain microRNAs, not all, regulate the same set of target genes in both the cell line and human cancer tissues.\n\nConclusions: Through systematically comparing the miRNA expression profile and the regulatory network, our study highlights the biological differences between cell line and tumor samples and provides resources for future miRNA and cancer studies.", "keywords": [ "microrna", "Cancer", "miRNA-mRNA Network", "CCLE", "TCGA" ], "content": "Introduction\n\nMicroRNAs (miRNAs) are endogenous, small non-coding RNAs that function in the post-transcriptional regulation of gene expression.1 Using the seed region, the mature miRNAs bind to the 3′ untranslated region (UTR) of the mRNAs via complementary base-pairing and suppress the expression of the targeted genes.2,3 Recently, miRNA has been found to be heavily dysregulated in cancer cells by functioning as either tumor suppressor or oncogenes.4,5 Many investigations suggest that abnormal miRNA expression levels could be used for diagnostic and prognostic biomarkers for lung, prostate, or breast cancers.6–8 Also, the usage of miRNAs as potential therapeutic targets has been shown to be promising in many cancer types.9–11\n\nRecent progress in high-throughput sequencing technology has offered researchers unprecedented opportunities to understand the molecular mechanism of cancer. The Cancer Genome Atlas (TCGA) collects molecular profiling of primary tumors of over 11,000 tumor samples and provides real-world cancer patient information.12 On the other hand, the Cancer Cell Line Encyclopedia (CCLE) characterize of around 1,000 human cancer cell lines for in vivo studies.12,13 Both datasets span across several tumor types and contain multi-omics sequencing data. A comparison between them can provide knowledge about the differences between the two sets and capture how well each cell line represents a specific type of primary tumor. K Yu et al. have previously conducted a pan-cancer comparison between TCGA and CCLE.14 Specifically, they used correlation analysis and gene set enrichment analysis (GSEA) to identify differences between cell lines and primary tumors and found that there is a strong correlation between TCGA and CCLE samples for each tumor type and that tumor purity is a main driving factor of cell line and primary tumor differences. They also identified cell lines representative of primary tumor samples in TCGA. While this study offered a comprehensive comparison of the gene expression profiles between TCGA and CCLE, whether miRNA expression differs between the two data sources is still yet unclear. Here, we seek to use similar correlation analysis to evaluate the concordance of miRNA expression profiles between TCGA and CCLE datasets.\n\nWe first obtained respective datasets and mapped miRNAs from TCGA to CCLE to provide consistency between cell lines and primary tumors. For both datasets, technical confounders were adjusted, as well as tumor purities, and the distribution of cell lines or tumor samples were visualized using the distribution in each dataset using t-distributed stochastic neighbor embedding (t-SNE). Then, we performed correlation analysis to evaluate the similarity between cancer cell line models and human primary cancer samples based on miRNA expression profiles. We found that the cell-type composition of cancer samples and the nature of cancer greatly affect the correlation strengths. Also, through studying the expression of miRNAs related to the hallmarks of cancer pathways, we found immune-related pathways were among the most differential expressed pathways between CCLE and TCGA. Lastly, we investigated whether the miRNA–mRNA regulatory networks in the human cancer samples could be accurately captured by the cell line models.\n\n\nMethods\n\nCCLE miRNA count matrix and the cell line annotations were downloaded from the CCLE Broad Institute data portal.15 The current version of CCLE data contains 954 cell lines across 31 tumor types based on TCGA cancer code, among which 258 were labeled as “NA” or “Unable to classify”. The miRNA expression data, as well as the clinical information of each TCGA sample, were downloaded from the GDC data portal using R package TCGAbiolinks, containing 11,082 samples across 32 tumor types.16 Due to the different sequencing technology, much more unique miRNA could be identified in TCGA comparing to CCLE. Thus, we first unified the miRNAs identified in two datasets by summing all raw miRNA counts in TCGA that correspond to a specific CCLE miRNA ID within each sample. Identical mature miRNA reads from different precursors at different genomic locations (eg, hsa-miR-1-1 and hsa-miR-1-2) were also summed in TCGA to match with CCLE (eg, hsa-miR-1), if no exact matching was found in CCLE. Also, for miRNA that is characterized as precursor in CCLE (lacking the -3p or -5p information) but as mature miRNA in TCGA, the corresponding mature miRNA reads from different precursors were summed in TCGA to match CCLE. Next, the counts were normalized using the trimmed mean of M values (TMM) and had log2 counts per million computed using EdgeR.17 MiRNA with an interquartile range equal to zero or a sum across samples equal or less than one was excluded.\n\nThen, since TCGA was sequenced on two different sequencing platforms, namely, 9,411 samples using HiSeq and 1,413 using GA (with 258 unannotated samples dropped), batch correction was done to eliminate differences caused by the difference of platforms using ComBat.18 Finally, as tumor purity has been proven to be a significant confounder in transcriptomic data of primary tumors by K Yu et al, TCGA miRNA expression profiles were additionally adjusted for their tumor purity scores using limma.19 Tumor sample purity scores calculated using the ABSOLUTE20 method were obtained from the TCGA PanCanAtlas website . 1,126 batch-corrected samples without tumor purity measurements were discarded, leaving 9,698 samples.\n\nTCGA and CCLE expression profiles consisted of the 327 miRNAs that passed the quality control. A Spearman correlation coefficient was calculated between each TCGA sample and CCLE cell line with the 327 miRNAs that passed the quality control in both datasets, resulting in a correlation matrix of 954 cell lines by 9,698 human tumor samples. The correlation coefficients were then Fisher-Z transformed and arranged with a hierarchical clustering by the cancer types of CCLE. Further, the coefficients obtained from correlation comparing CCLE to TCGA expression adjusted for tumor purity and not adjusted for tumor purity were compared using two-group t-test.\n\nIn the meantime, the correlation matrices between CCLE and TCGA based on gene expression were obtained from the github repository from K Yu et al.14 Then, the correlation coefficients between each cell line and TCGA of the same tumor type based on miRNA expression profiles were compared to that from mRNA expression profiles using Spearman correlation.\n\nThe miRNA set associated with either upregulation or downregulation of these hallmarks for cancer pathways were obtained from A Dhawan, et al.4 For each pathway, single-sample gene set enrichment analysis (ssGSEA) was used to summarize the miRNA expression in CCLE, and both adjusted and unadjusted TCGA. Afterward, the GSVA scores were compared between CCLE and TCGA, and between tumor purity adjusted and unadjusted TCGA samples using a two-group t-test.\n\nThere are very few cell lines included in CCLE for several certain types of cancer. Also, it is less appealing to study the miRNA–mRNA network when the expression level correlation is low between CCLE and TCGA. Thus, to increase the validity of network analysis based on miRNA–mRNA correlation, we first filtered the cancer type and cell lines based on sample size and miRNA expression level correlation strength. Among the 22 overlapping cancer types between CCLE and TCGA, seven tumor types with 30 or more cell lines strongly correlated (r ≥ 0.45) with TCGA (LUAD, COAD/READ, BRCA, SKCM, STAD, HNSC, SARC) were chosen for miRNA–mRNA network analysis.\n\nFor each cancer type, the Pearson correlations between each miRNA and mRNA expression levels were calculated for CCLE cell lines or TCGA primary tumor samples. Then, each negative and statistically significant (FDR < 0.05) correlation was retained to form an edge in the constructed miRNA–mRNA correlation network. For each cancer type, the miRNA-mRNA network was constructed within CCLE and for TCGA samples separately. The intersection between the resulted correlation network and the TargetScan21 predicted target network is taken to construct a regulatory network from miRNA to mRNA.\n\nFor each cancer type, the degree distributions of miRNAs of both CCLE and TCGA were calculated. Hub scores of all miRNAs and the correlation between the hub scores from CCLE and TCGA were obtained. Further, the hamming distance between networks of CCLE and TCGA was calculated for each cancer type.\n\nTo prioritize miRNA based on the target gene conservation, we compare the overlaps between the connected gene for each miRNA in the CCLE and the TCGA network for each cancer type. Using a one-tailed Fisher’s exact test, the significance of the overlap between targets of CCLE and TCGA as FDR was calculated.\n\nAll code for data processing and analysis is available at https://github.com/hanwenzhu/mir-tcga-ccle-paper.22\n\n\nResults\n\nThe TCGA miRNA data were sequenced via small-RNA sequencing, while Nanostring platform was used for CCLE. To avoid the confounding caused by the two drastically different platforms, we processed and normalized the two datasets separately and only compared the relative miRNA expression rather than the absolute expression level. MiRNA counts from 954 cell lines across 31 tumor types from CCLE and from 11,082 samples across 32 tumor types from TCGA remained after processing (Figure 1 and Extended Table 1). Note that there were significantly more samples in TCGA than in CCLE. A significant portion of the CCLE lacked specific annotation of tumor type in the format of TCGA disease code (“NA” or “Unable to classify”), which were excluded for the correlation analysis later on. Overall, the number of samples of each tumor type varied by a lot in TCGA, with multiple types from either dataset having less than 10 samples, such as GBM, which had only five samples. Also, the number of BRCA tumors was the highest and was much greater than any other type in TCGA.\n\nThe COAD and READ were merged in TCGA to match the classification used in CCLE. (a) The number of samples by tumor type in the TCGA data. (b) The number of cell lines by TCGA tumor type in the CCLE data. BRCA, breast invasive carcinoma; COAD/READ, colorectal adenocarcinoma; KIRC, kidney renal clear cell carcinoma; THCA, thyroid carcinoma; HNSC, head and neck squamous cell carcinoma; UCEC, uterine corpus endometrial carcinoma; LUAD, lung adenocarcinoma; PRAD, prostate adenocarcinoma; LGG, brain lower grade glioma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; STAD, stomach adenocarcinoma; SKCM, skin cutaneous melanoma; BLCA, bladder urothelial carcinoma; LIHC, liver hepatocellular carcinoma; KIRP, kidney renal papillary cell carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; SARC, sarcoma; ESCA, esophageal carcinoma; LAML, acute myeloid leukemia; PCPG, pheochromocytoma and paraganglioma; PAAD, pancreatic adenocarcinoma; TGCT, testicular germ cell tumors; THYM, thymoma; KICH, kidney chromophobe; MESO, mesothelioma; UVM, uveal melanoma; ACC, adrenocortical carcinoma; UCS, uterine carcinosarcoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; CHOL, cholangiocarcinoma; GBM, glioblastoma multiforme; ALL, acute lymphocytic leukemia; NB, neuroblastoma; MM, multiple myeloma; LCML, chronic myelogenous leukemia; MB, medulloblastoma; CLL, chronic lymphocytic leukemia.\n\nWhile the cell lines of each tumor type in the CCLE did not form perfectly distinct clusters by themselves, we did observe two large separate clusters (Figure 2). The lower cluster consisted of cell lines derived from lymphocyte or myeloid cells, such as those derived from ALL, DLBC, and LAML. The upper cluster contained cell lines derived from solid tumor tissues. This suggests that a large portion of variance within the CCLE miRNA profiles is reflecting the tissue origins of the cell lines. On the other hand, the imperfect distinction between clusters also indicates CCLE cell lines of the same tumor type could be very different from each other as a result of the immortalization or lab culturing process.\n\nThe t-SNE coordinates were calculated based on processed miRNA read counts and the points were colored by tumor types. The “UNABLE TO CLASSIFY” type was merged with NA in the figure above, shown in grey.\n\nIn the meantime, we did observe that the TCGA tumor samples clustered much better than the CCLE cell lines (Figure 3). The clusters of tumor types were more distinct after both batch correction and adjustment for tumor purity. Such observation is similar to the results based on gene expression from a previous study.14 The lack of clear cancer type clustering in the CCLE t-SNE might suggest that within each tumor type, the CCLE cell lines are more heterogeneous than the TCGA samples, and not all cell lines are good models for miRNA and cancer studies.\n\nThe figures above show the t-SNE of TCGA expression data, after normalization (a), batch correction (b), and purity adjustment (c), respectively. The samples formed highly distinct clusters by different tumor types after batch correction and purity adjustment.\n\nSeveral tumor types with similar pathological features were mixed with each other when being clustered using normalized miRNA counts or batch corrected counts, such as BRCA, HNSC and LUSC (Figure 3a,b). These samples could be distinguished after we adjusted for the tumor purity, as reported by other studies,14 suggesting that immune infiltration could affect not only the gene but also the miRNA profiles of related tumor types in a similar way (Figure 3c).\n\nNext, we investigated how well CCLE cell lines capture the miRNA expression profiles of TCGA human primary cancer samples. Generally, cell lines show a moderate to high correlation with their matched tumor samples across different cancer types (Figure 4a; see Extended Fig. 1-2 for correlation per each cell line). The median correlation is between 0.5–0.6. However, the correlation coefficient distribution within each tumor type was much larger than that calculated based on gene expression, such as for COAD/READ (0.23–0.83) and SARC (0.19–0.80). This indicates that the miRNA expression might be more prone to the heterogeneity within each tumor type.\n\nThe bars indicate the median value. Tumor types with FDR > 0.05 are labeled with “ns”, FDR ≤ 0.05 with one star, FDR ≤ 0.01 with two stars, and FDR ≤ 0.001 with three stars, and FDR ≤ 0.0001 with four stars. GBM is not shown since it lacks tumor purity estimates. (b) Heatmap of median Spearman correlation coefficients between each cancer type of TCGA and CCLE. CCLE cancer types are clustered based on their correlation with each TCGA cancer type. (c) Violin plot of the Spearman correlation between miRNA and mRNA data of the correlation coefficients between TCGA and CCLE in each cell line.\n\nBased on the correlation between CCLE and TCGA samples, samples of the same tumor type from cell lines and primary tumors are more correlated (Figure 4b). Also, similar to the observation from K Yu et al.14 cancer types with similar pathology cluster together. The two cancer types on the top of the heatmap are DLBC and LAML, both originating from immune cell types. Such separation indicates that miRNA expression could reflect the difference between hematopoietic and solid tumors. Other cancer types that are biologically related also tend to cluster together, such as LUSC and LUAD, or GBM and LGG.\n\nGiven that it is been previously reported that the cellular composition of a tumor sample has strong effects on the correlation analysis, we also compared the correlation calculated using tumor purity adjusted and the unadjusted TCGA miRNA expression profiles (Figure 4a). Most of the cancer types have a significant increase in the correlation coefficient distribution after tumor purity adjustment, such as BRCA, HNSC, LUSC, suggesting purity being a driving factor of this increase, which reflects findings based on mRNA from K Yu et al.14 These cancers, as well as the cell lines, are originated from the epithelial cells, and the amount of immune cell infiltration is expected to have a strong influence on their expression obtained from bulk RNA-sequencing. In the meantime, other hematopoietic cancer types such as LAML and DLBC, have small changes in the correlation coefficient distribution, suggesting their cellular composition is not influenced by immune infiltration.\n\nFinally, we compared miRNA and mRNA in terms of the similarity between CCLE and TCGA expression profiles (Figure 4c). Among the tumor types, the ones with the highest median correlations are PAAD, ESCA, and KIRC, and the lowest ones are HNSC, COAD/READ, and STAD, although all median correlations are positive. Interestingly, the cancer types with a higher correlation between CCLE and TCGA based on either miRNA or mRNA expression profiles are not necessarily the ones with higher concordance between miRNA and mRNA based correlation. For example, HNSC is highly ranked by mRNA and miRNA expression (median = 0.63 and 0.57) but ranked the last based on the correlation between miRNA and mRNA correlation coefficients, while LIHC, although showing lower correlation based on expression (0.50 and 0.49), has strong concordance. Such results show that the expression similarity between cell lines and human primary tumor samples does not directly reflect the concordance in the miRNA–mRNA regulatory relationship between sample types.\n\nTo further elucidate the biological difference between CCLE and TCGA miRNA expression profiles, we investigated the expression alteration in the miRNAs that are associated with the hallmarks of cancer pathways. Comparing the GSVA score of miRNAs associated with hallmarks of cancer pathways between CCLE and TCGA (Figure 5a and Extended Figure 3), we saw higher enrichment of pathways related to immune cell infiltration in the TCGA (up-regulation of up hallmark miRNA set and vice versa), such as inflammatory response and IL6-JAK-STAT3 signaling pathways,23 suggesting the absence of immune infiltration in CCLE pure cancerous cell compositions might be the major difference between two sample types. In the meantime, CCLE is more enriched in hallmarks related to tumor cells, such as more G2M checkpoint, p53 pathway, and epithelial-mesenchymal transformation pathways, revealing a higher portion of cancerous cells composing the cell lines. These results are consistent with GSVA scores of mRNA from K Yu et al,14 suggesting miRNA is similar to mRNA in terms of the difference in composition and hallmark enrichment between TCGA and CCLE.\n\nThe downregulated hallmarks are compared between CCLE and tumor purity-adjusted TCGA (a) and between TCGA before and after purity adjustment (b) using a t-test and visualized as heatmaps. It is possible to observe that immune infiltrate hallmarks are more present in TCGA, especially before adjustment.\n\nAlthough both purity-adjusted and unadjusted TCGA compare similarly to CCLE, there is a consistent difference across cancer types due to purity adjustment (Figure 5b). Inflammatory response and TNF-a signaling are significantly more enriched in TCGA before purity adjustment, and other hallmarks from cancer cells such as DNA repair and oxidative phosphorylation are more enriched in TCGA after adjustment. This demonstrates that the purity adjustment of TCGA effectively reduces the impurity of the gene expression data.\n\nTo further characterize the difference between cell line models and human primary tumor tissues in terms of transcriptomic profiles, we constructed the miRNA–mRNA network in CCLE and TCGA for each cancer type separately and compared the network metrics. The degree distribution shows the distribution of the number of targets of all 230 miRNAs for each cancer type, reflecting the connectivity of the target network. The results reveal differences in the global connectivity between CCLE and TCGA regulatory networks. CCLE miRNAs have, on average, fewer obvious targets than TCGA, with miRNA out-degrees concentrated at lower values (Figure 6a). Both CCLE and TCGA contain some outlying miRNA hubs with significantly more targets than average. However, since TCGA contains more samples than CCLE (1,030 vs 50 for BRCA in Figure 6b; Extended Fig. 4), there are expected to be more significant correlated miRNA–mRNA pairs in the TCGA data so the degree distribution comparison may be inconclusive.\n\n(b) The miRNA out-degree distribution of BRCA target networks. (c) Hamming distance between CCLE and TCGA target networks for each cancer type. (d) Pearson correlation coefficient of hub scores of all miRNAs between CCLE and TCGA for each cancer type. (e) Common targets of the five miRNA hubs with the most significant overlap of targets between CCLE and TCGA networks of BRCA. Each triangle represents a miRNA hub and each circle node represents a target. The FDR values of the one-tailed Fisher exact test of the overlap are labeled below the miRNA names.\n\nThe hamming distance offers a metric of the difference between target networks that also takes the edges to mRNA targets into account rather than merely the out-degree of each miRNA. The hamming distances between CCLE and TCGA miRNA–mRNA networks in Figure 6c measure the difference of miRNA regulation between CCLE and TCGA of each cancer type, and a small hamming distance indicates that the two networks are more similar to each other. Cancer types with more similar networks are COAD/READ (distance = 18,114) and SKCM (distance = 20,872), while more different ones are BRCA (distance = 34,677) and HNSC (distance = 28,047). The hamming distance, however, failed to adjust to the greater total number of edges in TCGA target networks than in CCLE, so an analysis focused on miRNA hub scores and without the influence of network size was conducted.\n\nWhile network metrics as the degree distribution and hamming distance provide a global view on the network similarity between CCLE and TCGA, it is unclear whether individual miRNA targets the same set of genes in both cell lines and human tumor samples. To further explore each miRNA, the Pearson correlations of 230 miRNA hub scores between CCLE and TCGA were calculated (Figure 6d). They reflect whether the network topology, especially the central regulatory miRNA, is conserved between CCLE and TCGA. All coefficients are positive, meaning the regulatory roles between CCLE and TCGA are largely similar, although the correlation strength is very varied in different cancer types. Cancer types with more pronounced similarity of miRNA centrality are STAD (r = 0.57) and HNSC (r = 0.53), while less similar ones are SKCM (r = 0.01) and SARC (r = 0.12).\n\nTo prioritize hub miRNAs based on the conservation of regulatory potential, networks of hub miRNAs with the most significant overlap of their targets between CCLE and TCGA were obtained (Extended Fig. 5). Taking BRCA as an example, the miRNAs of BRCA with the most significant overlap of mRNA targets between CCLE and TCGA are hsa-miR-200c, hsa-miR-141, hsa-miR-29c, hsa-miR-200b, and hsa-miR-221 (FDR < 0.0001; Figure 6e). They are miRNA hubs with conserved connectivity between CCLE and TCGA and are ideal regulatory hubs to be studied using cell line models. In fact, previous research involving cell line models has shown that hsa-miR-200 promotes the proliferation of breast luminal progenitor cells and facilitates the growth and metastasis of breast cancer, while hsa-miR-221 regulates breast cancer development and progression and serves as a promising biomarker.24,25 These findings highlight the value of our results for identifying miRNAs with conserved regulatory roles in both cell lines and human tumor samples and could guide cell-line-based research to model how miRNA regulates mRNA in primary tumors.\n\n\nConclusions\n\nWhile previous research has characterized how CCLE cell lines reflect the gene expression profiles of human primary tumors in TCGA, our work extends the analysis to miRNA expression profiles. We show that cell lines of the related cancer type cluster closer based on miRNA expression profiles, similar to the mRNA based analysis. In the meantime, there seems to be a larger variance in the correlation distribution within a cancer type, indicating miRNA affecting by the heterogeneity of the samples. There are two potential technical reasons for such observation: 1) different platforms were used to profile miRNA between CCLE and TCGA; 2) there are smaller number of unique miRNA species comparing the mRNA and could be more affected by the noise. Yet, the larger variance between cell lines and tumor samples based on miRNA profiles indicates that choosing the cell line most similar to the cancer type of interests is even more important when studying miRNAs.\n\nWe have also shown that tumor purity affects the correlation between CCLE and TCGA miRNA expressions. With the adjustment of tumor purity in the TCGA miRNA expression profiles, we observe an increase in the correlation between miRNA expression profiles of cell lines and human primary tumor samples. Also, through functional characterization of miRNA related to hallmarks of cancer pathways, we find that TCGA samples have higher expression of immune pathways and lower expression of cell-cycle and cell growth pathways. The discrepancy is particularly stronger for solid tumor comparing to blood cancer, such as AML or DLBC. The observation could be the result of immortalization or simply cells being removed from the original microenvironment. The loss of cancer cell communication with surrounding structural cells and particularly the lack of immune infiltration within cell lines could greatly affect the transcriptomic profiling. We believe extra cautions should be made when cell lines are being used to tackle question involving cell-to-cell interaction.\n\nMore importantly, we compared the miRNA–mRNA regulatory networks between cell lines and human tumor samples and observed a very different trend than comparing the expression profiles alone. This result indicates that the similarity in miRNA or mRNA expression profiles between cell line model and human tumor tissues does not guarantee a conserved miRNA regulatory pattern. To comprehensively examine the miRNA–mRNA network, we further evaluated whether each miRNA regulates the same set of genes in CCLE and TCGA samples. The top miRNAs from our analysis function as hub regulators of gene expression and have been previously shown to be biologically essential to cancer development. Such miRNAs with strong conservation in regulatory functions from tumor to cell lines are ideal candidates for in vitro analysis.\n\nThere are several limitations to our analysis. Firstly, the CCLE and TCGA miRNA expressions were measured using very different techniques. We tried to overcome such discrepancy by applying a stringent filter during data preprocessing and avoided direct expression comparison. Given the consistent observations between our results and previous work, we believed our analysis is valid. Second, we used CCLE cell lines of the same cancer type to calculate the correlation network even though there might be underlying differences. Given that there is only one replicate for each cell line in CCLE, there was no easy way to circumvent this issue. We filtered for cell lines with a high correlation to TCGA samples and cancer types with enough number of cell lines to ensure the reliability of the analysis. Finally, we understand miRNA-mRNA correlation based on normalized count data should not be interpreted as causal relationship. In a more rigorous setting, miRNA regulatory roles should be examined based on perturbation assays, in which we would observe whether target gene expression levels are altered following over-expression or knock-out of a miRNA. However, such task is beyond the scope of this project. Here, we only aim to objectively explore the differences between cell lines and human tumors samples from two public databases.\n\nIn summary, we conducted systematic comparison between CCLE and TCGA in terms of miRNA expression profiles and the regulatory landscapes. Our results highlight the importance of choosing appropriate cell lines to study miRNA in cancer research. The cellular composition heterogeneity, particularly for the solid tissue tumors, greatly affect whether the cell lines can accurately capture the miRNA expression profiles of the tumor. Certain miRNAs, not all, have preserved target gene regulatory roles in the cell lines and may be more suitable for in vitro investigation. We believe our results can provide valuable resources for selecting cell lines to study how a particular miRNA regulates cancer-related gene expression.", "appendix": "Acknowledgements\n\nWe would like to graciously acknowledge the Pioneers China Program (PCP) program which supported this research.\n\n\nAdditional information\n\nProf. Marc Lenburg confirms that the authors have an appropriate level of expertise to conduct this research and confirms that the submission is of an acceptable scientific standard. Prof. Lenburg declares they have no competing interests. Affiliation: Boston University School of Medicine.\n\n\nReferences\n\nBartel DP: MicroRNAs: Target Recognition and Regulatory Functions. Cell. 2009; 136: 215–233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJonas S, Izaurralde E: Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genetics. 2015; 16: 421–433. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "90434", "date": "29 Jul 2021", "name": "Chongzhi Zang", "expertise": [ "Reviewer Expertise computational biology", "cancer genomics and epigenetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Zhu and Ning performed a systematic and comparative analysis of transcriptomic profiling data from TCGA and CCLE across 22 cancer types to characterize the miRNA regulatory network. The results demonstrate high variances between primary tumor samples and cancer cell line models, reveal heterogeneity of cancer cells, and emphasize the importance of selecting the right cell line model when studying cancer gene regulation. The manuscript is written clearly, including a thorough discussion on the scope and limitations of the work. The computational methods are appropriate, and the authors provide sufficient details on the methods as well as source code and data, which not only ensure reproducibility but also can be useful resource to both cancer research and gene regulation research 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": [] }, { "id": "118670", "date": "04 Feb 2022", "name": "Vinayak C. Palve", "expertise": [ "Reviewer Expertise Drug discovery", "gene expression", "signaling mechanism", "prognostic biomarkers", "proteomics 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\nIn the present manuscript the authors aim to provide critical biological knowledge on the extent of consensus microRNA expression and regulation between cell line models and primary human tumours. The authors have utilised the CCLE cell lines and TCG tumour samples databased to compare the expression of miRNAs associating the hallmarks of cancer pathways a regulatory network. The authors have nicely shown the regulatory network and correlation between the miRNA expression between cell lines and tumor tissues. However, I believe there are some major points that needs to be addressed.\nMajor:\nThe present manuscript only concludes on the basis of co-relation of expressions from datasets to make conclusions. The authors should show some experimental evidence to validate their findings. The authors can discuss giving certain examples like targeting XYZ miRNA using RNAi or CRISPR studies in certain cancer cell lines to show validation of their network, etc. Adding this would strengthen the overall impact and quality of the manuscript.\nMinor: Most of the figures (Fig 2, 3 , 5 & 6) in the manuscript needs major updates as the resolution is not enough. Kindly change the figures to at least 300dpi.\n\nI believe the authors have utilised databases to compare the expression of miRNAs associating the hallmarks of cancer pathways a regulatory network but to improve the overall quality and impact of the manuscript the suggested experiments and data would be highly crucial. Without these changes the conclusions drawn are not adequately supported by the results.\n\nThank you.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-633
https://f1000research.com/articles/10-627/v1	21 Jul 21	{ "type": "Research Article", "title": "The paradoxes of love in the Spanish family: a sociological approach", "authors": [ "Juan Antonio Roche Cárcel" ], "abstract": "This article defines the Spanish family in the context of the “Mediterranean model” and the “individualization society”. The former is characterised by strong social interrelationships between family members and their emotional ties, while the latter is defined by the separateness of citizens and by institutionalising the basis of society in individuals rather than in the family. The work also describes how modern forms of love, both romantic and confluent, are institutionalized in this society, discussing if they coexist or not, how they exist, and which is the dominant form. Finally, it analyzes the degree of strength or fragility of the family institution and the affective relationships that sustain it. The work concludes that the Spanish family is balancing between the strong resistance to disappear as an institution and its eclipse, crisis, or complete end. This is because, although the Spanish family still retains a large part of its former functions, at the same time as divorce is on the increase and family members are decreasing, it is increasingly ephemeral and a plurality of family forms have emerged that have broken with the traditional dominant model of lifelong romantic marriage. Moreover, the Spanish family is also among the “familist” model and the individual, while the way of loving fluctuates between the traditional patriarchal and the democratic, individual, and communitary. Thus, the thesis I propose qualifies and questions the majority of theoretical works on love and the Spanish family, which argue that the family is inscribed in the “Mediterranean model”. As will be seen, the romantic relationships that have been institutionalised in the Spanish family are more paradoxical, insofar as they are still inscribed in that model, but they are rapidly approaching those of Northern Europe.", "keywords": [ "Spanish family", "society of individualization", "confluent love", "romantic love", "eclipse of family", "typology of Spanish families", "Family relationship models" ], "content": "Introduction\n\nSociology is concerned with love because of its enormous capacity to build bonds, ties, and social interrelations, as well as constituting a fundamental component of the family and the conjugal couple. The discipline has also classified the types of love characteristic of modernity. Thus, based precisely on some of the sociological, theoretical, and empirical work that has analyzed the interrelationship between the Spanish family and love, the objectives of this article are the following:\n\n1) To contextualize the Spanish family with regard to the society of individualization and define the basic features that characterize it.\n\n2) To reveal how modern forms of love, and above all the romantic and confluent forms, are institutionalized, formalized, or “familiarized” in Spanish society.\n\n3) To discuss whether different types of love coexist and how they do so, to reveal which is the primary and which is the secondary form.\n\n4) To provide evidence of the degree of strength or fragility of the family institution and the emotional relationships that support it.\n\nTo achieve these ends, I have structured this article in sections dedicated to the sociological treatment of love and modern types of love, including the five basic aspects of the Spanish family and the coexistence of these modern types of love, namely romantic and confluent. Finally, I will conclude that love in Spain is characterized by its individuality and its minimalism: that the five basic characteristics of the family express a strong tension between the resistance to disappear and its tendency to eclipse, crisis or end and the two types of modern love - the romantic and the confluent love - coexist with other models.\n\n\nThe sociological treatment of love and the two modern types of love: romantic and confluent\n\nLove constitutes one of the fundamental themes of human existence, understood in an individual and social way. It is, in fact, a multifaceted, multidimensional, and polysemantic issue that requires an inter- and transdisciplinary approach. As far as sociology is concerned, since its origins with Weber in a form of modern rationalism (Weber, 1975, 1979, 1987, 1997; Bellah, 2005, p.130-131), it neglected the general study of the field of emotions and, in particular, of love. Thus, although it certainly has classical antecedents that referred to this emotion, it was always considered a badly seen subject (Jónasdóttir, 2014). Before the publication of N. Luhmann’s book, El amor pasión, 2008 (originally published in 1985), sociology did not have a monograph exclusively dedicated to this essential and complex issue (García Andrade, 2015, p.56).\n\nSince then it is true that the road traveled in sociological research around the subject of love has been long and fruitful, to the point that numerous theoretical and empirical articles and important books dedicated to love have appeared; finally, love has assumed a central role in the discipline. However, this does not mean that a canonical paradigm has been established nor that there has been a homogeneous approach, but rather, on the contrary, that plurality, divergence, and contradictions are the norm (García Andrade, 2015, p.37).\n\nThis has not been an obstacle since, throughout the history of the subject, sociologists have referred to the capacity of love to produce bonds, ties, and social interrelations (García Andrade, 2015, p.56). For example, N. Elias in La sociedad Cortesana, 1996, highlights love’s axial role in social bonding, which is affective, has a strong intensity and builds a sphere of meaning (Sabido and García, 2015, pp.22, 38).\n\nI am particularly interested here in sociologists who have analyzed love as a fundamental component of the family and the conjugal couple (Cicchelli-Pugeautl, 1999, p.58). Thus, for Luhmann, the family is ordered based on a particular code of symbols, one being love (2008, pp.24, 203), and since the 18th century the unity between love and marriage has been observed as a principle for the perfect fulfillment of the human being. François de Singly, for his part, has drawn attention to the central role of the family in the process of revealing the self, of building individualized identity (2016, p.28) and, according to Ulrich and Elizabeth Beck, the family has moved from being a working community to one of feeling (Beck and Beck-Gersheim, 2008, pp.19-76). For the philosopher Alain Baidou, the family builds the state of love (Badiou, 2011, p.72) and according to Bruckner and Finkielkraut, behind this state of love there is the desire of the institution to conjure up chance (Bruckner and Finkielkraut, 2001, p.327). While other social thinkers have emphasized the transformations that the modern family is undergoing, as is the case with Marx, who prophetically anticipated that the family, once the process that will lead teleologically to communist society is concluded (in this he was wrong), will be formed by a couple united by pure erotic-individual love (González, 2009, p.513). Durkheim, for his part, understands that due to the ‘law of progressive contraction’, the family will diminish in size as the division of labor in society grows (Segalen, 1992, p.28). Although the Frankfurt School does not defend the disappearance of the family, it considers that the bourgeoisie has come to an end, which has led to the emergence of other family forms, basically expressive and egalitarian (González, 2009, p.524).\n\nTherefore, as suported in the claims above, the family constitutes a great expressive and equalizing force and builds on the state of love. Conversely, love is a fundamental component of the family and the conjugal couple and love then becomes its basis: love is family and family is love.\n\nAs for the types or styles of love, there have been various classifications made by social scientists. One of them proposes that, in summary, today there are two models of affective-sexual relationships - traditional and alternative - but they are paradigms of “ideal type” behavior (Gómez, 2008, pp.63 ff.). Another of the most accepted categorisations in sociology is that proposed by Anthony Giddens in The Transformation of Intimacy: Sexuality, Love and Eroticism in Modern Societies, where he highlights the two variants of modern love: the “romantic” and the “confluent” (2006, pp.43-63).\n\nThe first of these emerged at the end of the 17th century, and was defined by sociology as the First Modernity, from 1890 to 1973, after which the Second Modernity then begins (Bell, 1992, p.117). This is a legacy of the “courtly love” of the feudal system (Elias, 1994, p.325 ff.) and the “passionate love” characteristic of the absolutist aristocracy, linked in turn to the Platonic “eros” and the Christian “agape”. However, as will be discussed, this romantic form of love is still very strong today, particularly in Spain, where it relates to the majority of the population because the current patriarchal and capitalist system effectively reproduces it through the norms, beliefs, models, customs, myths, traditions, morals and ethics of the culture of which Spanish citizens are a part (Herrera, 2018, p.10). Moreso, literature, cinema, and television soap operas represent it, revive it, and re-actualize it (Elias and Dunning, 1996, pp.94-95), in the same way that the sacred rites of the church keep Catholic religiosity alive. It is not surprising that this type of love contains a literary component, even melodramatic, linked more to the mental than to the carnal, more to passion than to sex, and that, consequently, is an ideal, which usually is never completely fulfilled. Without forgetting that it constitutes a pure relationship that restructures intimacy and is characterized by the fusion of the couple, in such a way that the other ceases to represent otherness. The spouses renounce being individual beings and, simultaneously, separate themselves from their broad social context, for example, from institutions or the friendship group, and even from everyday life. In this way, romantic love creates a shared, relational, and non-individual story that, like fiction, flees or duplicates the world.\n\nHowever, paradoxically romantic love is interrelated with the idea of the free choice of the couple and with the consequent conception of freedom and individual self-fulfillment. This vision reaches its maximum ardor (i.e. this love is very intense) in women, who believe that they obtain maximum happiness through marriage, the ultimate sustenance of romantic love. The latter gives them independence and identity, while at the same time, with the affections and emotional ties it generates, it almost reaches the sublime, an almost sacred feeling. In contrast, in men, this idea of freedom and self-realization is manifested through success and professional development. That is, while the husband, with his external function in the family, mainly out at work, and the instrumental role that this work gives rise to does not concern himself with expressive roles, which instead are adopted by the wife. Hence, romantic love is feminized, not in vain, it seems - as Giddens (2006, p.43 ff.) points out - that it has been an invention of women, like motherhood and childhood, from the changes produced in the home itself, to the extent that it is transformed from a space of production and reproduction to another in which affection dominates. Women have indeed empowered themselves in the home, have taken control of its administrative and economic management, and have adopted a greater weight in the education of minors. These facts have led to the weakening of the traditional patriarchal functions and, ambivalently, to the parallel reinforcement by them of the reproduction of the patriarchal and capitalist system.\n\nFinally, romantic love implies a continuous search, a work (Gómez, 2008, p.49) and in Erich Fromm’s (2007) vision an artistic expression, a day-to-day work, a cultivation (Cruz, 2010, p.187), and a total dedication which constitutes a process that has no purpose. However, it is projected into the future, insofar as it is a love that seeks to last a lifetime and that, if it could, would even border on transcendence and immortality.\n\nConfluent or “liquid love”, characteristic of the Second Modernity, is defined sociologically with at least five characteristics (Giddens, 2006; Beck and Beck-Gersheim, 2008; Costa, 2006, pp.775-778; Bauman, 2007, pp.32 ff.; Illouz, 2009, p.205):\n\n1. It pursues the free and not closed construction of the individual members of the couple, not the fusion - as romantic love does - because what it seeks is to open up to the other and, therefore, to understand him or her. It is defined, therefore, by individualism (Simmel, 1986, p.96; Elias, 1999, p.49; García Andrade, 2015, p.55), by the self-confirmation of the self or by the self-realization of the individual (Berger and Kellmer, 1993, p.226). In short, it is defined by the defense of individual autonomy (Ayuso, 2015, p.90).\n\n2. It presupposes the equality of its members and requires that, in the interrelationships between them, there is reciprocity or “intrahuman interpenetration” (Luhmann, 2008, pp.233 ff.). For this reason, it is a more democratic, more “negotiating” love than the more aristocratic and authoritarian romantic version.\n\n3. It builds lighter personal ties and, consequently, fragile, “liquid” (Bauman, 2007), contingent, ephemeral, and uncertain relationships. Thus, although it may exist today, it does not necessarily do so tomorrow and hence is intense and active, without this meaning that it lasts forever; indeed, it is an instantaneous love. As undesired effects of this inconsistency, there is an increase in separations and divorces, while the loving subject becomes a disposable consumer object, i.e. a commodity determined by the frenetic rhythms of the capitalist system.\n\n4. It is a risky love, in that it assumes a whole series of dangers and insecurities that put its own survival at stake, since it escapes from the protection and control of the family and since those in love invest in feelings, in economic, symbolic and value capital, following the economic laws of supply and demand. However, there is neither profit nor happiness guaranteed - especially in the case of women (Jónasdóttir, 1995, p.314).\n\n5. It highlights the ars erotica, sexuality, although this decreases over time, with its short duration. However, it is not exclusively linked, like romantic love, to heterosexuality.\n\n\nThe Spanish family and the individualistic model of Northern Europe\n\nNext, I will discuss how the two modern forms of love - romantic and confluent - are institutionalized, formalized, or “familiarized” in Spanish society. Specifically, I will first contextualize these two types of love in the unique Spanish family model and its family structure. I will then explore if and how the two forms of love coexist, to reveal which is the primary and which is the secondary form.\n\nIt has been pointed out that, in Europe, there are two different family care models between the North and the South (Holdsworth, 2000, p.201). In the first, the “Bismarkian”, there is explicit government support, through specific fiscal and social policies, for the role of the family as a welfare and care provider, mainly through women’s unpaid care work. In the second, the “Mediterranean” model, families carry the burden, replacing the absense of public or state intervention. The existing literature so far has defined the Spanish model as Mediterranean or “familist”, due to the primacy of the family in the protection of dependents. However, recently the Spanish model, in general, and the family principle in particular, is being revised (León and Migliavacca, 2013, pp.25, 35). It has even been pointed out that the Spanish welfare state based on familism may be in crisis. This is the result of the fact that the Spanish family has undergone numerous changes linked to the transformations of society, principally with the economic and industrial development initiated in the 1960s and with the transition and consolidation of democracy. Specifically, there have been numerous external economic, social, cultural, and political variations that have had an impact on this institution. Next to these, there are also internal demographic metamorphoses, in attitudes and behaviors, in the rights and status of family members, in couple relationships and those of parents and children, in marital harmony and couple symmetry (Iglesias, 1990, p.236; Carrasco, 1997, p.90; Del Campo and Rodríguez-Brioso, 2002, p.104).\n\nThese profound economic, cultural, and social changes in Spanish families have influenced the transformation of the dynamics of family solidarity (Cais and Folguera, 2013, p.573). Indeed,\n\n[f]amily exchanges may be becoming less driven by duty and more open to individual variation, so that personal affection and attachment may be increasingly important for family cohesion and intergenerational ties. Normative obligations remain in place but may be increasingly being modified by affection and choice so that relationships within the family are being transformed (Lowenstein and Daatland, 2006, p.219).\n\nConsequently, while intergenerational relationships and solidarity within the family still enjoy social prestige and while a strong and ingrained social custom survives, people’s preferences for family care are less driven by duty and more dependent on affection and personal attachment. As a result, Spaniards no longer feel an inescapable moral duty towards their dependent parents, and they do not spend as much time caring for their relatives as they once did. On the contrary, normative beliefs have become more flexible and have adapted to new social realities such as gender equality and the increase in women’s participation in paid work (Cais and Folguera, 2013, pp.558-573).\n\nFrom the mid-1990s to the mid-2000s there has been strong growth in female employment in the country from 20% in 1992 to 52% in 2010, with the female employment rate (between 15 to 64 years old) increasing from 46% to 64% between 2004 and 2008. Besides this, there has also been a notable increase in their level of education, especially among the younger generation, from 8% in 1987 (plus another 16% of those in primary school) to 30% in 2007, without forgetting that those reaching tertiary education increased from 6% to 16% (Salido, 2011, p.190; León and Migliavacca, 2013, pp.29-31).\n\nThus, in 20 years Spain has seen an increase of 25% in female workers and has gone from being the country with the lowest number of women in the workforce in the European Union to having a volume of female labor comparable to other countries such as France, Germany or the United Kingdom. As a result, the number of women who are exclusively “employed” in the home and who form the basis of the family has halved over the last two decades. Thus, if in 1988, 42% of those women were dedicated only to household tasks, in 2008 this figure has fallen to 23% (Cais and Folguera, 2013, pp.561-562; León and Migliavacca, 2013, pp.29-30).\n\nConsequently, since the 1990s, and decisively facilitated by mass migration - a key element in the configuration of care systems in Southern Europe - a rapid process of commoditization of care work has also taken place, although still within the confines of the home. This is also in line with common trends in other European countries (León and Migliavacca, 2013, p.38).\n\nLikewise, the Spanish family model has converged with that of the European family due to its immersion in the individualization society (Meil, 2003, p.2 ff.; Meil, 2004, p.421). Indeed, there has been an increase in individualism and in the ethics of personal self-fulfillment (Flaquer, 1991, p.70), especially in women, who have moved from a life model oriented towards family service to one in which they assert their right to have their own professional career (Cais and Folguera, 2013, p.561). This has led to the predominance of individual interests and rights over those of the family institution (Mora, 2012, p.102). This means that the variety of lifestyles is wider (Cais and Folguera, 2013, p.561) and that what is really at stake is the place of the individual within society (Del Campo, 2004, p.452). In other words, the lives of citizens are no longer planned by society, which has forced them to build their own lives, with all the risks that this new situation entails. Therefore, from having a conventional biography, more robust and built over time, they have come to have a more flexible one, something which denotes a greater fragility in relationships and the loss of certainty (Beck, 1988, p.217).\n\nAs a result, over the last three decades, the ability of the family in Spain to care for its dependent members, especially the disabled and elderly, has been affected by two transformations. The first is that changes in family structure - particularly the increased participation of women in the labor market - have been accompanied by the changes experienced in family dynamics. This is because people’s preferences about how to care for dependents are now less motivated by moral duty and more by a sense of responsibility that can be addressed by different means, as well as by affection and personal attachment (Cais and Folguera, 2013, pp.560-561). Hence, it has been written that the inherited social rules that have defined the structure and roles of family members in the past have become obsolete and that the figure of the woman dedicated exclusively to domestic work and the care of children, parents, and husband is dying (Mari-Klose, 2006). It is not surprising that the merging of employment and education levels among the younger cohorts of women during the 1990s and the first half of the 2000s, which is much more pronounced in the Spanish case than in other European countries, has been interpreted as a move away from the “southern male breadwinner” model to the northern adult worker model; that is, Spain has moved away from “southern” norms and towards “northern” ones. In a similar vein, it has also been argued that Spain has made the transition from a “male breadwinner model” to a more diversified pattern of family formation concerning women’s participation in the labor market. It is further argued that this male ideal of a breadwinner has long since disappeared and that no one seems interested in regaining it (León and Migliavacca, 2013, pp.25-36).\n\nHowever, the process is not as linear as it seems, as it is much more nuanced and complex. First of all, despite the variations in Spain, as all the specialists insist are happening, the family continues to be one of the “friendliest” institutions in Spanish society and is shown to be the most highly valued aspect of life, as the degree of satisfaction with family life and marriage is high. In fact, it is the most important institution in the lives of Spaniards, insofar as 90% of them declare themselves to be fairly or very satisfied with their family life (Sánchez and Bote, 2008, pp.201, 204). Hence, living together as a family is still the most widespread living arrangement, with approximately 86.5 % of households being family-based, the highest rate in Europe (Del Campo and Rodríguez, 2002, p.108). Furthermore, the population’s complacency with the functioning of the couple’s relationship is quite considerable; conflict in marriage is relatively low, marital harmony is desirable, marital break-ups have not reached the European level (Del Campo and Rodríguez, 2002, pp.103-142) and marriage remains the step which comes before procreation (Delgado, 1993, p.126). This explains why the Spanish family continues to be determined by the intensity of relationships (Sarrible, 1995, p.48); by a type of interpersonal relation characterized by sociability, proximity, physical affection, and intergenerational family structures (Lavell et al., 2020, pp.6-8). Likewise the Spanish family is determined by an inherited cultural dimension, based on family solidarity and on the structure of kinship, in which societal norms continue to be important (Holdsworth, 2000, p.201; Moreno, 2001, pp.1-5).\n\nAlso, Spain, together with Greece and Portugal, is the European country where the percentage of people over 65 living in single-parent households is lowest: less than 20% in 2001. Moreover, the percentage of people over 65 living with their children is also higher in Spain (17%), Ireland (15%), Greece (15%), Italy (14%) and Portugal (12%) than in countries such as Germany (1%), Denmark (0.3%), France (5%) or the United Kingdom (6%). It can also be said that families continue to care for people in need. In 2008, for example, 55% of people in need of care had this provided by their families, mainly by children and, secondarily, by spouses (Cais and Folguera, 2013, p.562).\n\nTherefore, in Spain the family continues to be the source of the most support and care for the elderly, while public services are so scarce to the extent that some families are not familiar with them. Actually, the care expectations of elderly dependents are focused on the family and not on social services (Cais and Folguera, 2013, pp.562-564; León and Migliavacca, 2013, p.37). Finally, the 2008 crisis brought a clear decline in female employment, indicating that the level of change developed over the last two decades towards a model of adult worker has encountered a strong obstacle (León and Migliavacca, 2013, pp.32-35). We still do not know how this situation will end, particularly after the coronavirus crisis.\n\nIn sum, this transformation of the state of the family is likely to manifest not as an unequivocal trend towards individualization, but rather a reformulation of the concept of family in terms of roles, functions, and face-to-face relations with other institutions (Daly and Scheiwe, 2010 in Daly, 2011, p.18).\n\n\nFive basic structural aspects of the Spanish family\n\nWithin this complex general context, the structure of the Spanish family is characterized by at least five basic aspects: its progressive reduction and de-institutionalization; its heterogeneity; its privatization; its negotiating capacity; and the plurality of its forms (Meil, 1998, p.188; Meil, 2006, pp.11-38; Del Campo, 2004, pp.456 ff; Ayuso, 2015, p.76). Numerous works, both theoretical and empirical, confirm this basic mapping of the Spanish family and simultaneously deepen and nuance its structure while making it possible to reveal the type of love that prevails. I will now discuss these two concepts in further detail.\n\nThe Spanish family is becoming increasingly smaller. In 1970, the average size of a Spanish family was 3.81 people, in 2000 it was around 3.7, while in 2017, the Short-term Fertility Indicator is 1.31 children per family (Eurostat, in Del Campo and Rodríguez-Brioso, 2002, p.105). This means that, on average, the Spanish family has only one child, thus being made up of the father, the mother, and a child, and therefore, the population is not being renewed and is instead decreasing. However, this decrease in the number of members per family unit reflects the slow decline of the nuclear family and its replacement by new forms of cohabitation. Indeed, in addition to the traditional family, which in Spain could be made up of four children or more, other family forms emerge, e.g. families with one or two children or those with no descendants: in 2000, 19% of couples had no children (Del Campo and Rodríguez-Brioso, 2002, p.110). In this sense the decline experienced from 1998 onwards is the result of the increase in single-parent and single-child families. Therefore, the family in Spain is gradually becoming ‘minimal’, reduced to its minuscule expression, a process which seems to have reached its peak.\n\nIn any case, in the end this reduction is an exponent of the contemporary “eclipse” of the Spanish family, or its deconstruction. This is motivated by the passage from family to personal autonomy, by the diversification of types, by the evolution of marriage models, by the conversion of marriage into an individualistic one, with a life phase rather than a life project, and by dismantling or deinstitutionalizing the nuclear family (Flaquer, 1991, pp.69, 72). In fact, the latter has gone from being a public to a private institution, from closed to open, and from the single model to take a variety of forms (Del Campo, 2004, pp.454-8), similar to what has happened in Northern Europe (Meil, 1998, p.188). Together with this, the family has also suffered a loss of centrality in today’s society, since it has ceased to be the primordial institution and has become just another one among others, with few functions of its own: among them, fundamentally, that of being the seat of affection and the socializing agent of individuals (Del Campo, 2004, p.453).\n\nIn any case, this phenomenon affects the intensity and quality of the relations between its members and, particularly, between the spouses, who are, of course, focused on the education of a child, although they also have enough energy left – which is projected as love - to devote to themselves.\n\nThe Spanish family is also increasingly heterogeneous, mainly due to changes in their models and their formation processes (Delgado, 1993, p.123), in the formalization of couples (Iglesias, 2003, p.15), in gender roles and in the affective forms of love. This is expressed through four factors: the pluralization of the ways of living together, the greater heterogeneity in the participation of paid work, the superior heterogeneity in domestic tasks and in the care of the children, and the increasingly heterogeneous work roles inside and outside the home. Indeed, plurality is the most dominant form of cohabitation between couples. Thus, in Spain there are de facto couples, extended families, functional or flexible, nuclear or nuclear-marital, single parent, homoparental, bicultural or multicultural (transnational), simple mixed, complex mixed, single parent, and simultaneous or reconstituted (Rondón, 2011, pp.82-5). In this pluralization of ways of entering into, remaining in, and leaving family life, however, it should be pointed out that marriage, as a way of living a common life, has been losing its binding force - the marriage rate was 7.50 in 1950 and decreased to 5.12 in 2001 - and even appears to be void of ritual significance (Del Campo and Rodríguez-Brioso, 2002, pp.117-8, 161). However this does not mean that its social acceptance has deteriorated (Meil, 2004, p.430). In the same vein the number of nuclear Spanish families has seen a reduction: in 1968, 66% of families were comprised in the form of a nuclear family, in 2001 the percentage fell to 54%, in 2004 the number fell to 50% and in 2018 it reached a low of 33.9% according to the Spanish Office for National Statistics (INE). Despite the declining numbers, this family form is still the majority, although it is decreasing and approaching European levels -in 2011, the percentage of European nuclear families was 33.2% (Eurostat). The number of unmarried couples has also been increasing in recent years, approaching 12% in 1998 and 15.7% in 2019 (INE), although this may be associated with the absence of a family project, the lack of professional stability, and the fact that it is harder to own a home together. If this were proven to be the case, it would constitute a transitory form pending its regularization as a new form of nuclear family (Meil, 2004, pp.435, 446). As for single-parent families, those in which the responsibility lies with the mother have increased by 41%, whilst single-parent families under the responsibility of the father have increased by 60%: in total, in 2018 they accounted for 10.1% of families (INE). For their part, one-person families – who made up 25.5% of the total number of families in 2018 (INE) have grown by a high percentage (INE)1, especially in large cities where more people live alone2, especially older women (INE)3. Households that consist of only an adult couple without children make up 21.1% of the total and those households in which the family nucleus takes in people from outside the home constituted 4.3% of the total in 2018 (INE, 2018).\n\nWith regards to the participation of fathers in paid work, the incorporation of women into the labor market - particularly in sectors that have not traditionally been female friendly - increases the heterogeneity of family members present in the labor market, which until then had been composed only of men. Thus, as suggested by Alamillos Guardiola, in half of Spanish households, both spouses are working, but half of the women are unemployed, as is the case in all the Mediterranean countries, which mainly affects those women with less education. In contrast, urban and middle-class women have more work than those who live rurally and that of lower-class women. On the other hand, there are more part-time working mothers than fathers, due to the incompatibility between motherhood and their professional career (Alamillos Guardiola, 2016, p.219).\n\nWith regards to heterogeneity in domestic work and childcare, despite being in a country with one of the highest equality awareness rates – 83% of citizens agree with it – it is not put into practice (Meil, 2003, pp.1-16; Meil, 2006, p.11 ff.). Domestic work is still unevenly distributed (Ajenjo and García, 2014, p.467), with women spending 38 hours a week doing domestic chores and men barely reaching 23 hours (INE, 2016). Regarding childcare, the distribution of hours is more equal, as both parents dedicated 16 hours per week in 2016 (source INE). Therefore in general, women continue to devote more hours to domestic chores, although men have been able to devote more hours to childcare. It is precisely the education of children that is the task most accepted by parents and the best distributed of all, although it does not include all responsibilities such as going to school to talk to teachers (Meil, 1997, p.3). Moreover, another interesting observation is that in 40% of families where both spouses work outside the home, domestic tasks are shared, although not entirely equally. This generates conflicts within the couple, although the females do not demand the domestic participation of the males in order to prevent creating further conflicts because this would be considered as questionning the established gender roles so that they end up doing more domestic chores Finally, concerning the heterogeneity of paid and unpaid work, in short, women continue to spend the most time working at home, which means that those who work both domestically inside and professionally outside the home, in a large number of cases resort to taking anxiolytics. Indeed, various surveys indicate that more than 50% of Spanish women take anxiolytics and that one of the most important causes leading them to do so are gender asymmetries, work-related asymmetries between women and men, and the fact that women are more likely to take anxiolytics than men (Martínez, 2003, p.253 ff.).\n\nThe Spanish family has also privatized daily life, behavior, and morals (Flaquer, 1991, pp.68-71), as a result of the questioning of social norms and the relaxation of social control, which has created greater freedom and growing egalitarianism among its members (Rondón, 2011, p.87; Ocón, 2006, p.172). Within this privatization, sexuality stands out, a private expression and personal fulfillment that is not conditioned but rather disconnected from reproduction and marriage (Delgado, 1993, p.124; Del Campo, 2004, p.459; Sánchez and Bote, 2008, p.209). In this sense, there is a tendency in families to differentiate between love and sexual fidelity (Meil, 2004, pp.424-6).\n\nAll this has meant, on the one hand, that despite significant and profound progress in the cultural, economic, and political fields, Spain has retained a very traditional family system in the context of a democratic society (Flaquer, 2002, pp.84-85). But, at the same time this traditional family is also becoming more flexible (Mora, 2012, p.103) and is even being transformed from patriarchal or matriarchal authoritarian to democratic which entails a more peaceful and negotiating character (Ocón, 2006, p.172; Meil, 1998, p.193; Rodríguez-Brioso and del Campo, 2002, pp.133, 169). This transformation is due, in turn, to a variation in the relations between spouses and in the paternal-affiliation relations, moved by the increasingly emotional and affective interrelations between family members, as has been seen with the care of the elderly. The family of the 1950s was based on discipline, on family control rooted in an ironclad and authoritarian upbringing, as well as on the imposition of rules and the “domestication of beasts”. In contrast, today’s family is less rigid and more emotional and between the members of the couple and their children, the imposed rules have been replaced by negotiation and shared decisions taken jointly:this was the case for 64% of families in 1995 (Rondón, 2011, p.88).\n\nIn short, these five major transformations of the Spanish family have broken with the traditional system, which could be summarized in two major transformations: interculturality and gender equality (Rondón, 2011, p.88). However, for all that has been said both with regard to the family model of care and the structure in which it is implemented, the deepening of the social reality of our families is not without ambivalence or, as L. Flaquer, one of the most important experts in the sociology of the Spanish family, points out, it is “paradoxical”: a family paradox (2002, pp.84-85). This explains how, although the family has reduced its institutional charactertistics, it has retained its importance and significance on a factual and ideological level (Alberdi, 2005). On the other hand, its de-institutionalization, minimalism, and fracture in a multitude of forms may make us think that the family, although it refuses to disappear, does not seem to have much of a future in Spain. (Del Campo, 2004, p.451).\n\n\nThe coexistence of the two types of love - romantic and confluent - with other models in the Spanish family\n\nThis resistance and fragility of the Spanish family is not, however, the only ambivalence. As will be discussed, at least two other ambivalences are evident: love put into practice is ideal or real and the ideological system it expresses is, respectively, patriarchal, or democratic. For this reason, the analysis of the formalization, institutionalization, and “familiarization” of love is twofold. This is because permanence and change go hand in hand; the replacement of the old alliance between families by love as the constitutive foundation of marriage, has given rise to a dynamic in the social structure, towards an increasingly plural, negotiating, heterogeneous, privatized, less institutionalized and more minimalist family, as has just been described in the previous section. Thus, simultaneously, there has been both the preservation of the authoritarian patriarchal system and the opening up to a more democratic one, characterized by a mechanism of upward social mobility and by the weakening of the stability of the family institution itself (Meil, 1998, p.191). This explains why, on the one hand, the ideological structure of the Spanish patriarchal family has changed little or less rapidly in practice (García de León, 2009, pp.209 ff.; Castells and Subirats, 2007, pp.22-5). On the other hand, even though family culture is inserted into the Second Modernity, the same has not happened with affective behaviors (Meil, 2004, p.432), more characteristic of the First Modernity.\n\nI delimit the scope of this contradiction by analyzing several empirical investigations and contrasting them with the theoretical statements made, which I have echoed in the characterization of the care model and the Spanish family structure. It has been stated that the Spanish family has been transformed in the Second Modernity, since, in general terms, the first family modernization was characterized by permanence, by the conditioned choice of marriage, by cultural uniformity and homogeneity. On the contrary, the second modernization is imperishable, develops a personality, freely chooses the couple, and its members adopt a more egalitarian and shared responsibility (Rondón, 2011, p.89). As Anthony Giddens argues if the First Modernity is defined by romantic love, the second one it is defined by confluent love. It has also been claimed that the patriarchal family has changed to another more egalitarian and democratic one, detached from the structural parameters and transformed into a cultural form that coexists with diverse patterns of coexistence (Del Campo and Rodríguez, 2002, pp.103-133).\n\nHowever as we will see below, the situation does not evolve so linearly, without forgetting that in the Second Modernity both forms of familiarized love coexisted. In this sense, in Spain today we are faced with two basic family models: one more institutional - based on romantic love - and another in which there is more choice, which is emotional and unstable and more conditioned by the satisfaction of personal needs (Alberdi, 2005). The latter model corresponding to that of the “negotiating family” (Meil, 2003, pp.1-16; Meil, 2006, p.11 ff.) is founded mainly on confluent love.\n\nThe first model emerged at the end of the 19th century when the connection between the concepts of romantic love, marriage, and sexuality began and is still present today. This type of love constitutes the main reason for maintaining fidelity, for the intention to marry and to form a family (Del Campo and Rodríguez Brioso, 2002, p.119) and for marital relationships more broadly, since being in love becomes the foundation for initiating and maintaining a couple (Ferrer et al. 2008, pp.589-590). Thus, in 1995, 67% of the Spanish population understood that an authentic relationship lasts a lifetime, while 76% considered that true love is omnipotent, that when in true love one can cope successfully with any situation, no matter how negative it may be. On the other hand, in 2002, 70% of Spaniards agreed that romantic love can exist without marriage (Del Campo and Rodríguez Brioso, 2002, pp.119-120).\n\nIn Spain, the model of romantic love and the myths associated with it are those that prevail in our society. Indeed, this model continues to show great strength in female socialization, becoming its backbone and priority as a life project, as women turn to the private sphere, while men turn to the public sphere. For women then, the most important thing is social recognition, with love taking second place. As an example of this, in an empirical study by Ferrer et al. (2008, pp.589-592), it is concluded that the “Eros” style or passionate or romantic love is the most accepted by more than 80% of the people interviewed and this is true of all groups, both men and women. It is therefore evident that in in Spain there is a high valuation of romantic love (Ubillos et al., 2001).\n\nTo reinforce this concept, it is worth checking the degree to which romantic myths have become established in Spain. The fact is that these myths, which are beliefs with a strong emotional charge, which concentrate many feelings and tend to create and preserve group ideology, are resistant to change and reasoning. Besides, they constitute “the set of socially shared beliefs about the supposed “true nature of love”, and, therefore, normally the romantic myths are “fictitious, absurd, deceitful, irrational and impossible to fulfill” (Ferrer et al., 2010, p.7). In a 1995 interview carried out in Spain by the Centre of Sociological Research on the presence and social acceptance of myths about love, it was found that those consulted, in general, agreed or very much agreed with these myths (Barrón et al., 1999, p.66 ff.). Specifically, “the myth of the couple” is accepted by 95% of the population interviewed; that of “fidelity” by 80%; that of “omnipotence” by 75%; that of “marriage” by 67%; that of “eternal passion” by 63%; that of “exclusivity” by 55%; that of “the better half” by 51%; and the myth of “the equivalent” is accepted by 45% of the population questioned (Table 1).\n\nSource: (Ferrer et al., 2010, p.13-14).\n\n(*) This myth has not been analyzed in this paper.\n\nAnother empirical work4 (Ferrer et al., 2010, pp.16-29) about the establishment of romantic myths in Spain shows that the acceptance of the myths of “omnipotence” (73%), “the eternal passion” (72.3%), “marriage” (71.3%) and “the better half” (52.6%) dominate (Table 2). In this research conducted by Ferrer et al. (2010), the number of couples is measured through cohabitation and the acceptance of those myths of “omnipotence”, “marriage” and “pairing” and it shows the level of satisfaction with the couple’s relationship and the acceptance of these same myths. Consequently, romantic love is a very widespread experience, which is linked to the persistence of a series of clichés rooted in a traditional, romantic conception of love that helps to preserve the power structure and inequality of love relationships. It also connects that romantic vision with the majority of couples and marriages in Spain.\n\nThe total number of people interviewed is 1351 (Ferrer et al., 2010, pp.11, 16).\n\nIn a recent empirical sociological essay (Rodríguez-Santero et al., 2017, pp.1-13), addressed to university students in Seville about love, it is concluded that the interviewees have an idealized and romantic concept of love in which sexuality and the more passionate or attractive aspects are secondary. Thus, in this work, the most accepted style of love, the “Agape”, the altruistic love oriented to the good of the other and of renunciation, which understands the relationship as a denial of individualism and as a process of abnegation or voluntary relinquishment of one’s desires, affections or interests for the benefit of the loved one: surrender and sacrifice and the phrase “for life” corresponds to the parameters of romantic love. Again, it should be remembered that the ideology behind this type of love expresses a form of modern patriarchy. However, in their conclusions, the authors stress that “we cannot confirm that these statements correspond absolutely to reality”, that they are more “perceptions” (Rodríguez-Santero et al., 2017, p.11), that is, that they have a great imaginary component coated with a strong idealism.\n\nTherefore, according to these empirical works, romantic love dominates in the perception of Spanish behaviors and mentalities. Therefore, I will now try to corroborate, through the analysis of two other empirical studies, how romantic love is formalized, familiarized, and institutionalized, if at all, through the different family models existing in Spain. In the 2006 study by Navarro (2006, pp.123-135), the results show that of those surveyed, 45% identify more with the ideal model of the “symmetrical family” (see Table 3): an egalitarian, democratic and tolerant model, the negotiating family, with less weight on conventions and traditional customs. Of those included in the survey, 27% were in favor of the traditional model, the “lifelong” one, rooted in romantic love. However, the “intermediate” family - which makes up 23% of those included in the study - perhaps a mixture of the traditional family and symetric family, is a type of family that could be called paradoxical, since it simultaneously combines characteristics of the other types of family without overtly opting for either of them. In any case, the symmetrical, triumphant model is ideal, since day-to-day behavior and social practices, despite progress, do not conform to this desire for equality, as we have seen in the section devoted to the characteristics of the Spanish family. As is evident, the traditional model continues to be of considerable importance.\n\nSource: Own elaboration based on Navarro 2006, p.121.\n\nSource: Own elaboration based on Elzo 2004, p.220.\n\nThe second work devoted to Spanish family models is especially useful to the research on this topic. It was written by Javier Elzo (2004, pp.205-229), for the Foundation for Support for Drug Addiction, 2002 (FAD) and although it focuses on parent-child relationships, it also addresses issues that can help us to infer vital information on the couple’s emotional interrelationships. Specifically, it reveals the existence of four types of families in Spain (Table 3). In the “nominal” family, which I refer to as a “cold family” because of its non-emotional characteristic, which represents the majority of families in Spanish society (42.9%), the parent-child relationship is one of peaceful co-existence rather than one of participative cohabitation. Moreover, family members communicate little and do not participate in common objectives, as they have decided to do in order not to create conflicts. The “adaptive” family model (18.4% of those surveyed) is the most recent and the most modern (Second Modernity) and is the model that best reflects family tensions. This is the negotiating family I referred to earlier, as it is the one with good communication and continuous role review. However, there is a strong risk of ruptures due to misunderstandings within the couple and between the couple and the children. The most traditional model within this is that of the “family and familistic and endogamic” family (23.7% of those surveyed), in which the parents have strong identities. Furthermore, this model is closed and such families are unconcerned with the problems of the world, although good relations and a warm home enviroment full of affection among family members do also exist within this model. Finally, the “conflictive” family model (15% of those surveyed), which I call a “dramatic family”, is the one in which there is the most conflict (mainly due to drugs and sexual issues, relations between siblings, and between parents and children).\n\nJavier Elzo does not quote the term “romantic” to refer to the four models and, indeed, it does not seem appropriate to define them as such, except for one of them: the “family familistic and endogamic”, the second largest, whose characteristics resemble some elements of romantic love. The “adaptive” family (the third most extensive model) does clearly refer to the negotiating family, while the majority family, the “nominal” one, is not defined by its enthusiasm or love intensity, nor is this the case in the less established model, the “conflictive” family.\n\n\nConclusions\n\nIn this article, I have tried to confirm how the two types of modern love are institutionalized, formalized, or familiarized, firstly by putting them into the context of the Spanish family structure and, secondly, by revealing how the two coincide, as well as denoting which is the primary and which is the secondary form. In this respect, the Spanish care model and family structure tend towards individualism, but not without resistance from the old patriarchal system. Thus, the analysis of numerous works, both theoretical and empirical, has allowed me to confirm the five great transformations of the Spanish family structure: progressive reduction and de-institutionalization, heterogeneity, privatization, negotiating capacity, and a plurality of forms. All of these transformations demonstrate that the Spanish family is indeed immersed in the general European context of individualization but with unique nuances and markedly ambivalent characteristics since the Spanish family is situated halfway between the traditional and the modern, the communal and the individual.\n\nThe same goes for love, which is also subject to the same process of reduction and individuation. Concerning the two types of love in Spanish families, four final considerations have been inferred:\n\n1) It has been revealed that likely the most institutional love - the “familistic and endogamic family”, based on romantic love - and the prevalence of the myths associated with it are still common in Spain, according to five of the six studies. If these studies are correct, this love would manifest in a series of clichés rooted in a traditional romantic conception of love that contributes to preserving the power structure and the inequality of love relationships. Alongside romantic love, there is the majority form according to Ferrer et al. (2008) empirical works. This form corresponds to the “negotiating family”, “adaptive” or “symmetrical” model, and is mainly based on confluent love. Finally, there are two other institutional forms of love: the “nominal” and the “conflictive” family (Elzo, 2004).\n\n2) However, as those researchers who were consulted recognize, although the desire for romantic love is widely established and is the majority in Spanish society, it is more of an ideal, more of a perception than a practice. This is also the case with the “symmetrical” or negotiating family and confluent love, which expresses an ideal of equality not yet achieved in social practice. In the case of “nominal” and “conflictive” love, they do not seem to exhibit any idealization, but on the contrary, these depict a crude reality in which, although the family is sustained, it does so without the affective quality it would require, since either communication is annulled in the first case, or it is dramatized and problematized, in the second case.\n\n3) The familiarization of the forms of love in Spanish society (love is, by nature, ambiguous) are therefore determined by ambivalence, as the two basic types of love - the romantic and the confluent - coexist. With them also coexists two ideological conceptions of love, patriarchal and democratic; the first being the most accepted by Spanish society today, while the second continues to develop in terms of familiarity and popularity.\n\n4) This contradicts the evolutionary linearity expressed by some specialists who understand that the Spanish family has gone from being romantic in the First Modernity, to confluent in the second. However - as I have noted here - in the Second Modernity, both modern types of love and familiarity coexist, along with two others: the “cold family” and the “dramatic family”. Added to this is the contradiction that, although the evolution of the family structure places the Spanish family within the Second Modernity, the same does not seem to have happened with affective interrelationships, more typical of the First Modernity; that is, it is a legacy of the mentality and family structure of Spanish patriarchal culture. This means that - in matters of love - the old is not completely over and the present has not been definitively installed, that the present and the past coexist paradoxically without there being a clear differentiation between the two and neither becomes the dominant paradigm. Thereby, this is reinforced by the existence of the other two models, hybrid and undefined, and of poor emotional quality, which - if Elzo is right - could represent, as a whole, most of Spanish society. Nonetheless, they denote the deep disorientation and crisis in which family affairs find themselves today.\n\nIn conclusion, although most analysts believe in the strength of the nuclear family and the positive consideration of marriage in the Mediterranean and in Spain in particular, something is wrong. The fact is that a careful, contrasted analysis of the theoretical and empirical works consulted here has revealed nuances, ambivalences, and contradictions in Spanish society with regard to love and the ways in which it is familiarized. Thus, the Spanish family as a whole could be called “paradoxical”. Indeed, its reduction to the minimum expression of itself, its fragmentation and its emptying of meaning may lead one to think that, although the family institution resists this potential collapse, it is so weakened that it does not seem to have a great future. If I am right, then it would be worth reflecting on the future of the emotional ties of the spouses on which the current Spanish family is based. Therefore, the weakness of the institution implies - as has been proven - the fragility of the emotional interrelations - of love - that sustain it, since the intensity, quality, and consistency of the relations between its members and, in particular those between the spouses, have been affected.", "appendix": "Acknowledgements\n\nThis article in English is based partly on the French version: Cárcel, J. A. R. (2020). La coexistence de l’amour romantique et du confluent en Espagne. Societes, (3), 87-102. It involves an extension of the work and incorporates new ideas and concepts.\n\n\nData availability statement\n\nNo data are associated with this article.\n\n\nReferences\n\nAjenjo Cosp M, García Román J: Changes in the use of couples’ time: are we on the way to greater equality? RIS (Int J Sociol). 2014; 2 (72), 453–476. Publisher Full Text\n\nAlamillos Guardiola MC: La maternidad tardía: expresión contemporánea del patriarcado occidental. 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Documentos de Trabajo Social . 2012; 51: 99–120.\n\nMoreno L: “Supermujeres” y bienestar en las sociedades mediterráneas. Claves de Razón Práctica . 2001; 111: 49–53.\n\nNavarro Ardoy L: Modelos ideales de familia en la sociedad española. RIS (Revista Internacional de Sociología) . 2006; 64(43): 119–138.\n\nOcón Domingo J: Familia adoptiva y cambios en la organización familiar tradicional. Papers . 2006a; 81: 171–185.\n\nOcón Domingo J: Familia adoptiva y cambios en la organización familiar tradicional. Papers . 2006b; 81: 171–185.\n\nRodríguez-Santero J: Los estilos de amor en estudiantes universitarios. Diferencias de género. RIS (International Journal of Sociology) . 2017; 75(3): 1–13.\n\nRondón García L: Nuevas formas de familia y perspectivas para la mediación: la transición de la familia modelo a los diferentes modelos familiars. In: I Congreso Internacional de Mediación y Conflictología. Cambios sociales y perspectivas para el siglo XXI. Sevilla: UNIA; 2011.\n\nSabido Ramos O, García Andrade A: El amor como vínculo social: con Elias y más allá de Elias. Sociológica . 2015; 86: 31–63.\n\nSalido O: Female Employment and Policies for Balancing Work and Family Life in Spain. In: Guillén AM, León M (Ed.). The Spanish Welfare State in European Context. Farnham: Ashgate; 2011; (pp. 187–208).\n\nSánchez Vera P, Bote Díaz M: Redes sociales y familia en España. Coherencia y debilidades. Portularia . 2008; 8(1): 197–213.\n\nSarrible G: La solidaridad en familias atípicas. Papers. Revista de Sociología. 1995; 45.\n\nSegalen M: Antropología histórica de la familia . Taurus; 1992.\n\nSimmel G: Sociología . Alianza: Estudios sobre las formas de socialización; 1986.\n\nUbillos S, Páez D, Zubieta E: Relaciones íntimas: atracción, amor y cultura. In: Páez D, Fernández I, Ubillos I, et al. (Eds.). Social Psychology, Culture and Education. Madrid: Pearson-Prentice Hall; 2001; (511–535).\n\nWeber M: Economía y sociedad . FCE; 1975.\n\nWeber M: La ética protestante y el espíritu del capitalismo . Península; 1979.\n\nWeber M: Ensayos sobre sociología de la religión. 1987; (Vol. 3). Taurus.\n\nWeber M: Historia económica genera . FCE; 1997.\n\n\nFootnotes\n\n1 In 2015, there were 1,897.5 thousand single-parent households in Spain. In 2020 the number of thousands of single parent families has risen to 1,944.8.\n\n2 In 2020, of the 1,944.8 thousand single-parent families in Spain, 672.5 were from Madrid, and 586.9 from Barcelona.\n\n3 Of the total of 1944.8 thousand single-parent households in Spain in 2020, 1582.1 were women.\n\n4 In this work, the sample was selected from the general Spanish population through a sampling tool stratified by gender and, within each stratum, by quotas based on age. In total, 1,351 people were selected.\n\n5 The end values are the goals you have in life, while the instrumental values are the ones you use in your day-to-day life to reach the end values." }	[ { "id": "90111", "date": "12 Aug 2021", "name": "Adrián Scribano", "expertise": [ "Reviewer Expertise Sociology of Emotions", "Sociology of Body", "Social Theory" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 refers to the connections between the different modalities of love and the types of families associated with them and pointing out their impact on the Spanish social structuring process. In this context, the author intends to: characterize and define the basic features of the \"Spanish family\" in relation to the society of individualization; investigate the forms of institutionalization of modern forms of love, with special emphasis on those known as romantic love and confluent love; discuss if different types of love coexist and how this it's possible and if there is some kind of pre-eminence among them and analyze the strength / fragility of the family institution in relation to the affectivity that sustains them.\nThe author shows in the first section the conceptualization of the practices called romantic love and confluent love as modern modalities of instantiation of love as tensions between different ways of managing the formal / informal, proximity / distance, dogmatisms / flexibility that involves these various ways of affectivity. In this context, the author declares their special interest in love as a fundamental component of the family and the conjugal couple.\nThe section that follows involves the discussion of \"the Spanish family\" in connection with the individualistic family model of northern Europe, where it begins by presenting how the forms of romantic and confluent love are institutionalized in Spain through their familiarization. Said section ends by stating:\n“In sum, this transformation of the state of the family is likely to manifest not as an unequivocal trend towards individualization, but rather a reformulation of the concept of family in terms of roles, functions, and face-to-face relations with other institutions” (page 7).\nThen the article is dedicated to establishing the five structural characteristics of the Spanish family: progressive reduction and deinstitutionalization; heterogeneity; privatization; the modification of negotiating capacity; and the plurality of forms. It ends by holding that these five great transformations of the Spanish family have broken with the traditional system where interculturality and gender equality synthesize the most important modifications for the social system.\nIn the context of their argument, the author proposes three types of family modalities in connection with forms of love: a) Negotiating and democratic family with Confluent love; b) The two types of family are mixed paradoxical family with two types of love life together and c) Patriarchal family and Romantic love.\nThe article concludes that the state of the Spanish family as a whole could be called \"paradoxical\", provoking to think that it would be worth reflecting on the future of the emotional ties of the spouses, accepting that the weakness of the institution implies the fragility of the interrelations emotional - of love - that sustain it, since the intensity, quality and consistency of the relationships between its members and, in particular, between spouses, have been affected.\nThe article offers an adequate review of the literature and an organization of the theoretical instruments in connection with the objectives of the writing, in this sense, perhaps a greater approach to the works from other disciplinary fields of sociology and anthropology could have been included, but this does not affect the development of the argument.\nThe work presents an argumentative structure that goes from conceptualization, through the systematization of empirical information until it reaches a creative hermeneutic of the connection between family types and forms of love where the originality of the author's proposal can be observed.\nThe statistical data consigned are identifiable and easily accessible so that every reader is in a position to validate both its presentation and meaning, in this direction it can be said that in the context of a necessary plural framework of analysis the conclusions are appropriate.\nIn the context of the above, I believe that a way of understanding even more what the author argues in connection with the proximity / distance of the Spanish family organization, the individualization processes and the romantic and / or confluent modalities is possible to be captured by through qualitative information coming from the current “youth popular culture” in Spain:\nThe 'influencer' Naim Darrechi, who boasts of not using a condom and deceiving girls saying that he is sterile, has 26 million followers on TikTok and adds up to more than seven million followers on Instagram, which as is known are the most popular applications among young people.\n\nFarruko has more than 32,000,000 monthly listeners on Spotify and his hit, Pepas, had in June more than 64,000,000 reproductions where it is said repeatedly:\n\"I do not care what they say about me Life you your life, that I live Mine That is only one, enjoy the moment That time is running out and does not go back Drinking, smoking and f***ing\"\n\nIf you search Google Trends for the words love, sex and family between 2016 and 2021 in Spain you will find that the following order of importance love, sex and family and within love the most sought after is the Turkish series \"I rent my love to you\" that Google describes as follows:\n\"Six months is the time a young woman will have to fall in love, marry and leave a successful businessman. The protagonist, desperate to pay off a family debt, will accept the surprising challenge of love proposed by the aunt of the millionaire designer.\"\nThese simple brushstrokes from popular culture bring us closer to those that the author of the evaluated article indicates as a territory to explore the modalities of a politics of sensibilities in a normalized society in immediate enjoyment through consumption where the politics of perversion, the banalization of the good and the logic of waste are presented as its central axes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "90112", "date": "12 Aug 2021", "name": "Jose Miguel Beriain Razquin", "expertise": [ "Reviewer Expertise Sociological Theory", "Sociology of Religion and Cultural Sociology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is an excellent contribution to the sociology of the family highlighting the features of the \"Spanish family\" closer in its shape, and challenges to the families of the modern complex societies. The family in Spain has its own \"Mediterranean\" features but at the same time faces the challenges, beyond any communitarian background, of a fast process of modernization within the fields of market economy and the modern lifestyles.\nFamily in Spain is a symbolic substitute of the lacks of religion which has been quickly privatized and a functional substitute of the market which is not sufficiently dynamic in order to absorb huge amounts of labor force.\nTo my account the paper can be indexed in his present version without any change.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "90115", "date": "17 Aug 2021", "name": "Elaine da Silveira Leite", "expertise": [ "Reviewer Expertise Sociology", "family", "budget", "emotional ties", "intimicy." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 theoretical-methodological consistency and quality, and it also cited the current literature. The research evidence is the result of a review of the literature. The author demonstrates that the Spanish family and love are immersed in the general European context of individualization, but distinctly characteristics, in which the Spanish family is situated halfway between the traditional and the modern, the communal and the individual. The article presents a clear and objective writing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "90114", "date": "31 Aug 2021", "name": "Olga Alejandra Sabido Ramos", "expertise": [ "Reviewer Expertise Sociology of emotions", "Embodiment and sensory studies", "Sociological Theory." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “The paradoxes of love in the Spanish family: a sociological approach” presents a broad theoretical and empirical panorama to demonstrate the coexistence of two types of modern love (romantic and confluent) and the nuances around the notion of Spanish family based on “Mediterranean model.” According to the evidence, the Spanish family structure and love tend towards individualism but are in tension with traditional family forms. In conclusion, the romantic relationships in the Spanish family are more paradoxical and hybrid. The article has a rigorous critical apparatus and complete research of current literature. Likewise, it stands out for its sophisticated analytical and interpretive construction. This analytical horizon allows us to introduce nuances in the ideal types about family and love. This input is an excellent contribution to the research of these topics.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/10-627
https://f1000research.com/articles/10-625/v1	21 Jul 21	{ "type": "Research Article", "title": "Essential oil extraction from onion using ethanol and CO2 as an extraction fluid mixture", "authors": [ "Etiandra dos Santos", "Teresa Saleme Tingeira", "Vicencia de Fátima Cristovão da Costa", "Luana Marcele Chiarello", "António André Chivanga Barros", "Etiandra dos Santos", "Teresa Saleme Tingeira", "Vicencia de Fátima Cristovão da Costa", "Luana Marcele Chiarello" ], "abstract": "Introduction: Essential oils are volatile chemical compounds, widely known by their fragrance, as well as by antimicrobial and antioxidant activities. These oils are generally extracted from aromatic plants in procedures using conventional solvents. Methods: In this study, essential oil was extracted from onion (previously chopped and dried) using a mixture of ethanol and CO2 as the extraction fluid. The essential oil obtained from the extraction was collected and purified and the mass was determined (by weighing) to evaluate the effect of CO2 flow on the yield. The essential oil extracted and purified was characterized to determine the acid and refraction indexes, viscosity, and specific mass. Results: The values obtained for refraction and acid indexes are within limits and similar to the average reported in literature. In all cases, when the CO2 was used, there was an increase the essential oil recovery. In terms of quality, the products from this process were characterized to determine the density, acid index and refraction index. The results obtained were similar to those published in the literature. Discussion: The proposed apparatus and CO2 methodology can be considered a good alternative to boost the extraction of essential oil aiming the obtaining of new products for use as raw materials in different industrial processes. Since this apparatus presents more than double extraction yield than Soxhlet experiment.", "keywords": [ "Extraction", "Oil", "Solvents", "Supercritical Fluids", "Ethanol and CO2" ], "content": "Introduction\n\nFor Reddy (2019) and Tongnuanchan & Benjakul (2014), the natural essential oils are aromatic compounds present in different parts of medicinal plants, for instance: flowers, leaves, bark, roots and fruits that are separated after hydro-distillation or steam distillation as continuous solvent extraction techniques. Essential oils are a combination of low molecular mass chemical compounds, which contain: alcohols, polyphenols, terpenoids, carbonyl compounds, aliphatic substances, and they have distinct aromas and possess biological properties.\n\nCavalcanti (2013) and Sharmeen et al. (2021) noted that essential oils are used and applied as additives in the cosmetic, pharmaceutical, food, textile and perfumery industries to incorporate appropriate functional characteristics, ensuring a wider range of organic functions. In fact, most natural essential oils incorporate several functions, with emphasis on their use as natural dyes, nutraceuticals, functional foods, preserving agents, flavorings and fragrances, medicines, vitamin supplements, chemical standards and perfumes, among others.\n\nEssential oils incorporate vegetable oil and are known by having an elevated content of organic and natural substances when obtained from flowers, herbs, fruits and aromatic spices. The extraction procedures involve the use of an extractor and a conventional solvent or supercritical fluid, or both, known as a hybrid mixture, where the proportion can be adjusted to improve the extraction yield (Uwineza & Waśkiewicz (2020) and Chemat et al. (2019)).\n\nThe extraction procedure used directly reflects in the quality of the products obtained, mainly in relation to the degree of purity, and the recovery capacity. The concepts of diffusion and convection mass transference, based on interfacial equilibrium between solid–liquid or solid–gas phases, are used to assess the process efficiency and to define the appropriate extraction equipment.\n\nBoucard & Serth (2005) evaluated the extraction process based on diffusion and convectional mass-transfer phenomena to identify procedures that could enhance the quantity of the products. Shakir (2018) observed that a higher extraction temperature could increment the interaction of solvent and matrix and also the diffusion mass transfer.\n\nDanlami et al. (2014) observed that the fundamental of solvent extraction is based on the contact of solvent and the solid material, allowing the solid materials to be transferred to the liquid phase as soluble compounds. However, in the case of plants, this extraction occurs through concentration differences. In the moment when the mass transfer decreases by the concentration of active sites, the equilibrium is finally reached and therefore the mass transfer will no longer occur.\n\nIn recent decades, an increase in international demand for essential oils produced from plants has been observed. This has driven further research to develop new extraction techniques aimed at increasing the oil quality and reducing the production cost.\n\nIn general, essential oil extraction involves vapor flow in distillation, hydro-distillation or procedures based on organic solvents, mechanical extraction or supercritical fluids (generally carbon dioxide) to improve the mass transfer during the extraction (Cavalcanti, 2013).\n\nFilippis (2013) studied supercritical extraction with CO2 and noted the importance of the production and commercialization of essential oil globally, with a considerable variety of essential oils on the market (more than 90 types). Several important differences in the oils obtained arise from the characteristics of the process applied for the extraction.\n\nIn this context, hydro-distillation is characterized by direct contact between two phases, vapor and solid. When the vapor comes into contact with an aromatic plant, the essential oil is dragged into this phase and then recovered in a condensation system (Figure 1). In this process, to increase efficiency extraction, the processes parameters are controlled, mainly temperature, pressure, vapor flow rate and particle size of the solids. Optimizing the operational conditions in this way generally increases the extraction efficiency and yield. After this step, the essential oil produced can be purified to satisfy international standards (Golmohammadi et al., 2018).\n\nProcess adapted from Kusuma & Mahfud (2017).\n\nWhen oil extraction is applied in industrial processes, the particle size is reduced and the essential oil is extracted inside an extractor tank, using appropriate solvents. After this step, the liquid phase is passed to an evaporator to remove the solvent and recover the essential oil. These processes can be applied on an industrial scale only after the associated phenomena have been studied. The difference between the ebullition temperature of essential oil and solvents ensures rapid solvent removal, using a one-stage evaporation processes. The solvent can be recovered, recycled and re-used in same extraction process, leading to a reduced need for new solvents.\n\nFor Vieira de Melo et al. (2000), the conventional procedures for essential oils recovery from plant materials, as cited above, can present limitations due to instability of essential oils by heat and by residual organic solvent from extraction. Hence, the use of SCF for essential oils extraction is being evaluated as an alternative to traditional methods.\n\nSupercritical CO2 offers advantages because it is not toxic, not expensive, non-flammable and odor- and colorless. Also, the critical temperature is near to ambient temperature and it has low viscosity and high diffusivity in relation to liquids. Thus, it has transformed into a common solvent in the process of natural materials (Vieira de Melo et al., 2000)).\n\nThe extraction of compounds from solid matrices is associated with the internal mass transfer (dissolution and diffusion) and external mass transfer (solid particles). Thus, the solute diffused in the supercritical fluid-rich phase can be carried away by the bulk flow. Numerous authors have been published their attempts of model for these supercritical extraction process (Reverchon & Morone, 1997; Sovová, 1994; Sovová, 2005), and a broad discussion of modelling aspects is presented elsewhere (Capuzzo et al. (2013); Danlami et al. (2014); Khaw et al. (2017); Reverchon (1996); Vieira de Melo et al. (2000) and Lin et al. (2013)).\n\nEssential oil extraction using high pressure or supercritical fluids (SCF) is an alternative from organic solvent extraction or steam distillation, and has been studied by different industries, especially in the food, pharmaceutical and cosmetic industries (Ferreira et al. (1999); Pinto et al. (1999) and Cao et al. (2007)).\n\nKhaw et al. (2017) observed the influence of solubilization on fluid density. At a high pressure, SCF presents high density, allowing the dissolution of great quantities of organic compounds. When the density is reduced, those dissolved compounds can be recovered, and it can be performed by decreasing the pressure or increasing the temperature.\n\nFor the author cited below, by performing the separation process at lower temperatures it can avoid the degradation of compounds by decreasing the heat exposure, which occur in steam distillation.\n\nSupercritical fluid extraction provides better extraction efficiency, increases the production rate and improves the product quality. These processes, generally hybrid, involve the use of supercritical fluid, compressed air and a co-solvent, which, in combination, can intensify the fluid bubbling in the solid mass and increase the extraction efficiency (Figure 2).\n\nProcess adapted from Borges et al. (2017).\n\nTo increase the extraction efficiency with use the supercritical fluids, the extraction process must be optimized, guaranteeing the best operational conditions; mainly the temperature, pressure, flow rate, residence time, solid material size, and ratio between the material and the flow rate of the fluid used as the extractive solvent (organic solvent, supercritical fluid and compressed air).\n\nFor Manjare et al. (2019), the low polarity of supercritical CO2 is an important obstacle to its application, hindering the extraction of polar compounds. However, this could be surpassed by the addition of polar modifiers, as methanol or ethanol together with supercritical CO2, therefore increasing its solution power.\n\nUnder these conditions, it is possible to increase mass transfer rate and improve the performance of the process, guaranteeing the applicability of this type of fluid as an alternative for the improved extraction in industrial processes (Huang et al. (2016) and Silva et al. (2004)).\n\nDe Barros et al. (2014) studied supercritical extraction using laboratory and pilot scale processes. Their parametric studies supported the mathematical models developed for industrial-scale extraction, guaranteeing extraction products with high quality and greater added value. The parameters studied highlight the most appropriate and highest-performing operating conditions, allowing extraction curves to be constructed, and those arising from different procedures can be compared.\n\nBased on these considerations, this paper proposes the extraction of essential oil, using a hybrid solvent system, involving a conventional solvent (ethanol) and a supercritical fluid (CO2). In this study, the operational parameters (temperature, pressure and solvent flow rate and mass used ratio) were controlled in order to determine the optimal condition, to increase the mass transfer and yield of the essential oil production process. The procedure used in this paper is the same used by Capuzzo et al. (2013).\n\n\nMethods\n\nIn this study, for the experimental setup, an extractor system was built. The system involves the use of ethanol and supercritical fluid (CO2), as a hybrid fluid mixture, based on supercritical concepts, and the essential oil was extracted from onion. The best operational conditions were determined, aimed at market efficiency, considering the supercritical flow rate, temperature and mass of onion used in the extraction. The turbulence phenomenon was evaluated in terms of the intensity of the solid-fluid mixture, considered the velocity of the ethanol-CO2 mixture. The CO2 was bubbled in ethanol in a distillation balloon, where the mixture received the thermal energy needed to increase the solid to fluid phase transferring. The effect of onion mass on the mass transfer efficiency was also investigated.\n\nThe onion sample used in this study was previously chopped and dried in a horizontal dryer with controlled circulating air speed and the air was heated using electrical resistance, in order to improve the efficiency during the extraction process. The results are represented as graphs showing the mass loss over time.\n\nRaw material preparation. In the study reported herein, onion (Allium cepa) was used as the raw material, which was chopped into cubes to obtain an adequate size for essential oil extraction. The chopped onion was placed in a convective dryer, coupled to an analytical balance and temperature control sensors.\n\nThe mechanical ventilation system provided airflow and there was a humidity (water concentration) gradient between the air and the onion, to increase the mass transfer from solid particles to the air.\n\nThe operation parameters for drying, hot air and mass loss of raw material were controlled. The process was carried out to give an onion mass loss of 50%, with a temperature of 40±1.2ºC, hot air speed of approximately 1.7 m/s, extraction time for 240 min and average particle size of 4 mm.\n\nProcedure for essential oil extraction. The experimental apparatus shown in Figure 3 was used to carry out the extraction of essential oil from the dried onion.\n\nThe apparatus built for this study was comprised of three sections: a) section with fluid mixture of solvent and supercritical fluid (CO2) heated to the temperature required to provide the best conditions for the extraction process; b) the extraction of essential oil section, allowing interaction between solid and fluid phases, where the mass transfer from solid to fluid mixture occurs; c) section for recovery of essential oil, where the extraction fluid mixture carries the essential oil through the heat exchanger to the expansion tank for recover, by condensation, of the fluid mixture, ethanol and essential oil.\n\nThe experiments were performed using the apparatus (Figure 3), applying the following procedure: a) the raw material is placed inside the extraction tank and the water in the condensation system is cooled. Also, during this step, the ethanol inside the balloon is heated; b) cooled water begins to recirculate in the condenser system and when the temperature in the balloon reaches that suitable for extraction, the bottle of the CO2 valve was opened and the flow of supercritical fluid was bubbled into the ethanol to form the mixture which would pass to the extraction tank; c) the fluid mixture flows in contact with the solid phase and the mass transfer occurs. The flow then passes through the condenser to recover the ethanol and essential oil in the expansion tank.\n\nThe methodological sequence described herein was used to carry out the essential oil extraction using onion as the raw material, as well as the effect of operation parameters on the quality of the process was evaluated.\n\nTable 1 show the operational conditions used in this study, based on an experimental plan to evaluate the parameters and their effect on the process efficiency, based on the essential oil extraction capacity.\n\nThe performance of each experiment was evaluated based on Equation 1.\n\nThe performance of each extraction essay was related to the operation parameters mainly the bubble intensity of CO2 in ethanol, temperature and fluid mixture flow rate (measured at the inlet and outlet of the expansion tank).\n\nEssential oil purification. The essential oil produced in the extraction process describe herein was purified using simple distillation and filtration, supported by technical norms. The procedure applied provides products with a high degree of purity, suitable for application as a raw material in other industrial processes.\n\nCharacterization of essential oil. The essential oil extracted and purified was characterized to determine the acid and refraction indexes, viscosity, and specific mass. The analysis procedures are described in the standard test method ASTM D 974 and the European standard EN14103.\n\n\nResults and discussion\n\nThe results of the experimental assays described above are reported in this section along with a discussion, supported by data available in the literature, to better contribute to future scientific and technological developments.\n\nTable 2 shows the results obtained from the drying of the raw material (onion) used in this study, based on experiments conducted using the procedure described above. The results show the mass loss over time, characterized by a loss of the water present in the structure of the raw material. The runs were conducted with control of the operation parameters.\n\nThe data in Table 2 were obtained during the drying process (240 min) and the mass was measured at 30-min intervals. Globally, for this assay, a mass loss of 892.4g corresponds to 54.42% of the initial raw material mass. These data were used to construct the graphs in Figure 4, which show the mass loss during the drying time. The drying curve indicates that a greater mass was transferred from the solid to gas phase, occurred during first hour of drying, equivalent to 39.15% of the global mass loss. In the second hour, the mass loss represents 26.45% of the global mass, in the third hour 20.80% and in the last hour 13.5%.\n\nThe decrease in water mass during drying, according to Figure 4, was associated with the mass transfer phenomenon, where the flow rate is directly related to the concentration gradients of water, between the solid and gas phases, associated with this process. In this process, mass transfer by convection at the solid surface predominates, and this is proportional to the solid area through the transfer coefficients. Equation 2 can thus describe the mass transfer, where the flow transference is related with global coefficient and the concentration gradient.\n\nWhere NA is the flow rate of water (kmol/m2s), kc is the global coefficient and ΔCA is the concentration difference between the solid particle and the airflow.\n\nThe mass loss rate was greatest during the first 180 min when approximately 65.60% of the total loss occurred. The mass transfer rates then decreased substantially leading to relative stability. The reduction in mass transfer rates is related to the drying time, which can be adjusted to guarantee the optimal condition for the essential oil extraction, as described herein.\n\nExperimental apparatus. The experimental apparatus described above was used to carry out the procedure applied to obtain the essential oil extract from dried onion (Figure 4).\n\nOnce the apparatus had been built, we ascertained the limits operational based on experimental planning, mainly the characteristics of the mixture, supercritical fluid flow rate and the ratio of mass of conventional solvent and supercritical fluid that gives the best extraction performance.\n\nExperimental data acquisition. Initially, the conventional procedure tested the interaction of ethanol (vapor) rate and onion (solid) mass transference from solid to vapor phase. This process, established according the solubility of the solid in the solvent, occurs through mass transfer by diffusion and convection at the solid and vapor interface. In this initial study, a Soxhlet apparatus was used to extract the essential oil from onion particles, with a global yield of the 27.93%.\n\nUsing the same conditions (Table 3), the yield increased in non-linear fashion of CO2 performance operation and reach over 60% (yield).\n\nIn all of these experiments, the operating time was the same. Aris et al. (2019) and Hassanien (2019) evaluated the effect of fluid flow rate using supercritical CO2 and concluded that when CO2 flow rate increased, the extract yield increased by 3.698% for every 0.7 mL/min. According to the cited authors, under conditions of low pressure and high temperature, a rise in the flow rate of 8 mL/min would increase extract yield. Maran & Priya (2015) and Gadkari & Balaraman (2017) observed that with an increase in the flow rate, the film thickness surrounding the solid particle reduced, thus decreasing the external mass transfer resistance around the solid. The solute could thus easily move to the bulk solvent, enhancing the solubilization of the solute in the solvent and increasing the extract yield.\n\nÖzkal & Yener (2016) observed that when the flow rate of the supercritical fluid was increased considerably, there was an increase in the convection between solid and CO2. In this case, this causes damage to the weak parts of the solid particles, leading to freer solute being removed from the solid particle during extraction.\n\nTable 3 shows the yield effect by the CO2 flow rate in terms of essential oil recovery, related to the role of the solubility of the oil on the concentration in the supercritical fluid flow.\n\nIn a study by Inamuddin & Asiri (2020) and Da Silva et al. (2016), the extraction yield with ethanol, at high CO2 solvent pressures, increased with a decrease in pressure in the binary CO2-ethanol system, and the extraction yield was high.\n\nIn the study reported herein, a hybrid system was used, with supercritical fluid and ethanol as the extraction fluid. In this case, the bubbling of the supercritical fluid in a conventional solvent was used to form the mixture, which was applied in the extraction of essential oil. Interaction between the solid and vapor phases was verified.\n\nIn all cases evaluated, the essential oil was characterized in terms of the acid index and refraction index (Table 3). The values obtained for the three runs were 1.132; 1.130 and 1.127 g/mL, which are within limits reported in literature. The average value obtained for the acid index in these the experiments was 3.19 mg KOH/g, while data obtained from the literature show an average value 3.56.\n\n\nConclusions\n\nWe can conclude:\n\na) The essential oil from supercritical fluid and conventional solvent mixture extraction provided good yields;\n\nb) The use of CO2 in the extraction processes is a good alternative to improve the extraction performance, due to the turbulence imposed on the mixture in the extractor, which increases the mass transfer between solid and fluid phases;\n\nc) The mass transfer by convection in essential oil extraction, based on the results of this study, is more effective than that by diffusion, mainly with the use of a supercritical fluid in the system; and\n\nd) The apparatus proposed for this study represents a good alternative to improve the extraction of essential oil, with a view to obtaining new products for use as raw materials in other industrial processes.\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\nAris NA, Idrus NFM, Yian LN, et al.: The Effect of Fluid Flow Rate and Extraction Time in Supercritical Carbon Dioxide. J Adv Res App Sci Eng Technol. 2019; 16(1): 26–34. Reference Source\n\nBorges VM, Cunha M, Paiva M, et al.: Extração Supercrítica: Simulação do Sistema Solvente+Cossolvente com o Aspen Hysys. XV Encontro de Profissionais da Química da Amazônia; Belem Pará; 2017; 1. Reference Source\n\nBoucard GR, Serth RW: Practical design of a distillation plant for the separation of essential oils from aromatic row materials. Texarome Inc., 2005; 5. Reference Source\n\nCao H, Xiao JB, Xu M: Comparison of volatile components of Marchantia convoluta obtained by supercritical carbon dioxide extraction and petrol ether extraction. J Food Compost Anal. 2007; 20(1): 45–51. Publisher Full Text\n\nCapuzzo A, Maffei ME, Occhipinti A: Supercritical Fluid Extraction of Plant Flavors and Fragrances. Molecules. 2013; 18(6): 7194–238. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCavalcanti RN: Extração de antocianinas de resíduo de jabuticaba (myrciaria cauliflora) utilizando líquido pressurizado e fluido supercrítico: caracterização química, avaliação econômica e modelagem matemática. Dissertação de Mestrado; Universidade Estadual de Campinas (UNICAMP); Campinas, São Paulo, Brasil. 2013; 197. Reference Source\n\nChemat F, Vian MA, Ravi HK, et al.: Review of Alternative Solvents for Green Extraction of Food and Natural Products: Panorama, Principles, Applications and Prospects. Molecules. 2019; 24(16): 3007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDa Silva RPFF, Rocha-Santos TAP, Duarte AC: Supercritical fluid extraction of bioactive compounds. Trends Analyt Chem. 2016; 76: 40–51. Publisher Full Text\n\nDanlami JM, Arsad A, Zaini MAA, et al.: A comparative study of various oil extraction techniques from plants. Rev Chem Eng. 2014; 30(6): 605–626. Publisher Full Text\n\nDe Barros NA, von Randow de Assis A, Mendes MF: Extração do óleo de manjericão usando fluido supercrítico: analise experimental e matemática. Cienc Rural. 2014; 44(8): 1499–1505. Publisher Full Text\n\nFerreira SRS, Nikolovb ZL, Doraiswamy LK, et al.: Supercritical fluid extraction of black pepper (Piper nigrun L.) essential oil. J Supercrit Fluids. 1999; 14(3): 235–245. Publisher Full Text\n\nFilippis FM: Extração com CO2 supercrítico de óleos essenciais de Hon-sho e Ho-sho: Experimentos e modelage. Dissertação de Mestrado; Porto Alegre. 2013.\n\nGadkari PV, Balaraman M: Mass transfer and kinetic modelling of supercritical CO 2 extraction of fresh tea leaves (Camellia sinensis L.). Braz J Chem Eng. 2017; 34(3). Publisher Full Text\n\nGolmohammadi M, Borghei A, Zenouzi A, et al.: Optimization of essential oil extraction from orange peels using steam explosion. Heliyon. 2018; 4(11): e00893. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHassanien MFR: Fruit Oils: Chemistry and Functionality. Editors: Ramadan, Mohamed Fawzy (Ed.); 2019. Publisher Full Text\n\nHuang Z, Ye C, Li L, et al.: Measurement and Correlation of the Mass Transfer Coefficient for a Liquid-Liquid System with High Density Difference. Braz J Chem Eng. 2016; 33(4). Publisher Full Text\n\nInamuddin M, Asiri AM: Advanced Nanotechnology and Application of Supercritical Fluids. Editors: Inamuddin, Muenuddin, Asiri, Abdullah M. (Eds.) 2020. Reference Source\n\nKhaw KY, Parat MO, Shaw PN, et al.: Solvent Supercritical Fluid Technologies to Extract Bioactive Compounds from Natural Sources: A Review. Molecules. 2017; 22(7): 1186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKusuma HS, Mahfud M: The extraction of essential oils from patchouli leaves (Pogostemon cablin Benth) using a microwave air-hydrodistillation method as a new green technique. RSC Adv. 2017; 7(3): 1336–1347. Publisher Full Text\n\nLin TM, Ping TS, Saptoro A, et al.: Mass Transfer Coefficients and Correlation of Supercritical Carbon Dioxide Extraction of Sarawak Black Pepper. Int J Eng Res Appl. 2013; 10(1): 1–15. Publisher Full Text\n\nManjare SD, Dhingra K: Supercritical fluids in separation and purification: A review. Mater Sci Energy Technol. 2019; 2(3): 463–484. Publisher Full Text\n\nMaran JP, Priya B: Supercritical fluid extraction of oil from muskmelon (Cucumis melo) seeds. J Taiwan Inst Chem Eng. 2015; 47: 71–78. Publisher Full Text\n\nÖzkal SG, Yener ME: Supercritical carbon dioxide extraction of flaxseed oil: Effect of extraction parameters and mass transfer modeling. J Supercrit Fluids. 2016; 112: 76–80. Publisher Full Text\n\nPinto JSdS, Assis LM, Lanças FM, et al.: Accelerated Solvent Extraction (ASE): a new approach for environmental sample characterization. International Symposium on Capillary Chromatography and Electrophoresis. 1999.\n\nReddy DN: Essential Oils Extracted from Medicinal Plants and Their Applications. book: Natural Bio-active Compounds. 2019; 237–283. Publisher Full Text\n\nReverchon E: Mathematical modeling of supercritical extraction of sage oil. AIChE J. 1996; 42(6): 1765–1771. Publisher Full Text\n\nReverchon E, Marrone C: Supercritical extraction of clove bud essential oil: isolation and mathematical modeling. Chem Eng Sci. 1997; 52(20): 3421–3428. Publisher Full Text\n\nSharmeen JB, Mahomoodally FM, Zengin G, et al.: Essential Oils as Natural Sources of Fragrance Compounds for Cosmetics and Cosmeceuticals. Molecules. 2021; 26(3): 666. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShakir IK: Mass Transfer Coefficients of the Extraction Process of Essential Oil from Myrtus Communis L. Plants Using Different Solvents. The Eighth Journal International Chemical Engineering Conference (JICHEC, 2017). 2018. Reference Source\n\nSilva EO, et al.: Processamento Mínimo de Hortaliças no Brasil. Simposium Estado Actual Del Mercado de Frutos y Vegetables Cortados em Iberoamérica, San José, Costa Rica. 2004.\n\nSovová H: Rate of the vegetable oil extraction with supercritical CO2—I. Modelling of extraction curves. Chem Eng Sci. 1994; 49(3): 409–414. Publisher Full Text\n\nSovová H: Mathematical model for supercritical fluid extraction of natural products and extraction curve evaluation. J Supercritical Fluids. 2005; 33(1): 35–52. Publisher Full Text\n\nTongnuanchan P, Benjakul S: Essential Oils: Extraction, Bioactivities, and Their Uses for Food Preservation. J Food Sci. 2014; 79(7): R1231–49. PubMed Abstract \| Publisher Full Text\n\nUwineza PA, Waśkiewicz A: Recent Advances in Supercritical Fluid Extraction of Natural Bioactive Compounds from Natural Plant Materials. Molecules. 2020; 25(17): 3847. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVieira de Melo SAB, Costa GMN, Garau R, et al.: Supercritical CO2 Extraction of Essential Oils from Thymus Vulgaris. Braz J Chem Eng. 2000; 17(3). Publisher Full Text" }	[ { "id": "91263", "date": "20 Aug 2021", "name": "Benevides Costa Pessela", "expertise": [ "Reviewer Expertise Biotechnology", "Screnning of enzymes", "Enzyme purifications", "Reconbinant process on biotecnhology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the subject of this experiment is important. Even so, I think that the organization of the data should be improved.\nI believe very sincerely, even if I am not an expert in the English language, it must be somewhat improved for a better understanding of the work.\nThe authors should explain why they use 3 masses of the starting material for the extraction of essential oils in the experiment.\nAnother aspect that must be presented has to do with purification experiments. Some analytics that compare a commercial essential oil with those obtained in this experiment is necessary, for example an FTIR experiment or a thin layer chromatography would be essential to better understand this experiment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7061", "date": "25 Aug 2021", "name": "António Barros", "role": "Author Response", "response": "Thank you for your consideration and contribution on this paper. We evaluate your comment and I answer here. We make three experiments with better evaluation of the influence of CO2 flow rate on extraction essential oil yield (%). For this case, was used the range of CO2 flow rate, respectively 0,5; 1,44 and 3,6 ml/min, with one increase relation of 0,94 ml/min between the first and second CO2 flow rate and 2,66 ml/min between the second and last CO2 flow rate. In this increment, it is possible to evaluate the operational conditions used to relate it with mass transference associated with the essential oil yield. With this performance we can know the relevance of CO2 flow rate with the mass transference and its relation with the turbulence systems. The results showed that when the CO2 flow rate increase, the turbulence mixture in the system increases too, and that results in increased essential oil yield (%). For this case, the turbulence affect more effectively of mass transference than the contact time between the two phases involved in this study. We need to evaluate the quality of essential oil produced here using all tool or equipment commonly used for this type of study, but our laboratory has many limitations in term of special equipment for this ending. The laboratory, same with its big dimensions, don’t have FTIR or chromatography or infrared or other equipment. We use only, to evaluate essential oil produced in this study, the procedure associated with equipment’s disposed in laboratory. For this, we evaluated the density, viscosity and refraction index parameters associated with the laboratory conditions. Even so, the results from this study were compared with those referenced in the literature and all that gave the same performance. In these conditions we can confirm the quality of essential oil produced in this study. In future we will establish relation with other laboratory in Angola or in another country to develop joints projects to minimize these situations." } ] }, { "id": "257049", "date": "21 Mar 2024", "name": "Henrique Gasparetto", "expertise": [ "Reviewer Expertise Process separation", "green chemistry", "chemical engineering." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn my opinion, the article is very short and inconclusive. Although I believe it can be indexed after a major revision. Here are some considerations to improve the work:\n- Please provide recent references for oil extraction using ethanol and supercritical CO2. In my opinion, solvent extraction is better for industry than supercritical CO2. Although the results are worth studying.\n- It is unclear how the authors chose the operational conditions mentioned in Table 1; is it derived from some experimental planning design or solely from the prior literature?\n- Please mention the technical norms in the \"essential oil and purification\" section. It would be better to describe them also.\n- In Table 2, it's unnecessary to mention the mass loss in g over time; only the % is enough. Indeed, I recommend that the authors present only Fig. 4. as the drying kinetic is not the main result of the work.\n- Why do the authors not estimate parameters for the drying kinetics? Several models can be easily linearized to obtain mass transfer coefficients.\n- The extraction conditions are very unclear. Is it part of a central composite or rotational design?\n- How about the amount of ethanol and CO2? Is ethanol used as a co-solvent at which amounts?\n- The authors do not report the Soxhlet methodology in the procedure for oil extraction.\n- Some considerations about solvent flow are well compared with the literature, but in my opinion, they do not provide any exciting information. It is kind of trivial that increased solvent speed will increase oil extraction. However, what about the density and refraction indexes? What do they mean? How could the authors explain those differences concerning the operational variables? Besides, are temperature and pressure fixed?\n- Were experiments performed in multiple attempts?\n- I recommend the authors report the acid index separately for each experiment and not the average. The acid value and refractive index could respond to how the operational variables influence onion essential oil extraction, as there isn't any other characterization technique. FTIR could be an exciting tool for briefly screening the oils extracted, while GC-MS could offer a profound understanding of the essential oil characteristics.\n- The experimental results must be scrutinized cautiously and not just presented.\n- The article is inconclusive; no key conclusion is reported in the \"Conclusions\" 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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "257054", "date": "29 Mar 2024", "name": "Ruengwit Sawangkeaw", "expertise": [ "Reviewer Expertise Supercritical fluid extraction", "Biodiesel production", "Subcritical water extraction", "Lipid", "Essential oil" ], "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 proposed the extraction method using ethanol and carbon dioxide at atmospheric pressure to extract the essential oil from the dried onion. The extractions were conducted in three experiments with different flow rates of carbon dioxide. However, the characterization of extracted oil was doubtful. Because the extraction yield obtained at the flow rate of 3.6 mL/min were 2-fold higher than observed from the Soxhlet extraction, this statement is questionable. The reviewer suggests the comments for improvement of this article below. Introduction:\nCondense the introduction and ensure that it clearly highlights the importance of onion essential oil. Explicitly mention the significance of onion essential oil in various applications to provide context for the study.\nFigures:\nEnsure that Figure 1 accurately depicts the supercritical carbon dioxide extraction process. Verify that Figure 2 accurately represents the hydro-distillation process.\nAim of the Study:\nRevise the aim of the study to accurately reflect the extraction conditions. The extraction process at 760 mm Hg is not under supercritical condition of ethanol-carbon dioxide mixture.\nMethods and Experimental Procedure:\nAvoid repetition and redundancy in the Methods section. Provide clear and accurate methods for characterizing essential oil properties ASTM D974 and EN14103 are employed to determine acid number and the fatty acid methyl ester (FAME) content of oil feedstock used in biodiesel production, respectively.\nResults and Discussion:\nEnsure that data presented in Table 2 and Figure 4 are not redundant. If the drying curve is not directly relevant to the focus of this study, consider moving it to supplementary data. Clarify what is meant by \"non-linear fashion\" in the context of the results or discussion. Provide more detail about the Soxhlet extraction method used for comparison, including relevant parameters and conditions.\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? No", "responses": [] }, { "id": "257040", "date": "01 Apr 2024", "name": "Sitinoor Adeib Idris", "expertise": [ "Reviewer Expertise Extraction of vegetable oil", "extraction of essential oil", "supercritical fluid extraction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 focusing on extracting essential oil from onion using supercritical CO2 + ethanol as the fluid in supercritical extraction and comparing the yield with the one from the conventional method (Soxhlet). The introduction was enough as it highlighted the conventional and current trends and research on the techniques and methods used in extracting essential oil. However, no writing on onion in the introduction part which is also crucial as to highlight why onion is chosen for this research. Methodology was good written, however the layout configuration of Figure 1, Figure 2 and Figure 3 can be better. Process flow diagram (PFD) form or PNID would be better. The caption for Figure 1 and Figure 2 should be revised. The word pump was incorrectly spelled in Figure 1. Equation 1 should be type using correct symbol for multiplication. The term 'non-linear fashion' used in the second paragraph in Experimental data acquisition section should be validated with a statistical value. Conclusion (c) should be discussed more in Discussion part as the term diffusion was more stated in the Introduction part but not being discussed together with the results in the discussion part. Maybe a graph or a sketch can be done to highlight the diffusion and convection process during the extraction process.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": [] }, { "id": "257060", "date": "03 Apr 2024", "name": "Fajriyati Mas'ud", "expertise": [ "Reviewer Expertise Extraction", "food technology", "analytical 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 Introduction section is too long and doesn’t focus on the main point of the study, namely Essential oil extraction using ethanol and CO2 as an extraction fluid mixture. The introduction section should clearly highlight the importance of Essential oil extraction using ethanol and CO2 as an extraction fluid mixture. It’s best to cite several studies related to this extraction technique along with its advantages compared to conventional extraction in terms of essential oil yield and the physicochemical properties of the product. Please state the significance of this study, and also re-state the aim of the study. Why discuss drying onions? Just mention the % water content of the onion as a sample. Table 2 and figure 4 do not need to be displayed and discussed in this article, they must focus on the core problem, namely essential oil extraction using ethanol and CO2 as an extraction fluid mixture. In table 1 it’s stated that there were 3 unit experiments, but the operating conditions were different (temperature, ratio of sample and ethanol). Why? Unlike its current form, this article requires major revision to clarify the methods, results, discussion, and conclusions of this work. Cite several studies that support the statement “the yield increased in the non-linear fashion of CO2 performance operation and reached over 60% (yield).” Cite references that support the results of this work. Results and discussion are very short and unfocused, making it difficult to summarize the results of the work. Conclusions are unclear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "257046", "date": "06 May 2024", "name": "Djéssica Tatiane Raspe", "expertise": [ "Reviewer Expertise Emerging/unconventional extraction techniques." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1) Knowing that the term \"essential oil\" is only and exclusively permitted when obtained by hydrodistillation (aqueous distillation), do the authors think it is appropriate to use this term in the title of the work? The results were compared with Soxhlet using a solvent (other than water), however this is not appropriate for obtaining essential oil. The introduction cites \"vegetable oil\" as a result of these techniques. If this is correct, vegetable oil has volatile compounds that provide aroma to the sample, which is not essential oil. I suggest that the text be revised, as it is not adequate.\n2) The introduction is too long. The references cited are not current (5 years) and compare essential oil extraction with extraction of oils and plant extracts, hence the confusion in the terms used. I suggest revising the text.\n3) It is not necessary to highlight in the Summary that the onion was \"previously chopped and dried\".\n4) In the abstract the authors say \"The essential oil obtained from the extraction was collected and purified and the mass was determined (by weighing) to evaluate the effect of CO2 flow on the yield\". Was the ethanol evaporated?\n5) Where was the onion purchased? Add geographic location.\n6) Why were the brand and model of the equipment, and the origin of the solvents, not mentioned? It is not possible to reproduce the experiments without this information.\n7) How many replications were made of each experiment? I suggest adding the deviation of the replicas in the answers, as well as data from some statistical test.\n8) The work presents a very long introduction and poor discussion of the results. Why was the supercritical fluid flow rate evaluated? Is it relevant? Why? What physical influence does this variable have? Discuss this data further.\n9) \" the bubbling of the supercritical fluid in a conventional solvent was used to form the mixture, which was applied in the extraction of essential oil. Interaction between the solid and vapour phases was verified\", what is the physical explanation of this interaction? It is necessary to explore this topic further and better.\n10) Discuss the results of acid number and refractive index. Just citing is poor. What do these values mean? What does the legislation establish? Other authors, using what techniques, found \"similar\" values? For which oils? Were they really essential oils, or were they vegetable oils?\n11) In the abstract it is mentioned \"Since this apparatus presents more than double extraction yield than Soxhlet experiment\". Where is this data? Why were they not presented and discussed? Where was this information taken from?\n12) The conclusion only repeats phrases cited in the text. Prepare a coherent and appropriate conclusion for the work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-625
https://f1000research.com/articles/10-422/v1	27 May 21	{ "type": "Research Article", "title": "A simple optical pH sensor based on pectin and Ruellia tuberosa L-derived anthocyanin for fish freshness monitoring", "authors": [ "Nazaruddin Nazaruddin", "Nurul Afifah", "Muhammad Bahi", "Susilawati Susilawati", "Nor Diyana Md. Sani", "Chakavak Esmaeili", "Muhammad Iqhrammullah", "Murniana Murniana", "Uswatun Hasanah", "Eka Safitri", "Nurul Afifah", "Muhammad Bahi", "Susilawati Susilawati", "Nor Diyana Md. Sani", "Chakavak Esmaeili", "Muhammad Iqhrammullah", "Murniana Murniana", "Uswatun Hasanah", "Eka Safitri" ], "abstract": "A simple optical pH sensor using the active compound anthocyanin (ACN), derived Ruellia tuberosa L. flower immobilized in a pectin membrane matrix, was been fabricated and employed to monitor the freshness of tilapia fish at room temperature and 4oC storage. The optimum pectin weight and ACN concentrations were 0.1% and 0.025 mg/L. The sensor showed good sensitivity at 0.03 M phosphate buffer solution. The sensor’s reproducibility was evaluated using 10 replicate sensors where a standard deviation of 0.045 or relative standard deviation of 9.15 was achieved. The sensor displayed an excellent response after 10 minutes of exposure, possessing a response stability for 10 consecutive days. The decrease in pH value of the Tilapia fish from 7.3 to 5 was observed in a 48 hour test, which can be used as the parameter when monitoring fish freshness.", "keywords": [ "optical pH sensor", "matrix membrane", "pectin", "anthocyanin", "fish freshness" ], "content": "Introduction\n\nFish freshness assessment is the main concern for consumers nowadays as people are more cautious about what they put into their body. Eating spoiled products will cause food poisoning symptoms to various degrees. For example, eating spoiled fish may result in an almost immediate onset of diarrhea, nausea and vomiting. According to the United Nations, about 4.5 billion people rely on fish for 15% of their animal protein intake.1 Therefore, it is imperative to monitor the freshness and quality of fish. Currently, consumers rely on their own experience in determining fish freshness. This is mostly based on the physical condition of the fish like its color and smell. This method is very subjective; hence, there is a need for a more quantitative monitoring method for fish freshness. Heising et al.2 has produced a fish freshness monitoring method by detecting total volatile basic nitrogen using an ammonia ion-selective electrode. However, not all of the ammonia produced will dissociate in the aqueous phase, which is a challenge in the conductivity changes-dependent method. Determination of fish freshness can also be performed by measuring trimethylamine (TMA) levels using electrochemical sensing, as reported by Sondes et al.3 However, determining the freshness of fish via measuring TMA requires a complicated procedure and experts to operate the equipment. Beside these two methods, a pH sensor can also be employed to monitor fish freshness.4–7 There have been several methods proposed to determine pH levels of a fish sample. The most common methods used are optical sensors and ion-selective electrodes (ISEs).8 The measurement of pH using an ISE H+ is very much affected by interferences from samples. Therefore, the determination of pH through optics may be an excellent alternative for samples that contain interfering ions.\n\nSeveral organic pH-sensitive dyes, immobilized in synthetic membranes, have been utilized in the construction of optical pH sensors. Nonetheless, safer compounds derived from natural products have attracted the attention of researchers in developing pH sensors. An earlier report of optical pH sensors includes the construction of a pH sensor using phenol red as an active molecule.9 The further report had described the development of a pH sensor utilizing polyvinyl chloride as the matrix and the fluorescence compound fluorescein-O-methacrylate as the active molecule.10 Nevertheless, these aforementioned pH sensors could only be used on solutions with near-neutral pH as more basic or acidic solutions will give an insignificant response time. Pourjavaher et al.11 has designed an optical pH sensor based on cellulose nanofibers with red cabbage (Brassica oleracea) extract, while Rajan et al. (2018)12 has produced an optical pH sensor using peonidin pigment. However, this study did not report the working pH range of peonidin. The use of anthocyanin (ACN) from blackberries and chitosan membrane in an optical pH sensor has been established.13 The interaction and mechanical properties of chitosan membrane with entrapped ACN have also been reported.14\n\nA more recent study on fish freshness monitoring through optical methods was reported by Moradi et al.15 using nanofiber bacterial cellulose with ACN. However, this method requires a relatively long analytical time as the pH measurement could not be conducted in situ. Chen et al. (2020)6 has developed a sensitive novel film prepared from starch polyvinyl alcohol and starch polyvinyl alcohol glycerol. This membrane contains curcumin and ACN to monitor fish freshness by measuring volatile ammonia as the fish freshness indicator.\n\nHerein, we constructed a new optical pH sensor based on pectin (PC) matrix and ACN extract from the Ruellia tuberosa L flower. The ACN derived from the crude extract of Ruellia tuberosa L flower has been reported to be pH sensitive.16 PC is a natural polymer that can be crosslinked with the assistance of CaCl2. PC membrane is transparent, deeming it suitable as a matrix for optical measurements. In addition, PC is also a hydrogel that will enable easy diffusion of analytes leading to a faster response time compared to another hydrophobic matrices.17 ACN is well known to be pH sensitive and will undergo color changes at different pH.18 This compound is easily obtained from nature and is relatively cheap compared to other pH sensitive active molecules. In the present work, ACN has been extracted from the flower R. tuberosa L. The ACN was immobilized onto PC membrane to produce CAN/PC composite membrane which can be used for in situ detection of fish freshness without requiring a destructive procedure.\n\n\nMethods\n\nAll chemicals used in this research are analytical grade. Monopotassium phosphate (KH2PO4) and dipotassium phosphate (K2HPO4) were purchased from Merck (Merck Millipore, Darmstadt, Germany); PC, ethanol, and CaCl2 – from Sigma-Aldrich (Sigma Aldrich Chemie GmbH, München, Germany); and methanol and acetic acid – from Fluka (Fluka Chemie GmbH, Buchs, Switzerland). As for the plant sample, wild Ruellia tuberosa L. was collected from the area near Universitas Syiah Kuala in Banda Aceh, Aceh, Indonesia. To study the application of the optical pH sensor on the real sample, dead tilapia fishes were used and purchased from the traditional market in Banda Aceh, Aceh, Indonesia.\n\nThe procedure follows a previous report.19 Briefly, 200 g fresh R. tuberosa L. was macerated in 85 mL methanol for 24 h at room temperature (32-34°C). The residue was then separated from the filtrate by simple filtration. Finally, ACN was obtained after the solvent was removed from the filtrate by means of steaming at 50°C until the volume reached 50 mL.\n\nThe optical pH sensor was constructed by dissolving PC powder into a matrix solution (0.1% w/v) in 100 mL CaCl2 0.1 M solution, heated at 60°C. After the mixture was cooled down, the previously obtained ACN extract (1.503 mg/L) was added to 1.66, 2.49 and 3.33 mL PC matrix solution to produce three different 100 mL ACN/PC solutions with respective ACN concentrations of 0.025, 0.0375 and 0.05 mg/L. A total of 40 μL the ACN/PC solution was dropped onto a polyvinylchloride plastic mold surface with a diameter of 0.8 cm (Figure 1). The sensor was allowed to dry for 24 h at 4oC.\n\nFourier Transform Infrared (FTIR) Cary 630 Anti Agilent (Penang, Malaysia) was used to identify the structure and functional groups. The membrane morphology was observed under Zeiss Merlin/Merlin Compact/Supra 55VP Field Emission Scanning Electron (FESEM) (Selangor, Malaysia). Thermal stability of the constructed membrane was analyzed using Shimadzu DTG-60 Thermal Gravimetric Analyzer (Kyoto, Japan) and Differential Scanning Calorimetry (DSC) Shimadzu DSC-60 (Kyoto, Japan). Unless otherwise stated, the conditions for these characterizations followed that of reported work for film specimens.20,21\n\nTo test its response and evaluate its analytical performance, each sensor was dripped with 30 μL 0.1 M phosphate buffer solution with a variety of pH values ranging from 5.0 to 8.5 with 0.5 interval –the pH values of each phosphate solution on the sensor were checked by pH-meter Thermo Orion Star A2111 (Selangor, Malaysia). The sensor color changed corresponding to the different pH values of the administered buffer solutions. It consequently resulted in the difference of the absorbance that was then measured nm using UV-VIS Spectrophotometer (Shimadzu Uv-mini-1240, Kyoto, Japan) at λmax = 635,16 until the sensitivity value for pH determination was obtained.\n\nThe effect of PC concentration was tested based on % weight of PC in CaCl2 0.1 M solution; 0.05, 0.10, and 0.15%. In total, 40 μL of the three different PC solutions containing 0.025 mg/L ACN were casted as previously explained above. Finally, the pH sensor was pipetted with 30 μL phosphate buffer 0.1 M (pH 4-9), and its absorbance was measured.\n\nThe optical pH sensor with optimum ACN and PC concentrations was used to test its performance against phosphate and citrate buffers 0.1 M (pH 5.0-8.5) to select which buffer generated the best outcome. To select the optimum buffer concentration (once the best buffer had been chosen; phosphate), the best buffer solution was varied in concentration (0.01, 0.03, and 0.05 M) and used in the optical pH sensor performance with pH ranging from 6-8 following the previously explained procedure. The optimum concentration was selected based on its sensitivity and linearity of the absorbance versus pH plotting curve.\n\nResponse time of the optical pH sensor was determined by measuring the optimum absorbance of the pH sensor at a range of 5, 10, 15, 20, 25 and 30 minutes. For reproducibility, the performance was conducted 10 times using ten optical pH sensors. For the determination of the optical pH sensor’s lifetime, the absorbance measurement was carried out after 1, 2, 3, 4, 5, 10, 15 and 20 days after the sensor preparation. All of these studies were conducted under optimum buffer conditions.\n\nThe pH values of the tilapia fishes were measured by attaching the sensors onto the fishes' surface for 5 minutes before measuring the absorbance, as explained before. The fish were stored at 4°C and ambient temperature (32-34°C). The pH analysis was carried out every 7, 12, 24, and 48 h of the storage time.\n\n\nResults and discussion\n\nAnthocyanin (ACN) is one of the most important components in the construction of this optical pH sensor other than PC. ACN is obtained from the extract of R. tuberosa L. flower that displays different colors at different acidic or basic pH levels.22,23 FTIR analysis of the extract showed that the broadening vibrational band with medium intensity at the wavenumber, ranged between 3333 cm-1 and 3291 cm-1, indicating the presence of free O-H groups (Figure 2). The presence of the aromatic C=C vibrations at wavelength region 1644 cm-1 and 1454 cm-1 indicates the typical characteristics of an ACN compound.24 The vibrations by group C-O were recognized from wavelength range 1111 and 1015 cm-1. The FT-IR characterization shows that the ACN is in the form of cyanidin-3-glucoside; similar vibration patterns has been reported previously.25\n\nFT-IR characterization on PC displayed typical PC functional groups at wavenumber range of 1000-2000 cm-1. Spectral band at 1717 cm-1 and 1624 cm-1 are assigned to be vibrations of C=O stretching from ester and carboxylate. The presence of other spectral band at 3370 cm-1 is assigned to the vibrational absorbance of O–H functional groups. The ether bonds of C–O–C is observed by the presence of the absorbance peaks at 1219 and 1096 cm-1. In the case of ACN/PC, free O–H groups from the PC molecule were observed from the overlapping band at 3200-3650 cm-1. The other spectral bands at 1630 – 1850 cm-1 and 1050 – 1260 cm-1 are assigned to carbonyl groups (C=O) and symmetrical ether groups (C–O–C) from glycoside bonds, respectively.26,27\n\nTGA/DTGA and DSC profiles of PC membrane\n\nThermal stability is one of preferable characteristics when it comes to a bio-sensor as it may influence its performance. We conducted thermal gravimetry analysis (TGA) and differential scanning calorimetry (DSC) studies to assess whether the PC membrane has ideal thermal stability. The thermograms of TGA and its derivative (DTGA) and DSC have been presented in Figure 3a and b. At around 58°C, the release of solvent (water) was observed on the TGA and DTGA thermograms (Figure 3a). The second peak of DTGA suggests thermal degradation with 30% weight loss.28 A better insight regarding the thermal stability of the PC membrane can be seen in the DSC thermogram.20,21\n\nThe first endothermic peak that appears in the DSC thermogram (Figure 3b) agrees with the water content release observed in the TGA. Tonset = 83°C indicates the first observable thermal transition, in which it is assigned to melting temperature. It is because within the temperature range (83-118°C), the decrease in weight does not occur in the TGA thermogram. This finding is in line with a previous report investigating PC powder.29 The exothermal peak (Tpeak = ± 309°C) observed afterward indicates the degradation of the PC polymeric chain. From these data, we can conclude that the PC membrane is thermally stable at room temperature range.\n\nSEM images of PC membrane\n\nSEM images of PC (Figure 4a) and ACN/PC (Figure 4b) depict a clear difference of surface morphology between the two.17 PC surface has a morphology that is uniform and smooth. With the addition of ACN into the membrane, cracks are shown as the result of the presence of the liquid. A similar phenomenon was observed previously,30 where the cracks were associated with the presence of water. The surface of ACN/PC membrane was divided into darker and lighter areas. The water contents were gathered in areas with a darker color due to their interactions via hydrogen bond. It further results in the disturbance of compacted PC intramolecular interaction, pushing the molecules to have a dense structure resulting in line-like structures with lighter appearance. This change may lead to poorer sensor performance as a transparent membrane is preferred for optical sensor to allow the UV light passing through the membrane.\n\nThe constructed optical pH biosensor based on the ACN derived from R. tuberosa L flower has hydrogel characteristics. The advantage of a hydrogel membrane in an optical system is the quick interaction between analyte and active membrane which in turn will accelerate the response time.17,31 The PC membrane with the immobilized ACN is transparent, where the color change is sensitive against the pH value (Figure 5). This optical pH sensor is optimized by means of ACN variation to achieve the best sensitivity, observed by a wide linear range and good linearity. Further characterization is followed by the determination of sensor performance.\n\nColor change of ACN can be affected by several factors such as temperature, pH, light intensity, sugar moiety and different phenolic derivatives.17 Due to its solubility in aqueous solution, the color change of ACN is caused by structural transformations of carbon skeleton affected by the levels of H+. Four major anthocyanin skeletons have been reported in the literature at different pH values, namely the red flavylium cation (pH below 2), the blue quinoidal base (pH 8-10), the colorless carbinol pseudo base (pH 3-6), and the colorless chalcone (pH 3-6).32,33 The effect of ACN concentrations on optical pH sensors response has also been studied and shown (Table 1 and Figure 6).\n\nThe sensitivity of the sensor toward variations in ACN concentrations showed not significantly different, but the absorbance vs pH plot showed an increase in the value of the intercept. This indicates the intensity of the sensor color increases with increasing ACN concentrations. Furthermore, the ACN concentration of 0.025 mg/L will be used to construct the optical pH sensor for the next characterization.\n\nEffect of PC weight towards sensor sensitivity\n\nThe weight variation of PC (0.05, 0.1, and 0.15% w/v) was studied to find the best sensor sensitivity. At varied weights, PC was dissolved using CaCl2 0.1 M to construct cross linking between Ca2+ ion and galacturonate until a pectin solution in the form of gel was produced.34 The effect of PC weight towards the sensitivity of optical pH sensor has been presented (Figure 7). The optimal weight percentage of PC was found at 0.1 % w/v. The membrane with 0.1% w/v pectin has a flatter surface thus making it as the most suitable optical sensor. PC membrane with only 0.05% w/v PC possessed a gel like texture due to the excess of water which causes a longer time to form a solid membrane. This phenomenon is quite similar for membrane preparation using a phase inversion method.20,35,36 On the other hand, membrane with 0.15% PC is very dense and has a non-homogenous surface which is not preferred for optical pH membrane application.37\n\nThe performance of an optical pH sensor may be affected by the types and concentration of the buffer. Figure 8 shows that the sensitivity of the sensor with phosphate buffer was 0.0877 with an R-square value of 0.993. On the other hand, the ACN/PC sensor with citrate buffer had a sensitivity of 0.074 (R2 = 0.981). Through physical observation, the ANC in the sensor would display a higher color intensity when in phosphate buffer compared to citrate buffer even in the same pH range. This is due to the lower Ka value of phosphate buffer compared to citrate buffer. Altogether, we conclude that the phosphate buffer contributes to better sensitivity of our pH sensor as opposed to citrate buffer. Therefore, the effect of concentration was studied using the phosphate buffer.\n\nThe effect of phosphate buffer concentration towards this sensor’s sensitivity is shown in Figure 9. This pH sensor produces the best sensitivity of 0.1238 (R2 = 0.9989) when the phosphate buffer 0.03 M was used. Meanwhile, the sensitivities of the pH sensor using phosphate buffer with concentrations of 0.05 M and 0.1 M were found lower at 0.072 (R2 = 0.9745) and 0.084 (R2 = 0.9805), respectively. The pH sensor with phosphate buffer 0.03 M gave a more contrast in the color change at different pH levels, in comparison with that of citrate buffer. In comparison to other earlier studies,9,10 our ACN/PC optical pH sensor has a wider working range of pH.\n\nThe response time of this sensor was determined by the required duration (minutes) that the sensor achieves a stable result. Response time was determined at 0, 5, 10, 15, 20, 25, and 30 minutes (Figure 10). The absorbance increased drastically from the first 5 minutes, indicating a good diffusion of the sample onto the membrane. The increase was later observed at minute 10, but no observable significant change afterward. Therefore, the optimum response time of this optical pH sensor is 10 minutes.\n\nIn addition, the reproducibility measurement was conducted on 10 different sensors with the same condition, where the relative standard deviation (RSD) was 9.15. This shows that there is a small difference in the absorbance values obtained from the repetition using new sensors. However, RSD that is below 10% is still acceptable for qualitative measurement.37\n\nThe investigated optical pH sensor had a stable response until the tenth day of storage (Figure 11). Afterward, the sensor response fell as much as 8.3% from the initial response, in which further decline was observed on the 15th day. At the same time, the %RSD also become poor; increasing as much as 36.61% from its initial state. The decrease in sensor performance after particular days of storing depends on the stability of the anthocyanin in maintaining its color. The lifetime of the optical pH sensor in this study is worse in comparison to that of our previous study,17 in which the performance did not drop until the 15th day. However, previously we used the synthetic chromoionophore ETH 5294 (CI); unlike in this study where we used natural anthocyanin that can be considered more sustainable. Furthermore, in this study, the lifetime is better in comparison to our currently reported sensor using ACN from Dioscorea alata L.37\n\nOptical pH sensor with the optimal conditions was used to monitor the freshness of tilapia fish that was kept at 4oC. The pH profile of the fish at two conditions, namely room temperature and 4°C storage temperature, is shown in Figure 12. A living fish has a pH value of around 7.4, but after death the pH decreases.38 The pH of the fish samples was measured after 0, 7, 12, 24 and 48 h storage time at room temperature and 4°C. Fish freshness was measured based on the absorbance value that is converted to pH value based on the constructed calibration curve.\n\nFish samples kept at room temperature possess a higher pH compared to the fish sample stored at 4°C. Fresh fish that was measured at 0 hours displayed pH of around 7.3-7.4. Following that, the pH decreases to 5.5-5.9, indicating that the fish has reached rigor mortis or postmortem rigidity. After the rigor mortis phase, the fish will undergo putrefaction due to the microbial activity in the fish sample.39 This activity causes the pH to become more basic due to the breakdown of proteins in the fish sample to become ammonia and trimethylamine.22–24 Results achieved from pH measurements at 7, 12, 24 and 48 hours at 4°C using the optical sensor yielded results of pH 5.9, 6.9, 7.1 and 7.9. Based on these results, it can be said that fish that is kept at room temperature will undergo a faster decomposition. This is due to the exposure to sunlight thus a higher temperature that will accelerate the process of decomposition.\n\n\nConclusion\n\nACN extracted from Ruellia tuberosa L can be immobilized into a PC matrix to produce a sensitive optical pH sensor. The extracted ACN has a similarity over the FT-IR profile of cyanidin-3-glucoside. The amount of ACN and PC in the membrane composite affected the optical pH performance, which was largely indicated by intercept and linearity values. The constructed optical pH sensor works best in phosphate buffer with a long lifetime. Its application in monitoring the freshness of fish has been successfully conducted against the storing time, where the decrease in pH values after a specific storing time indicates that the fish has reduced freshness.\n\n\nData availability\n\nHarvard Dataverse: Data Set for Optical pH Sensor Based on Pectin and Ruellia tuberosa L-derived Anthocyanin for Fish Freshness Monitoring, https://doi.org/10.7910/DVN/ZYCXAM.40\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\nBéné C, Barange M, Subasinghe R, et al.: Feeding 9 billion by 2050 – Putting fish back on the menu. Food Secur. 2015 Apr 10; 7(2): 261–74. 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Publisher Full Text Reference Source\n\nSharifi KA, Pirsa S: Biodegradable film of black mulberry pulp pectin/chlorophyll of black mulberry leaf encapsulated with carboxymethylcellulose/silica nanoparticles: Investigation of physicochemical and antimicrobial properties. Mater Chem Phys. 2021; In Press: 124580. Publisher Full Text\n\nHasanah U, Sani NDM, Heng LY, et al.: Construction of a Hydrogel Pectin-Based Triglyceride Optical Biosensor with Immobilized Lipase Enzymes. Biosensors. 2019 Nov; 9(4): 135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorbowicz M, Kosson R, Grzesiuk A, et al.: Anthocyanins of Fruits and Vegetables - Their Occurrence, Analysis and Role in Human Nutrition. Veg Crop Res Bull. 2008 Jan; 68(1): 5–22.\n\nLevy R, Okun Z, Shpigelman A: The Influence of Chemical Structure and the Presence of Ascorbic Acid on Anthocyanins Stability and Spectral Properties in Purified Model Systems. Foods. 2019 Jun; 8(6): 207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHastuti B, Masykur A, Hadi S: Modification of chitosan by swelling and crosslinking using epichlorohydrin as heavy metal Cr (VI) adsorbent in batik industry wastes. In: IOP Conference Series: Materials Science and Engineering. IOP Publishing; 2016. p. 12020.\n\nRahmi IM, Audina U, Husin H, et al.: Adsorptive removal of Cd (II) using oil palm empty fruit bunch-based charcoal/chitosan-EDTA film composite. Sustain Chem Pharm. 2021; 21: 100449. Publisher Full Text Reference Source\n\nIqhrammullah M, Marlina NS: Adsorption Behaviour of Hazardous Dye (Methyl Orange) on Cellulose-Acetate Polyurethane Sheets. IOP Conf Ser Mater Sci Eng. 2020 Jun 18; 845: 012035. Reference Source\n\nSafitri E, Humaira H, Murniana M, et al.: Optical pH Sensor Based on Immobilization Anthocyanin from Dioscorea alata L. onto Polyelectrolyte Complex Pectin–Chitosan Membrane for a Determination Method of Salivary pH. Polymers (Basel). 2021 Apr 14; 13(8): 1276. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nLi K, Luo Y, Shen H: Postmortem Changes of Crucian Carp (Carassius auratus) During Storage in Ice. Int J Food Prop. 2015 Jan; 18(1): 205–12. Publisher Full Text\n\nLiu D, Liang L, Xia W, et al.: Biochemical and physical changes of grass carp (Ctenopharyngodon idella) fillets stored at −3 and 0°C. Food Chem. 2013 Sep; 140(1–2): 105–14. PubMed Abstract \| Publisher Full Text\n\nNazaruddin N, Afifah N, Bahi M, et al.: Data Set for Optical pH Sensor Based on Pectin and Ruellia tuberosa L-derived Anthocyanin for Fish Freshness Monitoring. V1 ed. Harvard Dataverse. Publisher Full Text" }	[ { "id": "86206", "date": "04 Jun 2021", "name": "Sagir Alva", "expertise": [ "Reviewer Expertise My specialty is the synthesis and characterization of materials such as polymers for the development of chemical sensors/biosensors." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAfter I read and reviewed this article, I found the theme of this article quite interesting. However, unfortunately, there are shortcomings in this article which make it unfit for indexing. Therefore, I suggest a Not Approval status for this article. However, it can be improved in the revision with the following comments:\n1. It is true, when fish begin to undergo a process of decomposition, in addition to producing H+, ammonia is also produced. In the first paragraph, the authors only compared it with ammonia-ISE, where basically, the concept of measuring ammonia-ISE is an indirect measurement of ammonia based on the dissociation of ammonia in solution to form NH4+. So naturally not all ammonia will be detected. However, there are actually a lot of research on ammonia optical sensors. In other words, the ammonia optical sensor is nothing new. So there needs to be an explanation added to the introduction why choosing an optical pH sensor in detecting the freshness of fish compared to an ammonia optical sensor. What are the advantages of an optical pH sensor compared to an optical ammonia sensor?\n2. At the end of the first paragraph, you stated that measuring pH using an optical sensor might be good for samples that have interfering ions. With fish, what ions are supposed to be can interfere with the pH-ISE sensor, so you end up choosing the optical pH sensor over the pH-ISE sensor? An explanation of this needs to be added in the introduction section.\n3. Basically, a lot of plants and fruits also have ACN, and here you have also given examples such as blackberries. But why in this study have you focused on the Ruellia flower? Instead, you can also use ACN from blackberries immobilized using Pectin. What are the advantages of ACN from Ruellia compared to other plants? It is worth mentioning in the introduction the reasons for this.\n4. The use of a hydrogel membrane will indeed facilitate the diffusion of the analyte. However, the hydrogel membrane has serious problems such as easy to swell and break, so that the dye used can be leached and the sensor life time is decreased. There needs to be some clarification on this. In addition, there needs to be additional experimental data on the % swelling index of the pectin membrane used.\n5. In optical sensor, leaching study is an important thing to do. However, in this article there are no leaching study data, so it is necessary to add experimental data for leaching study testing.\n6. There are several natural hydrogel polymers. It is necessary to add reasons why choose Pectin over other natural hydrogel polymers. What are the advantages of pectin over other natural hydrogel polymers?\n7. On page 7 and the beginning of the first paragraph, there is the sentence: “The constructed optical pH biosensor based on the ACN derived from R. tuberosa L flower has hydrogel characteristics.” - It need clarification, is this really an optical biosensor? Because here I don't see any use of enzymes, peptides, micro-organisms etc.\n8. On page 7 it is stated that the colour change is caused by a structural transformation of the ACN. It is necessary to add pictures of the changes in the chemical structure of ACN at various pH variations, such as acidic, neutral and basic.\n9. Still from page 7, you stated that one of the factors that caused the change in ACN colour was caused by light. You need to clarify, how do you control the light intensity during the test period, so that the colour of the ACN remains stable and how long can the light change the colour of the ACN?\n10. In the ACN variation data, the resulting absorbance will also decrease with the lower ACN concentration, and in the end you use a concentration of 0.025 mg/L as the optimum concentration of ACN. What if the concentration of the ACN is less than 0.025 mg/L? Is ACN still able to respond to changes in pH or not able to respond to changes in pH? Additional data are needed for testing less than 0.025 mg/L.\n11. In sensor development, validation testing is very important to ensure that the fabricated sensor performs at least the same as standard test equipment. Here, I don't see that. There needs to be additional validation data with standard methods to test the freshness of fish based on pH changes.\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? Yes", "responses": [ { "c_id": "6895", "date": "21 Jul 2021", "name": "Nazaruddin .", "role": "Author Response", "response": "Comment: It is true, when fish begin to undergo a process of decomposition, in addition to producing H+, ammonia is also produced. In the first paragraph, the authors only compared it with ammonia-ISE, where basically, the concept of measuring ammonia-ISE is an indirect measurement of ammonia based on the dissociation of ammonia in solution to form NH4+. So naturally not all ammonia will be detected. However, there are actually a lot of research on ammonia optical sensors. In other words, the ammonia optical sensor is nothing new. So there needs to be an explanation added to the introduction why choosing an optical pH sensor in detecting the freshness of fish compared to an ammonia optical sensor. What are the advantages of an optical pH sensor compared to an optical ammonia sensor? Response: The literature on the development of the NH3 optical biosensor was developed by Dan-Feng Lu and Zhi-mei Qi in 2019 using bromothymol blue and a porous glass membrane. This sensor can only work at low concentrations of ammonia. Another ammonia sensor has also been developed by Maximilian Maierhofer et al. (2020), who fabricated the sensor using fluorescence properties of aza-BODIPY dyes with a response time of 390 seconds. Detection of ammonia as a total volatile basic nitrogen (TVB-N) to determine fish spoilage requires a sample destruction process (Nathan Wells et al.(2019), Talanta 194: 830–836). Then, the standard curve was obtained from measuring the absorbance of the trimethylamine (TMA) compound that produces ammonia through a complicated procedure. On the other hand, this method was also based on pH measurements. So it can be concluded that ammonia is also correlated with changes in pH to determine the freshness of fish. The same concept has also been previously reported by T. Werner et al. (1995) Analyst 120 1627–1631 where the determination of ammonia was based on measuring pH using an ion-pair indicator. Therefore, the detection of fish freshness through pH measurements is more representative of the actual condition of in-situ tests. An explanation of the ammonia optical sensor and its drawbacks for determining fish freshness has been described in the introduction. Comment: At the end of the first paragraph, you stated that measuring pH using an optical sensor might be good for samples that have interfering ions. With fish, what ions are supposed to be can interfere with the pH-ISE sensor, so you end up choosing the optical pH sensor over the pH-ISE sensor? An explanation of this needs to be added in the introduction section. Response: Literature reported ISE H+ response is strongly affected by alkaline ions has been added in the Introduction. Comment: Basically, a lot of plants and fruits also have ACN, and here you have also given examples such as blackberries. But why in this study have you focused on the Ruellia flower? Instead, you can also use ACN from blackberries immobilized using Pectin. What are the advantages of ACN from Ruellia compared to other plants? It is worth mentioning in the introduction the reasons for this. Response: Mostly, coloured plants contain anthocyanins, including blackberries. Anthocyanins from blackberries can also be used as pH-sensitive active ingredients to develop optical pH sensors. On the other hand, sources of anthocyanins from blackberries are difficult to obtain in our area. In this study, Ruellia anthocyanins were used as a sensitive pH compound for optical pH sensor development because the flowers are easy to obtain. In addition, based on a preliminary study on the sensitivity of the anthocyanin at various pHs, we found that the anthocyanin has a great potential to be further applied in developing optical pH sensor. Comment: The use of a hydrogel membrane will indeed facilitate the diffusion of the analyte. However, the hydrogel membrane has serious problems such as easy to swell and break, so that the dye used can be leached and the sensor life time is decreased. There needs to be some clarification on this. In addition, there needs to be additional experimental data on the % swelling index of the pectin membrane used. Response: Firstly, the membrane use as optical pH sensor is not applied by immersion into aqueous samples therefore swelling index is not relevant. Secondly, there have been extensive research pertaining to the swelling profile of pectin, of which are Fong H. WEH et al. (2014) Lat. Am. J. Pharm. 33(3): 420-31 and Naziha Chirani et al. 2015. Journal of Biomedical Sciences. Vol. 4 No. 2:13. P 1-23. Below is swelling index of pectin in different media based on the reported study. [Figure] Comment: In optical sensor, leaching study is an important thing to do. However, in this article there are no leaching study data, so it is necessary to add experimental data for leaching study testing. Response: In our opinion, not all leaching tests need to be carried out in sensor or biosensor manufacturing studies. It depends on the sensor application. In this study, we did not immerse the sensor in the sample. The sensor is placed directly on the surface of the fish, and then the colour changes are measured. For a liquid sample, only a small amount of sample dropped onto the sensor surface. The sensor produced is a disposal sensor. Comment: There are several natural hydrogel polymers. It is necessary to add reasons why choose Pectin over other natural hydrogel polymers. What are the advantages of pectin over other natural hydrogel polymers? Response: Pectin was chosen because of: Its non-toxicity; because the application is for a foodstuff, the sensor should not be toxic. Its ability in forming membrane structure. Transparent and homogenous. In the case of optical pH sensor for fish freshness monitoring, other studies have reported chitosan, starch, and cellulosic materials; while pectin is scarcely reported. Hence, the use of pectine is a novelty. Those characteristics have been added in the last paragraph of introduction. Comment: On page 7 and the beginning of the first paragraph, there is the sentence: “The constructed optical pH biosensor based on the ACN derived from R. tuberosa L flower has hydrogel characteristics.” - It need clarification, is this really an optical biosensor? Because here I don't see any use of enzymes, peptides, micro-organisms etc. Response: It is a sensor not as a biosensor. Has been modified: “biosensor” to “sensor” Comment: On page 7 it is stated that the colour change is caused by a structural transformation of the ACN. It is necessary to add pictures of the changes in the chemical structure of ACN at various pH variations, such as acidic, neutral and basic. Response: Has been added, see figure 7 [Figure]. Comment: Still from page 7, you stated that one of the factors that caused the change in ACN colour was caused by light. You need to clarify, how do you control the light intensity during the test period, so that the colour of the ACN remains stable and how long can the light change the colour of the ACN? Response: The sensor has been made through a storage process in a dark condition and a temperature of 4oC. At the time of measurement, the sensor is also kept in the dark and needs a short time of exposure to light during the measurement process. We predict no significant colour change. In addition from our preliminary experiment, immobilized anthocyanins on the pectin matrix have good stability. Comment: In the ACN variation data, the resulting absorbance will also decrease with the lower ACN concentration, and in the end you use a concentration of 0.025 mg/L as the optimum concentration of ACN. What if the concentration of the ACN is less than 0.025 mg/L? Is ACN still able to respond to changes in pH or not able to respond to changes in pH? Additional data are needed for testing less than 0.025 mg/L. Response: The effect of anthocyanin concentration is not significantly different on sensor sensitivity and linear range. Anthocyanin concentrations less than 0.025 mg/L are predicted still to respond to pH changes. Due to the intensity of the colour decreases, the sensitivity will also decrease. Thus, the determination of sensitivity for anthocyanin concentrations lower than 0.025 mg/L was not determined. Comment: In sensor development, validation testing is very important to ensure that the fabricated sensor performs at least the same as standard test equipment. Here, I don't see that. There needs to be additional validation data with standard methods to test the freshness of fish based on pH changes. Response: We have validated the optical sensor method using H+ ion-selective electrodes. However, we do not report it. In this paper, we focus more on how the pH changes in fish stored at room temperature and 4oC. The following are the results of the validation of measurements carried out on fish measured using an optical pH sensor with H+ ISE. [Table] From the results obtained that the ISE measurement is influenced by temperature (as also suggested by other reported studies) so that the results obtained are different from the optical pH sensor. Due to the limited features available in this comment column, we choose to upload our full response in an accessible link. Please find it through this link." } ] }, { "id": "86281", "date": "18 Jun 2021", "name": "Nur Hamidah Abdul Halim", "expertise": [ "Reviewer Expertise Electrochemical biosensors" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper shows an experimental work on optical sensor using simple optical approach. This paper shows a good work with a potential study on optical pH sensor for fish freshness monitoring. However, it is suggested to elaborate further on discussion how the mechanism and reaction with illustrated figure. The novelty should be explicitly mentioned in the introduction, abstract and findings. A table on reported or published and comparison should also be made available to see the research contribution. It is suggested that the authors may revise based on few comments below:\nAbstract “The sensor displayed an excellent response after 10 minutes of exposure, possessing a response stability for 10 consecutive days. The decrease in pH value of the Tilapia fish from 7.3 to 5 was observed in a 48 hour test, which can be used as the parameter when monitoring fish freshness.“\nComment: The statement of decrease in pH value need to be elaborated to highlight the novelty of this research. The authors may add comparison in terms of performance and mechanism that differentiate this works and other reported work. E.g How pH value decrease mechanism is evaluated and correlated to observe the fish freshness.\nIntroduction Para 2: \"Nevertheless, these aforementioned pH sensors could only be used on solutions with near-neutral pH as more basic or acidic solutions will give an insignificant response time. Pourjavaher et al.11 has designed an optical pH sensor based on cellulose nanofibers with red cabbage (Brassica oleracea) extract, while Rajan et al. (2018)12 has produced an optical pH sensor using peonidin pigment. However, this study did not report the working pH range of peonidin. The use of anthocyanin (ACN) from blackberries and chitosan membrane in an optical pH sensor has been established.13 The interaction and mechanical properties of chitosan membrane with entrapped ACN have also been reported.\"\nComment: This paragraph should elaborate more on fish freshness and its correlation to pH based on previous study. The use of ACN should be illustrated for reader to understand more as the sentences is hanging (Referring to “The interaction and mechanical properties of chitosan membrane with entrapped ACN have also been reported.”). It is more helpful if a table or illustrated mechanism is shown to support this study and having a good flow of this paper.\nPara 3: \"A more recent study on fish freshness monitoring through optical methods was reported by Moradi et al.15 using nanofiber bacterial cellulose with ACN. However, this method requires a relatively long analytical time as the pH measurement could not be conducted in situ. Chen et al. (2020)6 has developed a sensitive novel film prepared from starch polyvinyl alcohol and starch polyvinyl alcohol glycerol.\"\nComment: Again, this paragraph does not add the value on published work with this work. The mechanism on optical pH to monitor fish freshness is still not addressed. No comparison on the electrochemical performance (LOD, Linear range, selectivity) was mentioned here. How long analytical time is related to pH measurement by having different material like nanofiber and optical properties coming from ACN dye. The ACN sensitivities towards pH correlation to ACN optical properties may need to be added here as well.\nResearch and Methodology Comment: The methodology shows a sufficient description a to give reader a good understanding on how this study is conducted. It is suggested that the authors may add process flow/illustration to complete the overall picture on steps and its mechanism.\nResults and Discussion Figure 4. SEM profile of (a) PC and (b) ACN/PC membranes.\nComments: The morphology of ACN/PC membrane does not seem like a crack. It seems to have a wavy layer of membrane that might be the contributed to the adhesion/stress tension or air gap of the ACN/PC compared to PC alone. Is there any study on different ration of CAN added to this PC, or is it already optimized? The caption should be more detailed.\n\"Color change of ACN can be affected by several factors such as temperature, pH, light intensity, sugar moiety and different phenolic derivatives. Due to its solubility in aqueous solution, the color change of ACN is caused by structural transformations of carbon skeleton affected by the levels of H+.\"\nComments: The color change mechanism is important to be introduced earlier in the introduction section and can be help with illustration. How different phenolic derivatives change this CAN, and which phenolic derivatives took place in this reaction? The authors may put or add this point to support the color change mechanism towards fish freshness from the finding.\nEffect of PC weight towards sensor sensitivity Comments: The pectin is a membrane that hold the ACN dye to improve the sensitivities. From Fig 4, the importance of having optimum load/weight of pectin is important the membrane with less surface tension, and this is the reason of having crack or wavy like membrane. It is very important optimum ratio of CAN/PC to have smooth ACN/PC membrane in this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6896", "date": "01 Sep 2022", "name": "Nazaruddin .", "role": "Author Response", "response": "Reviewer Nurhamidah Thank you very much for valuable comments Comment: The statement of decrease in pH value need to be elaborated to highlight the novelty of this research. The authors may add comparison in terms of performance and mechanism that differentiate this works and other reported work. E.g How pH value decrease mechanism is evaluated and correlated to observe the fish freshness. Response: The pH of fresh tilapia was 7.3, and the pH decreased to 5 after 7 hours of storage in two storage conditions (room temperature and 4oC). Changes in pH from 7.3 to 8.7 are the condition of fish monitoring within 48 hours. The post mortem glycolysis-derived lactic acid accumulation is also responsible for the pH decrease. Additionally, we have amended the manuscript with the following text: “Our method of measuring the change of pH is different to the most reported studies using colorimetric response. Indeed, one may argue that colorimetry could give the best practicality of the sensor use. However, it suffers from quantitative information, as it depends on the RGB profiles that requires complex model to convert the response into measured pH value. Moreover, the reported studies rely on the volatile basic compounds released from the meat. Taken altogether, the reported studies were unable to capture the decrease of pH during rigor mortis phase. In food industry, fish meat is best processed by the filleting machine during the pre- or post-rigor mortem. This is the novelty of our optical pH sensor which is useful for the quality control and processing of fish meat in industrial settings.” Comment: This paragraph should elaborate more on fish freshness and its correlation to pH based on previous study. The use of ACN should be illustrated for reader to understand more as the sentences is hanging (Referring to “The interaction and mechanical properties of chitosan membrane with entrapped ACN have also been reported.”). It is more helpful if a table or illustrated mechanism is shown to support this study and having a good flow of this paper. Response: The paragraph 2 has been elaborated: Nevertheless, these aforementioned pH sensors could only be used on solutions with near-neutral pH as more basic or acidic solutions will give an insignificant response time. Pourjavaher et al.11 has designed a pH sensor using bacterial cellulose (BC) nanofiber matrix to immobilize anthocyanin (CAN) from red cabbage (Brassica oleracea) extract. The sensor has a fairly wide pH range but it needs further characterization to evaluate the sensor performance, especially, for real foodstuff analysis. The use of ACN from blackberries and chitosan membrane in an optical pH sensor has been established.13 The interaction and mechanical properties of chitosan membrane with entrapped ACN have also been reported.14 Anthocyanins are flavonoids possessing a number of hydroxyl groups contributing a strong interaction with chitosan via hydrogen bonding. Comment: Again, this paragraph does not add the value on published work with this work. The mechanism on optical pH to monitor fish freshness is still not addressed. No comparison on the electrochemical performance (LOD, Linear range, selectivity) was mentioned here. How long analytical time is related to pH measurement by having different material like nanofiber and optical properties coming from ACN dye. The ACN sensitivities towards pH correlation to ACN optical properties may need to be added here as well. Response: The paragraph 3 has been elaborated: A more recent study on fish freshness monitoring through optical methods was reported by Moradi et al. 15 using nanofiber bacterial cellulose with ACN. However, this method requires a relatively long analytical time as the pH measurement could not be conducted in situ. Chen et al. (2020)6 has developed a sensitive novel film prepared from starch polyvinyl alcohol and starch polyvinyl alcohol glycerol. The study used curcumin from turmeric and anthocyanin from purple sweet potatoes. The results showed that the mixture of curcumin and ACN improved the stability than that of the individual active substances. As the consequence, the sensor could be employed to detect volatile ammonia as the fish freshness indicator. Comment: The methodology shows a sufficient description a to give reader a good understanding on how this study is conducted. It is suggested that the authors may add process flow/illustration to complete the overall picture on steps and its mechanism. Response: The steps have been added Methods: Study Design The first step in sensor fabrication was the extraction of anthocyanin from Ruellia tuberosa L. The extracted anthocyanins were then mixed with pectin solution and printed proportionally as an optical pH sensor. The optical pH sensor was then characterized and the optimized and then applied to monitor the freshness of tilapia. The image below is a schematic diagram summarizing research procedures conducted in this work. [Figure] Comment: The morphology of ACN/PC membrane does not seem like a crack. It seems to have a wavy layer of membrane that might be the contributed to the adhesion/stress tension or air gap of the ACN/PC compared to PC alone. Is there any study on different ration of CAN added to this PC, or is it already optimized? The caption should be more detailed. Response: The ratio of ACN has been optimized based on the sensitivity and R2, see Table 1 and Figure 8. The description for SEM images analysis has been revised per suggestion. Comment: The color change mechanism is important to be introduced earlier in the introduction section and can be help with illustration. How different phenolic derivatives change this CAN, and which phenolic derivatives took place in this reaction? The authors may put or add this point to support the color change mechanism towards fish freshness from the finding. Response: The anthocyanin structure under different pHs has been added in the manuscript as suggested (see Figure 7). [Figure] Comment: The pectin is a membrane that hold the ACN dye to improve the sensitivities. From Fig 4, the importance of having optimum load/weight of pectin is important the membrane with less surface tension, and this is the reason of having crack or wavy like membrane. It is very important optimum ratio of CAN/PC to have smooth ACN/PC membrane in this study. Response: We have optimized the PC weight and the optimum was reached for 0.1% PC to find optimum sensitivity. The membrane with 0.1% w/v pectin has a flatter surface thus making it as the most suitable optical sensor. SEM characterization was carried out on the optimum pectin weight. The wavy like surface structure was probably due to the addition of anthocyanin. Added as a recommendation in conclusion: More studies indeed need carried out to obtain smooth surface morphology to improve the optical sensor performance. Due to the limited features in this comment column, we have uploaded our full response through this link." } ] } ]	1	https://f1000research.com/articles/10-422
https://f1000research.com/articles/10-620/v1	21 Jul 21	{ "type": "Brief Report", "title": "Correlation between maternal variables and the onset and severity of preeclampsia", "authors": [ "Nafiu Amidu", "Moses Banyeh", "Stephen Justice Adusu", "Nafiu Amidu", "Stephen Justice Adusu" ], "abstract": "Background: The study sought to determine the correlation between the onset and severity of preeclampsia (PE) and maternal sociodemographic variables: age, parity and body mass index (BMI); medical history: systolic blood pressure (SBP), diastolic blood pressure (DBP), and proteinuria; fasting lipids: total cholesterol (TCHOL), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TRIG); liver enzymes: aspartate transaminase (AST), alanine transaminase (ALT) and gamma-glutamyl transferase (GGT), and other variables: serum neutrophil gelatinase associated lipocalin (sNGAL), endothelin-1 (ET-1) and creatinine (CRT). Methods: This was a case–control study from January–June 2018. The study involved 270 pregnant women aged 18–37 years. Half of the study population (n=135) had PE and were categorized into either early-onset PE (EOPE) or late-onset PE (LOPE) and whether the PE was characterized by severe features (PS) or without severe features (PNS). The cases (PE) were matched by maternal and gestational age to 135 women with normotensive and uncomplicated pregnancies. A single venous blood sample was collected after an overnight fast and analysed using ELISA or routine biochemistry technique. Results: Maternal blood lipids (except HDL), liver enzymes (except GGT), CRT, sNGAL, ET-1, proteinuria, and BMI were positively correlated while HDL was negatively correlated with the onset and severity of PE (P<0.050). Conclusion: There are significant correlations between maternal variables and the onset and severity of PE. These findings are useful for the early detection and management of PE, especially in resource-limited settings.", "keywords": [ "Preeclampsia", "Correlation", "Onset", "Severity", "Ghana" ], "content": "List of abbreviations\n\nALT, alanine aminotransferase\n\nAST, aspartate aminotransferase\n\nCRT, creatinine\n\nELISA, enzyme-linked immunosorbent assay\n\nEOPE, early onset preeclampsia\n\nET-1, endothelin-1\n\nGGT, gamma glutamyl transferase\n\nHDL, high density lipoprotein\n\nLDL, low density lipoprotein\n\nLOPE, late onset preeclampsia\n\nPE, preeclampsia\n\nPNS, preeclampsia without severe feature\n\nPS, preeclampsia with severe features\n\nTCHOL, total cholesterol\n\nTRIG, triglycerides\n\nsNGAL, serum neutrophil gelatinase-associated lipocalin\n\n\nIntroduction\n\nThis study sought to determine the correlation between the onset and severity of preeclampsia (PE) and maternal variables in a Ghanaian population. PE is a multi-system disorder of pregnancy with unknown aetiology. It is responsible for about 3–8% of the global burden of maternal and neonatal morbidity and mortality.1 Hypertension, with or without proteinuria, occurring after 20 weeks of gestation, is the cardinal symptom of PE. The occurrence of PE may be early, happening before 34 weeks (EOPE) or late (LOPE). Preeclampsia may also be characterized by severe features (PS) or without severe features (PNS).2\n\nAlthough the aetiology of PE is poorly understood, aberrant trophoblastic invasion of the placenta, placental hypoxia and endothelial dysfunction have been suggested. The generalized inflammatory process, angiogenic imbalance, endothelial injury and increased oxidative stress in PE, may cause organ and organ–system dysfunctions including the renal, the hepatic and the cardiovascular systems.2,3\n\nPrevious studies have demonstrated that maternal lipids, liver function, renal function, ET-1 and sNGAL are affected by the onset and severity of PE.4,5 However, the influence of genetic and environmental factors on PE may lead to population and sub-population variabilities in the correlations between the onset and severity of PE and maternal variables.6,7 There is therefore the need for population-specific studies to establish local reference data for the early diagnosis and management of PE.\n\n\nMethods\n\nThis was a case–control study from January to June 2018. The study was conducted at the Richard Novarti Catholic Hospital, located at Sogakope in the South Tongu District of the Volta Region of Ghana.\n\nThe study involved 270 pregnant women aged 18–37 years. The sample size was calculated following the recommendations of Fahim, Negida.9 The study population was divided into two groups: 135 women with PE who were further stratified based on the onset of PE as either EOPE (n = 71) or LOPE (n = 64) and also based on the presence or the absence of severe features as either PNS (n = 87) or PS (n = 48). The cases were matched by maternal and gestational age to 135 women with normotensive and uncomplicated pregnancies at sampling (serving as controls). The inclusion criterion was a diagnosis of PE, whether early or late onset, with or without severe features. Exclusion criteria included: all women with twin gestations; those who were previously diagnosed with chronic hypertension, sickle cell anaemia, diabetes mellitus, gestational diabetes, cardiovascular disorder, renal disease and those who were on antihypertensive or magnesium medication before the recruitment. Sociodemographic and clinical information was collected using a structured questionnaire and also from their medical records.\n\nThe definition of PE, its onset and severity was based on the recommendations of the American College of Obstetricians and Gynecologists.2 Preeclampsia was defined as the new-onset of hypertension (systolic ≥140 mmHg, diastolic ≥90 mmHg, taken 4 hours apart) with proteinuria (2+ on the dipstick at presentation) after 20 weeks of gestation. Early-onset PE was defined as PE occurring before 34 weeks of gestation and LOPE was defined as PE occurring at/after 34 weeks of gestation. Preeclampsia with severe features was defined as PE characterized by systolic (≥160 mmHg) or diastolic (≥110 mmHg ) blood pressure while PNS was defined as PE without severe features.\n\nBlood pressure was measured with the aid of a cuff sphygmomanometer according to the fifth Korotkoff sound. Urine screening for proteinuria was performed using a urine dipstick. A single peripheral venous blood sample was collected from each subject’s antecubital vein after an overnight fast (12 h) into an EDTA and a gel-separator tube. The serum samples were allowed to clot for 30 min at 4°C and both tubes were then centrifuged at 1500 rpm for 10 min to separate the serum/plasma. Serum and plasma samples were aliquoted into plastic cryotubes and then frozen at −25oC until analysis. Serum NGAL was analysed in duplicates using the ELISA kit from abcam (152 Grove Street Waltham, MA 02453., USA). The kit had a sensitivity of 14.8 pg/mL, within a linearity range of 46.9 pg/mL - 3000 pg/mL. The intra-assay and inter-assay coefficients of variation were ≤3.8% and ≤2.7% respectively. Serum ET-1 was analyzed in duplicates using Human ELISA kit (Biomedica Medizinprodukte GmbH, Divischgasse 4, 1210 Vienna, Austria). The sensitivity of the ET-1 ELISA kit was 0.086 pg/mL within a linearity range of 0.00–12.85 pg/mL. The intra-assay and the inter-assay coefficients of variation were ≤4% and ≤5% respectively. The TCHOL, HDL, LDL, TRIG, AST, ALT, GGT and CRT were assayed using the plasma/serum samples (as appropriate) through routine biochemistry analysis on the BT 1500 automated biochemistry analyzer (Biotechnica Instruments, SPA, Italy) following the manufacturer’s instructions and using the recommended reagents. The samples in this study were not previously thawed and refrozen.\n\nData were entered into a Microsoft Excel (RRID:SCR_016137) spreadsheet (Google Sheets (RRID:SCR_017679) is an open access alternative) and statistical analysis was performed using SPSS (v23) (RRID:SCR_019096), and GraphPad Prism (v8) (RRID:SCR_002798); JASP (RRID:SCR_015823) is an open access alternative. The normality of the data was checked using the Kolmogorov–Smirnov test. Continuous variables were reported as median (IQR) or mean ± SD (as appropriate) and number (%) for categorical variables. Differences between median and mean values were determined using the Mann–Whitney U test and the student t-test respectively. Spearman rank correlation and linear regression analysis were used to determine the relationship between the independent and the outcome variables. A probability level of P < 0.050 was considered statistically significant.\n\n\nResults\n\nThe general characteristics of the study population are summarized in Table 1. This was an observational study with only one stage and as such there were no loss of participants. The controls and the cases (PE) did not differ in maternal and gestational age (P > 0.050). The BMI, sNGAL, ET-1, TCHOL, LDL, TRIG, ALT, AST and CRT were significantly elevated while HDL was markedly reduced in PE as compared to the controls (P < 0.010). Also, the maternal and gestational age, BMI, GGT and CRT were significantly elevated in LOPE as compared to EOPE (P < 0.050). In comparing PNS to PS, maternal age was reduced while sNGAL, ET-1, TRIG, ALT, AST and GGT were considerably increased in PS (P < 0.050). The correlation and linear regression analysis between the onset of PE and maternal variables are shown in Figure 1. Aside from maternal age and GGT that showed no significant correlation with the onset of PE, HDL was inversely correlated (r = −0.125, P = 0.048), while, blood pressure, BMI, parity and proteinuria were all positively correlated with the onset of PE (P < 0.050). The correlation and linear regression analysis between the severity of PE and maternal variables are shown in Figure 2. Maternal HDL was inversely correlated (r = −0.199, P = 0.001), while the rest of the maternal variables (except maternal age, parity and GGT) were all positively correlated with the severity of PE (P < 0.050).\n\nThe control, EOPE and LOPE were dummy coded before linear regression and correlation analysis.\n\nSignificant at the 0.010 level, Significant at the 0.001 level.\n\nThe control, PNS and PS were dummy coded before linear regression and correlation analysis.\n\nSignificant at the 0.010 level, *Significant at the 0.001 level.\n\n Significant at the 0.010 level.\n\n** Significant at the 0.001 level compared to control.\n\n† Significant at the 0.050 level.\n\n†† Significant at the 0.010 level.\n\n††† Significant at the 0.001 level compared to EOPE.\n\n‡ Significant at 0.010 level.\n\n‡‡ Significant at the 0.001 level compared to PNS.\n\n\nDiscussion\n\nThe study aimed to determine the correlation between the onset and severity of PE and maternal variables. Maternal variables including lipids, liver enzymes (except GGT), creatinine, sNGAL, ET-1 were significantly correlated with the onset and severity of PE. Also, both systolic and diastolic blood pressure, proteinuria, and BMI were positively correlated with the onset and severity of PE.\n\nThe increased blood pressure in PE stems from the resistance in the vascular system and decreased intravascular volumes and cardiac output. There is a decrease in conduit artery compliance as well as the obliteration of the fall of nocturnal blood pressure. Hypertension in PE may contribute to organ damage including, acute kidney injury and renal dysfunction.10 Previous studies have shown that renal blood flow and glomerular filtration rates are decreased in PE as shown by the loss of podocytes, endothelial swelling and glomerular endotheliosis in biopsies from PE patients. Damaged endothelial cells in PE may induce clotting, as well as loss of anticoagulant ability as levels of nitric oxide and prostaglandin, plummet leading to kidney thrombotic microangiopathy.11,12 The associated renal dysfunction in PE may account for the elevated serum CRT and sNGAL. Serum NGAL is usually secreted directly into damaged renal tubular cells with the aim of re-epithelialization and has been shown to indicate renal injury much earlier than CRT. Serum NGAL is also involved in immune-system modulation as its levels increase in PE due to the generalized inflammation. The onset and severity of PE are also significantly correlated with proteinuria. Increased proteinuria in PE is due to increased permeability of the renal tubules partly due to the glomerular endotheliosis.10\n\nPE is also associated with hepatic dysfunction and abnormal lipid metabolism as shown by elevation in some serum liver enzymes and lipids. This may be due to endothelial injury which causes hepatocellular necrosis and hepatic microcirculatory deterioration. Hepatic dysfunction may affect the synthetic functions of the liver including abnormalities in blood coagulation.11,13 It is has been shown that particles of HDL and LDL are remodelled during normal pregnancy becoming smaller and denser with increased proatherogenic potential and this is worsened by PE.14 Although the exact mechanism is unknown, the changes in maternal lipid metabolism in PE has been suggested to be a compensatory process to increase energy supply to the developing foetus.15\n\nSerum levels of ET-1 were elevated in PE as compared to the controls and the levels increased with the severity of the symptoms of the disease.4,16–21 Several studies have demonstrated the role of ET-1 in the aetio-pathology of PE including the elevated circulating ET-1, increase in ET-1 converting enzyme (ECE) activity, the differential effect of systemic ECE inhibition and increased localized ET-1 production in tissues of maternal origin.16–18,22–24 Further evidence of the role of ET-1 in PE was the increased expression of markers of oxidative stress when placental tissue explant from normal pregnancy were incubated with exogenous ET-1 in-vitro. Also, ET-1 reduced JEG-3 cell lines proliferation, evidence of the possible role of ET-1 in preventing trophoblast invasion of the placenta in the early stages of PE.25 In the reduced uterine perfusion pressure (RUPP) model, where blood flow to the uterus was partially restricted, inducing placental hypoxia and ischemia, there was increased expression of preproendothelin mRNA, with the associated proteinuria, renal injury and endothelial dysfunction.26,27 However, issues have been raised regarding the interpretation of serum ET-1 results; firstly, ET-1 from syncytiotrophoblastic cells in the placenta would have been diluted by the time blood reaches the antecubital area for sampling, secondly, ET-1 is an autocrine/paracrine product whose serum levels does not reflect local tissue production. It has been suggested that an ET-1 receptor saturation study be performed, in addition to serum ET-1 estimation, to measure ET-1 receptor activation since a prolonged steady-state of ET-1 receptor saturation may be reached in PE through the continuous infusion of ET-1 from the uterine veins.28 This study is among a few studies coming from Ghana to have simultaneously determined the correlation between the onset and severity of PE with multiple maternal variables, including sNGAL and ET-1. However, the authors acknowledge some limitations; blood samples were collected after the manifestation of symptoms and diagnosis of preeclampsia. It is recommended that sampling is done in each trimester through to the post-partum period to determine the exact period changes in maternal variables begin to occur due to PE. Also, quantitative 24-hour urine protein or creatinine-to-protein ratio is preferred to the dipstick method in screening for proteinuria.\n\nIn conclusion, the onset and severity of PE are significantly correlated with maternal variables such as parity, proteinuria, blood pressure, lipids, liver enzymes, sNGAL, ET-1 and creatinine. There is therefore the need for regular monitoring of these maternal variables for the early detection and management of PE in the Ghanaian population.\n\n\nData availability\n\nOSF: Underlying data for ‘Correlation between maternal variables and the onset and severity of preeclampsia’. https://doi.org/10.17605/OSF.IO/CF8XB.\n\nThe project contains the following underlying data: maternal socio-demographics and anthropometric variables as well as serum/plasma fasting lipids, liver enzymes, creatinine, neutrophil gelatinase-associated lipocalin, and endothelin-1. The cases were grouped by the onset of PE and also by its severity.\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\nAuthors’ contributions\n\nNA conceived the idea and designed the experiment, SJA performed the experiment and collected the data, MB analysed the data and wrote the manuscript. All authors provided critical feedback, read and approved the final manuscript.\n\n\nEthics approval and consent to participate\n\nAll procedures performed in this study involving human participants were done following the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Institutional Review Board of the University for Development Studies (UDS), Tamale.\n\n\nConsent to participate\n\nInformed consent was obtained from all individual participants included in the study.\n\n\nConsent for publication\n\nConsent for publication was granted.", "appendix": "Acknowledgements\n\nThe authors will like to acknowledge the staff at the ante-natal clinic of the Novarti Catholic Hospital for helping in sample selection and data collection. We also acknowledge the staff of the Department of Biomedical Laboratory Science of the University for Development Studies for supervising the work. Lastly, we will like to thank all the participants for consenting to the study.\n\n\nReferences\n\nSaleh L, Danser JA, van den Meiracker AH : Role of endothelin in preeclampsia and hypertension following antiangiogenesis treatment. Curr Opin Nephrol Hypertens. 2016; 25(2): 94–99. PubMed Abstract \| Publisher Full Text\n\nACOG: ACOG Practice Bulletin No. 202: Gestational Hypertension and Preeclampsia. Obstet Gynecol. 2019; 133(1): 1. PubMed Abstract \| Publisher Full Text\n\nMalik A, Jee B, Gupta SK: Preeclampsia: Disease biology and burden, its management strategies with reference to India. Pregnancy Hypertens. 2019; 15: 23–31. PubMed Abstract \| Publisher Full Text\n\nWantania J, et al.: The Correlation of the Lipid Profile and Endothelin-1 with Severe Preeclampsia. Asian Res J Gynaecol Obstet. 2021: 7–16.\n\nJiang L, Zhou Y, Huang Q: Serum fibroblast growth factor 21 level is increased in pre-eclampsia patients: Association with blood pressure and lipid profile. J Obstet Gynaecol Res. 2021; 47(1): 375–381. PubMed Abstract \| Publisher Full Text\n\nRana S, et al.: Preeclampsia: pathophysiology, challenges, and perspectives. Circ Res. 2019; 124(7): 1094–1112. PubMed Abstract \| Publisher Full Text\n\nCui Y, et al.: Role of corin in trophoblast invasion and uterine spiral artery remodelling in pregnancy. Nature. 2012; 484(7393): 246–250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFahim NK, Negida A, Fahim AK: Sample Size Calculation Guide - Part 3: How to Calculate the Sample Size for an Independent Case-control Study. Adv J Emerg Med. 2019; 3(2): e20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCui L, et al.: Serum protein marker panel for predicting preeclampsia. Pregnancy Hypertens. 2018; 14: 279–285. PubMed Abstract \| Publisher Full Text\n\nMustafa R, et al.: A comprehensive review of hypertension in pregnancy. J Pregnancy. 2012: 2012. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIves CW, et al.: Preeclampsia-Pathophysiology and Clinical Presentations; JACC State-of-the-Art Review. J Am Coll Cardiol. 2020; 76(14): 1690–1702. PubMed Abstract \| Publisher Full Text\n\nSani HM, Vahed SZ, Ardalan M: Preeclampsia: a close look at renal dysfunction. Biomed Pharmacother. 2019; 109: 408–416. PubMed Abstract \| Publisher Full Text\n\nPankiewicz K, et al.: Insight into the Key Points of Preeclampsia Pathophysiology: Uterine Artery Remodeling and the Role of MicroRNAs. Int J Mol Sci. 2021; 22(6): 3132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArdalić D, et al.: Lipid profile and lipid oxidative modification parameters in the first trimester of high-risk pregnancies-possibilities for preeclampsia prediction. Clinical Biochem. 2020; 81: 34–40. PubMed Abstract \| Publisher Full Text\n\nBartha JL, et al.: Decreased mitochondrial fatty acid oxidation in placentas from women with preeclampsia. Placenta. 2012; 33(2): 132–134. PubMed Abstract \| Publisher Full Text\n\nAjne G, et al.: Endothelin converting enzyme (ECE) activity in normal pregnancy and preeclampsia. Hypertens Pregnancy. 2003; 22(3): 215–224. PubMed Abstract \| Publisher Full Text\n\nTaylor RN, et al.: Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancies. J Clin Endocrinol Metab. 1990; 71(6): 1675–1677. PubMed Abstract \| Publisher Full Text\n\nBaksu B, et al.: Plasma nitric oxide, endothelin-1 and urinary nitric oxide and cyclic guanosine monophosphate levels in hypertensive pregnant women. Int J Gynecol Obstet. 2005; 90(2): 112–117. PubMed Abstract \| Publisher Full Text\n\nNishikawa S, et al.: The relationship between serum nitrate and endothelin-1 concentrations in preeclampsia. Life Sci. 2000; 67(12): 1447–1454. PubMed Abstract \| Publisher Full Text\n\nGeorge EM, Granger JP: Endothelin: key mediator of hypertension in preeclampsia. Am J Hypertens. 2011; 24(9): 964–969. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAggarwal P, et al.: The relationship between circulating endothelin-1, soluble fms-like tyrosine kinase-1 and soluble endoglin in preeclampsia. J Human Hypertens. 2012; 26(4): 236–241. PubMed Abstract \| Publisher Full Text\n\nAjne G, et al.: Contribution of endogenous endothelin-1 to basal vascular tone during normal pregnancy and preeclampsia. Am J Obstet Gynecol. 2005; 193(1): 234–240. PubMed Abstract \| Publisher Full Text\n\nNapolitano M, et al.: Expression and relationship between endothelin-1 messenger ribonucleic acid (mRNA) and inducible/endothelial nitric oxide synthase mRNA isoforms from normal and preeclamptic placentas. J Clin Endocrinol Metab. 2000; 85(6): 2318–2323. PubMed Abstract \| Publisher Full Text\n\nFaxén M, et al.: Differences in mRIMA expression of endothelin-1, c-fos and c-jun in placentas from normal pregnancies and pregnancies complicated with preeclampsia and/or intrauterine growth retardation. Gynecol Obstet Invest. 1997; 44(2): 93–96. PubMed Abstract \| Publisher Full Text\n\nFiore G, et al.: Endothelin-1 triggers placental oxidative stress pathways: putative role in preeclampsia. J Clin Endocrinol Metab. 2005; 90(7): 4205–4210. PubMed Abstract \| Publisher Full Text\n\nAlexander BT, et al.: Reduced uterine perfusion pressure during pregnancy in the rat is associated with increases in arterial pressure and changes in renal nitric oxide. Hypertens. 2001; 37(4): 1191–1195. PubMed Abstract \| Publisher Full Text\n\nMakris A, et al.: Uteroplacental ischemia results in proteinuric hypertension and elevated sFLT-1. Kidney Int. 2007; 71(10): 977–984. PubMed Abstract \| Publisher Full Text\n\nCoffey CG: Issues in the interpretation of serum endothelin levels in preeclampsia. Med Hypoth. 2019; 133: 109400. PubMed Abstract \| Publisher Full Text" }	[ { "id": "151386", "date": "01 Nov 2022", "name": "Jussara Mayrink", "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\nIntroduction:\nThe third paragraph is really confused. Please, try to rewrite it, with some more data about the relationship between variables and preeclampsia, as well as, being very clear regarding the objective of this study. Reading all the text it is clear that the objective of the study is to establish the relationship between variables and preeclampsia, however, it must be clear in the introduction.\nDefinitions:\nConsidering this is a scientific article, which has many functions, as being instructive to readers, more than just inform the results, I think it would be interesting to add a sentence like “in this study, we consider the sign of severity present the arterial blood pressure above 160X110 mmHg”. This is not the only sign of severity, as the authors already know. This must be considered in the text. The same should be considered to the concept of preeclampsia. The authors choose a “partial” concept of preeclampsia in the reference applied (ACOG, 2019). However, the concept should be cited in the text in its totality.\n\nDiscussion:\nThe relationship between onset of preeclampsia and variables\nBMI: authors should add a sentence or a paragraph about this. The relationship of late-onset preeclampsia and higher BMIs has been already established and it is particularly interesting (see Roberts JM, Hubel CA, 2008).1\nThe relationship between severity of preeclampsia and variables\nSerum liver enzymes: at this point, authors should point the concept of HELLP syndrome, which is a validated sign of severity of preeclampsia.\n\nProteinuria: at this point, authors must be very careful with this result, in order to avoid the misunderstanding of consider high levels of proteinuria and severity of preeclampsia, which is not totally true, as demonstrated in the literature. I know this is a result of this study and of course, authors should point it out. However, consider adding a sentence pondering on this important discussion.\n\nConclusion:\n“There is therefore the need for regular monitoring of these maternal variables for the early detection and management of PE in the Ghanaian population.”\nIs it feasible to the Ghanaian context?\nMaybe the relevance of the findings should be considered in the light of the etiology of preeclampsia, in a treatment axis, in a prevention action, etc.\nProposing the measure of variables throughout the prenatal care in order to diagnose preeclampsia doesn’t seem to be a reasonable conclusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "165089", "date": "07 Mar 2023", "name": "Johnbosco E Mamah", "expertise": [ "Reviewer Expertise Maternal medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a short communication, this would guide the intensity of my review.\nOverall, the study is relevant in contemporary Obstetrics especially in sub-saharan Africa where the burden of preeclampsia is still quite high and there is limited resources for its investigation and treatment. With some improvement suggested below, the study would be a valuable addition to the body of knowledge regarding what is known about preeclampsia.\nThe authors need to review the article to improve the grammar and readability.\nAbstract: The abstract was well written. It was structured and concise. The methodology section of the abstract should include a summary of the statistical analysis performed. It is good practice to list the keywords in alphabetical order.\nIntroduction: As cited by the authors, preeclampsia affects about 3-8% of pregnancies globally but the incidence in sub-Saharan Africa including Ghana is even higher based on available studies. The authors should comment on the local burden of the disease.\n\nMethodology: This needs significant improvement to make the work reproducible elsewhere. I acknowledge that the authors were thorough in the methodology for the biochemical analysis of the blood sample taken and in describing the data analysis performed but that is as far as it goes.\nThe work could be improved upon by describing how the participants were recruited (sampling method), how the diagnosis of preeclampsia was made and at what point, what is the minimum and maximum gestational age for recruitment into the study, and how the sample size was calculated or arrived at. I note that the reference quoted in the methodology for the sample size calculation (ref 9) does not correspond to the listed citation in the list of references. Moreover, reference number 8 is missing in the body of the text.\nIn the definition of terms, the authors failed to list out the severe features of preeclampsia apart from severe hypertension. Severe hypertension (>=160/110mmHg) alone in the absence of proteinuria or other features of severe preeclampsia is not the same as severe preeclampsia (See ACOG practice bulletin number 222, December 2018).1 Listing out the clinical and biochemical features of severe preeclampsia would also validate the authors statement in the introduction that preeclampsia could be diagnosed as new onset hypertension after 20 weeks gestation with or without proteinuria.\n\nThe authors report there was no attrition in the study, were all the patients admitted overnight to collect the fasting sample? If some went home after the initial diagnosis, did they all return the next morning for a fasting sample to be taken? Were they medicated at the time of sample collection and was there repeat blood pressure check at this time? These need to be clarified.\nThe authors matched for gestational age and maternal age, did they correct for other confounders such as BMI, autoimmune diseases or parity which were not matched during recruitment? If not, then I suppose this a weakness and should be mentioned.\nDiscussion: The authors have written a robust discussion but there was little to no discussion of how this study compares with similar studies elsewhere. There was an objective acknowledgement of weaknesses and limitations of the study.\nConclusion: The authors have concluded as rightly found in the study that there was a significant correlation between maternal variable and preeclampsia. The results do not support their recommendation for regular monitoring of these variables for early detection of preeclampsia. They performed only a one off analysis with no follow-up to confirm consistency in their initial findings. The subjects on the case arm of the study already have the disease, I don't see how the variables predict preeclampsia in this case. It would have been good to know if any body in the control developed preeclampsia down the line and how this related to their initial biochemical findings. At best it can be concluded that the maternal variable correlated with the severity of the disease at the time of assessment.\nThe authors have recommended for these variables to checked regularly for early detection of preeclampsia, I would assume that these are expensive investigations which may not be affordable to the average patient in sub-Saharan Africa due to widespread poverty.[ref-2# I would recommend the authors perform a sub analysis of the data to determine which of the variable had the best correlation with preeclampsia and make a case for its wider use or more studies to confirm its utility. Performing all the assays listed in this study which might be difficult to afford and may not add much in terms of actual clinical practice.\nReferences: The references are well written but ref 8 is missing in the body of the text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-620
https://f1000research.com/articles/10-619/v1	21 Jul 21	{ "type": "Research Article", "title": "Vaping liquid flavour preferences, oral nicotine pouch and cannabis use: A survey of participants in the 2019 Oceania Vape Expo", "authors": [ "Marewa Glover", "Carl V. Phillips", "Kyro Selket", "Yolande Jeffares", "Carl V. Phillips", "Kyro Selket", "Yolande Jeffares" ], "abstract": "Background: New Zealand and other countries have introduced or are considering various restrictions on the sale and use of vaping devices and liquids used in vaping and smokeless tobacco products. This research aimed to assess the likely reactions of individuals who vape to proposed restrictions in New Zealand. Methods: A vape expo, a social and commercial weekend convention, provides convenient access to individuals with a strong interest in and or experience in vaping. A street intercept approach was used to survey attendees at a vape expo for adults aged 18 and over in Auckland, New Zealand in December 2019. Results: This research suggests restricting the sale of liquid flavours may have negative unintended consequences. 57% of respondents indicated they would circumvent a ban on popular liquid flavours by mixing their own and/or buying liquids from overseas or the black market. Over a third (36%) would likely be restricted in their choice of a low-risk substitute for tobacco smoking if products such as snus and oral nicotine pouches were banned. A further 36% had heard of these options but were currently not using them. Other results are in the areas of smoking behaviour, cannabis use, and responses to cannabis legalisation. Conclusions: Various legislative or policy initiatives proposed to regulate vaping may have unanticipated negative consequences for public health. The negative impacts are likely to be disproportional for groups with higher smoking prevalence such as Indigenous peoples, rural communities, and lower socioeconomic groups.", "keywords": [ "Vaping", "e-cigarettes", "flavours", "oral nicotine pouches", "tobacco harm reduction", "cannabis" ], "content": "Introduction\n\nA vape exposition is both a social and commercial event. A vape expo provides an opportunity for people who vape (uset electronic cigarettes (e-cigarettes) primarily with nicotine-containing liquids), and those considering vaping, to socialize over their shared interest, learn from one another and from vendors; sample and buy products. A vape expo is also a business-to-business event, providing manufacturers and distributors an opportunity to introduce their products to retailers who are exhibiting at or attending the expo.\n\nPeople who attend a vape expo are a useful target population for research about vaping, as they represent an invested cohort with advanced knowledge of vaping. An estimated 98 million people around the world have stopped smoking tobacco by switching to low and reduced-risk alternatives such as vaping, oral nicotine pouches, snus (tobacco pouches for oral use) and tobacco heating products (Knowledge-Action-Change (KAC), 2020). Despite this global trend, many governments have introduced restrictions and bans to limit this evolution (KAC, 2020).\n\nIn New Zealand (NZ), e-cigarettes have been sold as a common consumer product, controlled by generic consumer protection laws, since 2010. In 2015/16, daily e-cigarette use amongst New Zealanders aged 15 years and above was o.9%. By 2019/20 this had increased to 3.5% (Ministry of Health, 2020). Research suggests vaping is concentrated among adults who smoke or who have recently stopped smoking. In 2016/17, among NZ adults aged 18 and above who smoked, the prevalence of daily vaping was 8%. By 2018 this had increased to 11.4% (Edwards et al., 2020).\n\nFollowing a few years of debate and several iterations of draft legislation, in April 2020, the Smokefree Environments and Regulated Products (Vaping) Amendment (NZ Government, 2020) (hereafter referred to as the Vaping Amendment) introduced regulations to limit and control the import, marketing, sale, and use of herbal smoking products, smokeless tobacco products, and vaping products. The intent of the Vaping Amendment was to prevent the “normalisation” of vaping (clause 9). The Vaping Amendment banned the sale of all vape liquid flavours except tobacco, mint, and menthol-flavoured vape liquids or e-cigarettes in convenience stores, supermarkets and any other store not registered as a “specialist vape retailer”. The Vaping Amendment also banned the importation of all oral and chewing tobacco products including snus and oral nicotine pouches, for sale and distribution.\n\nThis study was conducted to inform the debate about the proposed content of the Vaping Amendment. We conducted a brief survey of adults aged 18 and above attending an Oceania region vaping exposition in Auckland, New Zealand in December 2019 with five objectives.\n\nThe first objective of our research was to develop a demographic profile of the participants attending the vape expo.\n\nThe second objective was to assess the likely reactions of people who vape to a ban of most flavours of vaping liquid (e-liquid). People who vape largely prefer non-tobacco cigarette flavours (Landry et al., 2019), both because they find one or more of the many options to be more aesthetically pleasing (which they are by design, as opposed to the taste that burning tobacco creates) and because some people actively want to avoid a flavour that reminds them of the taste of the cigarettes they gave up (Russell and McKeganey, 2018). In a 2016 NZ online survey of people who vaped, over 90% reported using a wide variety of flavours (Truman et al., 2018). While 42% had used tobacco or menthol flavours in the past, only 22% were still using these flavours in a follow-up round of the study, indicating that a shift to other flavours was a common transition (Truman et al., 2018).\n\nE-liquid flavour bans actually only ban some flavours since the base e-liquid has very little taste and thus “tobacco flavour” is as much an artificial flavouring as “strawberry”. The bans are therefore actually bans of flavour descriptors, since there are no legal definitions of the subjective experience of flavour itself. For example, an e-liquid might be called “pipe tobacco” but taste like fruit. Bans on e-liquid flavours that do not imitate the taste of cigarettes, either the taste of straight tobacco smoke or with menthol added, are intended to undermine the satisfaction and appeal of vaping. The ironic outcome, for governments committed to reducing the harms of smoking tobacco, is that reducing the appeal of vaping could slow the rate at which a population substitutes smoking tobacco with the risk-reduced alternative of vaping.\n\nOur third objective was to explore local awareness of and use of other harm reduction products, such as oral nicotine pouches and snus. Oral nicotine pouches were launched in NZ in early 2019. No data existed on their current use in the population.\n\nA fourth goal was to assess this population’s experience with cannabis use, in reaction to a pending legislative change in NZ. Small amounts of cannabinol (CBD) and/or tetrahydrocannabinol (THC) were legalised for medicinal use in December 2018. During the general election in October 2020, the Government ran a non-binding referendum for the Cannabis Legalisation and Control Bill, which proposed to legalise recreational marijuana. The referendum failed by a small margin.\n\nTen months prior to the referendum, our survey explored if people who currently vaped had an interest in vaping THC, now or in the future, if cannabis was legalised in NZ. This was relevant to the Government’s consideration of vaping due to a spate of deaths and hospitalisations of people in the United States with a lung injury incorrectly attributed to nicotine-containing e-cigarettes (Minton and Tanner, 2020). In fact, the injuries were caused by the use of contaminated black-market cannabinoid liquid cartridges containing Vitamin E acetate.\n\nA final objective of the study was to assess potential recruitment for the longitudinal VERITAS Cohort Study (veritascohort.org). The target population of this international study is people who vape and who have a very limited smoking history. Historically there were very few vapers who had not been long-term smokers, but that has changed to a degree that has largely been unmeasured.\n\n\nMethods\n\nThe study population was participants at the Oceania Vape Expo 2019 in Auckland, NZ’s largest city, on Saturday 7th December 2019 from 10 am to 5 pm and Sunday 8th December 2019, from 10 am to 4 pm. The Expo was organised by China E-Cigarette Media Online (CECMOL).\n\nWe aimed to recruit 200-500 participants, including Expo visitors and staff at commercial and other booths. The exact characteristics of this population were unknown, but it was almost certainly predominantly vaping enthusiasts. People were eligible for the survey if they were at least 18 years old (the Expo itself was restricted to people aged 18 or over), self-identified as vaping “regularly”, and gave informed consent.\n\nA street intercept approach was used because it is an efficient and cost-effective way to obtain a high response rate amongst a specific participant group in a public place (Henley and McCoy, 2018). The researchers walked around the Expo hall, lobby, and outside areas, approaching people to invite them to participate. If a person was interested, the interviewer gave them a printed participant information sheet (PIS) along with a brief verbal explanation of the purpose of the study; explaining that the survey was anonymous, as we were not collecting any identifying data, as well as how to complete the survey, how long it would take, that participation was voluntary, and that the participant could stop the survey at any chosen time without giving a reason. Participants were also informed that their data would be published on an online website and made available for other researchers to use. If participants gave verbal consent to the interviewer, they were handed an electronic tablet upon which to complete the survey. The first question of the survey asked participants to confirm that they understood the reason for the survey, had read the PIS, and had been given the opportunity to ask questions about the survey. Only participants who answered affirmatively progressed to the next question. We did not require participants to indicate consent electronically at the beginning of the survey as they had already given verbal consent to participate. It is not uncommon practice in NZ, (for example, see Wamamili et al., (2021)), and it is recognised as satisfactory demonstration of participant consent by the National Ethics Advisory Committee (2019), for anonymous minimal risk surveys like ours to consider return of a completed questionnaire as “implied consent” (p.71).\n\nThere were no subject selection limitations or preferences, and recruiters attempted to recruit the first person they noticed after the previous participant finished completing the survey. This was often a companion of the previous participant who was waiting for them. At any given time, there were two to four recruiters operating.\n\nPostcard advertisements about the survey were placed on stall countertops and announcements encouraging attendees to complete the survey were broadcast over the Vape Expo’s public address system. Survey staff wore an identification tag that showed their name, photo, and the name of the Research Centre conducting the study. Survey staff also wore vests marked with the wording “Vaper’s Survey”, “Spot Prize $30 Voucher” and the Centre’s website address. Because women are typically under-represented in surveys of people who vape (for example see Truman et al., 2018), female researchers were available for the whole period of the Expo to facilitate recruitment of women.\n\nThe voucher referred to a $30 grocery voucher that was randomly awarded to a participant after being interviewed, and this incentive for participation was noted in the recruiting materials and pitches. Depending on how many researchers were conducting the survey (two at quiet times and four when busy), every half hour per two researchers, the supervisor ran a random number generator on a mobile phone to determine which of the one to two or one to four researchers would hand out the voucher to the participant currently completing the survey. Researchers were notified by text. To ensure anonymity, recipients of the voucher were asked to sign their initials only, on a voucher receipt form.\n\nSuccessful recruits completed the survey (Extended data (Glover et al., 2021b)) in real time on an iPad supplied by the researcher, who stood nearby, but did not observe the screen during survey completion. The first three questions determined eligibility – they were informed about the survey, age, regular vaping, and an assurance that the participant could read English – and the survey was terminated for those who were not eligible.\n\nThere has been a history of enigmatic (Soule et al., 2017) and unethical (see Chen et al., 2017) research (e.g., a paper by Bhatta and Glantz (2019), which was published and then withdrawn from the Journal of the American Heart Association), which has misrepresented the significance of research at vape conventions (Phillips, 2016), and on vapers more generally (Siegel, 2015; McCausland et al., 2020). To ensure integrity of this research, a typical informed consent statement was used, and respondents had to affirm consent to participate in the survey. Participants were told that the survey was anonymous and that they could stop answering the survey questions at any time without having to give a reason. They were also advised that all legitimate data entered into the survey form would be used. Most importantly, several members of the target population (NZ vaping enthusiasts) and the Expo organisers were given the opportunity to express any concerns they had about the research plan in advance.\n\nTo prevent any misperception that the research was in any way connected with any vendor or the conference organiser, the researchers did not position themselves at any stand associated with any organisation, company, or brand in attendance at the Expo. We attempted to pay entry fees for all researchers for both days, but the conference organisers declined the payment. Some recruits reportedly expressed scepticism about the ethics of the survey, but they were reassured after being given the informed consent information and the name of the principal investigator, who is widely respected in the NZ vape community.\n\nAll survey questions are summarised in the univariate results reporting with the exception of two questions about nicotine content of e-liquid, which due to an error in the survey coding, produced data that could not be interpreted.\n\nThe survey was created using the Zoho: Survey Pro online app (version current at November 2019). Data were analysed using simple R (R Core Team, 2017) (version 3.3.3) code, using only default settings, run within R-studio (version 1.1) (a programming environment that runs R; RStudio Team, 2015). The code is available upon request. Every question asked is noted in the univariate results reporting. The reported multivariate results were pre-specified. Ethnicity shown represents single ethnicity selected using a common method used by NZ Statistics that prioritises Māori, then Pacific, then Asian, other, and European. All results that were calculated are reported.\n\nAn error in the skip patterns in the survey, in which participants who smoked were asked when they quit smoking, was corrected part way through the survey administration. Answers from those who should not have been asked that question were removed in data cleaning.\n\n\nEthics approval\n\nHealth and Disability Ethics Committee review in NZ is guided by the Government Standard Operating Procedures for Health and Disability Ethics Committees. The Health and Disability Ethics Committee (HDEC) assessed the study application for ethics review (19/STH/194) and deemed it out of scope because it was an observational study representing minimal risk to participants. As they explained, HDEC review is required “only if the study involves more than minimal risk (that is, potential participants could reasonably be expected to regard the probability and magnitude of possible harms resulting from their participation in the study to be greater than those encountered in those aspects of their everyday life that relate to the study).”\n\n\nResults\n\n391 recruits started the survey, and all gave informed consent. 48 recruits were eliminated when they indicated they did not vape regularly or refused to answer the age question (which required them to be 18+). Another three recruits were removed from the analysis because they abandoned the survey without providing tobacco use information (one participant who exited without providing cannabis use information was included in the rest of the analysis). This left 340 respondents for the tobacco products portion of the analysis, 291 of whom indicated their primary residence was in NZ.\n\nThere were no substantial differences between responses for non-NZ residents (n = 49) and NZ residents (n = 291). Our analysis focuses on the NZ residents, but extrapolates to the rest of the study population when relevant. The exception to their being no substantial differences was the ethnicity responses, where we included a “NZ European” category, which described a few of the white people who did not live in NZ.\n\nThe age, gender, and ethnic distributions of participants are shown in Table 1. A third (31%) of the respondents were women. Almost a quarter (22%) of the participants from NZ self-identified as Māori (Indigenous people of NZ). Other ethnic groups were NZ European (61%), Chinese (7%), and Pacific Island people (6%). Of the respondents from NZ, almost three-quarters (71%) were from the Auckland area.\n\nSix percent of the NZ respondents reported using standard cigarette flavours exclusively, although 37% used them (tobacco (15%); menthol/mint (22%)) amongst the other flavours they used regularly (see Table 2).\n\nIn the event of a ban on other flavours, 20% of the NZ residents said they would probably go back to smoking (see Table 3). A majority (57%) indicated they intended to circumvent the ban, by mixing their own liquids and/or buying from overseas or the black market. Only 29% indicated they would change their vaping consumption to comply with the ban, as is typically implicitly assumed will happen. Nine percent would try to stop vaping and 35% would buy only the legal products (which includes the 18% who were already buying only products that would not be banned).\n\nAbout two-thirds (65%) of the participants currently did not smoke and about one-third (35%) currently smoked at least occasionally (see Table 4). 70% reported having smoked at least every day for a year at some point in their lives, and almost all (86%) of them responded that they had smoked at least 1,000 cigarettes total.\n\nSlightly more than half (55%) of the respondents had smoked in the past but no longer smoked. Almost half (47%) of those had quit more than three years ago and the other half (53%) had quit more recently (Table 4).\n\nAbout one quarter of participants (n = 69, 24%) were in the intersection of having not smoked every day for a year in their life and having not smoked in a year or more suggesting they could be eligible for inclusion in the VERITAS study.\n\nAs shown in Table 5, more than a third (41%) of the 270 NZ residents who answered the question (and 40% of all respondents) indicated they had tried other low-risk substitutes for smoking – snus (smokeless tobacco) or oral (non-tobacco) nicotine pouches. However, very few (6%) participants currently used snus or pouches. An additional 36% had heard of these substitutes but had never tried them.\n\nOver half (57%) of those who responded to this question (one person did not, so n=290) said they smoked cannabis, and half (52%) of those who currently smoked cannabis did so at least once per week (see Table 6).\n\nAlmost everyone who said they currently smoked cannabis said they would almost certainly (57%) or maybe would (30%) vape cannabinoid liquids if they were legalised in NZ (Table 7). Even of those who did not currently smoke cannabis, 51% said they would almost certainly (11%) or maybe would (40%) vape cannabinoids if legalised. Half (51%) of those who currently smoked cannabis had already tried cannabinoid liquids (those who did not smoke cannabis were not asked this question) (Table 6).\n\n\nDiscussion\n\nConsistent with other studies on the role of flavours in vaping (Truman et al., 2018; Edwards et al., 2020) many participants used a variety of flavours and very few used only the tobacco or menthol/mint flavours. A recent USA study of people who both smoked and vaped found that about half used only non-tobacco, non-menthol/mint flavours (Kasza et al., 2020). Previous research in NZ found that people who smoked and vaped tended to be newer to vaping, and that with time people progressively moved from tobacco and menthol flavoured vape liquids to other flavours (Truman et al., 2018) suggesting that the availability of a range of non-tobacco smoke flavours are important to the success of vaping as a substitute for smoking. That flavours increase the appeal and enjoyment of vaping is a consistent finding as reported in a review of the impact of non-menthol flavours that looked at 51 articles (Meernik et al., 2019).\n\nIt is not clear to what extent participants’ predicted response to a ban on e-liquid flavours extrapolates to the average person who vapes, but who is not invested enough to attend a convention. However, it is a strong indicator that a flavour ban would not have the intended results, as previously suggested by others (e.g., Siegel, 2019). This is consistent with attitudinal research in other populations, as well as the demonstrated behaviour of people living under ban regimes. In particular, over a third of respondents indicated that they would mix their own e-liquid, a practice that is relatively easy, but which increases the risk of harmful mishaps, such as children inadvertently consuming high concentration nicotine solutions that are used in crafting.\n\nConsistent with other research (e.g., Bentley, 2020; Shahab et al., 2017), almost half of respondents said they would avail themselves of illicit markets. Such markets, once established, tend to increase their customer base over time and tend not to disappear if legal markets are reopened. For example, black market cannabis continues in legalised markets, especially if the legal domestic supply is unable to meet demand (Sen and Wyonch, 2018).\n\nWe observed that over half of respondents had recently stopped smoking and thus the results should not be extrapolated to the average NZ person who smoked in the distant past. However, this is what we would expect among those who quit by switching to vaping, because vaping has become a popular quitting strategy only recently (see, for example, Edwards et al., 2020).\n\nThe low awareness and trial of snus or oral nicotine pouches likely reflects the Ministry of Health’s historic claimed ban on the sale of snus in NZ and the very recent launch of the oral nicotine pouches. It could also be that people who used snus/oral nicotine pouch who do not vape would be less likely to attend a vape expo. We found that over a third of people, enthusiastic about vaping enough to attend a vape expo, had shown interest in other risk-reduced alternatives to smoking. Interest would likely be higher if the products were more accessible and their existence was communicated to people who smoke or vape. Limiting access to risk-reduced alternatives to smoking, for example, by allowing people access to only one type, reduces the opportunity for people who smoke to find a satisfactory substitute for smoking. The risk is that reduction of smoking-related mortality and morbidity will be delayed if vaping or tobacco heating products (legally for sale in NZ) are ineffective substitutes for smoking.\n\nOur study suggests that recruiting a cohort of people who currently vaped who had a limited history of smoking, difficult to imagine only a few years ago, is practical in Auckland and probably elsewhere. As might be expected, this would be a youthful cohort, with two-thirds (66%) under the age of 35.\n\nInterestingly, almost all (90%) of the respondents who had vaped cannabinoids had done so in NZ, where smoking cannabis is not legal. These results suggest that, for NZ residents who are familiar with vaping, (a) quite a few would become new users if vaping cannabinoid liquids was legal and (b) some could have been exposed to lung injury had the NZ black market been infiltrated by the harmful products that caused the USA outbreak.\n\nThe percentage of participants who said that they already had used cannabinoid vaping products indicated the existence of a black market for cannabinol liquids in NZ also. Resort to black market product could be ongoing as cannabis for medicinal use only, and available on prescription only, is all that is legal given that insufficient votes were received during the October 2020 referendum to support legalisation of recreational cannabis use.\n\nSurvey personnel believed that a greater proportion of women attended the Expo than are represented by the proportion of women who participated in this survey. They attributed this to women being more likely to decline to take the survey. However, among NZ adults aged 15 and over, males were 1.74 times more likely to vape at least once a month than women (Ministry of Health, 2019) so it is possible that women were well-represented in our study which would be a strength. It is also a strength that Māori and Pacific Island expo participants were well represented. Māori and Pacific Island people in NZ have disproportionately high rates of smoking. In 2019, 34% of Māori adults aged 15 and over, and 24% of Pacific Island adults smoked compared with 12% of NZ European/Others (Ministry of Health, 2019). It is promising for reducing this inequity in smoking prevalence that Māori were 1.65 times more likely to vape than non-Māori (Ministry of Health, 2019). Better promotion of risk-reduced alternatives is required to encourage Pacific Island people who smoke to switch. Though Pacific Island men and women are 1.68 times more likely to smoke than non-Pacific Island, Pacific Island men were only about half as likely to vape as non-Pacific Island men (Ministry of Health, 2019).\n\nIt was a strength that members of the research team were members of the local and international vaping community, and that vaping advocacy organisations or advocates were consulted about the survey questions and involved in conducting the survey at the expo. The study method respected the call from the vaping community for their expertise in vaping to be valued as a lay epidemiology and for studies to attend to questions of importance to them (Fraser et al., 2018).\n\nThe method also had to compensate for the understandable scepticism that the vaping community has towards researchers due to previous unethical and biased practices by researchers. For example, Spence and Stanbrook (2016) extracted quotes from social media posts by people who vape and displayed a selection of these with identifying information including the person’s profile picture (mostly real headshots) without permission at a conference. That our survey was conducted at an Expo with such transparency is a strength because previous researchers have malevolently collected data at expos without the organisers’ or any vaping community consent (for example see Soule et al., 2016; Bates, 2016).\n\nThe respondents in this study represent a younger population than those reported in the majority of previous research studies on people who have tried or who currently vape. This is likely a function of the specific venue for recruitment. It may be that younger people are more likely to work at, or spend part of their weekend at, a vape expo.\n\nBecause expo booth staff were present at the venue for longer than expo attendees a larger proportion of them could have completed the survey. The survey personnel perceived that between them most booth staff were likely invited to participate. Booth staff were a mixture of business owners, their regular staff, and casual retail staff likely recruited just for the expo. Several of the exhibitors were from overseas. Responses from booth staff cannot be identified from the data collected. This probably means that highly dedicated and experienced vapers were overrepresented in the sample, even compared to the average vape expo attendee.\n\n\nConclusion\n\nIf governments are committed to reducing smoking-related harm, they would encourage smoking cessation by providing a wider range of risk-reduced alternatives such as vaping, snus, and oral nicotine pouches. The Smokefree Environments and Regulated Products (Vaping) Amendment Act came into force in November 2020. From November 25 snus and oral nicotine could no longer be sold. Nine months was allowed for non “specialist vape retailers”, that is, convenience stores, petrol stations and supermarkets to stop selling vaping products containing other than mint, menthol, and tobacco flavours – the least used flavoured e-liquids among people who vape, according to our survey. A 2018 survey found that about 10% of vapers last purchased vaping consumables at a tobacconist and a further 5.4% had purchased consumables at a local convenience store (Edwards et al., 2020). Limiting convenience stores and supermarkets to just three of the tobacco cigarette-like flavours, could undermine access to, and thus impede transition to, vaping or quitting smoking.\n\nSmoking is inequitably distributed in New Zealand by ethnicity and socioeconomic level. The decline in smoking prevalence between 2018/19 and 2019/20 was very small (not even statistically significantly different from zero) and neither was any change in the prevalence of vaping detected (Ministry of Health, 2020). We have little idea how the Vaping Amendment's limitations will affect the prevalence of vaping among the groups with disproportionately high smoking rates (Māori, Pacific Island people, and lower socioeconomic groups), and only focused research will be able to determine that. A quarter of the NZ population lives in rural areas and small towns (Ministry of Health, 2019a). Māori, who have the highest smoking rates, are also over-represented in rural areas. People who smoke who live in rural areas or small towns may be disproportionately deterred from switching to vaping as the Vaping Amendment banned mobile vape vendors and various new regulations may make it prohibitive for small volume retailers to maintain a ‘bricks-and-mortar’ store.", "appendix": "Acknowledgements\n\nWe wish to acknowledge the generosity of the people who participated in the survey. Without you, this research would not have been possible. Many thanks go to Professor Riccardo Polosa for his feedback on the protocol, Andrew Thompson & QJ of NZ Vapor for comments on the draft survey, NZ Vapor for allowing us to pilot the survey in one of their shops, NZ Vaping Alliance for support of the study, CECMOL for permitting us to conduct the survey during their Expo, Steve Piner for Zoho Survey administration and supervision of data collection, and Kumar Paramanathen, Robin Shepherd and Andrew Thompson who assisted with conduct of the survey during the Expo. We are also grateful to Professor Viehland for copy-editing; and the comments on an earlier draft from three anonymous reviewers.\n\n\nData availability\n\nZenodo: Glover et al., 2021 Vape Expo Data. https://doi.org/10.5281/zenodo.5016186 (Glover et al., 2021a).\n\nThis project contains the following underlying data:\n\n- Data file 1. (Complete survey responses, CSV format).\n\nZenodo: Vaping liquid flavour preferences, oral nicotine pouch and cannabis use among participants in the 2019 Oceania Vape Expo: Copy of the online survey. https://doi.org/10.5281/zenodo.5034910 (Glover et al., 2021b).\n\nThis project contains the following extended data:\n\n- Copy of online survey used in vape expo survey 2019.\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\nReferences\n\nBhatta DN, Glantz SA: Electronic Cigarette Use and Myocardial Infarction Among Adults in the US Population Assessment of Tobacco and Health. J Am Heart Assoc. 2019 Jun 18; 8(12): e012317. [Retracted article]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCenters for Disease Control and Prevention: Outbreak of Lung Injury Associated with E-cigarette Use, or Vaping.Atlanta, GA: US Department of Health and Human Services; 2019 Feb 25. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShahab L, Goniewicz ML, Blount BC, et al.: Nicotine, Carcinogen, and Toxin Exposure in Long-Term E-Cigarette and Nicotine Replacement Therapy Users: A Cross-sectional Study. Ann Intern Med. 2017 Mar 21; 166(6): 390–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoule EK, Maloney SF, Spindle TR, et al.: Electronic Cigarette Use and Indoor Air Quality in a Natural Setting. Tob Control. 2017 Feb 15; 26: 109–112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTruman P, Glover M, Fraser T: An Online Survey of New Zealand Vapers. Int J Environ Res Public Health. 2018 Jan 29; 15(222): 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWamamili B, Lawler S, Wallace-Bell M, et al.: Cigarette Smoking and E-Cigarette Use Among University Students in Queensland, Australia and New Zealand: Results of Two Cross-sectional Surveys. BMJ Open. 2021 Feb 9; 11(2): e041705. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "307740", "date": "07 Aug 2024", "name": "Emke Brazier", "expertise": [ "Reviewer Expertise Nicotine use", "including e-cigarettes/ vapes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 surveyed vape users at a vape expo in Auckland in 2019 to understand future intentions around flavour use, cannabis use and other nicotine products to better understand the impact of proposed vape-related restrictions. The previous research included is relevant however a wider range would be beneficial to better support the statements made in the introduction. The methodology is appropriate for the survey aims; however, there could be more clarity about these methods in both the abstract and the methods section. Results are appropriately reported and interpreted in the discussion, although there could be more depth/ clarity at times to improve the discussion section. Overall the study has academic merit, a few smaller changes are required.\nAbstract - methods subsection needs more detail, it's not clear how many participants there were nor what methods were used apart from the recruitment method.\n\nIntroduction - typo \"aged 15 years and above was o.9%.\" should be 0.9% not o.9%\n\nIntroduction - There should be more evidence to justify the research aims/ objectives e.g. with the 2nd objective, I would expect to see research that shows limiting flavours may slow the rate of substitution, even if this is research is from other countries that have also made flavour bans.\n\nMethods - There is good depth about the recruitment methodology and sufficient detail about the analysis. There should be more detail about the specific measures/questions included and how participants could answer them (e.g. could they only select one answer, could they select multiple, did they have to rank their answers etc.).\n\nResults - Tables would be clearer if answers were listed in strength order (e.g. Table 2 is a good example of where this would be helpful).\n\nDiscussion - Results are not overstated so well done for capturing the key findings, I would have liked to see the aims/ objectives referred back to more explicitly to state how each one has been met/ answered by the findings.\n\nDiscussion - The statement \"This is consistent with attitudinal research in other populations, as well as the demonstrated behaviour of people living under ban regimes\" needs appropriate evidence to support it.\nDiscussion - The statement \"We observed that over half of respondents had recently stopped smoking and thus the results should not be extrapolated to the average NZ person who smoked in the distant past.\" is a little confusing and could be more explicit, do you mean those that stopped smoking a long time ago? if so, try to make this more explicit.\nDiscussion - Limitations -> State the implication of these limitations e.g. with the younger sample size, what does this mean in terms of the applicability/ generalisability of the 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?\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": "319936", "date": "09 Sep 2024", "name": "Ian Fearon", "expertise": [ "Reviewer Expertise Clinical and behavioural studies of tobacco and nicotine 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\nThe study described in this paper is an excellent study assessing vapers' reactions to a pending legislative ban on flavoured vaping products in New Zealand. The authors utilised a vape expo as a means of surveying participants who were current 'regular' vapers. The data are extremely timely; many countries around the world are considering implementing similar measures. While the intended purpose of such regulation is to try and reduce youth e-cigarette use (although evidence that such measures actually achieve that intended effect are rare and inconclusive), what this paper does (and what is ultimately necessary in order to understand the impact on tobacco harm reduction efforts) is examines the potential impact on adults who use vaping products. This population is all too frequently forgotten about when considering regulatory measures and as such the data in this paper provide an important factual input into the flavour debate. The study additionally examined awareness and use of other nicotine products such as tobacco-free nicotine pouches. Again, these data are timely given the rising use of such products and with many regulators considering regulatory proposals to restrict their use.\n\nPrimary findings of the survey include that significant proportions of vapers would either reinitiate smoking, mix their own liquids, or purchase by assessing the black market. It also appears that half of the vapers surveyed were former smokers, potentially suggesting that they has used e-cigarettes as a cessation aid. Additionally, approximately a quarter of participants had never heard of snus or nicotine pouches, and approximately one-third had heard of these products but never used them.\n\nOverall, I found that the methods were sound, and the findings interesting and well presented in the paper. Its strength is its timing (prior to the enactment of vaping legislation) and its participants (current regular vapers, whose vaping was likely to be affected by the legislation). The only weakness really, to me, is that no attempt was made to collect contact details for respondents to aid a follow up study assessing what these vapers actually did following the legislation. This doesn't impact the study findings but could have provided useful insight.\nMajor Comments\nThe study assessed the likely reactions of vapers to legislation which was implemented the following year and which banned flavoured vaping products aside from tobacco, mint and menthol. Although outside of the scope of this study, are there any published (or other) data to suggest whether the participants' proposed likely reactions became a reality?\n\nIs there any way you can further examine the data to assess likely reactions to the legislation among subgroups of participants? e.g., were flavoured vapers more likely to state they would access the black market? Cross-tabulating some of the findings way provide additional useful insight.\n\nThe 'Conclusion' section of the paper doesn't actually state any conclusions i.e., what was found. Please amend to state some conclusions, and to link these to the views presented in that section.\n\nMinor Comments\nFirst line of the introduction - change 'uset' to use.\n\nIn the 8th paragraph of the methods, I don't understand how a paper on vaping and MI (Bhatta and Glantz) relates to the ethics of surveying papers at conventions. Additionally, the Siegel reference here is not in the bibliography (please check that all citations are in the bibliography) though the link takes you to a blog post, and again I don't really see what this has to do with surveying vape expo attendees.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/10-619
https://f1000research.com/articles/10-505/v1	28 Jun 21	{ "type": "Research Article", "title": "Sleep quality among undergraduate students of a medical college in Nepal during COVID-19 pandemic: an online survey", "authors": [ "Dhan Shrestha", "Suman Prasad Adhikari", "Namrata Rawal", "Pravash Budhathoki", "Subashchandra Pokharel", "Yuvraj Adhikari", "Pooja Rokaya", "Udit Raut", "Suman Prasad Adhikari", "Namrata Rawal", "Pravash Budhathoki", "Subashchandra Pokharel", "Yuvraj Adhikari", "Pooja Rokaya", "Udit Raut" ], "abstract": "Background Good sleep quality is associated with a diverse range of positive outcomes such as better health, less daytime sleepiness, well-being, and proper psychological functioning. Sleep deprivation or poor sleep quality leads to many metabolic, endocrine, and immune changes. Many studies have shown changes in sleep schedule along with worsening of sleep quality during the COVID-19 pandemic. Methods This cross-sectional study was conducted among students of a medical college in Kathmandu, Nepal from January 13, 2021, to February 15, 2021. A stratified random sampling technique was used. Data were collected using the Pittsburgh Sleep Quality Index (PSQI). Questionnaires that were completely filled were included and analyzed using STATA vs. 15. Results 168 medical students filled out the questionnaires with a response rate of 88.42%. 30.36% (n=51) of respondents were having poor quality of sleep (PSQI total score of > 5) with an average PSQI score of 4.24±2.19. Unadjusted logistic regression analysis showed significantly higher odds of poor quality of sleep among females (OR, 2.25; CI, 1.14-4.43) comparing to male and the relation persists even adjusting with age and year in medical school (aOR, 2.81; CI, 1.35-5.86) Adjusting with age and gender 4th-year MBBS students were 82% less likely to have a poor quality of sleep compared to 2nd-year MBBS students (aOR, 0.18; CI, 0.04-0.76). Poor quality of sleep is common among medical students. Conclusions More than a quarter of medical students have a poor quality of sleep as per our study. So, education and awareness should be aroused among medical students regarding the detrimental effects of poor quality sleep on daily activities, physical and mental well-being, and the overall quality of life.", "keywords": [ "COVID-19", "Medical students", "Nepal", "Sleep", "Sleep Deprivation" ], "content": "1. Introduction\n\nThe novel coronavirus (COVID-19) that first appeared in Wuhan has been spreading around the world.1 The World Health Organization declared it a pandemic in March 2020.2 The pandemic has brought not only the fear of infection & death but also unbearable psychological pressure and disturbances.3,4 Good sleep quality is associated with a diverse range of positive outcomes such as better health, less daytime sleepiness, well-being, and proper psychological functioning.5 Sleep deprivation or poor sleep quality leads to many metabolic, endocrine, and immune changes.6 So, the importance of good quality sleep cannot be stressed more.\n\nMany studies have shown changes in sleep schedule along with worsening of sleep quality during the lockdown.7 Findings regarding sleep duration and sleep quality are mixed during the COVID-19 pandemic.7–13 Most studies examining differences in sleep quality have shown poorer sleep quality during the pandemic.12–16 Studies in different parts of the world showed that sleep quality was poorer during the lockdown period relative to the pre-lockdown period.7,10–12,14 Compared to the pre-lockdown period, there was a shift to a later bedtime and waking time, with a reduction in nighttime sleep and an increase in daytime napping during the lockdown period. Also, there was evidence of greater sleep latency and poorer sleep efficiency during the pandemic.13,17 Especially, undergraduate medical students have poor sleep quality, internet addiction, and depression.18–20\n\nSufficient studies have not been done in Nepal regarding the sleep quality of medical students during the COVID-19 pandemic. A study on Nepalese college students has shown the poor quality of sleep among the majority of them.21 Higher prevalence of poor sleep quality among medical students as compared to other non-medical students and the general population has also been reported and several factors including medical students’ attitudes, knowledge of sleep, and academic demands have been identified as the causative factors.22\n\nWe want to determine the quality of sleep in medical students during this pandemic. Thus, this study aims to assess the impact of this enormous change caused by the COVID-19 pandemic on the sleep quality of medical students of a medical college in Nepal during the COVID-19 pandemic.\n\n\n2. Methods\n\nThis is a cross-sectional study done among the undergraduate medical students (first to the fourth year) of Nepalese Army Institute of Health Sciences (NAIHS), Kathmandu, Nepal from January 13, 2021, to February 15, 2021. The Institutional Review Committee (IRC) of NAIHS approved the study (Reference no: 374). At the time of data collection, all the participants were informed about the study and its objectives. Consent was taken from the participants by incorporating the consent form in the questionnaire itself. So, all the participants are understood to have given consent (details of questionnaire and consent form attached as extended data).\n\nThe study participants were not recruited as front-liners to tackle the COVID-19 pandemic during or before the study period. All of them were restricted in their homes during the lockdown period. None of the participants had been infected by COVID-19 up until the study period. The stratified random sampling method was used. Data were collected from the participants after receiving their consent, via Google forms sent out by email explaining the objective of the study in the form itself. The participation was voluntary and anonymity was assured to the participants. The participants didn’t receive any incentives.\n\nThe sample size for the study was calculated using the Cochrane formula. Total students from 1st to 4th year in NAIHS are 423. The calculated sample size was 169. Considering and adding 10% as a non-response rate, the final sample size of 186. The questionnaire was emailed to 190 participants, among which only 168 responded giving a response rate of 88.42%. Detail of sample size calculation is as follow:\n\nSampling method: Stratified random sampling\n\nSample size: n = Z2pq/e2\n\n= (1.96)20.76(1-0.76)/0.052\n\n= 280.28\n\n= 280\n\nwhere,\n\nn = calculated sample size\n\nZ = 1.96 at 95% Confidence Interval\n\np = prevalence of poor sleep quality taken from previous study (76%) 10\n\nq = 1-p\n\ne = Margin of error (5%)\n\nTotal students from 1st to 4th year in NAIHS-COM (N) = 423\n\nAdjusted sample size (n′) = n/ [1+ {(n − 1)/N}]\n\n= 280/ [1+ {(280 − 1)/423}]\n\n= 168.72\n\n= 169\n\nConsidering and adding 10% as a non-response rate, our final sample size = 186.\n\nWe sent the questionnaire to 190 participants.\n\nParticipants were selected using stratified random sampling in such a way that every student from first to fourth has an equal chance of being selected. Firstly, a name list of students of the first to the fourth year was obtained from the administration of the institute, and each student was assigned a particular random number. An equal proportion of the male and female students from each year were selected for the study. Since the total number of students in each year was different, the total number of participants was different in a different year. Then the study participants were selected randomly using the computer random number generator maintaining equal proportion of students from each year and equal proportion of males and females in each year [(first year: 45; female = 15, male = 30); (second year: 45; female = 13, male = 32); (third year: 50; female = 17, male = 33); (fourth year: 50; female = 15, male = 35)].\n\nWe emailed the questionnaire to the participants as everyone was at their homes because of the COVID-19 pandemic imposed lockdown.\n\nWe used the standard and validated Pittsburgh Sleep Quality Index (PSQI), which was developed by researchers at the University of Pittsburgh in 1988 AD. The questionnaire included baseline variables like age, sex, academic year, and questions addressing participants’ sleep habits and quality i.e. PSQI. The PSQI assesses the sleep quality during the previous month and contains 19 self-rated questions that yield seven components: subjective sleep quality sleep, latency, sleep duration, sleep efficiency and sleep disturbance, and daytime dysfunction. Each component is to be assigned a scored that ranges from zero to three, yielding a PSQI score in a range that goes from 0 to 21. A total score of 0 to 4 is considered as normal sleep quality; whereas, scores greater than 4 are categorized as poor sleep quality.23 We typed the questionnaire in the Google form and sent them by email to the randomly selected study participants.\n\nData collected from students through the Google forms were extracted to Google sheets, cleaned in Excel, and then imported and analyzed using STATA software v.15. Simple descriptive analysis was performed to see the response for every PSQI variable. Then calculation performed following PSQI form administration instructions (attached in the extended data). We ran logistic regression analysis taking PSQI score-based category as the dependent variable and age, gender, and years in medical school as the independent variable. Poor quality of sleep was the outcome of interest so logistic regression analysis was run for the occurrence of poor quality of sleep to good quality of sleep. Firstly binary logistic regression analysis ran across the quality of sleep to gender, age, and year in medical school to estimate the unadjusted odds ratio (OR). Then multiple logistic regression ran to estimate adjusted OR across the quality of sleep to gender, age, and year in medical school. For logistic regression purposes, PSQI score-based quality of sleep was labeled as zero for good quality of sleep and one as poor quality of sleep, and odds of occurrence of poor quality of sleep to good quality of sleep estimated.\n\n\n3. Results\n\nAmong the mailed 190 individuals from 1st to 4th year of a medical school, 168 only filled the Google form making the response rate of 88.42%. The majority (n = 108, 64.29%) were male with a mean age of 21.57 ± 1.52 years. The majority (n = 110, 65.48%) of students were staying single while the rest used to share their room. The average sleep hour in the last month was 7:27:20.09 ± 1:25:49.79 hour and sleep latency was 24.92 ± 25.97 minutes. 30.36% (n = 51) of respondents were having poor quality of sleep (PSQI total score of > five) with an average PSQI score of 4.24 ± 2.19 (Table 1).\n\nSpecific questions on trouble sleeping in last month showed 24(14.3%) were having trouble getting sleep within 30 minutes three or more times a week. Similarly, 12(7.1%) mentioned they wake up in the middle of the night or early morning three or more times a week. Majority i.e. 44% did not use to get up to use the bathroom during the past month. Likewise, only 1.2% and 1.8% had trouble sleeping because they could not breathe comfortably and cough or snore loudly respectively for three or more times a week. When questioned if they had difficulty sleeping because of too cold or too hot feeling three or more times a week, 4.8% and 2.4% responded positively. The majority, 36.9%, and 75.6% did not have trouble sleeping because of bad dreams and having pain respectively during the last month. 98.8% of the participants did not have to take medicine to help him/her sleep during the last month. However, 1.2% had trouble staying awake while driving, eating meals, or engaging in social activities during the past month. Among those who had roommate or bed partner, 6.9%, 1.7%, and 3.4% snored loudly, took long pause between breaths while asleep, and twitched or jerked legs while asleep respectively for three or more times a week during the past month whereas none of them had episodes of disorientation or confusion during sleep (Table 2).\n\n\n\na. Cannot get to sleep within 30 minutes\n\n\n\nb. Wake up in the middle of the night or early morning\n\n\n\nc. Have to get up to use the bathroom\n\n\n\nd. Cannot breathe comfortably\n\n\n\ne. Cough or snore loudly\n\n\n\nf. Feel too cold\n\n\n\ng. Feel too hot\n\n\n\nh. Had bad dreams\n\n\n\ni. Have pain\n\n\n\na. Loud snoring\n\n\n\nb. Long pauses between breaths while asleep\n\n\n\nc. Legs twitching or jerking while you sleep\n\n\n\nd. Episodes of disorientation or confusion during sleep\n\nAmong 168 respondents, 111(66.1%) responded to have a fairly good quality of sleep while asking to rate their overall sleep quality in the last month (Figure 1).\n\nFifty-eight (34.5%) responded no problem at all when asking for a problem to keep up enough enthusiasm to get things done during the past month, while 27 (16.1%) responded a very big problem (Figure 2).\n\nUnadjusted logistic regression analysis showed significantly higher odds of poor quality of sleep among females (OR, 2.25; CI, 1.14-4.43) comparing to male and the relation persisted even adjusting with age and year in medical school (aOR, 2.81; CI, 1.35-5.86) (Table 3). There was no significant difference in the quality of sleep across years in medical school while running an unadjusted logistic regression analysis. But, adjusting with age and gender 4th-year MBBS students are 82% lesser likely to have a poor quality of sleep comparing with 2nd-year MBBS students (aOR, 0.18; CI, 0.04-0.76) (Table 3).\n\n\n4. Discussion\n\nThe COVID-19 pandemic in 2020 has imposed a substantial effect on psychological, health, and social issues all over the world. Medical students, like everyone else, have also suffered from these issues. Medical education is based more on a practical approach rather than just a theoretical core. Therefore, classes shifting from the classroom to online platforms have, without any doubt; affected medical education more than anything else. However, the impact of the pandemic on the psychological well-being and sleep quality of medical students has not been assessed and addressed to a good extent in Nepal. Therefore, this study uses the PSQI to determine the quality of sleep among the medical students of a medical college in Kathmandu, Nepal during the COVID-19 pandemic.\n\nOur study showed that 30.36% of medical students had poor quality of sleep (PSQI global score >5) but a study has done just before the pandemic in the Nepalese medical students showed that 44.23% of students had poor sleep quality.24 A study from India during the COVID-19 pandemic had 34.6% of medical students with poor sleep quality which was in line with the result of our study.25 Previous studies from Pakistan on medical students in 2013 and Nepal on undergraduate students in 2015 showed 39.5% and 35.4% poor quality sleepers respectively.19,26 Our findings were in contrast to a study in China which showed a high prevalence of sleep disorders among adolescents and young adult students due to the stress and anxiety caused by the pandemic.27 Previous studies in the Nepalese general population and health care workers had found a low level of psychological distress due to the COVID-19 pandemic in Nepal which may explain better sleep quality among medical students in Nepal.28,29\n\nOne of the positive findings of this study is that none of the participants had to take medication to help them sleep whereas 10.2% of Saudi Arabian physicians used sleeping pills once or twice a week during the COVID-19 pandemic.30 14.3% of our study participants had trouble falling asleep within 30 minutes thrice or more in a week, which was less compared to 24.7% in Saudi Arabian physicians during the COVID-19 pandemic.30 Our study showed significantly higher odds of poor quality sleep among female students (OR, 2.25; CI, 1.14-4.43). A study on Pakistani medical students in 2013 also showed that more females had poor quality sleep i.e. 44% as compared to 32.8% male poor sleepers.26 Similarly, Goweda RA et al. also found that sleep disorders were more common among female medical students based on a study done in Saudi Arabia during the COVID-19 pandemic.31 Increased incidence of poor sleep quality among females is justifiable as sleep disorder symptoms are usually greater in women as compared to men.28,32 So, more focus should be shed upon female students when programs are formulated to improve the sleep quality among medical students. Sleep quality among students across years in medical school was not significantly different while running unadjusted logistic regression analysis in the case of our study. Studies from Brazil and Saudi Arabia assessed the sleep quality of first and second-year medical students to be poor as compared to other years.30,33 We found that the fourth-year medical students were less likely to have a poor quality of sleep compared to second-year students which may be due to greater academic experience, and exposures during higher academic classes. In contrast, a previous study in China found that senior high school students had greater sleep problems due to increased academic burden and difficulty compared to junior school students.27\n\nThough our study shows less number of poor quality sleepers as compared to other studies discussed,19,24,25 30.36% is still a big number. We cannot stay still and satisfied based on the result of this study and the concerned authorities should try their best on bringing this number down to as minimum as possible. Because, poor quality sleep, without a doubt, will have a long-term impact on the mental well-being of the students. And no country would want future doctors who are not mentally healthy.\n\nThe number of students with poor sleep quality might decrease after the fear and restrictions because COVID-19 subsides and life will return to normality. Alternatively, the number might go up, as the students return to their hectic schedules of classes, clinical postings, and a lot of studying. Since the scenario post-COVID is still unpredictable, frequent monitoring of sleep health and habits in medical students for raising awareness about sleep quality and problems should be planned and implemented.\n\nOur study is not without limitations. We used a self-reporting questionnaire. Therefore, information bias is a major risk for this study. Our study was conducted among the students of a single institution. Therefore, the results might not be extrapolated to all the medical colleges of Nepal. Likewise, recall bias and subjectivity bias could have also affected the result of our study. Despite the limitations and biases, this study will surely provide a reference for further researches in this particular field.\n\n\n5. Conclusions\n\nPoor quality of sleep is prevalent in 30.36% of medical students of a medical college in Nepal during the COVID-19 pandemic. Further studies have to be done to delve deeper into the sleep habits and problems of the medical students to determine the risk factors and causation of poor sleep quality. Furthermore, awareness should be aroused about the importance of proper amount and quality of sleep as well as detrimental effects of poor quality sleep on daily activities, physical and mental well-being, and the overall quality of life.\n\n\nData availability\n\nFigshare. Sleep quality among undergraduate students of a medical college in Nepal during COVID-19 pandemic: an online survey. DOI: https://doi.org/10.6084/m9.figshare.14770182.v2.34\n\nThis project contains the following underlying data:\n\n- We used the standard and validated Pittsburgh Sleep Quality Index (PSQI), which was developed by researchers at the University of Pittsburgh in 1988 AD. The questionnaire included baseline variables like age, sex, academic year, and questions addressing participants’ sleep habits and quality i.e. PSQI. The PSQI assesses the sleep quality during the previous month and contains 19 self-rated questions that yield seven components: subjective sleep quality sleep, latency, sleep duration, sleep efficiency and sleep disturbance, and daytime dysfunction. Each component is to be assigned a scored that ranges from zero to three, yielding a PSQI score in a range that goes from 0 to 21. A total score of 0 to 4 is considered as normal sleep quality; whereas, scores greater than 4 are categorized as poor sleep quality.\n\n- Data collected from students through the Google forms were extracted to Google sheets, cleaned in Excel, and then imported and analyzed using STATA 15. Simple descriptive analysis was performed to see the response for every PSQI variable. Then calculation performed following PSQI form administration instructions.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).\n\nFigshare. Sleep quality among undergraduate students of a medical college in Nepal during COVID-19 pandemic: an online survey. DOI: https://doi.org/10.6084/m9.figshare.14770182.v2.34\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).\n\n\nAuthors’ contributions\n\nDBS, SPA, NR, PB, SP, YA, PR, and UR contributed to the concept and design, methodology, and data collection. DBS contributed to the analysis, and interpretation of data. DBS, PB, SP, YA, PR, and UR contributed to the literature search, and initial manuscript drafting. SPA, NR involved in the revision and intellectual interpretation of the manuscript.\n\nAll authors were involved in drafting and revising the manuscript and approved the final version.", "appendix": "Acknowledgments\n\nWe would like to thank all the participants in this research for their voluntary participation.\n\n\nReferences\n\nWHO\|Novel Coronavirus – China: WHO [Internet].2020 [cited 2021 May 18]. http://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/\n\nWHO Director-General’s opening remarks at the media briefing on COVID-19-11 March 2020 [Internet].[cited 2021 May 18]. Reference Source\n\nCao W, Fang Z, Hou G, et al.: The psychological impact of the COVID-19 epidemic on college students in China. Psychiatry Res. 2020 May 1; 287: 112934. 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Sleep Med. 2021 Jan; 77: 112–9. PubMed Abstract \| Publisher Full Text\n\nGao C, Scullin MK: Sleep health early in the coronavirus disease 2019 (COVID-19) outbreak in the United States: integrating longitudinal, cross-sectional, and retrospective recall data. Sleep Med. 2020 Sep; 73: 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenham G: Stress and sleep in college students prior to and during the COVID-19 pandemic. Stress Heal. 2020. PubMed Abstract \| Publisher Full Text\n\nAttal BA, Bezdan M, Abdulqader A: Quality of Sleep and Its Correlates among Yemeni Medical Students: A Cross-Sectional Study. Sleep Disord. 2021 Jan; 2021: 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhandari PM, Neupane D, Rijal S, et al.: Sleep quality, internet addiction and depressive symptoms among undergraduate students in Nepal. BMC Psychiatry. 2017 Mar 21 [cited 2021 May 18]; 17(1): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nAylie NS, Mekonen MA, Mekuria RM: The psychological impacts of COVID-19 pandemic among university students in bench-sheko zone, South-West Ethiopia: A community-based cross-sectional study. Psychol Res Behav Manag. 2020 Sep 30 [cited 2021 May 18]; 13: 813–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhadka R, Bista S, Baskota S, et al.: Sleep Quality among College Students in Kathmandu Valley, Nepal. Nepal Med J. 2019 Dec; 2(2): 1–4. Publisher Full Text\n\nAzad MC, Fraser K, Rumana N, et al.: Sleep disturbances among medical students: A global perspective. J Clin Sleep Med. American Academy of Sleep Medicine; 2015; 11: p. 69–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuysse DJ, Reynolds CF, Monk TH, et al.: The Pittsburgh sleep quality index: A new instrument for psychiatric practice and research. Psychiatry Res. 1989 May 1; 28(2): 193–213. PubMed Abstract \| Publisher Full Text\n\nSundas N, Ghimire S, Bhusal S, et al.: Sleep quality among medical students of a tertiary care hospital: A descriptive cross-sectional study. J Nepal Med Assoc. 2020 Feb 29 [cited 2021 May 18]; 58(222): 76–9. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nSaraswathi I, Saikarthik J, Kumar KS, et al.: Impact of COVID-19 outbreak on the mental health status of undergraduate medical students in a COVID-19 treating medical college: A prospective longitudinal study. PeerJ. 2020 Oct 16 [cited 2021 May 18]; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSurani AA, Zahid S, Surani A, et al.: Sleep quality among medical students of Karachi. Pak. J Pak Med Assoc. 2015 Apr; 65(4): 380–2. PubMed Abstract\n\nZhou SJ, Wang LL, Yang R, et al.: Sleep problems among Chinese adolescents and young adults during the coronavirus-2019 pandemic. Sleep Med. 2020 Oct 1 [cited 2021 Feb 20]; 74: 39–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShrestha DB, Thapa BB, Katuwal N, et al.: Psychological distress in Nepalese residents during COVID-19 pandemic: A community level survey. BMC Psychiatry. BioMed Central Ltd; 2020 [cited 2021 Feb 20]: 20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKafle K, Shrestha DB, Baniya A, et al.: Psychological distress among health service providers during COVID-19 pandemic in Nepal. PLoS One. 2021 Feb 1 [cited 2021 May 18]; 16(2 February): e0246784. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlnofaiey YH, Alshehri HA, Alosaimi MM, et al.: Sleep disturbances among physicians during COVID-19 pandemic. BMC Res Notes. 2020 Dec 1 [cited 2021 May 18]; 13(1): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoweda RA, Hassan-Hussein A, Alqahtani MA, et al.: Prevalence of sleep disorders among medical students of Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia. J Public health Res. 2020 [cited 2021 May 18]; 9(S1): 45–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlink M, Quan SF: Prevalence of reported sleep disturbances in a general adult population and their relationship to obstructive airways diseases. Chest. 1987 Apr 1 [cited 2021 May 18]; 91(4): 540–6. PubMed Abstract \| Publisher Full Text Reference Source\n\nde C CC, de Oliveira FK, Pizzamiglio DS, et al.: Qualidade de sono em estudantes de medicina: Comparação das diferentes fases do curso. J Bras Pneumol. 2017 Jul 1 [cited 2021 May 18]; 43(4): 285–9. Publisher Full Text\n\nShrestha D, Adhikari SP, Rawal N: Sleep quality among undergraduate students of a medical college in Nepal during COVID-19 pandemic: an online survey. figshare. Online resource. 2021. Publisher Full Text" }	[ { "id": "88340", "date": "01 Jul 2021", "name": "Roshan Acharya", "expertise": [ "Reviewer Expertise 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\nThis study addresses one of the important problems prevalent among medical students. It has been conducted following the proper research methods. The title of the study is appropriate. The manuscript seems to be technically sound. The statistical analysis has been done appropriately. All the data underlying the findings are also made fully available. The conclusion includes the results, and recommendations are given accordingly. The paper seems to be well-written despite a few minor errors.\nMy comments regarding the paper are as follows:\nMajor correction: None\nMinor corrections:\nAbstract:\n“Unadjusted logistic regression analysis showed significantly higher odds of poor quality of sleep among females (OR, 2.25; CI,1.14-4.43) comparing to male and the relation persists even adjusting with age and year in medical school (aOR, 2.81; CI, 1.35-5.86)”\n\nPlease put a full stop(.) after this sentence.\n\n“So, education and awareness should be aroused among medical students regarding the detrimental effects of poor quality sleep on daily activities, physical and mental well-being, and the overall quality of life.”\n\nIn this sentence, the replacement of the word ‘aroused’ with ‘raised’ would be better.\nIntroduction:\n“Studies in different parts of the world showed that sleep quality was poorer during the lockdown period relative to the pre-lockdown period.7,10–12,14 Compared to the pre-lockdown period, there was a shift to a later bedtime and waking time, with a reduction in nighttime sleep and an increase in daytime napping during the lockdown period. Also, there was evidence of greater sleep latency and poorer sleep efficiency during the pandemic.”\n\nIn the second paragraph, you have used past tense. I think it would be better to use present tense instead.\nMethods:\n\n“The stratified random sampling method was used.”\n\nThis statement has been mentioned thrice in the methods section itself, once in Study design and settings, twice in Study sample. Please mention it only once, wherever you think is suitable.\nStudy sample:\n“Detail of sample size calculation is as follow:”\n\nHere ‘as follows’ is the correct one, please correct it.\nStudy instrument:\n\n“Each component is to be assigned a scored that ranges from zero to three, yielding a PSQI score in a range that goes from 0 to 21.”\n\nIn this sentence, correct ‘assigned a scored’ to ‘assigned a score’\nDiscussion:\n“Our study showed that 30.36% of medical students had poor quality of sleep (PSQI global score >5) but a study has done just before the pandemic in the Nepalese medical students showed that 44.23% of students had poor sleep quality.”\n\nHere, two points; First, please write ‘study done’ in place of ‘study has done’. You have discussed and compared the findings of the 2nd and the 4th year, but not compared them to other years. Also, there is no discussion on why the score of the 3rd year is the highest. Please address this as well. Second: PSQI change it to ‘equal or greater than 5’. Please see my last point for further clarification.\nConclusion:\n“ Furthermore, awareness should be aroused about the importance of proper amount and quality of sleep as well as detrimental effects of poor quality sleep on daily activities, physical and mental well-being, and the overall quality of life.”\nIn this sentence, the replacement of the word ‘aroused’ with ‘raised’ would be better.\nTable 1: In the methodology it was mentioned that: A total score of 0 to 4 is considered as normal sleep quality; whereas, scores greater than 4 are categorized as poor sleep quality. But in Table 1, it is otherwise: PSQI category (sum score < 5 or > 5). It should be corrected in Table 1 for uniformity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "88342", "date": "06 Jul 2021", "name": "Samata Nepal", "expertise": [ "Reviewer Expertise Public health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract:\nPlease use punctuation wherever required, e.g. \"well-being and...\" instead of \"well-being, and...\".\n\n\"from January 13, 2021 to February...\" instead of \"from January 13, 2021, to February...\".\n\nThe sentence starting with a number is not appropriate in the Results section, second sentence. Please consider \"Around 30% (n=51) of respondents...\" or \"Thirty percent (n=51) of respondents...\".\n\nIntroduction:\nParagraph 2, line 5: \"Compared to the pre-lockdown...\", this sentence needs to be paraphrased. Consider \"When compared to pre-lockdown period, there was a later shift to bedtime and waking time, which reduced sleep during the night and increased daytime napping\".\n\nParagraph 4, line 1: The first sentence is not needed, please consider \"This study aims...\"\nMethods:\nParagraph 1, line 2: Consider \"January 13, 2021 to February 15\".\n\nParagraph 3, line 3: \"The questionnaire was...\", please remove this sentence as the same sentence is repeated in the next paragraph.\n\nThe last line of 2.2 is not required. \"We emailed the...\" has been mentioned already.\n\n2.3 Study instrument, line 5: Please consider \"component is assigned score...\".\n\n2.3 Study instrument, line 7: Please consider \"The questionnaire was typed in the Google form and...\". The use of passive format is better.\n\nPlease mention clearly if the questionnaire was emailed or the link of the Google form was emailed. Please consider using uniformity throughout the Methods section.\n\n2.4 Statistical methods: line 1: \"Google forms was extracted...\".\n\n2.4 Statistical methods, line 5: Please consider \"Logistic regression analysis was done...\". The use of passive format is better.\n\nRepeated use of the word \"ran\" in 2.4 can be avoided, e.g. \"logistic regression analysis was run...\", \"multiple logistic regression ran...\".\n\nPlease consider uniformity in writing either \"good/poor quality sleep\" or \"good/poor quality of sleep\".\nResults:\nTable 1: The table with the variable \"Do you have a bed partner or roommate?\" - the last row has a sub-variable: \" Partner/roommate in another room\". Please clarify this option.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/10-505
https://f1000research.com/articles/10-305/v1	20 Apr 21	{ "type": "Research Article", "title": "Analysing students’ environmental awareness profile using strategic environmental assessment", "authors": [ "Ahmad Khoiri", "Widha Sunarno", "Sajidan Sajidan", "Sukarmin Sukarmin", "Sajidan Sajidan", "Sukarmin Sukarmin" ], "abstract": "Background: Environmental awareness (EA) is a part of character education ignored by most students. This indifference tends to affect other students’ by not only in protecting and preserving the current environment but also in preventing and repairing the damage that occurs in the environment. This research analyses students' EA profile, based on the findings of LISREL 8.8 Confirmatory Factor Analysis. Methods: Research subjects included 131 students from Senior High School State 1 Selomerto Wonosobo, Central Java Province, Indonesia. Based on the Slovin formula, the number of samples is representative of the total population (N: 185; error tolerance: 0.05). Data were collected through non-test questionnaires and observation of Strategic Environmental Awareness (SEA). Quantitative descriptive data analysis on EA indicators (Care, Curiosity, Critical, Dependability, Responsibility, and Local Wisdom). Results: The EA profile of high school students was categorised sufficiently. This revealed the following results: a) the SEA instrument is effective in identifying students’ awareness about current environmental issues and meets model fit criteria (P-value 0.25>0.05; RMSEA 0.095; NFI 0.67); b) the SEA instrument is valid and reliable in accurately determining students’ EA profile; c) although the Responsibility profile was significant (t >1.96), other variables did not meet this significance criteria (EA 1a: ‘Care towards environmental damage’ under the Care profile; EA 3b: ‘Contributing towards preserving the environment’ under the Critical profile; and EA 6b: ‘Aware of local potentials’ under the Local Wisdom profile); d) evaluation of the expected changes in SEA is modified through an ethnoscience approach and the socioscientific issues strategy. Conclusions: Students’ lack of awareness of the environment and understanding of their regional potential fails to contribute towards creating a sustainable environment. Profile analysis in exploring attitudes, values, and ethics towards the environment are important, as it helps recognize students’ behaviour.", "keywords": [ "Confirmatory Factor Analysis", "Environmental Awareness", "Ethnoscience", "Strategic Environmental Assessment", "and Socioscientific Issues" ], "content": "Introduction\n\nHuman life cannot be separated from interactions with the environment. Rapid improvement of technology in various fields has left negative impacts on the environment. One such negative impact is environmental damage, which has led to decreased environmental quality. Hence the degradation of environmental conditions can worsen the development of science education. In addition, resources for environmental science learning are still limited. Despite being close to the surrounding environment, science learning is separated from its natural sources. Environmental issues remain under-studied and under-utilized in science learning.1,2 For instance, how students study the environment has not been integrated into science learning. There is lack of sufficient action to protect the environment, such as taking responsibility, exploring the local wisdom in a particular area, and maintaining and managing the environment.3,4 Taking care of the environment is an attitude that strives to prevent natural environmental damage, and is the best way to restore environmental damage.\n\nBased on the results of a preliminary survey of 15 teachers and 256 high school students in Wonosobo District, Central Java, Indonesia in March 2020,5 it is observed that schools have shown 75% progress in policies towards environmental awareness, 62% progress in terms of promoting curriculum-based environmental studies, 56% participatory-based development of environmental activities, and 55% management of environmentally friendly school support facilities. That research identified a low implementation of environment-based learning, resulting in a lack of environmental character for students.5,6\n\nEnvironmental care, as it has been taught in school, refers to definitions, terms, concepts, and knowledge about the natural environment. The absence of the implementation of contextual learning can limit meaningful experiences for students. Environmental care can be implemented if students develop relevant habits at home, at school, and in the community. This is the best way for schools to build environmentally friendly attitudes among students, that is, by integrating the environment into materials, methods, media, learning resources, and assessment. Hopefully, learning, combined with environmental care, can stir students’ concern for the environment and nature.7\n\nStudents’ knowledge of the environment has been positively associated with their environmental care behaviour, which can abate environmental damage. Nurwidodo et al.6 stated that students’ environmental care is low because there is a lack of intention to understand and study environmental problems. Therefore, teachers should facilitate students’ understanding of environmental issues in order to motivate them to addresses these problems.8 However, teachers only focus on students’ academic performance and not on how students try to keep the school environment clean.9 Therefore, strategic environmental awareness (SEA) analysis is essential so that students’ positive attitude towards environmental care can provide solutions to environmental problems.\n\nSchools should use an effective instrument to communicate about environmental issues, since environmental care is a crucial issue, especially for students. Learning associated with environmental care can enhance students’ awareness of their environment and surroundings. Creating a healthy ecological system requires long-term efforts.10 This is in congruence with Rahardjo’s11 study, which showed that learning should focus on promoting students’ positive attitude towards the environment.12\n\nSEA assesses environmental care and measures the environment awareness (EA) of students.1,13,14 Effective instruments should be valid and reliable.15,16 Therefore, SEA instruments are required to measure EA skills. EA indicators are determined based on healthy environmental problems, policies in preserving the environment, and the long-term SEA program.13 EA is incorporated in teaching Physics, including Care, Protection, and Conservation Component indicators.1\n\nBased on environmental care,17,18 EA assumes conscious thought in managing the environment is a factor in protecting and maintaining the environment.19,6,20 This does not only demonstrate the understanding of the natural environment but also the attitude, value, and, skills required to address environmental issues.21,22 Environmental education enhances people’s knowledge and awareness about the environment and induces environmental care behaviour.23 Phan Hoang and Kato24 and Mei et al.25 explain that knowledge and commitment are required to instigate EA. Students must gain knowledge and understanding of the fundamental environmental problems, which will initiate a change in attitude and awareness in their social life namely interactions with other humans and the surrounding environment.20,26,27 The impact of the students’ low environmental care can be assessed by evaluating the sustainable impact on condition, health, comfort and environmental benefits.28–30\n\nIn Indonesia, this attitude of caring for the environment has become integrated into academic culture.6 The Adiwiyata program in schools seems to be separated from the environmental-based education curriculum, even though EA should be integrated into the students’ learning process of environment friendly characteristics. The current research focuses on the importance of analysing the profile of students’ EA in facing challenges of globalisation. The profile analysis is adapted from the SEA with a holistic role and paradigm aimed at not only developing a caring attitude, maintaining cleanliness, and preserving the environment, but also making a real contribution through policy recommendations, planning, and environmental programs for sustainable practices.\n\nBased on previous literature,23,25 EA has a broad connotation, and this not only relates to knowledge about the environment but also the attitudes, values, and skills needed to solve environmental issues, which facilitate the ability to carry out responsible civic behaviour.22,31 Students have not acquired the skills needed to care for the environment, because teachers have not been able to facilitate the environment-based learning process effectively. Therefore, in this study, the researchers used SEA to perform a students’ profile analysis to address environmental problems.\n\nStudents from middle school that live between urban and rural areas were selected for this study.. Researchers’ assumption is based on analysing the profile of students from Senior High School State 1 Selomerto Wonosobo, Central Java Province, to study the EA profile of high school students in Wonosobo Regency. To build environment friendly characteristics among high school students in the era of globalisation, environmental education is crucial. The increasingly sophisticated flow of globalization has eroded pre-existing traditions and cultures because all human life activities are based on technological literacy.32 Thus, the EA profile of high school students will explain the lack of environmental awareness and care among students. Through an analysis of the findings, the criterion variable of each indicator, including those meeting and not meeting the Confirmatory Factor Analysis criteria, can be identified and a fair assessment of the solutions can be achieved.\n\n\nMethods\n\nA survey was conducted among students from Senior High School State 1 Selomerto Wonosobo, Central Java Province, Indonesia. The sample was determined based on the purposive sampling technique, meeting the research criteria, that is, the family background of students who lived in the central area of the city who all had similar attributes in terms of cultural and environmental recognition.\n\nThe inclusion requirements of this study were as follows.\n\n1) Male and female students who are actively registered as students at Senior High School 1 Selomerto, Central Java Province, Indonesia.\n\n2) Willing to become informants.\n\n3) Physically and mentally healthy.\n\n4) Students’ responses are not influenced by the opinions of teachers, friends, guardians of students or others.\n\n5) Students in class 10-12 only.\n\nA total of 143 students from the entire population in the school filled out the questionnaires after receiving a technical explanation from the researcher. Participation in this survey was voluntary, and no financial reward was offered. All survey procedures and data were guaranteed to be strictly confidential.\n\nSample size was determined based on the Slovin formula: n=N1+Ne2, where n is the number of samples; N is the total population (N = 185); and e is error tolerance (5%).33 The number of survey data represents the population with a minimum of 126 (131 > 126) samples collected.\n\nThe total number of students was 143, and 131 gave complete responses: class 10, 45 responses; class 11, 46; and class 12, 40. The questionnaire data collection process was adjusted to a 5-point Likert scale, namely: strongly agree, 5; agree, 4; neutral, 3; disagree, 2; and strongly disagree, 1.\n\nThe collected survey questionnaires had 131 complete responses out of a total 143 because 12 responses were incomplete and had to be deleted. Twelve samples had missing data, because they do not completely answer all the questions, and the remaining 54 samples were ignored because the sample size was reached.\n\nA questionnaire and a SEA instrument (see Extended data61) was used for data collection, consisting of 42 questions about environmental issues faced by the community. The questionnaire was filled in directly by students via the Google Form link (https://bit.ly/2MXA4HY) within a 2-month research period, from 8 April to 8 June 2020.\n\nThe profile of students’ environmental care attitudes was determined based on six EA indicators developed in the SEA instrument; each indicator had three sub-indicators. The assumption of EA is based on the conscious mind to regulate reason, which is a part of the attitude naturally forming social issues.6,21 This implies not only knowledge of the environment but also the attitudes, values, and skills needed to responsibly solve environmental issues.21 The indicators of students’ EA and the relevance of environmental learning are explained in Table 1.\n\nQuantitative descriptive data analysis was used to assess the students’ responses to the EA questionnaire.34 A t-test using LISREL 8.8 Second-Order CFA was used to check the measurement results to ensure that there are no offending estimates (the values that exceed accepted limits) on the variables. The EA indicators observed in each latent variable fulfilled the analysis requirements by linking and matching sub-indicators and indicator, one indicator with another, and combining the criteria components on EA indicators into one SEA model.\n\nLISREL 8.8 Second-Order CFA confirms variables based on factor analysis, so that students’ profiles are validly and reliably measured. The LISREL method was applied, and the results showed that output of solution standard in identifying the student’s EA profile problems are based on t-test scores, analyse expected changes to provide solutions, and provide recommendations based on results of the analysis. The LISREL 8.8 Second-Order CFA application identifies the relationship between complex environmental care attitude variables and the sub-indicators meeting statistical requirements. LISREL 8.8 Second-Order CFA analysis is very sensitive for ambiguous data and predicts every indicator of the question.\n\nThe dependence of one indicator’s data on other indicators may result in a mismatch, so the instrument was been validated by an expert in SEA instruments. The criteria for the validator of the SEA instrument are one environmental expert lecturer and one evaluation tool lecturer. The purpose of validation provides an assessment of the feasibility of the SEA instrument, and whether or not it can be used in the data collection process. The instrument was been revised based on the validator’s suggestions with the final decision that the SEA instrument is suitable for measuring students’ EA profiles.\n\nLISREL software is proprietary software; a freely available alternative software that can be used to perform the same analysis is lavaan: https://cran.r-project.org/web/packages/lavaan/.\n\nEthical considerations\n\nThe Universitas Sebelas Maret Surakarta gave permission for the study to be conducted on 7th January 2020 (letter number 40/UN27.02.9.2/DP/2020). This research was also approved by the Senior High School State 1 Selomerto, Wonosobo, Central Java Province, Indonesia (letter number 800/208/2020; dated 8 April 2020).\n\nWritten consent to participate was obtained from students, student guardians, and the principals of participating schools. If consent from student guardians was not obtained, the student was not allowed to participate. Respondents provided consent without any coercion from anyone. All forms of data obtained will remain confidential to protect the rights and privacy of the respondents.\n\n\nResults\n\nThe number of potential respondents who met the sample requirements was 143, but in the research process there were 12 students who did not give a complete response. Therefore, the data that were analysed were 131 respondents who gave complete answers. The results for the respondents’ answers to the questionnaire and observation of students’ EA profile35 are shown in Figure 1.\n\nBased on Figure 1, the observation variable (indicator) has a larger value of convergent validity (factor loading) than 0.5; the fit model in P-value is 0.25>0.05; Root Mean Square Error of Approximation (RMSEA) is 0.088; and Normed Fit Index (NFI) is 0.67. The value of the loading factor in indicators A1, A3, and A5 <0.5 and for indicators A2, A4 and A6 >0.5. The t-value is <1.96, so it fulfils the significance criteria. The results show that for indicators Care (EA I), Curiosity (EA 2), Critical (EA 3), Dependability (EA 4), Responsibility (EA 5), and Local wisdom (EA 6), the SEA instrument is valid. Therefore, the SEA instrument is valid and reliable to measure senior high school students’ EA. The development of the SEA instrument can be used as a standard measuring instrument to determine students’ EA through physical learning about temperature, heat, and global warming. The results of the t-value are presented in Table 2.\n\nTable 2 shows that the Care sub-indicator (EA 1a) does not fulfil the significance criteria 0.46 or less than 1.96 (0.46<1.96). The sentence needs to be revised from ‘care the environment’ to ‘Care towards environmental damage’. During testing of the questionnaire, there were sentences in the Care sub-indicator that were unclear. Therefore, it was necessary to revise the sentence about environmental damage by providing an example of an unpleasant smell in the Sikidang Crater area – this has been previously shown to worsen the health of society and requires global environmental care.\n\nThe Critical sub-indicator (EA 3b) does not fulfil the loading factor; it only amounts to 0.64. ‘Contributing towards preserving the environment’ as a form of critical thinking on environmental problems. The example given on the questionnaire was PranotoMongso, which has disappeared today because farmers in Dieng use a modern system of agriculture, using plastics on agricultural land (Figure 3). The farmers do not realise that the use of chemical fertilisers and modern farming tools is not environment friendly, and can harm the fertility of the land and destroy the ecosystem.\n\nThe Local Wisdom sub indicator (EA 6b) did not fulfil the factor loading 0,03. Therefore, re-evaluation is needed to obtain a good profile of EA. For the ‘aware of local potential’ indicator, the capability to understand the local potential and conserve local potential that does not match the indicator criteria. The example given on the questionnaire was the dew phenomenon or ‘Embun Upas’, as shown in Figure 2 (discussed further below). Due to the cold at night and freezing dew in several plateaus in Indonesia, like in Dieng, is caused by meteorological conditions and dry season. Java is now at the top of the dry season, which can be observed from the fact that several mountains have 0°C. This is because the air molecules in mountainous areas are more tenuous than the lowlands; they experience rapid cooling, especially when the weather is clear and not covered by clouds or rain, and water vapour in the air at night leads to condensation, sticking to leaves or grass and immediately freezing because of the temperatures.\n\nThree profiles are not qualified with a t-value of less than 1.96 from six indicators with three sub-indicators. There are 18 sub-indicators of EA with t-values in the highest responsibility indicators (EA 5a). It is a ‘Response towards the dangers of environmental damage’. The students agree that the environment is a part of their lives and must be protected and preserved. Science subjects and culture must be included in learning at school.\n\nThe care indicator (EA 1a) ‘care towards environmental damage’, critical indicator (EA 3b) ‘Contributing towards preserving the environment’, and local wisdom sub-indicator (EA 6b) to ‘Aware of local potentials’ are not fulfilled. The lack of EA and understanding about the potential make students unaware of the need to protect the environment, even though they know about the dangers of environmental damage. This means that students must have environmental care attitudes to initiate a reciprocation between students and nature. The profile of school students’ EA is good. However, it needs to be improved through the result of the expected change second-order CFA analysis. Furthermore, analysis of the suitability of the SEA model used in measuring students’ EA profiles is presented in Table 3.\n\nThe potential of the EA instrument is expected to be changed and revised in terms of measuring every good indicator from changes to the sentence on the questionnaire to make it clearer so that the research results are valid.\n\nBased on the result of descriptive counting, for environmental care data, the scores ranged from 59.4 sub-indicator ‘contributes towards environmental sustainability’ to 83.0 in the sub-indicator ‘know about preserving the environment’ as shown in Figure 4.\n\nBased on Figure 4, the environmental care of students show the value 72.4 on an average in ‘enough category’, with the highest indicator score for ‘local wisdom’ (75.4). Meanwhile, ‘curiosity’ has the lowest score (69.7). This is shown in Figure 5.\n\nThe value of the dominant tendency profile of environmental care is in the sufficient category with a score of 72.4 on average. Therefore, further analysis is required to identify sub-indicators that fulfil or do not fulfil the criteria determined in the SEA instrument.\n\n\nDiscussion\n\nThe results of confirmatory factor analysis showed an evaluation of the goodness of fit, t-value, and the expected change in identifying the EA profile of high school students. The change in the EA indicator (Table 3) is a crucial factor for creating a strategy to deal with environmental issues in society. The context-based learning benefits students and enhances their awareness regarding cultural preservation.38\n\nThe importance of strategy in an environment-based teaching model, while respecting local culture, is an ethnoscience approach. Ethnoscience is used as a reference to equip students with knowledge and character so that they respect their culture (Figure 2 and Figure 3). Ethnoscience learning is a means of self-development, increasing awareness to participate in preserving the environment and cultural traditions. This component has been shown in ethnoscience-based learning to improve students’ EA.1,6 Unfortunately, technology is becoming increasingly advanced and literacy has failed to improve social behaviour. Besides the effect on the education system that utilizes formal science from indigenous knowledge in the form of customs, local wisdom, and cultural traditions as learning resources, high school students do not focus on their interactions with the environment.35,36 The primary source of knowledge that can be effectively obtained is through direct interaction between students and nature, as opposed to studying concepts in classrooms.\n\nEthnography-based teaching will improve student’s skills in science, respect their achievements, and improve their skills to implement their knowledge. Through this teaching method, students’ understanding of science in a cultural context will be improved since they directly learn about the environment. Therefore, they will have a curiosity for, and give attention to, customs and culture. Thus, teaching improves their creativity.41\n\nSocioscientific issues (SSI) based on SEA is a teaching strategy that explains science subjects in terms of social problems involving moral or attitude components.39,40 SSI is a common conceptual or procedural problem related to science and has a rational solution, influenced by social aspects, such as culture, politics, economy, and ethics.39,41 The involvement of social aspects in SSI provides an opportunity to create conflict between scientific and social perspectives. Teaching can help develop moral reasoning and reflective assessment skills in terms of problem-solving.39,40,42\n\nEnvironmental education impacts society by enhancing students’ awareness of the environment.7,43,44 To integrate the ethnography approach as a source of science teaching in high schools using the SSI strategy to empower EA related to environmental damage uses a strategic environmental study48 in terms of learning.49\n\nA contextual aspect is required in environmental learning, since the scope of the problem is related to daily life, which pertains not only to knowledge but also to attitudes and skills to solve environmental issues.50 One of the potential learning strategies that can be implemented is SSI.\n\nCulture, as a part of the social life for different generations, also requires attention. Based on the purpose of learning to enhance students’ knowledge and understanding about life through values and attitude,51 the transition of students into future physics teachers requires excellent preparation.\n\nSkills can be gained from an ethnoscience learning model. Ethnoscience improves students’ knowledge and develops local wisdom and uses formal physics teaching as a source of learning in universities, where ethnoscience is a form of experience and culture.41 Further, it can improve students’ capabilities to implement their scientific knowledge.\n\nThe quality of science learning that reflects the social context as an environmental problem is indicated by the existence of authentic topics or issues that develop in society. The topic is relevant if students’ decisions influence their present and future lives. The science learning curriculum is reflected to point them in the direction of the impact. Moreover, SSI evaluation makes solving problems possible from various perspectives. An open discussion in specific forums helps students to understand social problems that develop, except for religious or ethnic issues.\n\nFurther, analysing science and technology as a tool for teaching, raises an informal logical question, which relates to scientific fact. It is explained either explicitly or implicitly as an argumentation subject.52,53 Therefore, the environmental-based science teaching process is more valuable than pure experimental activities.38,54 To design teaching based on socio-cultural problems, teachers need scientific knowledge and consideration of the social aspect. In addition, teachers also need to realise that in terms of teaching, there are uncertainties in the class. They must realise that teaching is not the only way to assert authority.55\n\nTeaching based on ethnoscience improves students’ skills, which are required to process science, appreciate, and protect the environment. The student also learns to use their scientific knowledge.40 Through this method, every student will understand the concepts of science in cultural context, since they learn directly from the environment.56 Therefore, there is a student’s curiosity and concern for the customs and culture that are learned to shape students’ environmental care profile, using strategic environmental studies, facilitated by modifying the ethnoscience approach to explore local potential, customs, and culture, which is crucial. Meanwhile, SSI is used to help them gain the knowledge to identify current issues in society and provide a positive response, protect the environment from danger, and understand and appreciate the local wisdom.\n\nEA is shown based on awareness, protection, and preservation, especially unfulfilled local potential.4,5,21 This study contributes to a critical analysis of EA profiles through LISREL Second-order CFA by revealing latent variables or indicators (Figure 1 and Tables 2-3) that affect other indicators. The factor analysis indicates that the students’ environmental care attitude is not only limited to caring, appreciating, and having good environmental ethics, but also includes making a real contribution and finding solutions to environmental problems that are integrated into the learning process.\n\nThere are rapid changes in the social behaviour, and the roles of humans are increasingly being replaced by sophisticated digitalization systems.45,54,55 Students are becoming increasingly indifferent about preservation of their surrounding environment.8,9 To avoid this, incorporating students’ direct experience acts as a useful learning resource, compared with learning where students only experience abstract concepts. Therefore, concerns about advances in technological literacy and culture, leading to an abandonment of environment in society can be resolved. Environmental literacy and competence are actively dedicated towards solving problems in human–environment interactions in an ecological and humanist way. The higher purpose is to balance the quality of life and the quality of the environment, with non-formal training in improving environmental literacy.58\n\nFilho59 explains that the perspective of life is the basis for moral formation and has a very complicated relationship with EA, environmental knowledge, and human behaviour. Simultaneously, our study holistically analyses the profile of students’ EA by considering policies, plans, and environmentally sustainable programs. Furthermore, according to Murniawaty5 and Widodo,60 knowledge and commitment are needed to realise environmental protection and awareness, but they have not revealed how practical solutions can be used to implement them.\n\nBased on the results of our analysis, the limitation of the study lies in the student bias data, namely the results are the same between the answers to ‘positive’ statements and ‘negative’ statements. It is assumed that there are irresponsible student answers. The results of this data cause several indicators and sub indicators to be not fulfilled (Figures 4 and 5). Furthermore, individually interviewing every student was not possible due to limited time and research costs. The study is significant in the sense that it illustrates how an increase in students’ EA profile can be determined through the interpretation of the LISREL 8.8 second-order CFA analysis, influenced by the relationship between EA indicators and sub-indicators with standardised SEA. Research recommendations in the form of measuring the EA profile of students using SEA are appropriate for considering the implementation of environmental education, integrated with ethnoscience and issues that develop in society. These are attained while realising the character of students who care for and respect the environment.\n\n\nConclusions\n\nThe profile of environmental awareness (EA) of high school students is in the category with a Care indicator (EA 1a). ‘Care towards environmental damage’, Critical (EA 3b), ‘Contributing towards preserving the environment’, and Local wisdom (EA 6b) to ‘Aware of local potentials’ are not fulfilled. Their lack of EA and understanding of potential fails to contribute to the environment. Their responses to environmental damage are good. It is highly necessary to implement SEA, which is modified with the ethnoscience approach and environmental issues strategy to build the character of students so that they develop an awareness of their environment. Further research should be conducted to study its contributions towards EA students who not only appreciate regional potential and ethics in ethnoscience studies but can also find real solutions to environmental problems with the socioscientific issues strategy. Furthermore, the synergy between teachers, students, and policymakers in the implementation of environment-based education is crucial for realising character through student EA.\n\n\nData availability\n\nFigshare: Environmental Awareness Questionnaire Score through SEA Assessment. https://doi.org/10.6084/m9.figshare.13977707.v1.34\n\nThis project contains the raw data of student’s response.\n\nFigshare: Environmental Awareness Result. Environmental Awareness Result. https://doi.org/10.6084/m9.figshare.14175563.v1.35\n\nThis project contains data that has analysed the scores obtained by each EA indicator, the actual score and the percentage.\n\nFigshare: Questionnaire for High School Student Environmental Awareness Profiles, https://doi.org/10.6084/m9.figshare.14254106.61\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 research team would like to thank Senior High School State 1 Selomerto, Wonosobo, Central Java Province, Indonesia for the ethical approval of the research. Furthermore, we wish to thank the academic community of Universitas Sebelas Maret, Surakarta, Indonesia, who have contributed ideas for the success of this research.\n\n\nReferences\n\nFischer TB, Jha-Thakur U, Hayes S: Environmental impact assessment and strategic environmental assessment research in the UK. 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Publisher Full Text" }	[ { "id": "83578", "date": "10 May 2021", "name": "Sarwi Sarwi", "expertise": [ "Reviewer Expertise The exact sciences", "environmental sciences", "innovative science learning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract, please add: Purpose is expressed in an operational sentence to reveal the topic referred to the title of the manuscript. Operational disclosure is required because it affects the adequacy of the required data.\nResearch discussing the importance of environmental issues associated with high school student attitudes is an interesting topic in education. The writing uses relevant and > 90% up-to-date article citations, according to the topics discussed. Please add: The theoretical framework that supports research is built so that the direction of the research is clear so that problems can be resolved.\nThe research design is appropriate, but qualitative data are still needed regarding the responses and results of in-depth interviews from respondents to answer research problems. The results of the research have good academic achievement, especially for high school and equivalent education levels.\nStudents from middle school that live between urban and rural areas were selected for this study (10th paragraph). Writing of that sentence in the text by the author is unclear, and even tends to have opposite meanings.\nMethods: The number of public high schools in Wonosobo Regency which are located in downtown or urban areas is at least 4 schools. Based on this data, it is necessary to refine or reconsider the subject or source of research data.\nStatistical analysis and interpretation can be applied in other institutions provided they have the same characteristics. However, it is still necessary to discuss aspects of attitude EA with low scores, why?\nAll data sources are available and can be used to ensure full reproducibility. There is a review that the data on the figure illustration (Figure 2 and Figure 3) is too large so that it is needs revision.\nThe conclusions drawn are supported by the results of data processing. Data processing requirements are adjusted to written objectives in an operational and measurable manner.\nReferences are used both as a guide for the path of ideas (state of the arts) and discussion. Reference is up to date and applicable to the problem.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6678", "date": "21 Jul 2021", "name": "Ahmad Khoiri", "role": "Author Response", "response": "Q.1: Abstract, please add: Purpose is expressed in an operational sentence to reveal the topic referred to the title of the manuscript. Operational disclosure is required because it affects the adequacy of the required data. A.1: I have added, this study aimed to analyses students’ EA profile of which consisted of six indicators, namely Care (EA 1), Curiosity (EA 2), Critical (EA 3), Dependability (EA 4), Responsibility (EA 5), and Local Wisdom (EA 6). Q.2: Research discussing the importance of environmental issues associated with high school student attitudes is an interesting topic in education. The writing uses relevant and > 90% up-to-date article citations, according to the topics discussed. Please add: The theoretical framework that supports research is built so that the direction of the research is clear so that problems can be resolved. A.2: The theoretical framework has been described in the 4th, 5th, and 9th paragraphs. Q.3: The research design is appropriate, but qualitative data are still needed regarding the responses and results of in-depth interviews from respondents to answer research problems. The results of the research have good academic achievement, especially for high school and equivalent education levels. A.3: Qualitative data has been obtained based on respondents' answers through the collected survey questionnaires had 131 complete responses out of a total of 143 because 12 responses were incomplete and had to be deleted. Twelve samples had missing data because they do not completely answer all the questions, and the remaining 54 samples were ignored because the sample size was reached. A questionnaire and a SEA instrument were used for data collection, consisting of 42 questions about environmental issues faced by the community. Q.4: Students from middle school that live between urban and rural areas were selected for this study (10th paragraph). Writing of that sentence in the text by the author is unclear, and even tends to have opposite meanings. A.4: that is, the research location is in the middle school (between urban and rural areas). Purposive sampling determination for the family background of students who lived in the central area of the city who all had similar attributes in terms of cultural and environmental recognition. Q.5: Methods: The number of public high schools in the Wonosobo Regency which are located in downtown or urban areas is at least 4 schools. Based on this data, it is necessary to refine or reconsider the subject or source of research data. A.5: Purposive sampling determination for the family background of students who lived in the central area of the city who all had similar attributes in terms of cultural and environmental recognition. Q.6: Statistical analysis and interpretation can be applied in other institutions provided they have the same characteristics. However, it is still necessary to discuss aspects of attitude EA with low scores, why?. A.6: EA indicators that score low and do not meet the good fit criteria are presented in Table 2 are (1) Care sub-indicator (EA 1a, 0.46 <1.96); (2) The Critical sub-indicator (EA 3b, 0.64<1.96); (3) The Local Wisdom sub-indicator (EA 6b, 0.03<1.96). Lisrel recommends creating error covariance between indicators and sub-indicators. If this procedure is performed, our new model will experience a decrease in the chi-square value, which of course makes the model better. Each item has a loading factor in measuring its latency factor. Q.7: All data sources are available and can be used to ensure full reproducibility. There is a review that the data on the figure illustration (Figure 2 and Figure 3) is too large so that it needs revision. A.7: The figure illustration has used guidelines. Q.8: The conclusions drawn are supported by the results of data processing. Data processing requirements are adjusted to written objectives in an operational and measurable manner. A.8: I have added the LISREL analysis result in score to the \"Care\" indicator (EA 1a, 0.46<1.96), Critical (EA 3b, 0.64<1.96), and Local wisdom (EA 6b, 0.03<1.96) are not fulfilled the significance criteria. Q.9: References are used both as a guide for the path of ideas (state of the arts) and discussion. Reference is up to date and applicable to the problem. A.9: Thank you" } ] }, { "id": "87791", "date": "01 Jul 2021", "name": "Latifah Abd Manaf", "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\nSome changes:\nConflict statement in the abstract (part results and conclusion) regarding students’ awareness.\n\nThe last paragraph in the introduction section is a method part, this can be removed.\n\nDiscussion about the students as the respondents should be in the method part. Rearrange.\n\nAdd info on the sample distribution in the table and add more discussion on the sampling method for the respondent selection.\n\nAdd more discussion on the instrument validation and pilot study.\n\nQuestions for each indicator were only 3? Is it valid to evaluate the SEA?\n\nInformation on the demographic background of respondents must be in the Table and discuss.\n\nIt’s better to discuss the results directly by supporting them with references, instead of putting them under a separate section.\n\nFigure 4 and 5 is a result part.\n\nThe conclusion can be improved.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6900", "date": "21 Jul 2021", "name": "Ahmad Khoiri", "role": "Author Response", "response": "Q.1: Conflict statement in the abstract (part results and conclusion) regarding students’ awareness. A.1: The EA profile of high school students was categorised sufficiently. Conclusions: Students’ lack of awareness of the environment and understanding of their regional potential fails to contribute towards creating a sustainable environment. Q.2: The last paragraph in the introduction section is a method part, this can be removed. A.2: The last paragraph has been moved to the methods section. Q.3: Discussion about the students as the respondents should be in the method part. Rearrange. A.3: Respondents have been described in the Study design and participants section. Q.4: Add info on the sample distribution in the table and add more discussion on the sampling method for the respondent selection. A.4: Table 1 has been added regarding the research sampling distribution. Q.5: Add more discussion on the instrument validation and pilot study. A.5: Added, The results of the revised SEA instrument validation are as follows. The instrument has been tested to find out content validation and construct validation on students' environmental awareness questionnaires and the results meet the valid criteria. Each question has presented social issues contextually to answer each question so that research focuses on environmental studies. The questionnaire grid has represented each indicator of the measured variable. Q.6: Questions for each indicator were only 3? Is it valid to evaluate the SEA? A.6: A valid criterion instrument that has been tested based on expert judgment and instrument testing. Q.7: Information on the demographic background of respondents must be in the Table and discuss. A.7: Added to table 1 about demographics of respondents based on gender were 44% male and 56% female. Age range 15-19 years with details of age 15 years, 26%; 16 years, 44%; 17 years, 24%; 18 years, 4%; and 19 years, 2%. Q.8: It’s better to discuss the results directly by supporting them with references, instead of putting them under a separate section. A.8: the article has been adapted to the journal guidelines for separate results and discussion. Q.9: Figures 4 and 5 is the result part. A.9: Figures 4 and 5 have been presented in the results section. Q.10: The conclusion can be improved. A.10: The conclusion has answered the research objectives and provided recommendations, added the criteria for the category of student environmental awareness are sufficient category." } ] } ]	1	https://f1000research.com/articles/10-305
https://f1000research.com/articles/9-1140/v1	16 Sep 20	{ "type": "Opinion Article", "title": "A multidimensional quality model: an opportunity for patients, their kin, healthcare providers and professionals in the new COVID-19 period", "authors": [ "Peter Lachman", "Paul Batalden", "Kris Vanhaecht", "Paul Batalden", "Kris Vanhaecht" ], "abstract": "Background: It is twenty years since the Institute of Medicine (IOM) defined quality in healthcare, as comprising six domains: person-centredness, timeliness, efficiency, effectiveness, safety and equity. Since then, a new quality movement has emerged, with the development of numerous interventions aimed at improving quality, with a focus on accessibility, safety and effectiveness of care. Further gains in equity and timeliness have proven even more challenging. The challenge: With the emergence of “service-oriented” systems, complexity science, the challenges of climate change, the growth of social media and the internet and the new reality of COVID-19, the original domains proposed by the IOM invite reflection on their relevance and possibility for improvement. The possible solution: In this paper we propose a revised model of quality that is built on never-ending learning and includes new domains, such as Ecology and Transparency, which reflect the changing worldview of healthcare. We also introduce the concept of person- or “kin-centred care” to emphasise the shared humanity of people involved in the interdependent work. This is a more expansive view of what “person-centredness” began. The delivery of health and healthcare requires people working in differing roles, with explicit attention to the lived realities of the people in the roles of professional and patient. The new model will provide a construct that may make the attainment of equity in healthcare more possible with a focus on kindness for all.", "keywords": [ "quality", "safety", "kin centered", "covid19", "person centered care" ], "content": "The rationale for change\n\nOver the past twenty years, since the defining of quality in healthcare by the Institute of Medicine (IOM)1, an industry has developed in the field of quality improvement and patient safety. This has included the academic study of the theory and methodology and the actual implementation of the studied theory. The result has been some improvement, but not to the extent that would allow a claim of success2,3. It has been said that there is insufficient evidence for the impact of quality improvement and more research is required4. In this paper, we take the opportunity to revise our basic framework and to redefine quality with the advantage of the experience gained over the past 20 years. One may ask why we need to redefine what is meant by quality in healthcare.\n\nThe actual work of healthcare service today struggles to meet the needs of people for better health. It has been designed to address failures in disease management, rather than in working with people to maintain health. It seems easier to focus on “standard work” and the “actions” in disease management, rather than on a more integrated view of the “relationships” that are required to maintain health. Furthermore, more advances in health have come from preventive measures in public health, such as immunisation, clean water, sanitation and housing5,6. In addition, the methods of assessing the impact of quality improvement have not lent themselves well to the standard way of assessing interventions in healthcare, nor have they bridged the gap between disease management and wellness or disease prevention7.\n\nCurrent healthcare service improvement has adopted many theories, methodologies and interventions from other industries, which have demonstrated important gains in quality, cost and safety. During the last century, one can discern two approaches on the creation, assessment, and improvement of the quality of healthcare delivery (see Table 1). Each approach has made important contributions to our abilities to make a better healthcare service and each has worked around a relatively common question. For convenience, we have named the first approach, Quality 1.0, “Q 1.0”. This began in the second decade of the 20th century in the USA, when the American College of Surgeons began their program of hospital standards. Three decades later, other national organizations of hospitals and professionals joined to form the “Joint Commission” for the Accreditation of Hospitals8,9. With the passage of the Medicare payment program, these certification efforts were linked to qualification for receipt of payment for hospitalisation.\n\nWith the advent of post-World War II improvement in systems thinking and system improvement methods, system- or enterprise-wide efforts to address quality emerged in many economic sectors. Initially, these improvement initiatives occurred outside of healthcare service, but increasingly from the mid-1980’s, improvement interventions spread to healthcare services. This new approach is termed Quality 2.0, “Q 2.0”. In this process the ideas of quality were defined by Donabedian as being system- and process-driven to produce the desired outcomes10. The early interventions to make quality a system or enterprise-wide endeavour were promoted with the introduction of the theories and methods of W. Edwards Deming, Joseph M. Juran and others11–14.\n\nThe IOM provided an important stimulus for the current focus on quality in healthcare with its reviews of the safety and quality of health care services1,15. The IOM defined six domains of quality, which have become the standard within the growing development of the science of improvement in healthcare: safe, efficient, effective, timely, equitable and patient-centred1. The theories and methodologies that had been successful in other economic sectors have been thought to be appropriate to the challenges of quality in health care delivery16–18. We have learned much, as a new language of systems, processes and outcomes has been added to the study and practice of clinical excellence, previously thought to be “quality in healthcare.” Attention shifted from a minimum “threshold” of quality to the concept of a “ceiling” of quality—not, “are you good enough to qualify?” but “how good can quality become?” Examples of success have been decreases in some infection rates, perceived increased access to healthcare, changes in person-centred care and improvements in aspects of safety19–23. System-wide improvement has been demonstrated at some institutions24.\n\nYet, for all these achievements, the persistence and the universal nature of the problem was highlighted in three key publications in 2018, which demonstrated that more than eight million people die from poor quality care in low and middle income countries25–27. In high income countries, at least 1 out of 10 patients is adversely affected during treatment, often resulting from persistent unwarranted variations in healthcare delivery, where a considerable proportion of patients did not receive appropriate, evidence-based care28.\n\nWe believe that the development of technical solutions helped connect improvement efforts to the earlier focus on “professional work.” These efforts allowed many gains. For example, specific safety initiatives have decreased pressure ulcers, falls in hospitals and hospital-acquired infections29–32. However, today we can also recognise the diminishment of attention to some very basic issues. For example, what does “quality” really mean to the person whose health it is? In our efforts to clarify desired professional roles, we may have inadvertently created a “product-dominant logic”: professionals making a quality healthcare service and then trying to “sell” it to patients. We think it is time to step back and reconsider what healthcare service is. How is it made and what does quality really means to the person whose health it is?\n\nThe approach has changed more recently, and the focusing question seems to have become something of the following nature: “How might we improve the value of the contribution that healthcare service makes to health?”33. This invites attention to who actually owns a person’s health: the healthcare provider or the individual receiving healthcare? In addition, we postulate that the concept of kinship extends to include both the care giver and the care provider, as they work together to make and improve services in support of an individual’s or a community’s health. The work of design, execution, assessment and improvement involves the integration of multiple systems of knowledge and skill.\n\nCo-productive work invites new models of value creation and attention to the basic architecture of those systems. Because these are different to those in the approach of “Q 2.0,” we have named this approach Quality 3.0, “Q 3.0”. Each of these approaches to quality offer important insights into the complex work involved in healthcare service. We think of each approach as adding to our capability to make better health, rather than “substituting” or “replacing” for the earlier approaches. The approaches are summarised in Table 1.\n\nIn this paper, we propose a new construct for defining quality of healthcare, where the aim is to meet the needs of the patient as a person, rather than meeting the needs of the healthcare system, which is as complex industry selling a product of disease management34. In proposing a new framework, it is tempting to dismiss earlier concepts. While we utilise the same dimensions, they have been reoriented with new ones added to invite a “service-dominant” logic. The new dimensions of quality will become even more relevant for the way we will facilitate health and make healthcare services in the future.\n\n\nWhy now?\n\nMany forces are at work today that seem to invite these changes. Information access has become more open, with the growth of the internet and social media, so it is much easier for any person to explore what is known about a problem or condition. “Making” and the maker-society invite a sense of personal agency more than traditional deference to “professional experts.” Healthcare professionals have been working to shed paternalistic legacies, creating a new construct, which we have named the commons, whereby all are working together towards the common good of health rather than simply managing disease. This is evidenced in some of the interventions to address the challenge of COVID-19. Historic conventions about payment and finance have given way to significant organizational financial stresses in all societies. The challenge of explicitly recognising the contributions of patients and families, in addition to those of professionals, while maintaining a person-centred focus during and after the pandemic for people who are affected and for those who are not, has invited a new model of quality for the future.\n\nConcurrent with the pandemic, the issue of the structural inequalities in society have become more prominent. A new model is required to address the way we, as healthcare providers, address issues in society that impact the health of the people. These include structural racism35 and the social determinants of health36, including food insecurity37, gender inequality38 and inherent violence39,40 within many societies. COVID-19 has unmasked these, and we think the new model is a response to the past failures of society to address these issues. Some may say that this is politicisation of health. Rather we see it as making the quality model socially relevant to our times and to the people who are most marginalised.\n\nOne of the early developers of modern Health Services Research, Kerr White, noted that the public’s health was not well served by the schism that developed during the last century between “medicine” (personal health) and “public health”41. He suggested that this separation was not serving the public’s health well and that the study of epidemiology might help. Today, the challenge of the COVID-19 pandemic has given us another clear view of the ways that this separation has had real consequences in unnecessary death and continues to serve us poorly. We believe that an appreciation of the common humanity—kin—amongst the people who act in the personal and in the public sectors, in addition to the study and contribution of epidemiology, can help. This focus on the relationships helps energise a bridge across the divide of the two sectors. By an explicit focus on the concept of kin, we can see a person as an individual and as a member of a population. This shared position of people helps us appreciate that kin-shipness or “kindness” can serve as a core value. It has helped us recognise the importance of kin, our fellow human beings, in our daily lives and that the absence of attention to these relationships—kin—, is a painful limitation to how we pursue health, not only in COVID-19, but also in numerous other ways, including in the end of life, for example. By kin we refer to the wider social construct around the people involved in receiving and providing care. Moreover, there is a need to develop a new way of thinking as one faces the challenges of measuring wellness, equity and good health42. The COVID-19 pandemic has exposed the failure of linear thinking to produce results when responding to a crisis. This has demonstrated that we need to see quality as part of a complex adaptive system with many competing linkages. Healthcare has many components, both within the formal structures of health service delivery and more importantly within the community and in other sectors. To produce health, these components need to interact in a way that benefits the people receiving care43,44.\n\nIn short, we can now see clearly that not only is it very difficult to outsource one’s health to someone else—the truth is that we have no real option but to work in new ways to coproduce a healthcare service that is capable of a greater contribution to better health. We believe that the impact of COVID-19 opens an opportunity not to return to the “old normal” or develop a “new normal” based on the old, but rather to conceptually redefine what we mean by quality in healthcare, how we define each other’s roles and how we define person-centred care for individuals and communities.\n\n\nAssumptions underlying a new quality movement\n\nUnderlying our thinking has been a recognition of the benefits of understanding systems as complex adaptive phenomena, of recognising that at some level all healthcare service is coproduced by persons we sometimes call professionals and persons we sometimes call patients. They are “kin” to each other in this interdependent work45.\n\nThe failure to link up the different parts of care during the pandemic, e.g. social care with healthcare, has exposed an underlying problem with the design of care. This has meant that many vulnerable people were placed at risk and potentially endured more harm. Healthcare quality and safety requires the interaction of these complex parts, continually adapting to the changing demands, each with its own complexity and each of which having to integrate at a specific time to deliver safe, good quality care. For example, the initial approach to patient safety (called Safety 1) focused on addressing adverse events and undertook linear assessments of unsafe events. These cause and effect assessments were often too simplistic to consider the complexity of causal systems at work. The progression has been to an understanding of complexity and resilience in quality and safety, with the building of resilience and constant learning, as we adapt to changing circumstances (called Safety 2). A different approach to quality is required as well46,47.\n\nThe quality and safety movement has been reactive to what has not been working and we believe that we now need to move to the concept of health and its coproduction. The concept of coproduction of quality in service systems is in its early phase of development48–50. There is a need to include people as partners and to move away from the correction of defects in disease management towards the creation of health. People, i.e. both the professionals and the patients interdependently involved, are not the problem, they are the key to a future quality model. While there has been a growing body of evidenced-based interventions, the problem has been one of implementation, spread and sustainability of interventions that have a firm evidence base51. We believe that organised efforts of quality improvement and safety, be it the practice or academic research of the practice has become too technical and people cannot relate to the challenge of actually fostering better health. We need a paradigm that works in today’s real world. One that facilitates better health for individuals and communities, so that the goal of better health will be achieved. In an era where shared creation of services is key, human resources in healthcare will become one of the major challenges. Quality should include care for both persons as patients and as professionals.\n\n\nThe model\n\nThe six domains of quality in the IOM model no longer fit the requirements of a person-centred approach to the facilitation of health and the delivery of universal healthcare. We suggest a focus on the co-creation of better health — a quality system for the people who are working together to co-produce services that contribute to better health (Figure 1).\n\nThe original model had person-centred care as one of the domains. We wish to further develop this by recognising the shared humanity of the people involved. The word “kin” is introduced to embody the social relationships and lived realities that surround the individuals involved, both those providing care and those receiving care. Healthcare service is not only about the person as patient or professional, but also about their family and wider social relationships. The dimension person/kin-centred surrounds every domain and is part of all that we do. The need for this approach has been demonstrated to be an essential component of the response to the pandemic. John Ballatt and colleagues suggest that “kindness [kinshipness] is ...not a ‘nice’ side issue, it is the glue of cooperation required for progress to be the most beneficial to the most people”45.\n\nWe place the person at the core of quality, rather than being a separate domain. At the core are the values of healthcare, based on kindness with compassion; partnership and coproduction; dignity and respect for people and each other; where people are seen from a holistic approach, in their totality and not as a disease or an organ of the body. The central tenet is kindness, so the dimension of person-centred care is kin-centred as well, involving all those who are related to the person receiving and the person providing care. This approach will facilitate the coproduction of quality and safety and achievement of the other domains. This emphasis invites and expands change from “installing” technical solutions to working with people and technical solutions. Telehealth efforts make it clear that more use of digital connectivity can work and possibly become part of the extended connectivity of kin52,53. The other domains remain in place. They are transfused with person-centred care. This new way of thinking also applies to the other person involved in making the service called “healthcare.” This means that among colleagues, and certainly with regards to relationships with hierarchical supervisors, there needs to be an understanding built on kindness, dignity, respect and partnership – and it includes the holistic person.\n\nA new domain, eco-friendly, is added to reflect the growing challenges of climate change and to introduce the need to address the challenges of sustainability, not only on organisation level, but in every contact in the micro-system54,55. We believe that being eco-friendly with a concern for climate change is central to the concept of kinship. The principle of transparency is included to surround all the technical domains, respecting the person’s right to privacy but also the right to know the data that specifically concerns themselves. Transparency is needed for providers, so that they can be open with themselves, as well as with the people to whom they deliver care. Transparency and resilience, i.e. the ability to operate with psychological safety, are the basis for the pursuit of truthful data collection, analysis and interpretation. Transparency with all our “kin” begins with professionals being transparent with each other56.\n\n\nImplication for current programmes\n\nWe believe that healthcare promotion and the delivery of healthcare must return to the core tenets of care—a form of “service”—and include the values that we have made central to the model in everything that we do. In the supplementary document we demonstrate the actions that are required to implement this new quality paradigm. Kin and person-centred care are infused in every effort to improve care, safety and effectiveness. The introduction of transparency will require a culture change in every sector of healthcare. Ecology is now a central domain, so all decisions and planning will require programmes to improve the impact on the climate and environment. Quality health services are based on what one human offers to another. These services are fundamentally a human activity, with attendant rights, responsibilities, and implications. To achieve this, we need to have high quality care for the professionals who deliver care and a redesign of systems, in order to facilitate true person and kin-centred care. In Table 2 the possible actions to be undertaken are suggested, these are not comprehensive and will be dynamic, changing in different contexts.\n\n\nConclusion\n\nOver the past few years, there has been a growing realisation that the current design of the system of healthcare has resulted in decreased wellbeing for the professionals involved in healthcare, with increasing reports of burn-out and “bore-out”57. The impact of safety events on clinicians has been documented and a meta-analysis of wellness and burn-out demonstrates the negative impact on care givers58,59. The review by the National Academies of Sciences concluded that the delivery of quality person-centred care will require a workforce whose wellbeing is paramount, which implies the dehumanisation of healthcare must be reversed60,61.\n\nThe recent focus on health inequalities and structural racism makes a change of focus more pressing with the concept of kinship reaching to the core of what it is to be a healer. This attention to relationship-as-fundamental is not new. In addition to the bridging energy for our use as we address the “schism”, we also recognise that numerous cultures across the globe have realised for centuries that this universal recognition of the importance of relationship is fundamental in all human life. Perhaps this is best known in the African philosophy of Ubuntu, where “I” am because “we” are. It is our contention that the new model of quality that we propose is the first step in this direction for policy makers, leaders and healthcare providers to explore and embrace this new way of thinking and to invite a return to a recognition of our shared humanity and the importance of kindness in healthcare for people and kin.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Acknowledgements\n\nThe graphic is based on one designed by Sinead McArdle at ISQua. Astrid Van Wilder proofread the paper.\n\n\nReferences\n\nInstitute of Medicine (US) Committee on Quality of Health Care in America: Crossing the Quality Chasm: A New Health System for the 21st Century. Washington (DC): National Academies Press (US): 2001. PubMed Abstract \| Publisher Full Text\n\nWells S, Tamir O, Gray J, et al.: Are quality improvement collaboratives effective? A systematic review. BMJ Qual Saf. 2018; 27(3): 226–240. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "71675", "date": "13 Oct 2020", "name": "Ross Baker", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article offers a substantial revision to the dominant model of healthcare quality and the measurement framework for that model, derived from the IOM Crossing the Quality Chasm report. The current model has been highly influential in the strategies and interventions developed to improve quality of care, yet frustratingly limited in practice. The authors acknowledge the ground-breaking nature of the current model (over the earlier framework that focused on standards and inspection.) They also point to a number of areas where that model has failed to yield substantive results because of poor implementation strategies that aim at technical changes rather than creating a more resilient and complex adaptive system. More profoundly, they also criticize the limitation of current themes reflected within the current model that aim at improving disease management rather than producing health, involve patients, but fail to create more effective co-production of services, and fail to create an environment that supports both providers as well as patients and caregivers.\n\nThe arguments are complex and thought-provoking, and ambitious in scope. Still, the reader is sometimes left uncertain about the requirements of the new quality framework; more details on several important constructs would aid in clarifying these ideas. In particular, the distinction between product dominant logic and service dominant logic is tied to the ideas of co-production and kinship but the linkages between these ideas is not fully developed for those unfamiliar with the sources, and examples might help to elucidate the ways in which these constructs are intertwined. The notion of “kinship of coproducing people” deepens the understanding of how providers and patients with their caregivers might more effectively identify appropriate care, but it is unclear what this might look like in practice, and how to develop closer connections and co-production in an environment where providers are stretched to the limit by growing needs.\n\nA critical issue for this new framework and the individual elements within is “how do we enable the work needed to achieve quality of care as defined in this way?” The details provided in Table 2 offer detail and definitions, but little guidance of what is needed to achieve quality in these domains. Full specification is likely beyond what could be addressed in the current article, but illustrative examples might provide guidance. Since the focus of the authors is aspirational, their conclusions are more speculative than can be documented from published literature. Nonetheless, their arguments provide a useful set of ideas and provocations to refocus current efforts to improve the quality of healthcare services.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6242", "date": "19 Jan 2021", "name": "Kris Vanhaecht", "role": "Author Response", "response": "Response to Ross Baker Thank you for the valuable review. We have identified three main issues to be addressed. Distinction between product logic and service logic We accept that the explanation of the distinction between product and service logic had not been as well developed as it could have been. We have added more detail to support this part of the argument . We have added a table which can provide can easier way to distinguish between n the two We have added as follows as well as provided a table to explain the point. Changes on page 6-7 With the Industrial Revolution came the development of the goods/product dominant logic for manufacturing. This logic separated the producer and the consumer with progressive specialization of the producer and the production of homogenous goods with progressively more efficient methods of production. This logic became a pervasive model for the operations of organized work and was transposed into the design of healthcare where the clinician held all the knowledge and skills and provide care to the person as a patient. Today the internet connects across “separated” functions and fosters networking that obliterates the earlier separation of producer and consumer. Service-dominant logic fosters integrated resources and interactivity and collaborative work of producers and consumers for mutual value-creating work.[33] For healthcare this implies that the distinction between clinician as the holder of knowledge no longer holds and the patient is now a person who can share in finding the solution. If one considers the study of the process of production of an outcome, the logic behind the making of products, or “goods” involves linked processes. Efforts to improve those processes often uses “standardization” of the processes and their linkages. The output of the processes is usually tangible. The logic behind the making of a “service” usually involves interactive steps of professionals and beneficiary users working in dyads or networks that are needed to solve a problem, on an individual or group basis.[34,35] Therefore the service will require interaction between all parties involved. t. Requirements of the new framework What the interventions may look like in practice especially where providers are stretched due to growing need These two points are related. A theoretical model needs to be tested and implemented. We believe the model brings together some of the current concerns regarding wellness of healthcare workers, of people who receive care and the concepts of kindness. As the model is new it has been tested in a limited way in Belgium where it has been adopted by the local policy makers as a framework. One could also look at different quality frameworks developed in other organization where elements of the framework are included in their definition of quality, e.g., in British Columbia. . https://bcpsqc.ca/what-is-quality/ We contend that the framework is being implemented in part and it brings together many of the current activities in to one construct. We have expanded on this in the text and have added more on leadership as any changes will require a leadership responsive to both people receiving care and those providing care. The model can be used to define and translate your own view on quality or integrate different visions and ideas into one overall framework. The multidimensional model has been piloted recently in different types of care organizations and it helped the involved clinicians and managers to define and specify the organization specific goals for the six technical domains, the umbrella domain of person and kin centred care and how to focus on the four core values. For example, to demonstrate application of the new eco-friendly domain one could preserve energy, water, resources, improve are using digital interventions and decrease carbon footprint. We invite clinical teams to use the model to examine how they can become person centred and then publish their experience so that we can coproduce the future." } ] }, { "id": "73483", "date": "09 Nov 2020", "name": "Gro Rosvold Berntsen", "expertise": [ "Reviewer Expertise Health service researcher with a focus on digitally supported", "person-centered", "integrated and proactive care." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\nThe paper is an opinion piece which analyses the development of the concept of “Quality of care” over time:\nQuality (Q) 1.0 – Accreditation and Improvement cycles\n\nQ 2.0 – IOM. The study and practice of clinical excellence\n\nQ 3.0 – New model in the current paper.\nAn acknowledgment of several shortcomings in the current IOM dominating quality of care model inspired this new quality of care model. The current IOM model consists of six goals: Effective, efficient, patient-centered, safe, timely, and equitable care. The authors claim that the following challenges make it necessary to review the IOM model:\nThe Quality Chasm is 20 years after the publication of IOM’s model for quality, not yet closed.\n\nThe current model of care is too focused on disease and disease management and less on health.\n\nThere is no focus on “relationships” and co-production of care in the IOM model, which is necessary for patients and professionals to “work” together towards a common good.\n\nThe focus on general prevention is lacking.\n\nWhat added value health care provides is not explicitly addressed.\nA new model for quality must attempt to correct these shortcomings. The new model builds on the IOM model but adds \"Eco-friendly\" as a 7th quality domain in addition to the original six IOM dimensions. It also provides a strong argument for Kinship as a core value in health care. The authors argue that Kinship is related to kindness – the sort of action you would confer upon someone of your kind. The authors underscore the importance of how both patient and professionals are persons and that although they have different roles, they have the same fundamental human needs for values that are core to the new model:\nKindness and compassion.\n\nDignity and respect\n\nHolistic\n\nPartnership- and co-production\nThe new model also has an outer \"shell\" consisting of:\n\nTransparency and resilience\n\nPerson/ Kin- Centered.\nAll the terms of the model are described more in detail in table 2, which shows how the authors expect each of the following “roles”: Patient-kin, Health provider, Organization, to work, or act in alignment with the goals that make up the model.\nStrengths:\nI agree with the authors that the current IOM-model of quality of care has not brought about the expected improvements. In practice, the IOM-domains of effective and efficient, and safe care have gotten all the attention and become the primary \"measures\" of care quality. The softer goals of person-centered, timely, and equitable care have also received some attention, but not nearly as much as the three former.\nThis paper adds some genuinely new ideas in the following areas:\n\nThe authors emphasize Kin-ship and how we are all first humans. It underscores even more than the many other papers on Person-centeredness in care that the foundation for success is a relationship built on trust between the person who needs care and the person who provides it. Without a robust trusting relationship, the common goal(s) of health can not be reached.\n\nAlso, I believe that the cross-table between roles and care goals is genuinely new. It bridges the gap from the desired outcome, e.g., safety (the row-headline), the most critical resources (column headlines), and the process (Cell value). The essential resources in every care process are those mentioned in the column headings: the patient, the provider, and the Organization.\n\nThe domain Eco-friendliness is not commonly highlighted in other publications on quality of care and adds a novel dimension to the complex concept of quality.\n\nThe strong emphasis on the well-being of the person who provides care is not entirely new 1. Still, this paper gives it a more decisive role than most other similar publications on quality of care, and I believe this to be an essential contribution.\nComments The authors present their quest as a novel approach: “Assumptions underlying a new quality movement.\" However, most of the features of Lachman's quality model, such as relationship-based care, patient-centeredness, and transparency, were highlighted in the original IOM \"Quality Chasm” report (See “Simple Rules for the 21st-Century Health Care System, p. 67”). Many other authors have also pointed out many of the same shortcomings of current care systems 2,3,4,5. The terms of kindness, compassion, dignity and respect, holism, partnership, and co-production, are desired outcomes highlighted before by different authors6,7,8,9,10. These authors share the concern, with Lachman et al., that the current care systems are not adopting the quality chasm report's recommendations well enough, nor fast enough. It would strengthen the paper to acknowledge how this paper builds and adds to the many previous publications that have pointed this out.\nThe many prior authors on this topic show that practice worldwide builds on a “too technical understanding of care quality” over a long period. The bigger question then is: Even though many express the need for qualities similar to those presented in this new model, such as person-centeredness, compassion, and empathy in current care, these authors concern does not seem to make an impact on care practices. Why are these compelling publications and analyses, many with strong arguments and support from empirical data, not making a difference? I would welcome the authors' reflection upon the following question: How will this model make a difference when other publications did not? What is it with this model that makes it stand out from previous models?\nThe paper has a paragraph concerned with complexity and how linear solutions may not provide the answers we need. The illustrated version of the quality model reflects the complexity in the arrows that link every component to other components. I share this view. Furthermore, I support the authors' recommendation of co-production, where a plurality of voices participates in the change implementation. However, change management in complex adaptive systems rely heavily on a shared vision and iterative trial-and-error implementation efforts, where measurement/ observations of \"the desired change\" are central 11. I want to challenge the authors on the consequences of making a complexity science approach to improvement: How should we identify the measurements or observations that will guide change?\nDonabedian's Structure => Process => Outcome model for understanding a care process's outcomes is still relevant. In their quality model, the authors have given us several domains that should be reflected in the care process. I would like to see the authors reflect upon the following question: If this is the process we would like to see, what changes, if any, do we need in care structures that would support such an approach?\nThe authors strongly argue the need to honor the relationship between patient and provider. However, in many care processes, the only stable person is the patient. Only those conditions that are managed by the general practitioner (GP) have continuity in terms of \"persons\" present in the relationship. Even in the GP office, there are temps and health secretaries and nurses, so that personal continuity might not be ensured even there. For patients with more complex issues, the care process consists of a string of care providers, who, more often than not, are not fully aware of what happened last with the last provider they saw. I invite the authors to reflect upon the \"patient-provider relationship\" in a situation where many different persons share the role of \"the provider.\"\n\n1.\n\nBodenheimer, T. and C.J.T.A.o.F.M. Sinsky, From triple to quadruple aim: care of the patient requires care of the provider. 2014. 12(6): p. 573-576.1 2.\n\nWHO. Framework on integrated, people-centred health services. Report by the Secretariat. 2016 08.08.2019]; 1-12]. Available from: Accessed at: http://apps.who.int/gb/ebwha/pdf_files/WHA69/A69_39-en.pdf?ua=12 3.\n\nWHO, The World Health Report 2000. Health Systems: Improving Performance. 2000.3 4.\n\nD’Avena, A., et al., Normalizing High-Value Care: Findings of the National Quality Task Force. NEJM Catalyst Innovations in Care Delivery, 2020.4 5.\n\nRich E, et al. Coordinating Care for Adults With Complex Care Needs in the Patient-Centered Medical Home: Challenges and Solutions. White Paper 2012 08.08.2019]; Available from: Archived at: http://www.webcitation.org/73DWky5eg.5 6.\n\nPerson-centered care made simple. 2014 08.08.2018]; Available from: Archived at: http://www.webcitation.org/73DX9aYtT6 7.\n\nMcWhinney, I.R., Family medicine in perspective. New England Journal of Medicine, 1975. 293(4): p. 176-181.7 8.\n\nJewson, N.D., The Disappearance of the Sick-Man from Medical Cosmology, 1770-1870. Sociology, 1976. 10(2): p. 225-244.8 9.\n\nMead, N. and P. Bower, Patient-centredness: a conceptual framework and review of the empirical literature. Social Science & Medicine, 2000. 51(7): p. 1087-110.9 10.\n\nCoulter, A., et al., Personalised care planning for adults with chronic or long-term health conditions. Cochrane Database of Systematic Reviews, 2015(3): p. 1-129.10 11.\n\nSnowden, D.J. and M.E. Boone, A leader's framework for decision making. harvard business review, 2007. 85(11): p. 1-9.11\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6243", "date": "19 Jan 2021", "name": "Kris Vanhaecht", "role": "Author Response", "response": "Response to Gro Berntsen Thank you for your valuable comments We have responded to each as follows. Features such as relationship-based care, patient-centeredness, and transparency, were highlighted in the original IOM \"Quality Chasm” report would strengthen the paper to acknowledge how this paper builds and adds to the many previous publications that have pointed this out We agree with this point . With reference to the IOM report while this has been so, the focus has been more on the product logic as we have noted and called Quality 2.0. We think that the new model addresses this issue and brings the person into the centre rather the process. Thank you for the references which we agree will ground the paper better within the literature. We have added as follows: In this paper, we propose a new construct for defining quality of healthcare, where the aim is to meet the needs of the patient as a person, rather than meeting the needs of the healthcare system, which is as complex industry selling a product of disease management.[38] The construct builds on an often overlooked emphasis in the original IOM concept, namely that person centredness is central to quality.[1] Some authors have focused on the need for compassion and person centredness to have a greater position in a quality framework and have noted the shortcomings of many initiatives.[39,40,41,42] In the person centred care literature the lack of kindness and respect has been raised as failing in our healthcare systems.[43,44,45,46,47,48] Despite the focus on the need for person centred care to be a central part of the quality system there has not been the traction required to make a difference. We believe that this is because person centred care is seen as a separate domain rather than one that is a precondition in every domain of quality. How will this model make a difference when other publications did not? What is it with this model that makes it stand out from previous models? This is an important question that we think is difficult to answer as only time will tell. However we think there are two difference from past models. Firstly, person centred care is expanded to kin centred so is an overtly broader; and secondly we do not see it as a separate domain of quality by rather in every domain. We contend that other models have focussed more on the technical – e.g. patient safety is a good example of this problem in the past. How should we identify the measurements or observations (of complexity) that will guide change? Table 3 provides many different possible interventions, each of which could be developed into a measure of the complexity and the changes required. This noted in the text If this is the process we would like to see, what changes, if any, do we need in care structures that would support such an approach? I invite the authors to reflect upon the \"patient-provider relationship\" in a situation where many different persons share the role of \"the provider.\" In response to the above two question, We agree there needs to be some consideration of the Donabedian model and the different relationships and have amended as follows: We believe that healthcare promotion and the delivery of healthcare must return to the core tenets of care—a form of “service”—and include the values that we have made central to the model in everything that we do. As one reflects on the Donabedian construct of “Structures and Process leading to Outcomes,” neither the structures nor processes we currently have designed are able to deliver a care model that could encompass the domains of quality nor kin centred approach. Healthcare will require a considerable redesign in which power is transferred to the person rather than remaining in the system. This would entail placing the people who receive care in positions of power in deciding how care should be delivered. And services be planned. As the complexity of care has redefined the way care is delivered with several providers often being involved in the delivery of care, the concept of integrating care around the person receiving care will be required with partnership and collaboration being core. We have reflected on the need for leadership and have added a reference to this point. We believe that this will be ongoing work in process. As we see the model being adopted so we will learn about the complexity of implementation. Nonetheless, the outcome will be looking at health from the view of the person and kin rather from the current model based on disease." }, { "c_id": "6286", "date": "22 Jan 2021", "name": "Gro Berntsen", "role": "Reviewer Response", "response": "Dear Authors, I think you have responded well to my comments. I especially liked the following response which i think is key to the potential impact of this paper: “Firstly, person centred care is expanded to kin centred so is an overtly broader; and secondly we do not see it as a separate domain of quality by rather in every domain. We contend that other models have focussed more on the technical – e.g. patient safety is a good example of this problem in the past.” I would expect to see this point outlined in the abstract, because this is the central change that you are advocating. Good luck." } ] } ]	1	https://f1000research.com/articles/9-1140
https://f1000research.com/articles/10-611/v1	20 Jul 21	{ "type": "Research Article", "title": "Towards recognizing the mechanisms of effects evoked in living organisms by static magnetic field. Numerically simulated effects of the static magnetic field upon simple inorganic molecules.", "authors": [ "Wojciech Ciesielski", "Tomasz Girek", "Zdzisław Oszczęda", "Jacek Soroka", "Piotr Tomasik", "Tomasz Girek", "Zdzisław Oszczęda", "Jacek Soroka", "Piotr Tomasik" ], "abstract": "Background: Recognizing effects of static magnetic field (SMF) of varying flux density on flora and fauna is attempted. For this purpose, the influence of static magnetic field upon molecules of water, nitrogen, ammonia, carbon dioxide, methane and molecular oxygen was studied. Methods: Computations of the effect of SMF of 0.1, 1, 10 and 100T flux density were performed in a computer vacuum involving advanced computational methods. Results: It was shown that SMF polarizes molecules depending on applied flux density but it neither ionizes nor breaks valence bonds. Three-molecular conglomerates of very dense packing form systems involving supramolecular orbitals. These orbitals deteriorate with an increase in the SMF flux density developing highly polarized structures. They are entirely different from these originally formed out of SMF. Conclusions: Small inorganic molecules commonly present in living organisms of flora and fauna can substantially influence functioning of those organisms when exposed to SMF.", "keywords": [ "ammonia", "carbon dioxide", "methane", "nitrogen", "organisms", "oxygen", "static magnetic field", "water" ], "content": "Introduction\n\nThere are a considerable number of monographs1–3 and papers4–12 characterizing the effects of all kinds of magnetic field upon flora and fauna. Currently, a focus is noted on the effect of magnetic resonance imaging on humans13–16. There are several reports with well documented biological effects of magnetic field. However, their interpretation and considerations on mechanisms of those effects on the molecular level are scarce16–20. Because of increasing environmental pollution with magnetic fields21,22 and considerable involvement of magnetic fields in current and future technologies23–25 this problem should attract particular attention.\n\nRecently, Jaworska et al., reported the stimulating effect of water treated with static magnetic field (SMF) of up to ~0.2T upon the growth and pathogenicity of entomopathogenic organisms both nematodes26 and fungi27,28. These results suggested a modification of the water macrostructure. Thus, one might assume that SMF can influence hydration of biological membranes and objects within the living cells. Turning the water macrostructure into smaller clusters29,30 could facilitate the penetration of water across membranes into the cells. Also structures of nitrogen, ammonia and carbon dioxide molecules present in living organisms could be modified by SMF and these processes might be involved in the observed effects. Białopiotrowicz et al.29 reported formation of singlet oxygen on a treatment of water saturated with air with a low-temperature, low-pressure glow plasma of low frequency. The singlet oxygen molecules were stabilized by their incorporation into aqueous clathrates of the nanometric dimensions.\n\nThese factors strongly suggested that water and small inorganic molecules could participate in the observed effects of magnetic field and control mechanisms and kinetics of phenomena induced by SMF.\n\nThis paper presents results of computational simulations of the structure of molecular oxygen, nitrogen, water, carbon dioxide, ammonia and methane in SMF of the flux density from 0 to 100T.\n\n\nMethods\n\nMolecular structures were drawn using the Fujitsu SCIGRESS 2.0 software31 (open-access alternative software: GaussView 6). Their principal symmetry axes were oriented along the x-axis of the Cartesian system. The magnetic field was fixed in the same direction with the south pole from the left side. Subsequently, the molecules were optimized involving Gaussian 0.9 software equipped with the 6-31G basis32 (open-access alternative software: Spartan 1.0.0). For those optimized single molecules bond lengths, dipole moments, heaths of formation and bond energies were computed. Additionally, computations of HOMO/LUMO energy level for single molecules as well as HOMO/LUMO energy level and total energy for systems built of three molecules were also performed.\n\nIn the consecutive step, influence of SMF upon optimized molecules were computed with Amsterdam Modeling Suite software33,34 (open-access alternative software: ORCA 5.0). and the NR_LDOTB (non-relastivically orbital momentum L-dot-B) method35,36. Following that step, using Gaussian 0.9 software equipped with the 6-31G basis32 values of bond length, dipole moment, heath of formation equal to the energy of dissociation, bond energy HOMO/LUMO energy level for single molecules were again computed using the single-point energy option key word.\n\nVisualisation of the HOMO/LUMO orbitals and changes of the electron density for particular molecules and their three molecule systems was performed involving the HyperChem 8.0 software37 (open-access alternative software: GaussView 6).\n\n\nResults and discussion\n\nProperties of the single molecule of oxygen (O2) situated along the x-axis of the Cartesian system in SMF of the flux density of 0 to 100T are presented in Table 1. Isosurfaces resulting from computations for molecules of the triplet and singlet molecules are presented in Figure 1 and Figure 2, respectively. Energy of electrons situated on particular orbitals in the single molecule of oxygen (O2) placed in SMF of the flux density of 0 to 100T are collected in Table 2. Table 3 and Table 4 collect data characterizing properties of the system of three molecules of oxygen and HOMO/LUMO energy of the molecules of oxygen building the three molecular system, respectively. Influence of the application of flux density and resulting distribution of corresponding maximum positive and negative charge density in case of three triplet and singlet oxygen molecules are presented in Figure 3 and Figure 4, respectively.\n\naUpper and lower (in italics) values are for the triplet and singlet states, respectively.\n\nIsosurface along the x-axis of the Cartesian system of the single molecule of triplet oxygen placed in the magnetic field of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nIsosurface along the x-axis of the Cartesian system of the single molecule of singlet oxygen placed in the magnetic field of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\naUpper and lower (in italics) values are for the triplet and singlet states, respectively.\n\naValues in normal font and italics are for the triplet and singlet states, respectively.\n\naUpper and lower (in italics) values are for the triplet and singlet states, respectively.\n\nIsosurface along the x-axis of the Cartesian system of three molecules of triplet oxygen placed in the flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nIsosurface along the x-axis of the Cartesian system of three molecules of singlet oxygen placed in the flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nMolecular oxygen in its ground state resided as triplet. Its excitation transformed it into singlet states38,39. They differed from one another in the heat of formation (Table 1). For the triplet molecule its value was slightly negative whereas for the singlet molecule it was considerably positive. Considerable difference between the values of heat formation of the triplet and singlet molecules reflected the fact that the singlet oxygen was a specific short leaving excited state of oxygen40. The bond energy for the triplet as well as singlet molecules slightly decreased with an increase in applied flux density. Corresponding values for the singlet molecules were lower than those for the triplet molecules. The O-O bond length in the triplet molecule was slightly shorter than in the singlet molecule. Therefore, the singlet molecule was more stretched, hence more unstable and reactive. In both cases the bond length fairly regularly increased with an increase in applied flux density. In both cases it resulted in developing residual electric dipole moment more readily in the triplet molecule. Comparison of the energy gaps ΔHOMO/LUMO revealed that SMF only slightly influenced the excitation energy of the triplet oxygen molecule but considerably decreased it in the singlet oxygen molecule.\n\nFigure 1 and Figure 2 present numerically simulated electronic density of so-called isosurfaces, for single molecules of the triplet and singlet oxygen, respectively, exposed to the magnetic field of 0.1 to 100 T along the x-axis. On these and subsequent figures green and pink colours represent positive and negative charge, respectively. The intensity of both colours associated with the value of the charges is scaled on the left side of every isosurface. An increase in applied SMF flux density resulted in polarization of the molecules. The original shape of isosurface of the triplet molecule only slightly changed but in case of the singlet molecule an essential change of the isosurface shape could be noted, particularly at 10 and 100 T.\n\nValues of the maximal negative and positive surface charge densities are grouped in Table 1. In the molecule of triplet oxygen, the maximum of the positive charge density rose regularly against T whereas, simultaneously, the maximum of the negative charge density rose irregularly. The same parameters found for the singlet oxygen molecule also varied irregularly with increase in T. Above 10T a complete shift of the surface charge was observed.\n\nEnergy of corresponding LUMO and HOMO of triplet and singlet molecules fairly regularly declined with increase in T (Table 1) but energy of electrons situated on particular orbitals of those molecules varied nonlinearly (Table 2). It might suggest that some flux density dependent hybridizations of corresponding orbitals were involved leading to resonance like structures.\n\nThe distribution of the charge presented in structures d and e might suggest formation of ozone.\n\nLike in case of single molecules placed in SMF of 100T also in the group of three singlet as well as triplet molecules the positive and negative charges were distributed symmetrically. Maximal positive charge density for the systems built of three triplet and singlet molecules changed irregularly (Table 3). An initial increase observed for the systems placed in SMF of 0.1 T declined reaching the lowest value for the system placed in SMF of 1 T. This irregularity was well illustrated with specific shapes of corresponding isosurfaces (Figure 3 and Figure 4). In contrast to the maximal positive charge density, maximal negative charge density declined with increase in applied flux density. The irregular pattern of those changes resembled these observed for the maximal positive charge density. Again, the irregularity was found in the data for the molecules placed in SMF of 1T.\n\nAn increase in the SMF flux density shortened and elongated distance between the excited (LUMO) and ground (HOMO) states in the system of three triplet and singlet molecules, respectively (Table 4).\n\nCorresponding results for single molecules of nitrogen are presented in Table 5 and Table 6 and Figure 5. Data for the system of three molecules of nitrogen are given in Table 7 and Table 8 and in Figure 6. Molecular nitrogen in the ground state is in the singlet state but under specific conditions it can be excited into triplet states41,42. SMF of the flux density increasing up to 100T only slightly changed properties of that molecule retaining its singlet character (Table 5). The interatomic distance slightly increased and the residual dipole moment generated already at 1T remained practically unchanged up to 100T. As the applied T increased, heat of formation of the molecule considerably increased and, simultaneously, bond energy decreased. The energy of LUMO initially slightly decreased in order to increase the flux density of 10 and 100T. It was paralleled with a regular decrease in the energy of HOMO. An insight into energy of electrons situated on particular orbitals in the single molecule of nitrogen (N2) (Table 6) revealed that, with some exceptions, they changed regularly against increasing values of T. The irregularities were noted in case of the π2p, σ2s and σ*1s orbitals at 0.1 and 1T. They might be associated with the SMF promoted hybridization of those orbitals. An increase in ΔMOMO/LUMO informs that an increase in SMF flux density resulted in inhibiting excitation of the molecule. It also increased and decreased maximum of the positive and negative charge density, respectively (Table 4), however, as shown in Figure 5 the size and shape of the isosurface along the x-axis remained fairly stable.\n\nIsosurface along the x-axis of the Cartesian system of the single molecule of nitrogen placed in the SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nIsosurface along the x-axis of the Cartesian system of three molecules of nitrogen placed in the SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nIn the system of three nitrogen molecules its total energy rose with applied flux density. Energy of LUMO and HOMO of the whole system increased and decreased, respectively (Table 7). Difference between energy of HOMO and LUMO was independent of applied SMF. Maximum of the positive and negative charge density distribution quoted in Table 7 reflected substantial changes of the shape and size of the isosurface presented in Figure 6.\n\nOn increase in applied T maximum of the positive charge on the surface increased by 0.220 units. Simultaneously, maximum of the negative charge density decreased by 0.221 units. Thus, the charge shifts proceeded solely on the surface. Because the sum of both values was close to zero the charge did not delocalize.\n\nDepending on the applied flux density, the system of three nitrogen molecules reoriented in the Cartesian system as shown in Figure 6. It was accompanied with a fluctuation of the value of the maximum negative charge density (Table 8).\n\nTable 9 and Table 10 and Figure 7 contain results of computations for a single molecule of water and Figure 8 shows its isosurface. Subsequently, data for the system of three molecules of water are given in Table 11 and Table 12. and the isosurface for that system is presented in Figure 9. In nature, above 0K, water molecules vibrate like other molecules. The O-H bond lengths change either symmetrically or asymmetrically. It is reflected by changes of their dipole moment. In consequence, their hydrophilic/hydrophobic properties are accordingly modified. Such vibrations also affect dissociation of the molecules influencing their ability to build the macrostructure with involvement of intermolecular hydrogen bonds. Additionally, bending modes change the H-O-H bond angle of these molecules43.\n\naSee Figure 7 for notation.\n\nIsosurface along the x-axis of the Cartesian system of the single molecule of water placed in the SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nIsosurface along the x-axis of the Cartesian system of three molecules of water placed in the SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nComputations for the water molecule placed under computer vacuum in SMF could potentially suggest a facile H-O-H bond break and the ability to form intermolecular hydrogen bonds. If lone electron pairs at the oxygen atom were activated an increase in basicity of the water molecule could be considered.\n\nAs shown in Table 9, SMF of the flux density of 0.1T symmetrically elongated both O-H bonds and that symmetry ceased at 1 to 100T. That treatment provided moderate increase in dipole moment of the molecule, monotonous decrease in the bond energy and stability of the molecule in terms of increasing its heat of formation. However, it should be noted that starting from 10T the length of the first of the O-H bonds decreased. It pointed to an involvement of decrease in the symmetry of vibrations, whereas vibrations of the second O-H bond remained nearly constant.\n\nAn increase in the flux density was paralleled with increase in the positive energy of LUMO and negative energy of HOMO. Monotonous increase in ΔHOMO/LUMO values showed that an increase in the SMF flux density reduced a possibility of excitation of the water molecule. Energy of electrons on particular orbitals in the single water molecule varied with the flux density in the manner presented in Table 10. Solely the positive energy of the empty 3a1 orbital monotonously increased against increasing flux density whereas the negative energy of electrons occupying remaining orbitals slightly monotonously declined. It pointed to inhibiting excitation of the water molecule.\n\nIsosurface along the x-axis of the Cartesian system for the single molecule of water (Figure 8) demonstrated that SMF only slightly changed orientation of the molecule with increase in the surface density but polarization of the molecule considerably increased.\n\nTotal energy of the system built of three molecules of water was fairly insensitive to an increase in applied flux density. Moreover, there was considerable response from the energy of LUMO and HOMO. The energy of LUMO monotonously increased and, simultaneously, energy of HOMO in the same manner decreased (Table 11) as demonstrated in terms of an increase in ΔHOMO/LUMO. Thus, SMF inhibited the excitation of the molecule of water.\n\nMaximum of the positive and negative charge density distribution quoted in Table 12 reflected substantial changes of the shape and size of the isosurface presented in Figure 9.\n\nOut of SMF, three water molecules arranged into an irregular ball. At 0.1T it polarized with simultaneous reorientation along the x-axis. Its shape was retained but as the flux density increased, it gradually expanded into a more linear shape in which the number of intermolecular hydrogen bonds declined. A considerable irregularity in the distribution of the maximum charge density at 10T should be noted. It was reflected by a specific shape of the corresponding isosurface (Figure 9).\n\nTable 13, Table 14 and Figure 10 and Figure 11 present relevant data for a single CO2 molecule. and Table 15, Table 16 and Figure 12 show computed data for the system of three CO2 molecules.\n\naSee Figure 10 for notation.\n\nIsosurface along the x-axis of the Cartesian system of the single molecule of CO2 placed in SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nIsosurface along the x-axis of the Cartesian system of three molecules of CO2 placed in SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nCarbon dioxide represents a π-electron system. Hence, because of the mobility of the π-electrons44,45 unexpected behaviour of the molecule placed in SMF might be anticipated. Thus, the length of both C=O bonds changed with increase in T but at 0.1 and 100T both bonds had slightly different length, perhaps mainly because of limited precision of calculations. Dipole moment of the molecule increased with T whereas the heat of formation and bond energy practically linearly decreased against T (Table 13). Energy of LUMO and HOMO also monotonously increased and decreased, respectively. The energetic ΔHOMO/LUMO gap reached maximum at about 1T.\n\nIrregularity was observed for changes of maximum of positive and negative charge density (Table 13). Maximum of positive charge density increased up to 1T in order to strongly decrease at 10 and 100T. Except electrons situated at 2b2u, level energy of electrons situated on particular orbitals of the single molecule at increasing SMF flux density only slightly changed. Energy of electrons located at the 2b2u jumped suddenly at 1T (Table 14).\n\nShape of isosurface of the SMF treated molecule (Figure 11) resulted in polarisation of the molecule in varying manner tending to localize the negative charge on the oxygen atoms. The effect was dependent on the applied flux density. The molecules did not reorient in the Cartesian system holding their initial orientation.\n\nIn the system composed of three CO2 molecules, total energy of the system increased with an increase in applied flux density. The LUMO energy reached maximum at 0.1T in order to decrease up to 100T. Energy of HOMO changed in the same way (Table 15).\n\nMaximum of the positive and negative charge density distribution (Table 16) reflected substantial changes of the shape and size of the isosurface presented in Figure 12. Three molecules of CO2 out of SMF formed a ball. In the SMF of 0.1T a tendency to separate that ball into two parts perpendicularly to the x-axis was observed. This tendency increased with increase in applied T in order to afford a separation at 100T.\n\nResults for a single molecule of ammonia are given in Table 17 and Table 18 and in Figure 13 and Figure 14. Data for the system of three molecules of ammonia can be found in Table 19 and Table 20 and in Figure 15.\n\naSee Figure 13 for notation.\n\nIsosurface along the x-axis of the Cartesian system of the single molecule of ammonia placed in SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nIsosurface along the x-axis of the Cartesian system of three molecules of ammonia placed in SMF of the flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nThe molecule is the shape of a pyramid with the orbital occupied by the lone electron pair and it is also capable of excitation46. When placed in SMF the bond lengths responded depending on the applied flux density. At 1 to 100T their lengths gradually compressed to an extent individual to each bond (Table 17). Nevertheless, the N-H bonds turned shorter, dipole moment slightly increased paralleling applied flux density. These changes were paralleled by increase in heat of formation and decrease in bond energy.\n\nAn increase in the SMF flux density resulted in an increase in the ΔHOMO/LUMO energy, Thus, SMF deactivates ammonia. Simultaneously, energy of LUMO and HOMO proportionally increased and decreased, respectively.\n\nMaximum of the positive charge density rapidly jumped after application of 0.1T and then rose regularly with increase in applied T (Table 17). This jump was accompanied by a similar jump of the maximum of the negative charge density. However, as the applied flux density increased, the value of the maximum charge density decreased. From the data for energy of electrons placed in SMF (Table 18), electrons situated on orbitals 1e could be involved. The molecule situated in the Cartesian system along the x-axis only slightly rotated under the influence of increasing flux density (Figure 14).\n\nIn the system of three ammonia molecules its total energy increased with increase in the SMF flux density whereas energy of LUMO monotonously decreased. Simultaneously, energy of HOMO monotonously increased up to 10T in order to slightly decrease at 100T (Table 19). A monotonously decreasing ΔHOMO/LUMO showed that an increase in the SMF flux density favoured excitation of the system. Maximum of the positive and negative charge density distribution quoted in Table 20 reflected substantial changes of the shape and size of the isosurface presented in Figure 15.\n\nThe change of the shape of isosurface of the system of three molecules of NH3 (Figure 15) suggested that in the system out of SMF the molecules interacted involving polar and van der Waals forces. Increasing flux density deteriorated these interactions leading to separated mutually non-interacting molecules.\n\nComputed data for a single molecule of methane are placed in Table 21 and Table 12 and in Figure 16 and Figure 17. whereas corresponding data for the system of three molecules of methane are given in Table 23 and Table 24 and in Figure 18.\n\nIsosurface along the x-axis of the Cartesian system of the single molecule of methane placed in SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\naSee Figure 16 for notation.\n\nIsosurface along the x-axis of the Cartesian system of three molecules of methane placed in SMF flux density of 0T (a), 0.1T (b), 1T (c), 10T (d) and 100T (e).\n\nMethane presents a symmetric tetrahedr with the carbon atom in its geometrical center and four hydrogen atoms in its corners. Some low-laying electronic excited states of the molecule were computed with the ab initio method47.\n\nLow polarization of the C-H bonds makes the whole molecule fairly resistant to SMF. Just the flux density of 10T slightly expanded the C-H bonds from 1.0932 to 1.0936 (two of four C-H bonds) and to 1.0938Ǻ (remaining two bonds). Increase in the flux density to 100T produced further, non-symmetric elongation of the C-H bonds (Table 21). It resulted in generation of a residual dipole moment of the molecule. However, an increase in the heat of formation could be noted already on exposure of the molecule to 1T. It was accompanied with a slight decrease in the bond energy. Changes of maximum positive and negative charge density appeared peculiar. They were accompanied neither by specific changes of the LUMO and HOMO energy (Table 21) nor energy of electrons situated on particular orbitals of that molecule (Table 22). A monotonous increase in ΔHOMO/LUMO with increasing SMF flux density pointed to reduced affinity of methane to activation. The 3a1 level corresponded to its first unoccupied orbital. Its energy expressed the energy required for the excitation of the methane molecule.\n\nPecularity mentioned above was rationalized in terms of the shape of isosurface of the molecule in particular values of flux density (Figure 17). At 1T, positive charge density concentrated almost completely on the carbon atom and its traces could be observed on two of four hydrogen atoms. The total positive charge density resided solely at one hydrogen atom. Increase in the applied T shifted the negative charge density completely to one of four hydrogen atoms. At 100T only one hydrogen atom carried the negative charge density and another one held the positive charge density.\n\nTotal energy of the system built of three methane molecules slightly decreased when placed in SMF and a more considerable decrease was noted just at 100T (Table 23). Energy of LUMO and HOMO gradually increased and decreased, respectively. Maximal positive and negative charge density changed irregularly with increase in applied T (Table 24). Value of the ΔHOMO/LUMO energy increasing with the SMF flux density pointed to reduced affinity of the system to the excitation.\n\nInvolving polar and van der Waals forces three molecules of methane formed various condensed structures. At 100T the system spread into three separate molecules (Figure 18).\n\n\nConclusions\n\nPerformed numerical simulations delivered strong evidence that small inorganic molecules commonly present in the living organisms of flora and fauna can substantially influence functioning of those organisms residing in SMF.\n\nStatic magnetic field polarizes molecules of oxygen, nitrogen, water, ammonia, carbon dioxide and methane depending on applied flux density. Static magnetic field of up to 100T causes neither ionization nor breaking valence bonds of molecules placed in that field. In modelled computer vacuum three-molecular conglomerates of very dense packing form systems involving supramolecular orbitals.\n\nStatic magnetic field deteriorates supramolecular orbitals of three-molecular conglomerates developing highly polarized structures.\n\nStatic magnetic field reduces affinity of the molecules and their conglomerates to the excitation.\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\nUeno S: Biological Effects of Magnetic and Electromagnetic Fields. New York: Plenum Press, 1993. Reference Source\n\nCarpenter DO, Ayrapetyan S: Biological Effects of Electric and Magnetic Fields. Beneficial and Harmful Effects. New York: Academic Press, 1994; 2. Publisher Full Text\n\nZhang X, Yarema K, Xu A: Biological Effects of Static Magnetic Fields. Singapore: Springer Nature, 2017. 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J Phys Conf Ser. 15th Int Congr Plasma Phys (ICPP2010) & 13th Latin American Workshop Plasma Phys, (LAWPP2010) 8–13 August 2010, Santiago, Chile. 2010; 511. Publisher Full Text\n\nGil GS: Excited states of ammonia and the Jahn-Teller effect ab initio and force field calculations. MSc Final Project. University of Bristol, UK. 2015. Reference Source\n\nMontagnani R, Riani P, Salvetti O: Ab initio calculation of some low-lying electronic excited states of methane. Theor Chim Acta. 1973; 32: 161–170. Publisher Full Text" }	[ { "id": "266811", "date": "09 May 2024", "name": "Hakki Gurhan", "expertise": [ "Reviewer Expertise Bioelectromagnetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript provides a detailed exploration of the effects of static magnetic fields (SMFs) on small inorganic molecules, and their potential impact on living organisms. The research utilizes numerical simulations to investigate how SMFs influence the behavior and properties of molecules such as methane, oxygen, nitrogen, water, and carbon dioxide. After reviewing the manuscript, I have identified some concerns and potential areas for improvement. Here are the concerns and suggestions for each:\nQuestions for the Authors: Can you provide more details on the computational models used for the numerical simulations, including any assumptions made and the level of accuracy achieved? How did you validate the computational results obtained from the simulations? Were any experimental validations conducted? The paper mentions changes in supramolecular orbitals under SMF flux densities. Could you discuss the significance of these changes in terms of molecular stability and reactivity? How do the observed alterations in molecular properties, such as charge density distribution and bond energies, correlate with the behavior of molecules in different environmental conditions or biological systems? Considering the potential influence of SMFs on living organisms, have you conducted any preliminary studies or experiments to assess the physiological effects of these changes? How might the findings of this study be applied to fields such as biomedicine, pharmacology, or environmental science? More references could be added for possible SMF effects [Ref-1,2]. What are the main limitations of the current study, and how might these limitations be addressed in future research? Are there any specific areas or aspects of the research that you believe warrant further investigation or exploration? While the paper is generally well-written, I have a few concerns where the language could be clarified for better understanding: Concern: The phrase \"Involving polar and van der Waals forces\" could be clarified for readers unfamiliar with these terms. Revision: \"The interaction of polar and van der Waals forces influenced the formation of various condensed structures by three methane molecules.\" Concern: The phrasing \"Static magnetic field deteriorates\" sounds awkward and might benefit from clarification. Revision: \"The static magnetic field affects the supramolecular orbitals of three-molecule conglomerates, leading to the development of highly polarized structures.\" Concern: The phrase \"Pecularity mentioned above\" is grammatically incorrect and unclear. Revision: \"The peculiarity mentioned earlier was rationalized in terms of the shape of the molecule's isosurface at specific flux density values.\" Concern: \"Energy of LUMO and HOMO gradually increased and decreased, respectively.\" The phrasing is a bit awkward and lacks clarity. Revision: \"The energy of the LUMO increased gradually, while that of the HOMO decreased, as the strength of the static magnetic field varied. Concern: \"At 1T, positive charge density concentrated almost completely on the carbon atom and its traces could be observed on two of four hydrogen atoms.\" The sentence structure is slightly awkward and could be rephrased for clarity. Revision: \"At 1T, the positive charge density was predominantly concentrated on the carbon atom, with traces observable on two out of four hydrogen atoms.\" Please update the descriptions in Table 17 and Table 23 by replacing 'SFM' with 'SMF'.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-611
https://f1000research.com/articles/10-609/v1	20 Jul 21	{ "type": "Research Article", "title": "Interpreting airborne pandemics spreading using fractal kinetics’ principles", "authors": [ "Panos Macheras", "Athanasios A. Tsekouras", "Pavlos Chryssafidis", "Athanasios A. Tsekouras", "Pavlos Chryssafidis" ], "abstract": "Introduction The reaction between susceptible and infected subjects has been studied under the well-mixed hypothesis for almost a century. Here, we present a consistent analysis for a not well-mixed system using fractal kinetics’ principles. Methods We analyzed COVID-19 data to get insights on the disease spreading in absence/presence of preventive measures. We derived a three-parameter model and show that the “fractal” exponent h of time larger than unity can capture the impact of preventive measures affecting population mobility. Results The h=1 case, which is a power of time model, accurately describes the situation without such measures in line with a herd immunity policy. The pandemic spread in four model countries (France, Greece, Italy and Spain) for the first 10 months has gone through four stages: stages 1 and 3 with limited to no measures, stages 2 and 4 with varying lockdown conditions. For each stage and country two or three model parameters have been determined using appropriate fitting procedures. The fractal kinetics model was found to be more akin to real life. Conclusion Model predictions and their implications lead to the conclusion that the fractal kinetics model can be used as a prototype for the analysis of all contagious airborne pandemics.", "keywords": [ "fractal kinetics", "COVID-19", "airborne pandemics", "herd immunity policy", "SI model" ], "content": "Introduction\n\nRecently, Jewell et al.1 criticized the predictive models of the COVID-19 pandemic. This rigorous analysis justifies the first portion of the famous quote by George Box2 “All models are wrong, some of them are useful”. All epidemiological models used in practice have a common origin, namely, the famous Kermack–McKendrick model.3 We argue in this work that their poor predictive power originates from the erroneous hypothesis of the “well-mixed” epidemiological system; this hypothesis is crucial for the validity of the differential equations, which describe the “reaction” between susceptible (S) and infected (I) subjects. We also argue that the violation of this hypothesis results in a wrong perception and definition of the basic reproductive number R04,5 of epidemiological models, which denotes the number of secondary infections produced by a single infection.\n\nPeople worldwide are concerned about the uncontrolled “exponential” spread of a disease, yet it is not clear or justified if this description is correct. In fact, an alternative “power” model based on an adjustable exponent of time has been proposed.6 We expand this approach by first questioning the ‘well-mixed” hypothesis and introducing a “fractal kinetics’” approach7 which yields, as a special case, the “power” model. This model7 relies on fractal kinetics’ principles that are suitable for the study of reactions and diffusion processes in insufficiently mixed media.8,9 In the same vein, we explored all theoretical aspects of the fractal kinetics’ SI model and applied it for the description of the time evolution of the COVID-19 pandemic in several countries. Our results support that this “conceptual change” from classical to fractal kinetics principles offers a novel, useful approach for the analysis of airborne pandemics data and justifies the second portion of George Box2 quote above.\n\n\nTheory\n\nThe “reaction” of susceptible-infected individuals under homogeneous conditions.\n\nIn the Kermack–McKendrick model,3 the studied population is divided into susceptible, S, infectious, I and recovered, R, sub-populations while the relevant terms SI and SIR model were coined a long time ago. For each one of the sub-populations, specific ordinary differential equations are written based on the principles of chemical kinetics. These equations rely on the law of mass action10 which states that the rate of the chemical reaction is directly proportional to the product of concentrations of the reactants. However, this law applies under the strict hypothesis that the studied chemical reaction takes place under well-stirred conditions. This dogma applies well in chemical systems and validates the use of time-independent reaction rate constants and molar concentrations of the reactants in the reaction rate expressions. Obviously, the well-mixed hypothesis cannot be applied to epidemiological models since individuals, unlike molecules in a stirred solution, do not mix homogeneously; this is particularly so when preventive measures are applied. This, in turn, makes the mathematical formalism used so far questionable and the derived estimates of the relevant parameters, e.g., R0, a very rough approximation of reality. In fact, R0 cannot capture time-dependent variations in the transmission potential; the time course of an epidemic can be partly described by the effective reproduction number, R(t), which is a time-dependent parameter defined11,12 as the actual average number of secondary cases per primary case at time t:\n\nwhere S(t) and S(0) are the numbers of susceptible subjects at time t and zero, respectively. Eq. 1 shows that R(t) relies on an estimate of R0, which is usually derived from the early phase data of the pandemic. R0 is also crucial for the calculations of herd immunity.5\n\nThe current SIR models for the ongoing COVID-19 epidemic include additional features to the classical SIR model,3,5 namely, the probability of death in the vulnerable fraction of the population, infectious period, and a time from infection to death are included.13,14 The basic reproduction number, R0, and all variables and parameters of the model are expressed as Gaussian distributions around previously estimated means.2,4,14 However, R(t) is used extensively as a reliable measure of a pathogen’s transmissibility.15,16\n\nThe “reaction” of susceptible-infected individuals under heterogeneous conditions.\n\nIn 1988, Kopelman8 introduced the concept of fractal reaction kinetics for reactions taking place under topological constraints. Under these heterogeneous conditions, time-dependent coefficients k(t) and not rate constants govern the rate of the reaction process.8 Numerous disciplines9,17–28 study rate processes with this approach. It is also very appropriate in studying the “reaction” of susceptible-infected individuals under “real-life” conditions.\n\nConsider two rooms of the same size shown in Figure 1 with the same “concentration” of 10 unmovable susceptible subjects and two COVID-19 infected subjects. The probability for SARS-CoV-2 transmission is much higher in the left-hand side room, because the distance for seven of the susceptible subjects from the two infected subjects is much smaller than the “critical distance” associated with the bimolecular reactions of fractal kinetics.8 On the contrary, only one of the susceptible subjects is within “critical distance” from infected subjects of the right-hand side room. The static picture depicts the equivalency of the social distancing (1.5 meters applied during the Covid-19 pandemic) with the “pair up” and “critical distance” concepts of fractal kinetics.8 Intuitively, if the subjects in the two rooms start moving, virus transmission will increase as a function of time and will be dependent on the trajectory of each individual. Obviously, continuous movement of the subjects in the two rooms sweeping the available space would result in the transmission of the disease to all susceptible subjects in accordance with the “well-mixed” hypothesis. This means that the “well-mixed” system is just a single limiting case of the myriad heterogeneous space/time configurations of the individuals in a population.\n\nAlthough the number of infected (red) and susceptible subjects (green) is the same in both rooms, the instantaneous probability for virus transmission is 7/10 and 1/10 for the left- and right-hand side room, respectively.\n\nThese considerations lead us to the following very important conclusions relevant to airborne pandemics.\n\n\n\na. The time evolution of pandemics described by the classical SI and SIR models,5 which are based on the well-mixed hypothesis, are very crude approximations of reality.\n\nb. The use of a fixed R0 value,4,5 is inadequate for capturing the transmission dynamics. The use of R(t) can capture time-dependent variations in the transmission potential,11,12,15,16 but is heavily dependent on the R0 estimate. In real-life conditions, the transmission of the disease is not only dependent on time, but also on the topology/movement associated with susceptible/infected individuals.\n\nc. The importance of the “initial conditions” for fractal reaction kinetics has been delineated.8 In pandemics, the corresponding “initial conditions” are “patient zero” at the epicenter of the country of pathogen’s origin as well as “patient zeros” of the first humans infected in different countries. For the COVID-19 pandemic specifically, since most of the infected subjects are asymptomatic during the initial phase of the disease spreading, no precautions are taken. During this initial period, which lasts until social distancing measures are applied, disease spreading follows a “herd immunity”5 style, which we call “herd kinetics”. Similarly, we coin the term “fractal kinetics” for the disease spreading when containment measures are imposed.\n\nThe fractal kinetics’ SI model7 for epidemic spreading relies on the following equation:\n\nwhere I(t) is the cumulative fraction of infected individuals at time t, β is a parameter proportional to the probability of an infected individual to infect a healthy one and h is the fractal dimensionless exponent associated with fractal kinetics.8 The core assumption of the model is that societies as complex systems will exhibit self-organization as a reaction to the emergence of a pandemic wave, enforcing preventive measures and increasing public awareness. Thus, instead of an infection rate constant, the fractal SI model uses a rate factor β/th decreasing of time. The solution of Eq. 2 gives I(t) as a function of time:7\n\nwhere c is a parameter which determines the fraction of individuals that will become infected eventually.\n\nBy substituting β=a1−h we introduce parameter α of inverse time dimension,29 which changes Eq. 3 into 4, namely:\n\nIn the limit t → ∞ we find:7\n\nThe “well-mixed” model, described by Eq. 2 with h = 0, has a limiting value of I(t) equal to one as a result of a completely susceptible population. However, this is not a realistic feature for all pandemics that have appeared so far. Eq. 5 reveals that the plot of I(t) versus time for h > 1 reaches a plateau equal to 1/(1 + c) (see Figure 2), which is a reasonable feature for all pandemics. For the special case h = 1, Eq. 6 (also plotted in Figure 2) is derived which describes what we call “herd kinetics” not only because no precautions or measures are taken, but also because the rate of increase of infected subjects progressively diminishes in a similar fashion when a “herd immunity”5 policy is implemented:\n\nParameter values used: h = 0, β = 0.064, c = 790; h = 1, β = 3, c = 4.4 × 105; h = 2.5, α = 0.018 (time)−1, c = 6. Marks on the curves are inflection points.\n\nA linearized form of Eq. 6 is as follows:\n\nwhere the slope β is an “apparent” dimensionless transmissibility rate constant during the “herd kinetics” period; the term “apparent” is used to underline its proportional dependency to the probability of an infected individual to infect a healthy one (see Eq. 2). At t = 1, hence ln t = 0, we get:\n\nTheoretically, the value of I(t = 1) corresponds to the “initial conditions”, i.e., the fraction of infected individuals at the first day of the pandemic; since the real “time zero” is unknown, c1 is proportional to the number of total (asymptomatic and symptomatic) infected cases from the real time zero to time t = 1 day, when the first case was confirmed. We use the notation c1 to distinguish it from c appearing in Eqs. 5, 6 and 7.\n\nIn all pandemics, a characteristic time is observed when the daily number of confirmed infected cases does not increase anymore and starts declining; this corresponds to the inflection point tip. When h > 1, an estimate for tip can be obtained by equating the second derivative of Eq. 4 to zero and solving the resulting equation for time. Lacking an analytical solution, this equation can only be solved numerically.\n\nFor the special case h = 0, tip, can be derived from Eq. 4:\n\nThe following t.ip can be derived from Eq. 2, under “herd kinetics” conditions (h = 1):\n\nThe inflection points for the three examples considered, h = 0, h = 1 and h = 2.5 are shown on the simulated curves of Figure 2. Inflection points denote when a curve changes from being convex (upwards) to concave (downwards), i.e., the confirmed infected new cases remain temporarily constant and then start to drop.\n\nIf the value of parameter c is low, all cases reach the asymptotic limit of 1. However, in real-life conditions the limiting value of the cumulative fraction of infected individuals, I(t) is always much smaller than 1. This epidemiological evidence (fact) can be explained only by the fractal kinetics SI model as shown in Figure 2. The curve of the example considered using h = 2.5 reaches the plateau value of 0.125, i.e., 12.5% of the population will be infected eventually.\n\nFor h > 1, the I(t) corresponding to the inflection time point, I (tip) can be derived from Eq. 4 using the tip estimate in the denominator of Eq. 4. The tip estimate is obtained by equating the second derivative of Eq. 4 to zero and solving numerically the resulting equation.\n\nFor h = 0:\n\nwhile for h = 1:\n\nDuring the time course of the pandemics, an estimate for the time of the termination or close to the termination of the spreading is desperately needed as early as possible. An estimate for the time of 90% termination, t90% for h > 1, can be derived from Eq. 4 using I(t) = 0.90/(1 + c):\n\n\nMethods\n\nThe best fits of Eqs. 4 and 6 to the data30 were obtained by maximizing the R2 of the two adjacent periods. By anchoring the date of each country’s lockdown decision (or any similar form of draconian measure) and moving forward in time, the Levenberg–Marquardt algorithm of least squares was implemented. The lockdown dates are close or very close to the transition from herd kinetics to fractal kinetics and vice versa. A minimum value of R2 = 0.985 was set as a criterion of goodness of fit and every value higher than that was accepted. The turning time data point at which the best R2 value began to diminish was rejected and its prior time data point was accepted. From that time segment and further on the consequent kinetic profile was fitted to the data points until the plateau of quasi-steady state was reached. The fitting discontinuities observed in the kinetics between the distinct periods (e.g., from second to third period for France) are associated with the fact that I(t) values at the boundary of the two periods were not equalized in our fitting methodology. Between the quasi-steady state and the beginning of the second herd period a 10% change of the number of cumulative infected cases at one week interval was sought in order to establish the commencement of a second viral wave and the reproduction of the according fitting procedure. Data acquisition, modelling and simulations were programmatically implemented with Python language31 and its respective libraries.\n\n\nResults\n\nIn our previous studies7,32 on COVID-19 data analysis, we applied the fractal kinetic SI model (Eq. 3) assuming that fractal kinetics commences at time zero. However, reconsideration of the topological characteristics of the virus transmission in the light of Eq. 4 led us to the realization that a “herd kinetics’” period precedes the “fractal kinetics’” period. Exponent β drives the kinetics during the “herd kinetics” stage and is the analogue of R0 for a not well-mixed system. But, unlike R0, β is not associated with the expected number of cases directly generated by one case in a population. During the “fractal kinetics” period, parameter α in Eq. 4 governs the rate of the disease, while the prevailing spatial conditions are reflected on the h value. During this period, a meaningful parameter for the rate of the process is the half-life, t½ = ln2/α.\n\nInitially, virus transmission takes place under “herd kinetics’” conditions (Eq. 6, Figure 3A). This prevails until the first preventive measures are imposed; these can be followed by a lockdown decision. The preventive measures and the lockdown status induce a gradual reduction in the rate of the disease spread, i.e., “fractal kinetics” starts operating (Eq. 4, h>1, Figure 3B). The transition from herd kinetics (Eq. 6) to fractal kinetics (Eq. 4, h>1) can be gradual during this fuzzy period, with both equations operating concurrently. The prevalence of fractal kinetics during the lockdown period results in an asymptotic approach of I(t) to the steady state, i.e., I(t) = (1+c)-1 (see Eq. 5, Figure 3B); according to Eq. 4 the higher the value of the fractal exponent of time h, the more rapid is the approach of I(t) to the steady state. This pattern we call “Herd-Fuzzy-Fractal” (HFF) kinetic motif. When the confirmed new cases reach a steady state, governments relax lockdown rules. In theory, when such a decision is taken, the termination of the first wave of the pandemic has been accomplished. However, the relaxation of lockdown measures in conjunction with the large number of infected individuals at steady state can, after a while, initiate a second wave of the pandemic leading to the application of new preventive measures and new lockdown rules. Consequently, a second wave of the disease emerges (Figures 3C and 3D); hence, the (HFF)2 kinetic motif.\n\nThe gray line segments indicate fuzzy periods. A–D. Subplots correspond to the four distinct periods of the kinetic motif. Equations and parameter values used: A: Eq. 6, β = 8, c = 3 × 1016; B: Eq. 4, h = 4.5, α = 0.012 (time)−1, c = 240; C: Eq. 6, β = 1.18, c = 119350; D: Eq. 4, h = 8, α = 3.35 × 10−3(time)−1, c = 20.\n\nWe focused on the data30 of four model countries, namely, France, Greece, Italy, and Spain. Figure 4 shows for each one of the four countries, the fittings of Eqs. 6 and 4 to herd- and fractal-kinetics’ periods’ data, respectively. Parameter estimates derived are listed in Table 1. High R2 values listed in this Table indicate that the model of Eqs. 4 and 6, for all four countries, is in excellent agreement with the disease data except Italy’s fourth fractal kinetics’ period data.\n\nThe data correspond from time zero up to 10 December 2020. The gray line segments indicate fuzzy periods.\n\nEstimates of the secondary parameters tip, t90%, steady state infected fraction are also listed.\n\nFor the first herd kinetics’ period, the estimate for βherd 1 in Greece was found to be 2.38 ± 0.06, which is much smaller than for the other three countries. This is in agreement with the remarkably lower initial I(t) profile of Greece in Figure 4. We should emphasize the valid estimation of the parameter βherd1 for all countries studied. This is clear proof that the initial phase follows a power of time function (Eq. 6) which is contrary to the general belief that the initial phase increases exponentially. This subexponential increase has been observed in the early phase of COVID-19 spreading in different parts of China.6 During the first fractal kinetics’ period, the estimate for αfractal 2 in Greece was also higher, 0.02 ± 2 × 10−4 (days)−1 compared with 0.010–0.012 (days)-1 found for the other three countries. This leads to a shorter half-life of 42 days for Greece compared with an average of 63 days for the three other countries; this, coupled with the earlier lockdown rules imposed in Greece, explains the more rapid approach to the steady state (Figure 4). The fractal exponent hfractal 2 was smaller in Greece, 2.93 ± 0.06, while for France, Italy and Spain it was 4.71 ± 0.09, 4.39 ± 0.02, 5.14 ± 0.06, respectively (Table 1). On the contrary, the estimate for cfractal 2 in Greece 3227 ± 30.19 was roughly ten-times higher than in the other three countries, resulting in much lower I(t) steady-state value.\n\nAll countries remained in a slightly moving upwards quasi-steady state for 2–3 months (Figure 4). This period was followed by a gradually increasing phase in the number of confirmed infected cases. Relaxed rules led to higher population mobility. All countries re-entered a herd kinetics’ period (blue concaving upwards segment in Figure 4). The estimates for βherd 3 were found 1.17 ± 0.02, 3.56 ± 0.04, 3.80 ± 0.04, 5.08 ± 0.08 for Italy, Spain, Greece and France, respectively, in full agreement with the visually increasing “curvature” of the blue concaving upwards segment of the four countries. All countries imposed preventive measures and lockdown rules several times (Figure 4). For France, Greece and Spain a remarkably similar reliable estimate for αfractal 4, 0.003 (days)−1 was found; this is indicative of a slow process with a half-life of 231 days. However, different hfractal 4 estimates, 16.11 ± 0.54, 9.01 ± 0.42 and 6.28 ± 0.43 were found for France, Greece, and Spain, respectively. Assuming that the conditions will not change in the next time period, predictions, based on the parameter estimates of the fourth fractal kinetics period for the steady-state value and t90%, can be made for the three countries (Table 1). On the contrary, the fitting of Eq. 4 to Italy’s fourth fractal kinetics’ period data was not equally successful and reliable parameters estimates for hfractal 4, αfractal 4 and cfractal 4 were not derived (Table 1). This is due to the fact that the point of inflection has not been reached yet and therefore the fitting algorithm cannot converge to reliable parameters estimates.\n\nThe estimates for tip reported in Table 1 for France, Greece and Spain correspond to time (days) from the commencement of the fourth fractal kinetics’ period. These estimates were found to be in agreement with the observed values, which is an additional piece of evidence for the validity of the fractal model. An estimate for Italy’s tip was not obtained for reasons mentioned above. Besides, the fourth fractal kinetics period data were used to predict the t90% (expressed in days from the commencement of this period) and the final steady-state (1/(1+c)) for France, Greece and Spain (Table 1).\n\nA large number of countries, besides the four analyzed, followed the (HFF)2 kinetic motif shown in Figure 4, e.g., Australia, China, Germany, Austria, United Kingdom.30 Yet, several countries did not exhibit the (HFF)2 motif, lacking a second wave and followed a “herd-fuzzy-fractal” (HFF) kinetic motif. Argentina and Brazil are examples of countries where strict/mild preventive measures were either not applied or did not work effectively. Both countries exhibit an (HFF) kinetic motif, Figure 5. Parameter values determined: Argentina, 1st stage: (h = 1), β = 1.929 ± 0.028; 2nd stage: h = 2.189 ± 0.043, α = (1.754 ± 0.067) × 10−3 (days)−1, c = 3.61 ± 0.38; Brazil, 1st stage: (h = 1), β = 4.633 ± 0.017; 2nd stage: h = 2.892 ± 0.023, α = (4.044 ± 0.017) × 10−3 (days)−1, c = 21.86 ± 0.24.\n\nXs mark the implementation of mild preventive measures. The gray lines indicate the fuzzy period after the X point.\n\nOn the other hand, some countries exhibited a more complex pattern, which deviates from the (HFF)2 and (HFF) motifs. Infection data for USA did not follow either the (HFF)2 or the (HFF) kinetic motif. The I(t) time profile never reached a steady state and the shape of the curve indicates a deformed three-wave like kinetic profile (Figure 6). Probably both types of kinetics (herd and fractal) run concurrently for most of the time throughout the course of the pandemic, with the contribution of each varying with time. This is most likely due to different COVID-19 policy containment measures followed in different states around the country. Sweden intentionally applied the herd immunity strategy30 during the COVID-19 pandemic. An initial herd-kinetics type continuous increase in the number of total infected cases reached a point of inflection around 20 July 2020, followed by a slower rate of increase of infected cases, (Figure 6). Since neither strict measures nor lockdown rules were applied at that time, the shape of the curve should be attributed to a fractal kinetics-like self-organization of the society. A rather sharp increase after 10 September 2020 can be attributed to the increased mobility of the individuals since no relaxation measures were taken close to this date.\n\nConfirmed infected cases for USA and Sweden.30\n\n\nImplications\n\nThe above results (Figures 2, 3, Table 1) demonstrate that the fractal kinetics SI model is more akin to real life. Since the well-mixed hypothesis is the crux of the matter of the epidemiological models,5 the use of not well-mixed hypothesis has important implications, which can metamorphosize airborne pandemics; these implications are discussed and itemized (designated with italics) below.\n\nThe reproductive number, R0 is not needed for the initial growth of the disease4 being incompatible with the not well-mixed hypothesis, Figure 1. Limitations associated with the estimation of R0, can be found in numerous publications. Our results show that the time exponent β of Eq. 6 controls the time evolution of the disease throughout the initial herd kinetics’ period. In other words, β drives the initial phase of the disease spreading being the slope of Eq. 7, i.e. a linearized form of Eq. 6. The predominant role of β during the herd kinetics’ period can be also concluded from Eq. 12, which explicitly shows that the infected population fraction at the inflection point, I(t)ip is solely dependent on β. Although R0 and β are different, however, they can be used complementary to each other during the initial stages of the pandemics. Estimates for β derived from the analysis of herd kinetics’ period data at two time points from 100 countries are shown in Figure 7 and Table 2. The degree of uncertainty (standard deviation) for the estimates was found in most cases small; this was accompanied with high correlation coefficients (not shown). Overall, the estimates derived from the longer period of 35 days seem to be either similar or higher or significantly higher than these derived from the analysis of the shorter period (10 days) data. For some countries, the small number of confirmed infected cases in the first 10 days did not allow the estimation of β. In view of the diversity and variability of data presented in Figure 7, we quote the median values derived from the analysis of 100 countries, 2.44 (0.25–12.24) and 1.34 (0.20–6.13) for the β estimates corresponding to 35 and 10 days, respectively.\n\nData of 10 and 35 days, after the first reported case, were analyzed. See also Table 2.\n\nData of 10 and 35 days, after the first reported case, were analyzed. Part of these results is shown in Figure 7. Blanks are due to fragmented data that prevented the fitting procedure to converge.\n\nThe classical phraseology “the exponential growth of the disease” used by medical doctors, scientists and laymen is questionable. This phrase is related to the approximate solution of the SIR model, which is an exponential function, when the parameter of the recovery rate is equal to zero.5 Based on our theoretical results and the good fittings of Eq. 6 to data of herd kinetics’ period (Figure 4), “the herd kinetics’ period seems to obey a power of time function”. According to Eq. 2, β drives the disease spreading when h = 1 and the rate of infection is inversely proportional to time. This is in agreement with the real-life conditions because of the continuous reduction of the probability of infection as a function of time (β/t). However, the resemblance of the I(t) profiles of the classical, h = 0 and the special case h = 1 in Figure 2 makes the discernment of the kinetics of the initial phase difficult.\n\nHerd immunity\n\nHerd immunity5 calculations rely on an estimate for R0 and syllogisms based on the relative magnitude, λ = R(t)/R0, which is the proportion of the population that is susceptible to catching the disease. If preventive measures are not applied, an estimate for the time needed to reach a certain level of the infected population fraction, e.g., I(t) = 0.6 ensuring herd immunity can be obtained from Eq. 7. Assuming an infected individual at time t=1, i.e., 1It=1=N, where N is the population of the country, then, from Eq. 7, we get c = N − 1 ≈ N. Hence, the time thi needed to reach a certain level of herd immunity I (thi) under non preventive measures is\n\nFigure 8 shows thi as a function of β and N assigning I (thi) = 0.6. It can be seen that population size has a mild effect, whereas the apparent transmissibility constant β severely reduces thi. Eq. 14 can be used at the initial stages of the pandemics and requires only a valid estimate for β. This will certainly provide valuable information for authorities, if coupled with estimates of the mortality rate and deaths, prior to a decision for a herd-immunity policy.33 Caution should be exercised with the use of Eq. 14, since it can be applied only under the strict assumption of herd kinetics operating throughout the entire period of the disease spreading. The example of Sweden (Figure 6) shows that societies can exhibit self-organization and move to a fractal kinetics’ mode.\n\nA contour plot based on Eq. 14 showing the time required to reach herd immunity level I (thi) = 0.6 for various values of parameter β and population size N.\n\nDeviation from the herd kinetic profile after the imposition of lockdown\n\nCumulative data of infected people from nine countries (Austria, Belgium, Denmark, France, Germany, Italy, Spain, Switzerland, United Kingdom) were gathered and analyzed under two different prisms. Analysis was broken down into two parts, before and after imposition of strict preventive measures (lockdown) (Figure 9). For the first period, the herd kinetic motif where h = 1 (Eq. 6) was found to be adequate, whereas after lockdown clearly fails. The latter period was also analyzed using the fractal kinetic motif of h > 1 with very persuasive goodness of fit (Figure 9). In all cases, R2 was greater than 0.98. This pictorial divergence shows that after implementing mobility restrictions the evolution of the pandemic could not be captured by a power law expression, but rather by a fractal kinetic one (Eq. 4) which eventually leads to a plateau of cumulative cases.\n\nThe blue dots represent cumulative infected cases up to lockdown datum points.16 The orange lines depict the power fit to these data. Purple dots represent data after lockdown imposition whereas the purple lines are their superimposed fractal fits. Red lines depict the hypothetical power fit to the aforementioned data points in the event that Covid-19 propagation followed a power law pattern. R2 values for all nine countries were measured higher than 0.98.\n\nModel predictions\n\nAccording to Jewell et al.,1 the ability of current models to predict is very poor. Our work demonstrates that the herd kinetics’ period is described by Eq. 6, while the kinetic motif “herd-fuzzy-fractal” should be taken into account in the modeling work. Apparently, these approaches have not been implemented so far. Roughly, predictions during the herd kinetics’ period can be based on a valid estimate for β, Eq. 6. Under preventive measures, valid estimates for the parameters of the model (Eq. 4, h>1) can be derived and used for predictive purposes provided that data beyond the point of inflection are available (see Table 1).\n\n\nConclusions\n\nSince the early days of epidemics’ modeling,3 a great deal of work has been done and now there is a change of paradigm. Interestingly, the results of our work are in full agreement with the basic conclusion of the most recent, extensive and elegant COVID-19 study16 based on the effective reproduction number R(t), “… that major non-pharmaceutical interventions—and lockdowns in particular—have had a large effect on reducing transmission”. Our approach quantifies this large effect on the basis of Eq. 4, which captures the dynamics of the disease under “herd kinetics’” and “fractal kinetics’” conditions. In addition, our herd kinetics’ period results are in full agreement with the observations of the distinctive subexponential increase of confirmed cases during the early phase of the epidemic in China, contrasting an initial exponential growth expected for an unconstrained outbreak.6 The present fractal SI model can be extended to its SIR analogue, with the caveat that the corresponding differential equations require numerical solution. In conclusion, the fractal kinetics SI model with the kinetically established herd period as well as the (HFF)2 or (HFF) kinetic motifs opens up a new era in the field of epidemiological models for airborne pandemics.", "appendix": "Acknowledgements\n\nThis work is dedicated to physicians, nurses and paramedical personnel of the Greek public hospitals as well as to the elementary and high school teachers of public and private schools of Greece for their unceasing devotion and work commitment during the COVID-19 pandemic.\n\n\nData availability\n\nThe underlying data are publicly available from https://www.ecdc.europa.eu/en/covid-19/data. 30\n\n\nReferences\n\nJewell NP, Lewnard JA, Jewell BL: Predictive Mathematical Models of the COVID-19 Pandemic: Underlying Principles and Value of Projections. JAMA. 2020; 323: 1893–1894. PubMed Abstract \| Publisher Full Text\n\nBox GEP: Science and Statistics. J. Am. Stat. Assoc. 1976; 71: 791–799. 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Chichester: Wiley; 2000.\n\nLourenco J, Paton R, Ghafari M, et al.: Fundamental principles of epidemic spread highlight the immediate need for large-scale serological surveys to assess the stage of the SARS-CoV-2 epidemic. medRxiv 2020.03.24.20042291. 2020. Publisher Full Text\n\nKissler SM, Tedijanto C, Lipsitch M, et al.: Social distancing strategies for curbing the COVID-19 epidemic. medRxiv 2020.03.22.20041079. 2020. Publisher Full Text\n\nPatel P, Athotra A, Vaisakh TP, et al.: NCDC COVID Incident Management Team, Impact of nonpharmacological interventions on COVID-19 transmission dynamics in India. Indian J Public Health. 2020; 64: S142–S146. PubMed Abstract \| Publisher Full Text\n\nFlaxman S, Mishra S, Gandy A, et al.: Estimating the effects of non-pharmaceutical interventions on COVID-19 in Europe. Nature. 2020; 584: 257–261. PubMed Abstract \| Publisher Full Text\n\nMacheras P: Carrier-Mediated Transport Can Obey Fractal Kinetics. Pharm. Res. 1995; 12: 541–548. PubMed Abstract \| Publisher Full Text\n\nAuclair J, Gagné F: The influence of polystyrene nanoparticles on the fractal kinetics of lactate dehydrogenase. Biochem. Biophys. Rep. 2020; 23: 100793. Publisher Full Text\n\nWang Z-W, Xu F, Manchala KR, et al.: Fractal-like kinetics of the solid-state anaerobic digestion. Waste Manag. 2016; 53: 55–61. Publisher Full Text\n\nMoiny F, Dumont M, Dagonnier R: Fractal kinetics and surface reactions. J. Chem. Phys. 1998; 108: 4572–4581. Publisher Full Text\n\nMacheras P, Argyrakis P: Gastrointestinal Drug Absorption: Is It Time to Consider Heterogeneity as Well as Homogeneity? Pharm. Res. 1997; 14: 842–847. PubMed Abstract \| Publisher Full Text\n\nKalampokis A, Argyrakis P, Macheras P: A Heterogeneous Tube Model of Intestinal Drug Absorption Based on Probabilistic Concepts. Pharm. Res. 1999; 16: 1764–1769. Publisher Full Text\n\nMacheras P, Dokoumetzidis A: On the Heterogeneity of Drug Dissolution and Release. Pharm. Res. 2000; 17: 108–112. PubMed Abstract \| Publisher Full Text\n\nKosmidis K, Argyrakis P, Macheras P: Fractal kinetics in drug release from finite fractal matrices. J. Chem. Phys. 2003; 119: 6373–6377. Publisher Full Text\n\nKosmidis K, Macheras P: On the dilemma of fractal or fractional kinetics in drug release studies: A comparison between Weibull and Mittag-Leffler functions. Int. J. Pharm. 2018; 543: 269–273. Publisher Full Text\n\nFuite J, Marsh R, Tuszyński J: Fractal pharmacokinetics of the drug mibefradil in the liver. Phys. Rev. E. 2002; 66(21904). PubMed Abstract \| Publisher Full Text\n\nKosmidis K, Karalis V, Argyrakis P, et al.: Michaelis-Menten kinetics under spatially constrained conditions: application to mibefradil pharmacokinetics. Biophys. J. 2004; 87: 1498–1506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVasilescu C, Olteanu M, Flondor P, et al.: Fractal-like kinetics of intracellular enzymatic reactions: a chemical framework of endotoxin tolerance and a possible non-specific contribution of macromolecular crowding to cross-tolerance. Theor. Biol. Med. Model. 2013; 10: 55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMacheras P, Iliadis A: Modeling in biopharmaceutics, pharmacokinetics and pharmacodynamics: homogeneous and heterogeneous approaches. 2nd Ed. Springer; 2016.\n\nEuropean Centre for Disease Prevention and Control: COVID-19 datasets. 2021. Reference Source\n\nMacheras P, Tsekouras AA, Chryssafidis P: Python language code used for data computations. Zenodo. 2021. Publisher Full Text\n\nMacheras P, Kosmidis K, Chryssafidis P: Demystifying the spreading of pandemics I: The fractal kinetics SI model quantifies the dynamics of COVID-19. medRxiv 2020.11.15.20232132. 2020. Publisher Full Text\n\nSridhar D, Gurdasani D: Herd immunity by infection is not an option. Science. 2021; 371: 230–231. PubMed Abstract \| Publisher Full Text" }	[ { "id": "93405", "date": "22 Sep 2021", "name": "Raoul Kopelman", "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\nOverall this is an important contribution, pointing out the occasionally fallacious assumption in epidemiological theories of a “well stirred” population. While in many chemical reactions “well stirred” conditions are met, or taken care of to be met, the adoption in the standard epidemiology approaches of a similar condition is questionable. While it may be a good approximation for an animal model, or for a primitive society where no precautions against infection are practiced, this is questionable for a modern society, where isolation of the sick and preventive measures by the healthy, e.g., wearing masks, are a standard, or even mandated modus operandi. This justifies rejecting the “classical reaction kinetics” model and replacing it by a “non-classical reaction kinetics” model, which was originally called “fractal reaction kinetics” (Ref 8).\nWhile with the new model more adjustable parameters are introduced, which should be emphasized by the authors, the agreement with the spread of Covid in a large number of countries is impressive. So is the agreement of the classical limit (“h=0”) with the spread of the pandemic in Sweden, where no preventive restrictions were adopted, with the aim of reaching “herd immunity” (an animal model).\nMinor Comments:\nExplain better the definition of the last factor in Eq. 1.\n\nExplain better the definition of the last factor in Eq. 2. Also discuss the positive vs. negative values of h.\n\nStraighten out and explain eq. 3.\n\nCarefully define “alpha” in eq. 4.\n\nIn Eq. 6, explain wherefrom comes “11”.\n\nEq. 8, ditto.\n\nEq. 10, ditto.\n\nEq. 12, explain wherefrom comes “12”.\n\nEq. 13, explain wherefrom comes “11”.\n\nClarify eq. 14.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7321", "date": "25 Oct 2021", "name": "Pavlos Chrys", "role": "Author Response", "response": "Response to comments by R. Copelman. We thank the reviewer for his constructive critique. Regarding the minor comments and questions, we have the following replies to each point raised. Explain better the definition of the last factor in Eq. 1. Response: This equation is presented in detail in Ref. 11. Explain better the definition of the last factor in Eq. 2. Also, discuss the positive vs. negative values of h. Response: [1-I(t)] is the fraction of the susceptible population. Negative h values don't come up in actual data. Straighten out and explain eq. 3. Response: Eq. 3 is described in Ref. 7. Carefully define “alpha” in eq. 4. Response: See line above Eq. 4. In Eq. 6, explain wherefrom comes “11”. Response: What does \"11\" stand for? There is no \"11\" in Eq. 6. Eq. 6 is derived from Eq. 3 by taking the limit h->1 and implementing L'Hopital's rule, i.e., taking the derivative of numerator and denominator in the exponent and then taking the limit. We will clarify the point in the next version of the text. Eq. 8, ditto. Response: Eq. 8 is derived from Eq. 7 after doing some straightforward algebra. Eq. 10, ditto. Response: The derivation of Eq. 10 is described right before Eq. 9 and repeated before Eq. 11. Eq. 12, explain wherefrom comes “12”. Response: Eq. 12 is derived from Eq. 6 following the procedure described before Eq. 9. Eq. 13, explain wherefrom comes “11”. Response: Eq. 13 is derived from Eq. 4 by setting the right-hand side of the latter equal to 0.9 times I(t->infinity) and solving for t. Clarify eq. 14. Response: Eq. 14 tells us when the fraction of infected individuals will reach a certain level given the size of the population and exponent β. Here again, we will add a brief explanation in the updated version of the text." } ] }, { "id": "100027", "date": "07 Dec 2021", "name": "Jinjun Zhang", "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\nThanks for the opportunity to review this manuscript. This article by Panos Macheras, Athanasios A. Tsekouras and Pavlos Chryssafidis gives a consistent analysis for a not well-mixed system using fractal kinetic principles. They derived a three arameter model and show that the fractal exponent of time larger than unity can capture the impact of preventive measures affecting population mobility, and lead to the conclusion that the fractal kinetics model can be used as a prototype for the analysis of all contagious airborne pandemics. Overall, The study is well-designed and data analyses are well performed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "100029", "date": "16 Dec 2021", "name": "Juan J Nieto", "expertise": [ "Reviewer Expertise Pure and Applied Mathematics", "Biomathematics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 timely and interesting contribution to the study of the dynamics of the COVID-19. Since some models do not capture well the dynamics, it is considered a fractional principle.\n\nFractal and fractional models have considered, for example in Ndaïrou et al. (20211) and Bushnaq et al. (20212).\n\nI would recommend to continue this approach with updated data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/10-609
https://f1000research.com/articles/10-253/v1	30 Mar 21	{ "type": "Method Article", "title": "CODECHECK: an Open Science initiative for the independent execution of computations underlying research articles during peer review to improve reproducibility", "authors": [ "Daniel Nüst", "Stephen J. Eglen" ], "abstract": "The traditional scientific paper falls short of effectively communicating computational research. To help improve this situation, we propose a system by which the computational workflows underlying research articles are checked. The CODECHECK system uses open infrastructure and tools and can be integrated into review and publication processes in multiple ways. We describe these integrations along multiple dimensions (importance, who, openness, when). In collaboration with academic publishers and conferences, we demonstrate CODECHECK with 25 reproductions of diverse scientific publications. These CODECHECKs show that asking for reproducible workflows during a collaborative review can effectively improve executability. While CODECHECK has clear limitations, it may represent a building block in Open Science and publishing ecosystems for improving the reproducibility, appreciation, and, potentially, the quality of non-textual research artefacts. The CODECHECK website can be accessed here: https://codecheck.org.uk/.", "keywords": [ "reproducible research", "Open Science", "peer review", "reproducibility", "code sharing", "data sharing", "quality control", "scholarly publishing" ], "content": "Abbreviations\n\nACM: Association for Computing Machinery; ECRs: Early Career Researchers; RCR: Replicated Computational Results; TOMS: Transactions on Mathematical Software.\n\n\nIntroduction\n\nMany areas of scientific research use computations to simulate or analyse their data. These complex computations are difficult to explain coherently in a paper1. To complement the traditional route of sharing research by writing papers, there is a growing demand to share the underlying artefacts, notably code and datasets, so that others can inspect, reproduce or expand that work (see Figure 1). Early proponents of this initiative were Buckheit and Donoho2,3, who noted: “An article about computational science in a scientific publication is not the scholarship itself, it is merely advertising of the scholarship. The actual scholarship is the complete software development environment and the complete set of instructions which generated the figures.”\n\nThe left half of the diagram shows a diverse range of materials used within a laboratory. These materials are often then condensed for sharing with the outside world via the research paper, a static PDF document. Working backwards from the PDF to the underlying materials is impossible. This prohibits reuse and is not only non-transparent for a specific paper but is also ineffective for science as a whole. By sharing the materials on the left, others outside the lab can enhance this work.\n\nIf researchers start sharing more artefacts, how might these artefacts be examined to ensure that they do what they claim? For example, although scientific journals now require a data sharing statement that outlines what data the authors have (or will) share, journals implement this differently. On one hand, journals have been created to accept “data papers” (e.g., Scientific Data, Earth System Science Data, Geoscience Data Journal, Biodiversity Data Journal, Journal of Open Psychology Data, Open Data Journal for Agricultural Research, Journal of Open Health Data); these journals have established rigorous procedures by which data are validated according to standards in each field. On the other hand, many journals still allow authors to state “Data available upon reasonable request”. Authors, while possibly well intentioned at the time of writing the article, often cannot provide data when requested as data disappears over time4.\n\nGiven that data are not routinely shared, what hope might there be for sharing computer programs? Both data and software are required to validate a computational analysis; data can be seen as inert whereas code requires an environment to be run in. This makes software harder to share. Our experience is that researchers offer several reasons for why code is not shared, e.g., “there is no documentation”, “I cannot maintain it”, or “I do not want to give away my code to competitors”. Our view is that sharing code, wherever possible, is good for the community and the individual5,6. Having code and data openly available, and archived, provides a valuable resource for others to learn from, even if the code is broken or lacks documentation. However, with a little effort, we believe that if an independent person can re-run the programs, this is worth documenting and that this reduces the barrier to evaluating non-text research materials. Just as data journals’ validations of data and all journals’ peer review provides a “baseline reassurance”, i.e., that a paper has been checked by someone with an understanding of the topic7, the same baseline could be provided for the workflow underlying a paper. With this in mind, we have developed a set of principles and an example workflow that provides a pragmatic way of checking that a paper’s code works, i.e., it is reproducible following the Claerbout/Donoho/Peng terminology8.\n\nHere we offer a thorough description of a process and its variations to integrate a much-needed evaluation of computational reproducibility into peer review, and we demonstrate its feasibility by means of 25 reproductions across scientific disciplines. We call this system CODECHECK.\n\n\nWhat is a CODECHECK?\n\nCODECHECK is best demonstrated by way of our example workflow, and later we expand on the underlying principles. The workflow involves three groups of people: (1) the author of a paper providing the code to be checked, (2) the publisher of a journal interested in publishing the author’s paper, and (3) the codechecker, who checks that the author’s code works. The six-step workflow we have refined is shown in Figure 2. In this article, we also refer to a peer-reviewer who is independent of this process, and performs the traditional academic review of the content of an article.\n\nCodecheckers act as detectives: They investigate and record, but do not fix issues. Numbers in bold refer to steps outlined in the text.\n\nStep 1: The author submits their manuscript along with the code and data to the publisher. The code and data need not be openly available at this point. However, in many cases the code and data may be published on a code hosting platform, such as GitHub or GitLab. Ideally, the author is expecting the CODECHECK and prepares for it, e.g., by asking a colleague to attempt a reproduction, and providing a set of instructions on how to re-run the workflow.\n\nStep 2: The publisher finds a codechecker to check the code. This is analogous to the publisher finding one or more peer-reviewers to evaluate the paper, except we suggest that the codechecker and the author talk directly to each other.\n\nStep 3: The codechecker runs the code, based on instructions provided by the author. They check if some or all of the results from the paper can be reproduced. If there are any problems running the code, the codechecker asks the author for help, updates, or further documentation. The burden to provide reproducible material lies with the author. The codechecker then tries to run the code again. This process iterates until either the codechecker is successful, or the codechecker concludes the workflow is not reproducible. As part of this process, the codechecker could work entirely locally, relying on their own computing resources, or in the cloud, e.g., using the open MyBinder infrastructure9 or alternatives, some of which are more tailored to scientific publications while others offer commercial options for, e.g., publishers (cf. 10). A cloud-based infrastructure allows for the codechecker and author to collaboratively improve the code and enforces a complete definition of the computing environment; but, unless secure infrastructure is provided, e.g., by the publisher, this requires the code and data to be published openly online. Note that the task of the codechecker is to check only the “mechanics” of the workflow. In the context of mathematics, Stodden et al.11 distinguish between verification and validation; following their definition, a CODECHECK ensures verification of computational results, i.e., checking that code generates the output it claims to create, but not a validation, i.e., checking that the code implements the right algorithm to solve the specific research problem. Nevertheless, simply attempting to reproduce an output may highlight a submission’s shortcomings in meeting a journal’s requirements (cf. 12) and may effectively increase transparency, thereby improving practices (cf. 13) even if the check does not go into every detail.\n\nStep 4: The codechecker writes a certificate stating how the code was run and includes a copy of outputs (figures or tables) that were independently generated. The certificate may include recommendations on how to improve the material. The free text in the certificate can describe exactly what was checked, because each workflow is unique. Since no specific tool or platform is required, such that no authors are excluded, it is futile for the codechecker to use automation or fixed checklists.\n\nStep 5: The certificate and auxiliary files created during the check, e.g., a specification of a computing environment, data subsets or helper scripts, and the original code and data get deposited in an open archive unless restrictions (data size, license or sensitivity) apply. Currently, codecheckers deposit the material on Zenodo themselves, but a publisher may complete this step after integrating CODECHECK into its review process. A badge or other visual aid may be added to the deposit and the paper and link to the certificate. Although a badge simplifies the CODECHECK into a binary value and risks introducing confusion regarding the extent of the check, a badge provides recognition value and acknowledges the completed CODECHECK. The badge and the actual check are incentives for undertaking the effort needed to provide a reproducible workflow.\n\nStep 6: The publisher can, depending on the timing, provide the certificate to peer-reviewers or editors or publish it and link between certificate, paper, and any repositories. Currently, the codechecker creates these connections on Zenodo. They appear as links with a relationship type on the Zenodo landing page for a certificate, e.g., the “related identifiers” and “alternate identifiers” of certificate 2020-02514. The publisher also credits the codechecker’s work by depositing the activity in scholarly profiles, such as ORCID (see peer review contributions in ORCID records). The publisher also ensures proper publication metadata, e.g., links from the certificate repository to the published paper or the original code repository.\n\nDimensions of CODECHECK workflows. Our workflow is just one of many possibilities of a CODECHECK workflow. Here we consider several dimensions in a space of possible CODECHECK workflows (Figure 3). These aspects touch on timing, responsibilities, and transparency.\n\nWhen to do a CODECHECK and with what importance? The time at which a CODECHECK is done and its ascribed importance are closely connected, so we describe the dimensions When and Importance together. The earlier a CODECHECK happens in the publishing process, the more it can affect editorial decisions: Is a paper published, sent back for revisions, or rejected? Even earlier checks, i.e., a CODECHECK of a preprint, may help to improve the workflow itself, even before a publisher is involved. As such, codechecking papers could be part of a preprint server’s policy or initiated by interested authors.\n\nPublishers could introduce a CODECHECK as a strict prerequisite. As this can reduce the workload of reviewers, such a check should occur early in the review process. Yet, the later in the review process the check happens, the easier is it to allow bidirectional communication between the author and codechecker, e.g., because the author might already be notified of the paper’s acceptance and may be more willing to share materials online closer to the paper’s publication date. A pre-review CODECHECK means editors would send a submission for peer review only if it passes the check, or include the certificate in the submission package provided to peer-reviewers. Peer-reviewers may then judge the relevance of the computations for the results of the work.\n\nA CODECHECK may also be conducted in parallel to the academic peer review. This puts less burden on the turnaround time for the CODECHECK, yet it only makes the outcomes available during the final consideration by the handling editor. The check could also be assigned after suggestion by a reviewer, which would remove the need for submissions to undergo a pre-review screening. However, soliciting such a “specialist review” is much less desirable than having a regular CODECHECK, thus avoiding the situation in which some submissions get special treatment. In both cases, the editor’s decision could be based both on CODECHECK and peer-review reports.\n\nA post-acceptance CODECHECK would have the smallest impact on editorial decisions and may simply provide extra merit on top of the submission’s acceptance. This is the least impactful solution in which all material is still evaluated and the results of the check are properly acknowledged, because the check can be completed before publication of the paper. The GIScience checks (see below) falls into this category: by displaying a badge on the volume and article landing pages, the AGILE conference highlights articles whose reproducibility was confirmed. Similarly, in collaborations with journals, some GIScience articles were checked whilst authors worked on revisions.\n\nA CODECHECK may also be conducted post-publication, though this requires an update to the article and article metadata to reference the check so that readers can find the CODECHECK. In general, publishers hesitate to make such revisions to published articles. We do not prefer this option as it has the least impact on current publishing practices and downplays the importance of reproducible workflows for ensuring good scientific practice.\n\nEnhancing existing processes with CODECHECKs allows communities to gradually transition towards more open practices. When integrating a CODECHECK into existing review and publication processes, the turnaround time is crucial. Depending on when and who conducts the check, it might be done quickly or it might delay publication. We found that a CODECHECK generally takes 2–5 hours, with some outliers on the higher end. This time includes writing and publishing the certificate but excludes actual computation time, some of which took days. These efforts are comparable to the time needed to peer review a submission, which aligns with the efforts some volunteer codecheckers are willing to make. Currently, there is considerable amount of communicating about the process, especially regarding who publishes which document when, so that proper cross-referencing between paper and certificate is ensured via persistent identifiers. When integrated into a peer review platform, this handling of documents should become much more streamlined.\n\nOpenness, or “Who knows who?” Anonymity is broadly discussed, especially in the push towards open peer review as part of the Open Science movement (cf. 15). Without taking a strong stance on this topic, our motivation behind CODECHECK for higher transparency and reproducibility does indeed favour a more open review process. However, anonymity can protect individuals16, e.g., junior scientists. The negative effects of a signed review may be reduced if a CODECHECK is not relevant for a journal’s decision to accept or reject, but that is, of course, not desirable when the goal is higher transparency and reproducibility. Instead, CODECHECK is a technical process that should generally find fixable problems; it is not aimed at giving an opinion or identifying a faulty approach. If passing a CODECHECK becomes mandatory, full transparency may need revisiting as the relations between authors and codecheckers would fall under the same social and community challenges as open peer review (cf. 17).\n\nThe technical nature of the check and the challenge of providing sufficient documentation is why we see great benefits in bidirectional communication between author and codechecker. Instead of trying to fix problems or guess the next step, the codechecker can ask the author to rework the documentation or update code. Instead of struggling to provide perfect instructions and as a result possibly not sharing any code or data, the author can make a best effort to document sufficiently. Authors and readers can profit from a codecheckers’ experience and approach, as during the check they may create useful and instructive files, e.g., a machine-readable computing environment specification. While communication between author and codechecker may be anonymised via the publisher, it most likely only helps to protect the identity of the codechecker, because code is hard to anonymise. Therefore, the most effective and desirable situation for the stakeholders is to hold a open and collaborative CODECHECK. The contributions by the codechecker may even be integrated into the code of the workflow and be acknowledged as code commits. This way, proper credit can be given within the research software development community.\n\nWho does the CODECHECK? Just as with peer-reviewers, a potential codechecker should have the right skills and availability to do the work. Ideally, the codechecker has a matching code and domain expertise to the paper, although a well-documented workflow should be executable by any computationally-competent person. Naturally, the more prerequisite knowledge the codechecker has, the quicker they can understand the goals and mechanics of an analysis. From our experiences, the priority should be given to matching technical expertise first, as lacking knowledge in setting up a computing environment with a particular language or tool is much more of a problem than assessing the outcome, e.g., comparing created figures with the original, without an in-depth understanding of the domain. The depth of the check will mostly be driven by the time required and expertise of the checker, though in general, we expect a CODECHECK to consider reproducibility of the results above performance of the code.\n\nCodecheckers could be drawn from a regular pool of peer-reviewers, or from a special group of reproducibility reviewers via specific roles such as reproducibility editors, or editorial staff with a publisher. One codechecker is sufficient to verify the workflow since it is mostly a factual process. Code usually harbours systematic and repeatable mistakes and is thereby more reliable and auditable than processes controlled by humans18, e.g., in a laboratory. If however publication of the paper depends on the CODECHECK, a second opinion may be required.\n\nWe also see a great opportunity to involve early-career researchers (ECRs) as codecheckers. ECRs arguably have a high interest in learning about new tools and technologies, to build up their own expertise. CODECHECK offers a way for ECRs to gain insights into new research and highlight the importance of reproduction. ReScience X, a journal devoted to reproduction and replication experiments19, shares an interest in this combination. ECRs are also often familiar with new technologies, thus also making them likely to author CODECHECK-ready manuscripts. A supporting data point for ECRs as early adopters is that they are responsible for 77% of 141 registered reports that were submitted20. As ECRs are introduced to peer review as codecheckers, they may transition into the role of peer-reviewer over time. Overall, we see several opportunities and benefits to setting up a new process for codechecking with a clear commitment to openness and transparency, independent of the current peer review process (see Openness dimension).\n\nThe codechecker could be a member of editorial staff; this is the most controlled but also resource-intensive option. Such a resource commitment would show that publishers are investing in reproducibility, yet this commitment may be hard for small publishers. These codecheckers could be fully integrated into internal workflows. Credit for doing the codecheck is also achieved, as it is part of their duties. By contrast, it is useful for researchers to be publicly credited for their reviewing activity. A regular review may be listed in public databases (e.g., ORCID, see Step 6 above, or commercial offerings such as Publons, and ReviewerCredits); a codechecker could be similarly listed. The codechecker community has over 20 volunteers who signed up in the last year, see https://github.com/codecheckers/codecheckers/. Their motivations, mentioned in the registration information, include: supporting reproducible research and Open Science, improve coding skills, gaining experience in helping scientists with their code, encouraging a sharing culture, and learning from other people’s mistakes; many are also motivated simply by curiosity. We see benefits to an open shared list of codecheckers across journals rather than a private in-house group, as this may allow for better matches regarding expertise and workload sharing. This community can establish CODECHECK as a viable option for independent no-cost Open Access journals.\n\n\nCore principles\n\nThe workflow and variations outlined describe our current views on how code could be checked. They are not immutable, but we believe the following core principles underpin our CODECHECK process:\n\n1. Codecheckers record but don’t investigate or fix.\n\nThe codechecker follows the author’s instructions to run the code. If instructions are unclear, or if code does not run, the codechecker tells the author. We believe that the job of the codechecker is not to fix these problems but simply to report them to the author and await a fix. The level of documentation required for third parties to reproduce a workflow is hard to get right, and too often this uncertainty leads researchers to give up and not document it at all. The conversation with a codechecker fixes this problem.\n\n2. Communication between humans is key.\n\nSome code may work without any interaction, e.g. 21, but often there are hidden dependencies that need adjusting for a particular system. Allowing the codechecker to communicate directly and openly with the author make this process as constructive as possible; routing this conversation (possibly anonymously) through a publisher would introduce delays and inhibit community building.\n\n3. Credit is given to codecheckers.\n\nThe value of performing a CODECHECK is comparable to that of a peer review, and it may require a similar amount of time. Therefore, the codechecker’s activity should be recorded, ideally in the published paper. The public record can be realised by publishing the certificate in a citable form (i.e., with a DOI), by listing codecheckers on the journal’s website or, ideally, by publishing the checks alongside peer review activities in public databases.\n\n4. Workflows must be auditable.\n\nThe codechecker should have sufficient material to validate the workflow outputs submitted by the authors. Stark22 calls this “preproducibility” and the ICERM report11 defines the level “Auditable Research” similarly. Communities can establish their own good practices or adapt generic concepts and practical tools, such as publishing all building blocks of science in a research compendium (cf. https://research-compendium.science/) or “repro-pack”23. A completed check means that code could be executed at least once using the provided instructions, and, therefore, all code and data was given and could be investigated more deeply or extended in the future. Ideally, this is a “one click” step, but achieving this requires particular skills and a sufficient level of documentation for third parties. Furthermore, automation may lead to people gaming the system or reliance on technology, which can often hide important details. All such aspects can reduce the understandability of the material, so we estimate our approach to codechecking, done without automation and with open human communication, to be a simple way to ensure long-term transparency and usefulness. We acknowledge that others have argued in favour of bitwise reproducibility because, in the long run, it can be automated (e.g., https://twitter.com/khinsen/status/1242842759733665799), but until then we need CODECHECK’s approach.\n\n5. Open by default and transitional by disposition.\n\nUnless there are strong reasons to the contrary (e.g., sensitive data on human subjects), all code and data, both from author and codechecker, will be made freely available when the certificate is published. Openness is not required for the paper itself, to accommodate journals in their transition to Open Access models. The code and data publication should follow community good practices. Ultimately we may find that CODECHECK activities are subsumed within peer review.\n\n\nImplementation\n\nTo date we have created 25 certificates (Table 1) falling into three broad themes: (1) classic and current papers from computational neuroscience, (2) COVID-19 modelling preprints, and (3) GIScience.\n\nAn interactive version is available at http://codecheck.org.uk/register.\n\nThe first theme was an initial set of papers used to explore the concept of CODECHECK. The idea was to take well-known articles from a domain of interest (Neuroscience). Our first CODECHECK (certificate number 2020-001) was performed before publication on an article for the journal GigaScience, which visusalized the outputs from a family of supervised classification algorithms.\n\nThe second theme was a response to the COVID-19 pandemic, selecting papers that predicted outcomes. The checks were solicited through community interaction or by our initiative rather than requested from journals. Some certificates were since acknowledged in the accepted papers24,25. In particular, we codechecked the well-known Imperial college model of UK lockdown procedures from March 2020, demonstrating that the model results were reproducible26,27.\n\nThe third theme represents co-author DN’s service as a Reproducibility Reviewer at the AGILE conference series, where the Reproducible AGILE Initiative28 independently established a process for reproducing workflows at the AGILE conference series29. While using slightly different terms and infrastructure (“reproducibility reports” are published on the Open Science Framework instead of certificates on Zenodo) AGILE reproducibility reviews adhere to CODECHECK principles. A few checks were also completed as part of peer reviews for GIScience journals.\n\nAfter running the workflow, the codechecker writes a certificate stating which outputs from the original article, i.e., numbers, figures or tables, could be reproduced. This certificate is made openly available so that everyone can see which elements were reproduced and what limitations or issues were found. The certificate links to code and data used by the codechecker, allowing others to build on the work. The format of the certificates evolved during the project, as we learnt to automate different aspects of the certification. The metadata is stored in a machine-readable structured file in YAML, the CODECHECK configuration file codecheck.yml. The technical specification of the CODECHECK configuration file is published at https://codecheck.org.uk/spec/config/latest/. The configuration file enables current and future automation of workflows and meta-analyses.\n\nFigure 4 shows pages 1–4 (of 10) of an example certificate to check predictions of COVID-19 spread across the USA51,52. Figure 4A shows the certificate number and its DOI, which points to the certificate and any supplemental files on Zenodo. The CODECHECK logo is added for recognition and to denote successful reproduction. Figure 4B provides the key metadata extracted from codecheck.yml; it names the paper that was checked (title, DOI), the authors, the codechecker, when the check was performed, and where code/data are available. Figure 4C shows a textual summary of how the CODECHECK was performed and key findings. Figure 4D (page 2 of the certificate) shows the outputs that were generated based on the MANIFEST of output files in the CODECHECK. It shows the file name (Output), the description stating to which figure/table each file should be compared in the original paper (Comment), and the file size. Page 3 of the certificate, Figure 4E gives detailed notes from the codechecker, here documenting what steps were needed to run the code and that the code took about 17 hours to complete. Finally, page 4 of the certificate shows the first output generated by the CODECHECK Figure 4F. In this case, the figure matched figure 4 of 52. The remaining pages of the certificate show other outputs and the computing environment in which the certificate itself was created (not shown here).\n\nWe use freely available infrastructure, GitHub and Zenodo, to run our system. The codecheckers GitHub organisation at https://github.com/codecheckers contains projects for managing the project website, the codecheckers community and its discussions, code repositories, and the main register of CODECHECKs. Both the project website https://codecheck.org.uk/ and the register at https://codecheck.org.uk/register are hosted as GitHub pages. The register database is a single table in CSV format that connects the certificate identifier with the repository associated with a CODECHECK. Each of these repositories, which currently can be hosted on GitHub or Open Science Framework, contains the CODECHECK metadata file codecheck.yml. The register further contains a column for the type of check, e.g., community, journal, or conference, and the respective GitHub issue where communications and assignments around a specific check are organised. No information is duplicated between the register and the metadata files. The continuous integration infrastructure of GitHub, GitHub Actions, is used to automate generation of the register. Zenodo is our preferred open repository for storing certificates. It mints DOIs for deposits and ensures long-term availability of all digital artefacts related to the project. The CODECHECK community on Zenodo is available at https://zenodo.org/communities/codecheck/. It holds certificates, the regularly archived register78, and other material related to CODECHECK.\n\nA custom R package, codecheck, automates repetitive tasks around authoring certificates and managing the register. The package is published at https://github.com/codecheckers/codecheck under MIT license79. It includes scripts to deposit certificates and related files to Zenodo using the R package zen4R80 and for the register update process outlined above. Codecheckers can ignore this package, and use their own tools for creating and depositing the certificate. This flexibility accommodates different skill sets and unforeseen technical advances or challenges.\n\nThese tools and resources demonstrate that a CODECHECK process can be managed on freely available platforms. Automation of some aspects may improve turnaround time. Our main resource requirements are the humans needed for managing the project and processes and the codecheckers. All contributions currently rely on (partly grant-based) public funding and volunteering.\n\n\nRelated work\n\nThe journal ACM Transactions on Mathematical Software (TOMS) recently established a “Replicated Computational Results” (RCR) review process81, where “replicable” is the same as our use of “reproducible”. Fifteen RCR Reports have been published so far (search on https://search.crossref.org/ with the term \"Replicated Computations Results (RCR) Report\" on 2020-12-10). and the process is being extended extended to the ACM journal Transactions on Modeling and Computer Simulation. The TOMS RCR follows CODECHECK principles 1–4, although our work was independently developed of theirs. The TOMS editorial81 shares similar concerns about selection of reviewers, as we discussed above. Unlike existing CODECHECK certificates, the RCR reports undergo editorial review. Publication of the RCR report recognises the efforts of the reproducing person, while the potential for this motive to be a conflict of interest is acknowledged. TOMS also recognises reviewer activity in a partnership with Publons (see https://authors.acm.org/author-services/publons).\n\nThis activity in the ACM journals can be seen as one possible workflow within a CODECHECK system, and clearly shares much in spirit. CODECHECK, however, specifically aims to give codecheckers recognition as reviewers. In our view, the reviewer role removes the possible conflict of interest while keeping the public acknowledgement. Specific to the field of mathematics, the RCR is also expected to apply a review of the software itself if the system it runs on cannot be evaluated by an independent party. The TOMS RCR creators concur with the importance of communication, expect collaboration between author and RCR reviewers, share the considerations around reviewer selection, and also put trust in reviewer judgement over numerical bit-wise perfection. A key difference is that for TOMS RCR, authors opt-in with an RCR Review Request and the RCR reports are published in the TOMS journal next to the actual papers.\n\nSeveral journals provide special article types for reproductions of published papers. Information Systems has an invitation only Reproducibility Section for articles describing the reproducibility efforts of published articles, which are co-authored by the original authors and the reproducibility reviewer(s) (see https://www.elsevier.com/journals/information-systems/0306-4379/guide-for-authors).\n\nNature Machine Intelligence recently introduced a new type of article, the reusability report82. Inspired by the detailed and nuanced submissions to a reproducibility challenge, the reusability report focuses on the exploration of robustness and generalizability of the original paper’s claims82. This answers the specific community’s challenges around computational reproducibility and also values these kinds of contributions as independent publications, which goes beyond the goals of CODECHECK. The journal Cortex has a special article type Verification Reports, which are actually about replication of results and are very well designed/reasoned83. In a similar vein, the CODECHECK certificates could also be published as a special article type within journals.\n\nGoing beyond individual articles, the journal ReScience C publishes only replications, also requiring open code and replication by a third party. ReScience also relies on free infrastructure (GitHub and Zenodo).\n\nFor research with high stakes, where reproduction would be too weak and post-publication replication possibly too late because of policy impact, Benjamin-Chung et al.84 propose internal replication. A workflow that has undergone internal replication would likely be of high quality and relatively easy to check. Similarly, internal CODECHECKs may be used, with the same limitations such as group think84, to ensure reproducibility before submission. Such internal checks are professionalised in local reproduction services, such as CISER R-squared or YARD, or in communities such as Oxford’s code review network.\n\nGavis and Donoho85 propose a new discipline and infrastructure for reproducible computational research. Their specific packaging format, provenance record, and cryptographic Verifiable Result Identifier would indeed provide excellent reproducibility. However, the system is also complex and since its creation in 2011 we are not aware of any publisher using it; also, the system is not open source. In comparison, CODECHECK is less powerful but also much more flexible and less dependent on specific tools or infrastructure. If data and code are deposited properly, i.e., very unlikely to disappear, then the certificate’s DOI is practically close to the cryptographic identifier.\n\nAnother platform for publishing results of reproductions is SciGen.Report. It is a community-run independent platform to foster communication on reproducibility. People can report on fully, partially, or failed reproductions of articles after publication.\n\nCODECHECK is uniquely designed to be adopted across journals or events and to build a community of codecheckers. CODECHECK shares its interdisciplinary nature with other community initiatives concerned with reproducibility awareness, education, and support, such as ReproHack, Code Copilot, or Papers with Code. The latter recently announced a collaboration with the preprint server arXiv on providing data and code supplements for machine learning manuscripts and runs a reproducibility challenge. Likewise, different disciplines and journals provide reproducibility checklists, e.g., science and engineering86 or GIScience87, which naturally share some aspects while addressing particularities as well as addressing researchers from different fields. Regarding the education and guidance for authors, we see CODECHECK’s role as referencing and linking educational efforts and helpful material, not as creating and maintaining such content.\n\n\nLimitations\n\nIsn’t CODECHECK what peer review should be doing already? On the surface, yes, but peer reviewers are overburdened enough and asking them to do more work around peer review is not likely to succeed. When an editor (Tsuyoshi Miyakawa) requested raw data from n=41 authors before reviewing, 21 authors withdrew their manuscripts; 19 of the 20 remaining articles were rejected after peer review88. Such basic checks require effort from editors, yet they only rely on the availability of data files and the content of the paper. These availability checks can be enhanced by having more complex CODECHECKs request the code and then execute it. This might fall within idealistic expectations of peer review, but is rare. Establishing a CODECHECK process acknowledges that peer reviewing practices have been unable to adapt to the challenges of computational papers. The concept of a CODECHECK, just as the concepts of reproducible research and Open Science, may be transitional by nature. If the activities described here as being part of a CODECHECK are integrated into the publication process the initiative will have succeeded.\n\nShould CODECHECK requirements be more demanding? CODECHECK by design does not require authors to provide (and sustain) an eternally functional workflow nor suggests a specific software stack or practical approach. Creating something that anyone can reproduce has been called a fool’s errand and we tend to agree. However, the package of data, code, and documentation collaboratively created by authors and codecheckers is a snapshot of a working analysis that greatly increases the likelihood of a successful reproduction and the possibility that a workflow can be extended by third parties in the future, if they have access to suitable resources and matching skill set. Concrete implementations of CODECHECK workflows, especially for specific disciplines, may reify much more helpful guidelines for authors on how to create reproducibility packages. Our author-friendly “low bar” should not stay low forever, but cultural change takes time and the encouragement and guidance that CODECHECK, as part of the widely accepted peer review concept, can provide may eventually allow the bar to be raised much higher, e.g., with executable research compendia89, “Whole Tales”90, or continuous analysis91. However, considering that missing artefacts and lack of documentation have repeatedly been identified as key barriers to reproducibility (e.g., 29,92), we would not underestimate the power of a simple check. For example, ModelDB curation policies require that only one figure need be manually reproduced93, but that has not limited the usefulness nor success of the platform.\n\nA codechecker does not fulfil the same role as a statistical reviewer, as it is applied by some journals in the biomedical domain (cf. 94,95). The statistical reviewer evaluates the appropriateness of statistical methods95 and can support topical reviewers if, e.g., complex methods or sophisticated variants of statistical tests are applied94. The codechecker may go equally deep into the review, but only if they have the expertise and time. We can imagine a tiered workflow where a codechecker could, just as a conventional reviewer could, recommend a detailed code review (see next paragraph) to the editor if they come upon certain issues while examining the work.\n\nA codechecker does not conduct a code review. Code reviews are valuable to improve reproducibility and reusability, and their proponents even believe they can improve the research96. Code reviews, however, have quite different structural challenges and require even more resources. That said, a well-reviewed codebase is likely to be easier to codecheck, and the awareness of high-quality code raised through CODECHECK may lead to more support for code reviewing. Initiatives and journals that conduct software reviews independent of a specific publication or workflow include ROpenSci, PyOpenSci, and JOSS. Furthermore, the codechecker’s task list is intentionally not overloaded with related issues such as ensuring proper citation of data and software or depositing material in suitable repositories. Nevertheless, codecheckers are free to highlight these issues.\n\nHow are failures during checks handled? We do not yet have a process for denoting if a reproduction fails, as our case-studies were all successful. In the case that a journal adopts CODECHECK for all submissions, the question remains as what to do if a check fails, after exhausting efforts between author and codechecker to reproduce the workflow. A negative comment in a CODECHECK certificate or a failed check does not necessarily mean the paper or research is bad (cf. discussion on negative comments in 17). We doubt that publicly reporting failures (i.e., the code would not run) will increase overall reproducibility, and may prohibit authors from sharing their work, which is always more desirable than nothing shared. Therefore, we recommend sharing interim reproduction efforts only with the authors, even if that means that volunteer efforts may go unnoticed if no certificate is published. Rosenthal et al.97 discuss such incentives for different actors around the implementation of reproducibility. We see CODECHECK as one way for organisations to invest in reproducibility by creating incentives until reproducible computations become the norm.\n\nWho will pay for the compute time? For papers that take significant compute time (days, not minutes), it is unclear who will pay for it. One must carefully consider the sustainability of rerunning computations and the environmental impact large calculations, such as training machine learning models, have. A pragmatic workaround is to request that authors provide a “toy” example, or small dataset that can be quickly analysed to demonstrate that the workflow runs correctly.\n\nWhat about my proprietary software and sensitive data? Given the prevalence of proprietary software, e.g MATLAB, we pragmatically decided that we should accept code as long as we could find a machine with suitable licences to run it. However, this prohibits us from using open infrastructure for reproducibility (cf. 10,98) and requires the codechecker to have access to that particular software. Non-open software also considerably hampers reuse, especially by researchers from the global south. Therefore, allowing proprietary software is a compromise that should be reconsidered.\n\nSolutions for proprietary and sensitive data exist. Authors can provide synthetic data (cf. 99), some data can effectively be redacted100, and publishers or independent entities can provide infrastructure for sharing data and workflows confidentially101 or with access to derived results but not raw data99, i.e., data enclaves102, or domains of reproducibility103.\n\nCan’t someone cheat? Yes. We simply check that the code runs, not that is correct or sound science. This “mechanical” test is indeed a low bar. By having code and data openly deposited, third parties can later examine the code, and we hope that knowing the code will be open ensures that authors will not cheat. It also allows researchers, potentially with new methods, to look for errors. This is more effective than engaging in an arms race on building methods to detect malicious intent now with closed datasets and code. This is analogous to storing blood samples of sport champions today to possibly detect doping in the future with more sensitive methods (cf. 104). Another comparison that helped us define the scope of a CODECHECK is that we think of the codechecker as forensic photographer, capturing details so that an investigator may later scrutinise them.\n\nWho’s got time for more peer review? Agree; codechecking takes time that could otherwise be used for traditional peer review. However, a CODECHECK is different from peer review. First, the technical nature of a CODECHECK sets clear expectations and thereby time budget compared to conventional peer review. For example, authors are told what to provide and the codechecker can be told when to stop. Codecheckers can always directly ask the author when clarification is required, thereby increasing efficiency. Second, the specific skill set of a codechecker allows for different groups to participate in the review process. ECRs might be attracted to learn more about recent methods, peer review, and reproducibility practices. Research Software Engineers who might not regularly be involved in writing or reviewing papers might be interested in increasing their connection with scholarly practices. An extra codechecker may simplify the matchmaking an editor does when identifying suitable reviewers for a submission, as technical and topical expertise can be provided by different people. Third, recall that CODECHECKs should always be publicly available, unlike peer review reports. With code and workflows, the codechecker’s feedback may directly impact and improve the author’s work. The public certificates and contributions provide peer recognition for the codechecker. Fourth, we found that focusing on workflow mechanics and interacting with the author makes reproductions educational. It also is a different role and, as such, could be a welcome option for researchers to give back their time to the community.\n\nWhile such benefits are also part of idealistic peer review, they are mostly hidden behind paraphrased anonymous acknowledgement.\n\nDo workflows need to be codechecked multiple times? If a paper is checked at the start of peer review, it might need re-checking if the paper is modified during peer review. This is inevitable, and happened to us51. This is desirable though, if interactions between author, reviewer, and codechecker led to improvements. Checking the manuscript the second time is likely to be much less work than the first time.\n\nWhat does it mean for a figure to be reproducible? Automatically detecting if a codechecker’s results are “the same” as an author’s is more challenging than it might appear. That is why we do not require results to be identical for a CODECHECK to pass but simply that the code runs and generates output files that the author claims. Stochastic simulations mean that often we will get different results, and even the same versions of libraries can generate outputs that differ by operating system105. While reproducibility practices can mitigate some of these problems, e.g., by using a seed, the flexibility of the human judgement is still needed, rather than bitwise reproducibility. The codechecker is free to comment on visible differences in outputs in their report.\n\nShouldn’t the next step be more revolutionary? CODECHECK’s approach is to acknowledge short-comings around computational reproducibility and to iteratively improve the current system. It remains to be proven whether this approach is welcomed broadly and if involving publishing stakeholders helps to further the cause. We have discussed more stringent rules at length, e.g. only considering fully free and open source software, diamond Open Access journals, but we eventually decided against them on the level of the principles. For the CODECHECK community process, documented at https://codecheck.org.uk/guide/community-process, and the volunteer codechecker community, these requirements can be reconsidered.\n\nWe have deliberated requiring modern technologies to support reproducibility (cf. 10), focusing instead on the human interface and the judgement of experienced researchers and developers as a more sustainable and flexible approach. All types of research can adopt CODECHECK due to its flexible design. CODECHECK could include automated scoring (e.g., 106), yet automation and metrics bear new risks. The focus of the CODECHECK principles on code execution allows journals and publishers to innovate on financial models and peer review practices at their own pace.\n\n\nConclusions and future work\n\nCODECHECK works — we have created a considerable number of certificates to demonstrate it. The creation of certificates and interactions with authors and editors shaped the principles and the workflow and also confirmed the approach taken. This result corroborates findings from similar evaluations of reproducible computational research in journals and conferences. CODECHECKs increase transparency of the checked papers and can contribute to building trust in research findings. The set of shared principles and common name, through recognition value, will allow researchers to judge the level of scrutiny that results have faced. CODECHECK requires direct acknowledgement of the codechecker’s contributions, not indirectly via citations of reproductions or informal credit.\n\nCODECHECK however harbours the same limitations as peer review in general and is closely connected to larger disruptions and challenges in scholarly communication7,107,108, including the tensions between commercial publishing and reviewers’ often free labour, and a global pandemic that has jumbled up academic publishing and exposed a broader general audience to preprints109. Establishing CODECHECK workflows must be seen as interconnected with much larger issues in research, such as broken metrics or malpractice triggered by publication pressure110,111. We certainly do not want the binary attribute of “code works” to become a factor in bibliometric approaches for performance assessments. While developed for the current “paper”-centric publication process, the CODECHECK principles would also work well with novel publication paradigms, e.g., peer-reviewed computational notebooks112, iterative and granular communication of research outputs, articles with live-code113 such as eLife’s ERA, decentralized infrastructure and public reviewer reputation systems114, and completely new visions for scholarly communication and peer review, such as described by Amy J. Ko in A modern vision for peer review. An explicit segmentation of research steps could even make the focus of a CODECHECK easier by only checking the “analysis” sub-publication. The discovery of CODECHECKs could be increased by depositing certificates into public databases of reproductions, such as SciGen.Report. Public researcher profiles, such as ORCID, may consider different types of reviewer activity to capture how independent code execution contributes to science. Notably, the discussed limitations are largely self-imposed for easier acceptance and evolutionary integration, as to not break the current system and increase demands gradually without leaving practitioners behind.\n\nA CODECHECK system, even if temporarily adopted as a sustainable transition towards more open publication and review practices, can contribute to increased trust in research outputs. Introducing CODECHECK should be informed by lessons learned from (introducing) open peer review15. Our conversations with publishers and editors indicate a willingness to adopt open practices like these, but that it is hard to innovate with legacy infrastructure and established practices.\n\nMore reproducible practices initiated by CODECHECKs could lead communities to reach a state where authors provide sufficient material and reviewers have acquired sufficient skills that peer reviewers will generally conduct a CODECHECK-level of checking; only in especially sophisticated cases will a specialised codechecker be needed. The main challenge for us remains getting journals to embrace the idea behind CODECHECK and to realise processes that conform to the principles, whether or not they use CODECHECK by name. We would be keen to use the flexibility of the principles and cooperate with journals to learn more about the advantages and yet unclear specific challenges – e.g do CODECHECKs really work better with open peer review? To facilitate the adoption, the CODECHECK badge is, intentionally, not branded beyond the checkmark and green colour and simply states “code works”.\n\nFuture CODECHECK versions may be accompanied by studies to ensure codechecking does not fall into the same traps as peer review did16 and to ensure positive change within the review system. This cultural change, however, is needed for the valuation of the efforts that go into proper evaluation of papers. Journals can help us to answer open questions in our system: What are crucial decisions or pain points? Can authors retract code/data once a CODECHECK has started? What variants of CODECHECKs will be most common? How will open CODECHECKs influence or codevelop with the scope and anonymity of conventional review over time?\n\nThe question of training codecheckers is also relevant. We expect a mentoring scheme within the CODECHECK community (experienced codecheckers will provide on-the-job training or serve as fall-back advisors). Codecheckers may also be found by collaborating with reproducible research initiatives such as ReproHack, ReproducibiliTea,115, and Repro4Everyone,116. The initial reaction of researchers to these ideas shows that scholarly peer review should continue on the path towards facilitating sharing and execution of computational workflows.\n\n\nData availability\n\nZenodo: codecheckers/register: CODECHECK Register Deposit January 2021 http://doi.org/10.5281/zenodo.448655978.\n\nThis project contains the following underlying data:\n\nregister.csv. List of all CODECHECK certificates with references to repositories and reports.\n\nData are available under the terms of the Creative Commons Attribution Share Alike license (CC-BY-SA 4.0 International).\n\n\nSoftware availability\n\nCodecheckers GitHub organisation: https://github.com/codecheckers\n\nCODECHECK community on Zenodo: https://zenodo.org/communities/codecheck\n\ncodecheck R package: https://github.com/codecheckers/codecheck\n\nArchived R package as at time of publication: http://doi.org/10.5281/zenodo.452250779\n\nLicense: MIT", "appendix": "Acknowledgements\n\nWe are grateful to the following individuals for discussions regarding the work presented here: Andy Collings, Melissa Harrison, Giuliano Maciocci, Naomi Penfold, Emmy Tsang (eLife), Rebecca Kirk (PLOS Computational Biology), Scott Edmunds (GigaScience), and Andrew Hufton (Scientific Data). Iain Davies and Yuhao (Sebastian) Wang developed code and example certificates. We thank Antonio Páez (Journal of Geographical Systems) for enabling CODECHECKs, Carlos Granell and Frank Ostermann for contributing certificates as reproducibility reviewers at the AGILE conference, and all authors of auditable workflows for their participation. We thank Celeste R. Brennecka from the Scientific Editing Service, University of Münster, for her editorial review.\n\n\nReferences\n\nMarwick B: How computers broke science – and what we can do to fix it. 2015. Reference Source\n\nBuckheit JB, Donoho DL: WaveLab and Reproducible Research. In Anestis Antoniadis and Georges Oppenheim, editors, Wavelets and Statistics. number 103 in Lecture Notes in Statistics, Springer New York, 1995; 55–81. Publisher Full Text\n\nClaerbout JF, Karrenbach M: Electronic documents give reproducible research a new meaning. In SEG Technical Program Expanded Abstracts 1992. SEG Technical Program Expanded Abstracts, Society of Exploration Geophysicists, 1992; 601–604. 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Publisher Full Text\n\nLarisch R: [Re] Connectivity reflects coding a model of voltage-based STDP with homeostasis. ReScience C. 2019; 5(3). Publisher Full Text\n\nEglen SJ: CODECHECK certificate 2020-006. Zenodo. 2020. Publisher Full Text\n\nDetorakis G: [Re] A Generalized Linear Integrate-And-Fire Neural Model Produces Diverse Spiking Behaviors. ReScience C. 2017; 3(1): #7. Publisher Full Text\n\nEglen SJ: CODECHECK certificate 2020-007. Zenodo. 2020.\n\nHathway P, Goodman DFM: [Re] Spike Timing Dependent Plasticity Finds The Start Of Repeating Patterns In Continuous Spike Trains. ReScience C. 2018; 4(1): #6. Publisher Full Text\n\nEglen SJ: CODECHECK certificate 2020-008. Zenodo. 2020. Publisher Full Text\n\nDavies NG, Kucharski AJ, Eggo RM, et al.: Effects of non-pharmaceutical interventions on COVID-9 cases, deaths, and demand for hospital services in the UK: a modelling study. 2020. Reference Source\n\nEglen SJ: CODECHECK certificate 2020-009. Zenodo. 2020. Publisher Full Text\n\nKucharski AJ, Klepac P, Conlan AJK, et al.: Effectiveness of isolation, testing, contact tracing and physical distancing on reducing transmission of sars-cov-2 in different settings: a mathematical modelling study. 2020. Reference Source\n\nFerguson N, Laydon D, Nedjati Gilani G, et al.: Report 9: Impact of non-pharmaceutical interventions (NPIs) to reduce COVID19 mortality and healthcare demand. 2020. Publisher Full Text\n\nEglen SJ: CODECHECK certificate 2020-011. Zenodo. 2020. Publisher Full Text\n\nFlaxman S, Mishra S, Gandy A, et al.: Estimating the effects of non-pharmaceutical interventions on COVID-19 in Europe. Nature. 2020; 584(7820): 257–261. PubMed Abstract \| Publisher Full Text\n\nEglen SJ: CODECHECK certificate 2020-012. Zenodo. 2020. Publisher Full Text\n\nUnwin H, Mishra S, Bradley VC, et al.: Report 23: State-level tracking of COVID-19 in the United States. 2020. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNüst D: CODECHECK certificate 2020-016. 2020. Publisher Full Text\n\nBrunsdon C, Comber A: Opening practice: supporting reproducibility and critical spatial data science. J Geogr Syst. 2020. Publisher Full Text\n\nNüst D: CODECHECK certificate 2020-017. 2020. http://www.doi.org/10.5281/zenodo.4003848\n\nBivand RS: Progress in the r ecosystem for representing and handling spatialdata. J Geogr Syst. 2020. Publisher Full Text\n\nNüst D: Reproducibility review of: Integrating cellular automata and discrete global grid systems: a case study into wildfire modelling. 2020. Publisher Full Text\n\nHojati M, Robertson C: Integrating cellular automata and discrete global grid systems: a case study into wildfire modelling. AGILE: GIScience Series. 2020; 1: 1–23. Publisher Full Text\n\nNüst D, Granell C: Reproducibility review of: What to do in the meantime: A service coverage analysis for parked autonomous vehicles. 2020. Publisher Full Text\n\nIllium S, Friese PA, Müller R, et al.: What to do in the meantime: A service coverage analysis for parked autonomous vehicles. AGILE: GIScience Series. 2020; 1: 1–15. Publisher Full Text\n\nNüst D, Ostermann F: Reproducibility review of: Window operators for processing spatio- temporal data streams on unmanned vehicles. 2020. Publisher Full Text\n\nWerner T, Brinkhoff T: Window operators for processing spatio-temporal data streams on unmanned vehicles. AGILE: GIScience Series. 2020; 1: 1–23. Publisher Full Text\n\nOstermann F, Nüst D: Reproducibility review of: Comparing supervised learning algorithms for spatial nominal entity recognition. 2020. Publisher Full Text\n\nMedad A, Gaio M, Moncla L, et al.: Comparing super- vised learning algorithms for spatial nominal entity recognition. AGILE: GIScience Series. 2020; 1: 1-18. Publisher Full Text\n\nNüst D: Reproducibility review of: Extracting interrogative intents and concepts from geo-analytic questions. 2020. Publisher Full Text\n\nXu H, Hamzei E, Nyamsuren E, et al.: Extracting interrogative intents and concepts from geo-analytic questions. AGILE: GIScience Series. 2020; 1: 1–21. Publisher Full Text\n\nOstermann F, Nüst D: Reproducibility review of: Tracking hurricane dorian in gdelt and twitter. 2020. Publisher Full Text\n\nOwuor I, Hochmair HH, Cvetojevic S: Tracking hurricane dorian in GDELT and twitter. AGILE: GIScience Series. 2020; 1: 1–18. Publisher Full Text\n\nEglen SJ: CODECHECK certificate 2020-024. Zenodo. 2020. Publisher Full Text\n\nVieira DM, Fernandes C, Lucena C, et al.: Driftage: a multi-agent system for concept drift detection and an application on electromyography. Gigascience. (under review), 2020. Reference Source\n\nCarrer F, Kossowski TM, Wilk J, et al.: The application of Local Indicators for Categorical Data (LICD) to explore spatial dependence in archae- ological spaces. J Archaeol Sci. 2021; 126: 105306. 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PubMed Abstract \| Publisher Full Text\n\nNosek BA, Alter G, Banks GC, et al.: SCIENTIFIC STANDARDS. Promoting an open research culture. Science. 2015; 348(6242): 1422–1425. ISSN 0036-8075, 1095-9203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEarthCube: New EarthCube Peer-Reviewed Jupyter Notebooks. Now Available. 2020. Reference Source\n\nPerkel JM: Pioneering ‘live-code’ article allows scientists to play with each other’s results. Nature. 2019; 567(7746): 17–18. PubMed Abstract \| Publisher Full Text\n\nTenorio-Fornés A, Jacynycz V, Llop-Vila D, et al.: Towards a Decentralized Process for Scientific Publication and Peer Review using Blockchain and IPFS. 2019. ISBN 978-0-9981331-2-6. Accepted: 2019-01-03T00:29:12Z. Publisher Full Text\n\nFitzgibbon L, Brady D, Haffey A, et al.: Brewing up a storm: developing Open Research culture through ReproducibiliTea. Report. Central Archive at the University of Reading. 2020. Publisher Full Text\n\nAuer S, Haelterman N, Weissgerber T, et al.: Reproducibility for everyone: a community-led initiative with global reach in reproducible research training. OSF Preprints . 2020. Publisher Full Text" }	[ { "id": "82470", "date": "19 Apr 2021", "name": "Nicolas P. Rougier", "expertise": [ "Reviewer Expertise Computational Neuroscience", "Open Science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, authors propose to implement a procedure to check for the code accompanying a submission to a journal. To do so, they describe a pipeline made of 6 steps that ultimately lead to the delivery of a code check certificate meaning that someone external to the author's lab has managed to re-run the code. At this point, no checking that the results are correct is necessary. The authors already issued several codecheck certificates in different disciplines. I find the idea really nice and certainly necessary but I've a few questions (even though some of them are already addressed in the \"limitations\" section). Given the structure of the paper, I'll just list my questions here:\nHow does CODECHECK compare to ACM Artifact reviews badges? (https://www.acm.org/publications/policies/artifact-review-and-badging-current)\n\nWhat would be the incentive for someone to code check the code? Being aware of the increasing difficulty in finding reviewers, I don't think it would be easy to recruit people to perform a task that can rapidly become very technical and time consuming.\n\nHow do you handle the case when specific hardware is necessary (e.g. NVidia GPU)? Is it documented somewhere such that code-checkers might first verify if they have the necessary hardware to run the code?\n\nHow do you establish a check has failed? For example, what happens if a code-checker gets a segfault (for some unknown reason) and the author is unable to help. Is it deemed failed?\n\nWho will pay for the computing resources needed to run heavy simulations and/or to acquire necessary software such as e.g. Matlab? When a simulation consumes a lot of resources, it might wise to give the checker access to computing resources. This could be paid for by the journal.\n\nI did not see in the report example a description of the environment necessary to run the software. How did you solve the \"dependency hell\"? Since the code might break at some point in the future because of incompatibility in some libraries or environments, it would be necessary to have a mechanism describing the running environment such that it can be re-run later.\n\nWhat do you recommend if the reviews are both excellents but the code check failed? Does this mean the paper is blocked until code check passes or rejected or else?\n\nThe code check proposal is close to some extents to the Journal of Open Science Software where each reviewer is assigned a list of things to check during the review. Do authors consider this pipeline when establishing their own pipeline?\n\nTo what extent the codecheck certificate can be updated automatically via some kind of \"manual continuous-integration\"? I mean that when reading a paper online, would it be possible to click a button to test if the code still runs considering the latest versions of libraries? (for example, the certificate has been issued for Python 2 but I want to know if this is usable with Python 3).\n\nWhen you look at journals advertising open data policies, it is unfortunately not rare to find articles in these same journals without the actual data. Do you have some suggestion for educating editors to actually enforce the code check a journal adopt it?\n\nSome suggestions:\nThe badge that is delivered would need some time information since the check is valid at one point in time (with a given software stack) and does not guarantee future runs.\n\nFor specialized journals, you could consider to offer a common generic environment where a code could be first tested. It this fails, then you would need only to slightly modify the environment to add missing dependencies. For example, in neuroscience, a Neuro Debian would probably suit the needs of a large number of models.\n\n- As editor-in-chief of ReScience C, I would like to inform authors that the journal now accepts \"reproduction report\". The idea it to try to re-run the code accompanying a published article and to report if it succeeded or failed. Our own procedure to check for reproduction is not standardized and we'll certainly benefit from the code check initiative.\n\nOverall, it's nice to have a clean description of a pipeline to check for code even though some questions need to be addressed. Also, I'm not too confident that journals will adopt it immediately and I'm afraid such initiative will take time to be generalized. But we have to start somewhere.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6805", "date": "20 Jul 2021", "name": "Stephen Stephen", "role": "Author Response", "response": "> 1. How does CODECHECK compare to ACM Artifact reviews badges? > (https://www.acm.org/publications/policies/artifact-review-and-badging-current) These badges, introduced in August 2020, show whether code is available (different levels) and reproduces same results. In principle CODECHECKER could award these badges (artifacts evaluated, functional). We have made a note to this effect in the manuscript (Related work, end of paragraph 1.) > 2. What would be the incentive for someone to code check the code? > Being aware of the increasing difficulty in finding reviewers, I > don't think it would be easy to recruit people to perform a task > that can rapidly become very technical and time consuming. This was addressed in our section \"Who's got time for more peer review?\" We would however note that we have a pool of about 20 volunteers currently willing to do codechecks. > 3. How do you handle the case when specific hardware is necessary > (e.g. NVidia GPU)? Is it documented somewhere such that > code-checkers might first verify if they have the necessary hardware > to run the code? This was handled in the limitation \"What about my proprietary software and sensitive data.\" but we now mention hardware too in the first paragraph of that section. > 4. How do you establish a check has failed? For example, what > happens if a code-checker gets a segfault (for some unknown reason) > and the author is unable to help. Is it deemed failed? We hope that codechecker and author can resolve problems, but in the end there may be problems that cannot be solved. Open infrastructure could help as both author and codechecker can work together in the same environment to minimise these failures. Ultimately however, there may be failures, which are noted in the section \"How are failures during checks handled?\". > 5. Who will pay for the computing resources needed to run heavy > simulations and/or to acquire necessary software such as > e.g. Matlab? When a simulation consumes a lot of resources, it > might wise to give the checker access to computing resources. This > could be paid for by the journal. In the section \"Who will pay for compute time?\" we mention this problem, and that toy examples might alleviate the need to re-run resource-intensive computations. We agree that one model might be that a journal provide some resource for this service. Likewise, in the following paragraph, we describe that our pragmatic approach for now is to find codecheckers that have access to particular software, e.g. MATLAB. > 6. I did not see in the report example a description of the > environment necessary to run the software. How did you solve the > \"dependency hell\"? Since the code might break at some point in the > future because of incompatibility in some libraries or environments, > it would be necessary to have a mechanism describing the running > environment such that it can be re-run later. The short answer is \"we didn't\". In the paragraph \"Should CODECHECK requirements be more demanding?\" we note our low bar of simply getting a codecheck to run once. We do, however, encourage CODECHECKERS to describe the environment in free text form in their report. Moving towards machine-readable descriptions would be a natural extension. > 7. What do you recommend if the reviews are both excellents but the code > check failed? Does this mean the paper is blocked until code check > passes or rejected or else? This is up to the editor of the journal -- see the \"Importance\" dimension of Figure 3. At one end, it could indeed be a \"strict requirement\" to get a codecheck certificate for the paper to be accepted. On the other hand, it could be entirely optional. > 8. The code check proposal is close to some extents to the Journal > of Open Science Software where each reviewer is assigned a list of > things to check during the review. Do authors consider this pipeline > when establishing their own pipeline? We have not considered this pipeline, nor do we have an explicit idea. We now note this reviewer list at the end of the third paragraph of \"Related Work\". > 9. To what extent the codecheck certificate can be updated > automatically via some kind of \"manual continuous-integration\"? I > mean that when reading a paper online, would it be possible to click > a button to test if the code still runs considering the latest > versions of libraries? (for example, the certificate has been > issued for Python 2 but I want to know if this is usable with Python > 3). To follow on from point 6, this would make a natural extension, but for now we are still considering one point in time, and keeping the requirements as close to the authors as we can. > 10. When you look at journals advertising open data policies, it is > unfortunately not rare to find articles in these same journals > without the actual data. Do you have some suggestion for educating > editors to actually enforce the code check a journal adopt it? We share this concern, and unfortunately have no simple suggestions for helping editors. At this early stage, we think the approach should be one of encouraging uptake, rather than mandating it. We also hope that having specific in-house experience, e.g. editorial staff to examine for code and data availability, can note this. But at the end of the day, this again is dependent on the journal's workflow. > 11. The badge that is delivered would need some time information > since the check is valid at one point in time (with a given software > stack) and does not guarantee future runs. Great idea. we could add the certificate number to the URL, or add the certificate number. We will try to implement this when revising our workflows. Nevertheless, the point in time and software stack should be documented via the certificate already now. > 12. For specialized journals, you could consider to offer a common > generic environment where a code could be first tested. It this > fails, then you would need only to slightly modify the environment > to add missing dependencies. For example, in neuroscience, a Neuro > Debian would probably suit the needs of a large number of models. Yes. We will certainly bear this in mind in future work, especially for author guidelines. > 13. As editor-in-chief of ReScience C, I would like to inform > authors that the journal now accepts \"reproduction report\". The idea > it to try to re-run the code accompanying a published article and to > report if it succeeded or failed. Our own procedure to check for > reproduction is not standardized and we'll certainly benefit from > the code check initiative. Thank you for noting this. We now mention the reproduction report in the manuscript where we describe Rescience C. > Overall, it's nice to have a clean description of a pipeline to > check for code even though some questions need to be > addressed. Also, I'm not too confident that journals will adopt it > immediately and I'm afraid such initiative will take time to be > generalized. But we have to start somewhere. We share your realistic assessment that (a) journals may be slow to adopt but that (b) we should start somewhere." } ] }, { "id": "82472", "date": "21 Apr 2021", "name": "Sarah Gibson", "expertise": [ "Reviewer Expertise As a Research Software Engineer", "I don't have a specific area of research any more. I have skills and expertise in software best practices", "computational reproducibility and cloud computing infrastructure", "which I have gained through the open source communities Project Binder (running mybinder.org) and The Turing Way (a pedagogical resource which includes a volume on reproducibility) alongside working on a range of projects within the Alan Turing Institute." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPaper Summary This paper outlines a set of principles and a community of practice for verifying computational analyses can be run and research artefacts reproduced as part of, or in addition to, traditional peer review processes. The ongoing scientific reproducibility crisis and current lack of many (or any) standards for checking computational research in the publishing industry makes this an important, new framework to share with the community. The authors demonstrate a deep and thoughtful knowledge of the cultural barriers surrounding such technological checks for peer review, such as time, expertise, and bitwise comparative reproducibility. They acknowledge that the specific incarnation of the CODECHECK practice outlined in this paper is limited to provide a low barrier for entry in order to encourage adoption, but do detail the scope in which such a workflow could be adapted and built upon to raise that bar and perform more stringent checks. Specifically, the principles are not technology-based to allow for flexibility in the complexity and domain of computational research to be checked. I particularly appreciated the authors’ recommendation/suggestion that CODECHECKs become a platform for engaging Early Career Researchers in the peer review process. Alongside CODECHECK’s own workflows (which are openly published on GitHub and Zenodo), the paper outlines many similar and related initiatives that fall within the CODECHECK framework providing a wealth of examples for the community to draw inspiration from when designing and applying their own CODECHECK workflows.\nIs the rationale for developing a new method clearly explained? The authors show a deep knowledge of the pitfalls of traditional peer review of static research artefacts and clearly identify and outline the rationale for a peer review-like system capable of assessing computation-based research.\nIs the description of the method technically sound? I’m going to answer a slightly different question of “Is the description of the method culturally sound?” This is because the authors have intentionally not provided a technological methodology for completing a CODECHECK so as to avoid vendor lock-in (e.g. cloud platform providers) and to provide flexibility for applying the methodology to a range of computational research domains. Instead, the focus of the methodology is on building a community of practice around having code mechanically checked by someone with comparable technical expertise from outside the project. The authors demonstrate a considerate knowledge of the burden of verifying computational reproducibility on both authors and peer reviewers and aim, not to increase this burden, but to provide an entry point into a world where checking research code can be run and produces the artefacts as they are presented in the paper is normalised. I think their recommended approach focussing on communication between codecheckers and authors, codecheckers will check and not fix, and codecheckers being an additional role to the traditional peer reviewer will aid early adoption of this framework.\nAre sufficient details provided to allow replication of the method development and its use by others? The concept of CODECHECK is intentionally presented as a set of principles and example workflows, as opposed to fixed, step-by-step actions, to allow for flexibility across computational complexity and research domains. The principles, example workflow, and potential variations under this framework are explained in depth and examples of workflows that fall under the CODECHECK framework from other publishers and/or conferences are provided, alongside CODECHECK’s own community. From this wealth of detail, I believe that others would be able to replicate, adapt and apply a CODECHECK-like workflow in their journal or community.\nAre the conclusions about the method and its performance adequately supported by the findings presented in this article? It is encouraging to see that the community feedback from authors and publishers shaped the workflow and principles that uphold CODECHECK and a number of certificates have already been issued under this framework. This shows that the workflow of a CODECHECK as outlined in the paper is achievable in partnership with current peer review operations. However, I would like to see the impact of the CODECHECK certificates issued. Is there any community feedback on the transparency and reusability of research published with CODECHECK certificates? This is perhaps too big of an ask this early in the initiative as research reuse and citations are independent factors of the publication and peer review of this specific paper - but I’d still be interested in any insights the authors have to offer on this topic.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6806", "date": "20 Jul 2021", "name": "Stephen Stephen", "role": "Author Response", "response": "> 1. Is there any community feedback on the transparency and reusability of research published with CODECHECK certificates? This is an excellent question. It is perhaps too early for us to assess this given most certificates are under a year old, but we are not aware of any reuse yet of the codecheck-deposited material. However, we certainly think it interesting to try and monitor this over a longer (3-5 year) timescale if possible. As well as looking for citations of certificates, we could also check for forks of our repositories, and download statistics from Zenodo. We note this as a good closing point for our article." } ] } ]	1	https://f1000research.com/articles/10-253
https://f1000research.com/articles/10-601/v1	19 Jul 21	{ "type": "Brief Report", "title": "Experiences of Hijra (transgender) communities during the COVID-19 pandemic in Bangladesh", "authors": [ "Faria Ahmed", "Ridwan Islam Sifat", "Faria Ahmed" ], "abstract": "In this paper, we study the transgender or Hijra communities to recognize and discuss the ongoing and long-term economic, mental, and emotional effects of lockdown on the most vulnerable who are worse off than daily wage earners. Hijras are a group of transgender people, non-binary and intersex women who have been assigned to be male at birth. In Bangladesh, they are deprived of basic human rights such as access to health care because of discrimination. Hijras in Bangladesh, especially during the national lockdown, have been adversely affected by the pandemic. There is a need for support and awareness to improve mental health awareness and eliminate stigma and prejudice. This article stands for the impact of COVID-19 on the Hijra or transgender community in Bangladesh.", "keywords": [ "COVID-19", "hijra", "transgender", "economic crisis", "health care" ], "content": "Introduction\n\nBangladesh’s population density, poor health care system, dysfunctional local government, and restricted civic education make it a huge concern to curb community transmission of the COVID-19 virus.1 The emphasis on individual actions makes it difficult for the poorest to obey national guidelines. In Bangladesh, the total affected as of 16 June 2021 is 837,247 people, according to the Bangladesh government’s official records. Bangladesh is the 32th most COVID-19 affected country globally, with a total of 13,282 deaths (Bangladesh COVID (worldometers.info)). The lockdown was introduced by western or developed economies with stronger economic bases and improved social safety nets.2 Bangladesh exposes the realities of lockdown for the poor and vulnerable. For many, the imminent survival kit is food and health comes later. Although the restrictions are there to save lives, the lockdown creates a whole new set of risks for others. With uncertain and limited incomes, the poor and vulnerable continue to struggle and prosper, displaying remarkable resilience.3\n\nIn Bangladesh, “transgender” are referred to by the historically adopted appellation “hijra”, which is often translated to mean transgender. People who identify as hijra are among those who are faced with more severe risks due to the nature of their work added to fear and prejudiced ideas against them.4,5 The objective of this study was to examine the ongoing and long-term economic, mental, and emotional effects of the COVID-19 lockdown on the hijra community people.\n\n\nMethods\n\nThis paper adopted qualitative methods, including interpretive approaches, content analysis and represented secondary data to get the possible research outcomes. The secondary data was collected from articles, newspapers, research studies, and websites. The interpretative approach was derived from a subjective perspective of imaging and in this paper, the imaging of the transgender person was studied. This paper used secondary survey data to triangulate the interpretative approach for verification and reliability for comparable results in a broad setting. We searched databases of organizational survey data, PubMed, Scopus and Web of Science for population-based original studies prior to March to June 2020. We included studies that are related to economic impact, psychology and also health impacts of the hijra people during the COVID-19 lockdown period. Data were obtained from Covid-19: Quick Survey for Community Response for transgender and hijra from the Bandhu Social Welfare Society. The survey was conducted among 80 community members covering all eight divisions of Bangladesh. Additionally, five in-depth interviews were conducted to understand the detailed situation. A total of 51 respondents participated in this rapid survey. Data were also used to understand the economic impact of the COVID-19 crisis on the third gender community in Bangladesh (innovision-bd.com). The data were analyzed in this paper in terms of theme, pattern, and perspective and also to compare and contrast the information and survey by linking them with the relevant literature. Finally, a sense-making approach6 was used for searching information or data and to obtain an objective understanding of the phenomena.\n\n\nResults\n\nFrom the quick survey for community response for transgender and hijra by the Bandhu Social Welfare Society, we found that 100% of transgender communities know about coronavirus, 80% wash their hands, 47.5% wear masks, 21.3% use hand sanitizer, 15% use gloves, only 8.8% eat a balanced diet and shield themselves while sneezing, and even less, that is 2.5%, keep themselves clean (https://www.bandhu-bd.org). Additionally, 71.3% of respondents knew that they need treatment if coronavirus infects someone, and 41.3% think they must be alone at home. Also, 26.3% of respondents call the government helpline for medical services, and 3.8% received help from the police.\n\nA significant aspect of the pandemic response is initiatives that bridge the divide between vulnerable groups and the government. Common income-generating practices for disadvantaged communities have evaporated with the nation on lockdown.7 A rapid perception survey8 showed no food stored at home for 18% and 10% of urban and rural respondents respectively. Extended shutdowns and the added stress of the pandemic intensified desperation and hopelessness.8 To counter the extreme economic and industrial fallout from the pandemic, Bangladesh has introduced an economic stimulus plan. The assistance package was inefficiently administered for the weakest.8\n\nDuring a crisis, many transgender people cannot get help because they are not publicly identified. From the Bandhu Social Welfare Society survey, 74% of the transgender respondents received packages of rice, pulses, oil, etc. and 14% faced gender discrimination while receiving the aid (https://www.bandhu-bd.org). In addition, 61% of respondents needed food, 59% needed money, 20% needed a hand washing agent, 11% needed a face mask, 9% needed safe and secure work, and medicine, and finally, 8% needed personal protective equipment. They are experiencing financial difficulties due to a lack of or limited access to social protection. Due to lockdown, they do not have access to ensure shelter and proper sanitation. They cannot afford hand sanitizers and masks in this kind of crisis. The authorities seem to remain blind to the miseries of the hijra community.4 According to a survey on the impact of COVID-19 on hijra or transgender communities, 82% of respondents had earned no money in two weeks, and 59% had received no assistance from aid programs or families (innovision-bd.com). Members of the hijra community typically make money by requesting voluntary donations in return for their good wishes.5 However, many of them have lost their income from alms collection, religious ritual engagement, sex work, and lockdowns, worsening their difficulties.4\n\nWhile several public and private organizations have provided support services and assistance programs to help financially vulnerable people, it is feared that such programs will not benefit transgender diverse people due to discrimination. However, a few citizen-led youth projects have supplied some relief to the hijra community.4,9 The aim is to raise money through crowdfunding platforms, raise public awareness about the community’s plight, raise money to source basic food supplies, or facilitate direct bank transfers to beneficiaries.7\n\nThe government, non-governmental organizations (NGOs), and voluntary organizations should work together to meet the needs of the hijra community, both socially and financially, to have proper access to their necessities during this pandemic.\n\nThe majority of transgender or hijras earned their money through street work and ritual work. They are working in informal workplaces, for example, as sex workers and in recreation. Hijras earn their livelihoods via a small range of available choices as they are still not socially accepted in daily employment.5 The lockdown meant they had all the doors locked as they were viewed as unclean and virus carriers. Therefore, shops and homes declined to support them.9 For sex workers, there were no customers. There is no community record of virus infections, but it is feared that social stigma has discouraged transgender people from obtaining medical care.10\n\nThe poorest who survive on daily wage labor show that economic mortality overshadows health mortality.3,11 In this pandemic, day-wage earners have struggled the most, but hijra people do not survive on a daily wage as they do get into jobs in the mainstream industries owing to their outdoor activities and lifestyle.11 Hijra groups have been accused as being carriers of the virus. Many hijra people are shunned by local elites and mosques and are left out of conventional support for relief (https://www.dhakatribune.com).\n\nEconomic impact transcending to mental health impact\n\nMarginalized societies around the world, including Bangladesh, are disproportionately affected by the COVID-19 crisis.3 With inadequate access to basic resources, minority groups such as Biharis and transgender people live on the fringes of society with a 95% decrease in daily income of transgender people, 71% have borrowed money, and 81% have a decreased diet (https://www.democracyspeaks.org/; https://www.bandhu-bd.org).\n\nThe average daily income of transgender people was Bangladesh taka (BDT) 296, and now it has dropped to BDT 14 (https://www.bandhu-bd.org). In a study we found 51 transgender people were surveyed on the economic impact of the COVID-19 crisis on the population in Bangladesh (innovision-bd.com). For the remaining crisis period, 100% of the respondents want food and monetary aid. During the economic slump, 86.36% need to postpone their loan payments due to low wages, 9.09% will borrow cash to repay a loan and 4.55% will sell their properties to repay a loan. Some are going to go to look for work, and some are going to start begging. A further 81% do not believe that funding from the government and individual private initiatives is going to continue (innovision-bd.com).\n\nBy contrast, hijra community people were previously aided by NGOs and community groups, which stopped during the lockdown. The transgender community cannot go for health care, diagnosis of sexually transmitted infections (STIs), and other illnesses amid COVID-19 due to the extreme strain on the Bangladesh medical service during the pandemic. Furthermore, many people create discomfort for them when they visit health care facilities as they are feared and disliked by larger communities.\n\nHijras are more vulnerable to mental health issues, anxiety, and depression due to the various and intersecting forms of discrimination they face. The pandemic’s severe economic and health consequences and increased transphobia could exacerbate the situation.8 Around 93% of hijra have anxiety due to uncertainty about food availability in the future, most survey respondents were psychologically anxious about money (94%) and food (68%), 16% endured mental abuse, but very few were exposed to physical torture and brutality (https://www.bandhu-bd.org).\n\nDiscrimination, stigma, and fear causing transgender people not to pursue physical health care\n\nTransgender people are viewed as having ill health and are seen as risky due to participation in sex work.12 They are apparent carriers of STIs. This results in social exclusion when they appear in public health services. In public or private hospitals and clinics, the doctors and the nurses sometimes feel uncomfortable treating hijra people. They are subject to violent acts from the staff, including physicians.5\n\nWhen it comes to medical services, this group of people is still disadvantaged and fails to gain access to our social system’s basic amenities due to discrimination. There is a distinction in fundamental health care. The medical infrastructure is still overburdened; testing and treatment for the transgender community are made more difficult by discrimination. Although the wards are divided into male-female categories, there is no center for transgender men and women.10\n\nRespondents from the hijra community alleged that they could not obtain medical health care due to discrimination, a lack of legal identity papers, and violations of patients’ rights in healthcare settings. Health care professionals fear and are stigmatized when it comes to accessing healthcare services, putting them at a higher risk of not being tested or monitored for COVID-19.4,10\n\n\nDiscussion\n\nFrom this study, we understand that there is a dire need to ensure an inclusive transgender awareness to remove the stigma and create a plan in line with their gender-based needs difference and demand for regulation and prevention of this coronavirus disease. Funding is needed for capacity building by community leaders. An increased communication platform has to be created such as community-based organizations (CBOs) for participation to mitigate the difference in information. The Ministry of Disaster Management and Relief can also collaborate with humanitarian aid initiatives and CBOs. As the transgender community’s main revenue-generating operations have been interrupted due to the coronavirus outbreak, the presence and adaptation of new Income Generating Activities (IGAs) and the introduction of alternative jobs have to be created to protect their means of survival. Besides, they need support from institutional funding such as banks and government support for entrepreneurial activities and empowerment to reduce their propensity to take loans directly from informal sources. They should be supported for mental and physical health practices via hotline numbers to ensure their access and use of services available to the public.\n\nFurthermore, it is salient to broaden aid by providing a transgender expert or social inclusion advisor to comprehend their needs. Reinforcement of the development of capability, and preparation for the ability can be discussed by experts or partnerships with other countries with diverse abilities. It is needed to build a welcoming policy or action to ensure fair access and respect during aid distribution. Separate budget allocation is required for the transgender and/or hijra community for their financial security in the forthcoming budget to protect them.\n\n\nConclusions\n\nMany people are living in a vulnerable state of life during the COVID-19 pandemic, and so are the hijras or transgender people. Sympathy is not exactly what one expects, but sensitivity is. This group of people is unnoticed when the world is busy talking about the pandemic’s effects on the poor, the middle-class, the industries, and the economy. They were a community that was heavily affected, too.4,10\n\nWith most of the nation in lockdown, Bangladesh is bracing for the effects of COVID-19. Some people have means to stock food in their homes. However, for many Bangladeshis, in a country where one in five lives below the poverty line, these precautions are unlikely. People who identify as hijra and/or transgender are among those who are acutely facing those risks. Many of these families, relying solely on revenue from their everyday jobs, live hand to mouth. The Ministry of Social Welfare should ensure enough funds and social safety net programs for the hijra community. In order to guarantee the prosperity of marginalized communities, the government must use a fair solution to resolve this crisis. For many Bangladeshis who consider themselves as hijra and/or transgender, profoundly rooted stigma also poses an obstacle to accessing critical health services.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nAl-Zaman MS: Healthcare Crisis in Bangladesh during the COVID-19 Pandemic. Am J Trop Med Hyg . 2020; 103(4): 1357–1359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSifat RI: Social Safety Net (SSN) Programs in Bangladesh: Issues and Challenges. J Soc Ser Resear . 2020; 1–3. Advance Online Publication. Publisher Full Text\n\nAhmed F, Sifat RI: The Impact of the COVID-19 Pandemic on the Mental Health of the Rickshaw-Puller in Bangladesh. J Loss Trauma. 2020; 1–8. Advance Online Publication. Publisher Full Text\n\nSifat RI: COVID-19 and mental health challenges among the hijra people in Bangladesh Int J Soc Psychiatry . 2020; 1–2. Advance Online Publication. PubMed Abstract \| Publisher Full Text\n\nSifat RI, Shafi FHY: Exploring the Nature of Social Exclusion of the Hijra People in Dhaka City. J Soc Ser Resear . 2020; 1–11. Advance Online Publication. Publisher Full Text\n\nTuryahikayo E: Using Sensemaking Technique to Construct Scientific Explanations in Organizational Research. Int J Philosop . 2019; 7(4): 167–172. Publisher Full Text\n\nChatterjee S, Basu S, Bhardwaj YA, et al.: The Health Crisis of Marginalized Populations during COVID-19 Pandemic: Challenges and Recommendations. Int J Soc Sci . 2020; 9(03): 185–191. Publisher Full Text\n\nRashid SF, Theobald S, Ozano K: Towards a socially just model: balancing hunger and response to the COVID-19 pandemic in Bangladesh. BMJ Global Health . 2020; 5: e002715. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkhter N: Transgender Realities in the Context of COVID-19 In Bangladesh. Eub J Asian Int Bioethic . 2021; 31(1): 35–36. Publisher Full Text\n\nSifat RI: The effect of COVID-19 on hijra (third gender) people in Bangladesh. Lancet Psychiatr . 2020; 7(12): 1015–1016. PubMed Abstract \| Publisher Full Text\n\nAhmed F, Sifat RI: Strategic assessment of mental health and suicide amid COVID-19 pandemic in Bangladesh. Int J Health Plann Manage . 2021; 1–6. Advance Online Publication. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan SI, Hussain MI, Parveen S, et al.: Living on the extreme margin: social exclusion of the transgender population (hijra) in Bangladesh. J Health Popul Nutr . 2009; 27(4): 441–451. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "150850", "date": "04 Oct 2022", "name": "Apurvakumar Pandya", "expertise": [ "Reviewer Expertise Public Mental Health", "Gender", "Sexuality and Health", "Health Promotion", "Counselling", "Health Technology Assessment", "Public Health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is relevant and important. However, it has many methodological and analytical weaknesses that should be addressed. In the Introduction, it is said that transgender people are referred to \"hijra\". Provide reference. Transgender is an umbrella term and multiple gender identities exist within that but it remains unclear in the context of Bangladesh. Kindly provide more details of Hijra community, how they live, where they live, what they do for a living, and their social status. Details of Transgender population, cultural identities, and legal status should be elaborated on for audiences from other countries. Further, polito-legal environment, people's attitudes towards transgender population must be elaborated. The justification of the study is missing. Statement of objective need to be revised: it states...\"to examine the on-going and long-term economic, mental and emotional effects of COVID-19 lockdown on the hijra community people.\"\nAs the study is qualitative, it would explore not examine (which is quite common when a quantitative approach is used). Second hijra community people phrase should be revised to hijra community.\nMethods: This section is very weak. Once it is said qualitative approach but later talks about rapid survey. It also talks about secondary data collection. It is confusing which method authors have adapted. It looks like a mixed method approach and authors must clarify its sequence and approach with proper reference.\nData analytic approach needs to be specified and steps must be articulated. Sense making approach was used but is not reflected in the results. Authors must expand on sense-making approach, and its steps. Ethical considerations for the study is missing.\nResults section needs to be more analytic. No specific themes (and sub-themes) are observed in the results section. It can be strengthened with verbatim. In text-citation should be avoided in the results section.\n\nThe discussion section does not compare and contrast current findings with existing literature. Not a single reference of existing literature is cited. This section must compare and contrast current findings, and discuss key results.\n\nConclusion: This write up is more aligned with discussion. Many of the recommendations are broad and out of the scope of the research findings. Finer nuanced understanding should be highlighted and recommendations should be specific. The recommendations section can be integrated into the discussion. There is a huge scope of recommendations for community based organizations.\nThe conclusion section summarizes key findings and the authors' conclusions based on findings should be represented.\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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-601
https://f1000research.com/articles/10-598/v1	19 Jul 21	{ "type": "Research Article", "title": "Whole-genome sequencing of SARS-CoV-2 in Uganda: implementation of the low-cost ARTIC protocol in resource-limited settings", "authors": [ "Gerald Mboowa", "Savannah Mwesigwa", "David Kateete", "Misaki Wayengera", "Emmanuel Nasinghe", "Eric Katagirya", "Ashaba Fred Katabazi", "Edgar Kigozi", "Samuel Kirimunda", "Rogers Kamulegeya", "Jupiter Marina Kabahita", "Moses Nsubuga Luutu", "Patricia Nabisubi", "Stephen Kanyerezi", "Bernard Ssentalo Bagaya", "Moses L Joloba", "Gerald Mboowa", "Savannah Mwesigwa", "David Kateete", "Misaki Wayengera", "Emmanuel Nasinghe", "Eric Katagirya", "Ashaba Fred Katabazi", "Edgar Kigozi", "Samuel Kirimunda", "Rogers Kamulegeya", "Jupiter Marina Kabahita", "Moses Nsubuga Luutu", "Patricia Nabisubi", "Stephen Kanyerezi", "Bernard Ssentalo Bagaya" ], "abstract": "Background: In January 2020, a previously unknown coronavirus strain was identified as the cause of a severe acute respiratory syndrome (SARS-CoV-2). The first viral whole-genome was sequenced using high-throughput sequencing from a sample collected in Wuhan, China. Whole-genome sequencing (WGS) is imperative in investigating disease outbreak transmission dynamics and guiding decision-making in public health.\nMethods: We retrieved archived SARS-CoV-2 samples at the Integrated Biorepository of H3Africa Uganda, Makerere University (IBRH3AU). These samples were collected previously from individuals diagnosed with coronavirus disease 2019 (COVID-19) using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). 30 samples with cycle thresholds (Cts) values <25 were selected for WGS using SARS-CoV-2 ARTIC protocol at Makerere University Molecular Diagnostics Laboratory.\nResults: 28 out of 30 (93.3%) samples generated analyzable genomic sequence reads. We detected SARS-CoV-2 and lineages A (22/28) and B (6/28) from the samples. We further show phylogenetic relatedness of these isolates alongside other 328 Uganda (lineage A = 222, lineage B = 106) SARS-CoV-2 genomes available in GISAID by April 22, 2021 and submitted by the Uganda Virus Research Institute.\nConclusions: Our study demonstrated adoption and optimization of the low-cost ARTIC SARS-CoV-2 WGS protocol in a resource limited laboratory setting. This work has set a foundation to enable rapid expansion of SARS-CoV-2 WGS in Uganda as part of the Presidential Scientific Initiative on Epidemics (PRESIDE) CoV-bank project and IBRH3AU.", "keywords": [ "COVID-19", "Makerere University", "Uganda", "SARS-CoV-2", "Whole-genome sequencing", "PRESIDE", "RT-PCR" ], "content": "Introduction\n\nThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) which has now spread throughout the entire world, causing more than 175 million infections and over 3.7 million deaths globally.1 During early 2020, whole-genome sequencing (WGS) enabled researchers to rapidly identify SARS-CoV-2, and knowing the genome sequence allowed rapid development of diagnostic tests and other appropriate tools needed for the response to this novel infection. Continued genome sequencing supports the monitoring of the disease’s spread, activity, viral evolution as well as emerging new viral variants.2 The COVID-19 pandemic is still ongoing and the global response will have to continue for the foreseeable future; the World Health Organization (WHO) has recommended WGS to be further adopted as well as implemented in new settings and new uses to better understand the world of emerging pathogens and their interactions with humans in a variety of climates, ecosystems, cultures, lifestyles, biomes2 and genetic backgrounds.\n\nUganda has had approximately 60,250 COVID-19 cases and 423 deaths by June 12, 2021.3 As more countries move to implement genome sequencing programmes, Uganda is among those embracing SARS-CoV-2 WGS. Of the 131 Uganda full SARS-CoV-2 genomes analysed in December 2020, 50 (38%) belonged to lineage A and the rest belonged to a variety of B lineages with the majority lineages being B.1 (N = 30; 23%) and B.1.5 (N = 17; 13%) which were found predominantly in cross border truck drivers seeking to enter the country.4 As of April 26, 2021, a total of 328 SARS-CoV-2 samples had been sequenced and deposited in the GISAID5 by the Uganda Virus Research Institute (UVRI).6 This represented 0.8% of the total COVID-19 cases that had been detected in the country at that time. This situation is very similar to almost all other African countries, yet this ongoing global pandemic has already demonstrated the importance of widespread access to rapid novel pathogen discovery and subsequent surveillance, as well as comprehensive pathogen information sharing.\n\nWe piloted sequencing of SARS-CoV-2 samples at the Molecular Diagnostics Laboratory located in the Department of Immunology and Molecular Biology, Makerere University for two reasons: (i) to test the feasibility of ARTIC amplicon-based genome sequencing at our local institution; and (ii) to extend genomic analyses for COVID-19 surveillance in Uganda. ARTIC protocol was selected due to its low cost and high sensitivity, as well as its scalability compared to other sequencing methods.7 Routine SARS-CoV-2 genome sequencing in many places still faces difficulties such as an unreliable supply-chain for WGS reagents, since many of these are imported from western countries, limited technical expertise as well as genomic infrastructure, and relatively high costs of genome sequencing. Consequently, this work also served as a feasibility study to assess the implementation, practicality, and adoption of ARTIC amplicon-based sequencing in Ugandan’s resource-limited settings, allowing future efforts to integrate and expand into routine laboratory diagnostic pipelines. This current study served as a proof-of-concept to extend genomic capacity for COVID-19 surveillance at Makerere University, College of Health Sciences (MakCHS) Molecular Diagnostic Laboratory. This laboratory has been performing SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) since March 2020 and was among the first facilities in Uganda to be accredited by the Ministry of Health to carry out routine SARS-CoV-2 testing. We sought to perform WGS of 30 SARS-CoV-2 RT-qPCR positive samples using the COVID-19 ARTIC v3 amplicon-based sequencing protocol in our settings using the Illumina MiSeq sequencing platform. We considered samples based on the criteria below;\n\nCycle thresholds (Cts) values below 25. This is because the ARTIC SARS-CoV-2 sequencing protocol produces longer and high-quality genomes with Ct values below 30.8\n\nWe sequenced samples collected after September 2020 to increase the chances of detecting any of the emerging circulating SARS-CoV-2 variants including the high mortality9 variant originally referred to as the UK variant or B.1.1.7, and the highly transmissible10 variant originally referred to as the South Africa variant or B.1.351/501Y.V2 which had been reported on 14 December and 18 December, 2020 respectively.11\n\nAll our sequenced isolates in this study were therefore selected from those archived samples collected after September 2020.\n\n\nMethods\n\nThe Integrated Biorepository of H3Africa Uganda (IBRH3AU) received ethical approval from Makerere University School of Biomedical Sciences Research and Ethics Committee (SBS-REC) and from the Uganda National Council for Science and Technology (UNCST) to collect, process, store, and share biospecimens including COVID-19 specimens. Additionally, the IBRH3AU obtained ethical approval from the Mulago National Hospital REC (Protocol Number MHREC 1868 and approved on March 27th, 2021). Participants consented in writing to sample storage and subsequent use of their samples in current and future studies related to understanding SARS-CoV-2 infection in Uganda.\n\nThis was a cross-sectional study design.\n\nSamples for this study included nasopharyngeal swabs collected from individuals who had a positive COVID-19 test at the Molecular Diagnostic Laboratory. COVID-19 samples processed at this facility were archived at the Integrated Biorepository of H3Africa Uganda – IBRH3AU. These samples were collected between September 2020 and February 2021 from individuals coming from Kampala metropolitan area, which consists of Kampala city itself and the neighboring Wakiso, Mukono, Mpigi, Buikwe and Luweero districts of Uganda.\n\nTotal nucleic acid was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) at the MakCHS Molecular Diagnostic Laboratory, as per the manufacturer’s protocol. All patient samples had initially been assessed by RT-qPCR for SARS-CoV-2 viral RNA using a triplex approach that targets the N, ORF and S viral genes. Therefore, this study used samples that were diagnostically SARS-CoV-2 positive with amplification of the targeted region(s) crossing the threshold before 25 PCR cycles. In total, 30 SARS-CoV-2 positive samples were selected randomly for high-throughput genome sequencing at our facility using a MiSeq Illumina platform.\n\nThese samples had been collected and previously stored from patients diagnosed with COVID-19 using real-time RT-qPCR. Metadata associated with the patient samples included the date of sample collection, gender, nationality, and purpose of testing (Routine, Contact, Alert, Travelers, Quarantine, Case, and Professional jobs requiring COVID-19 PCR test). ARTIC amplicon-based sequencing was used to generate 400 bp amplicons with 75 bp overlaps covering the length of the ~ 29.9 kB SARS-CoV-2 genome as described elsewhere.12 Briefly, cDNA synthesis was carried out with random primers (Protoscript II First Strand cDNA Synthesis Kit, E6560S) followed by PCR amplification using ARTIC primers. Genomic library preparation was carried out using the Nextera XT DNA Library Preparation kit (15032355) according to manufactures' recommendations, and sequencing was carried out on the Illumina MiSeq platform (Illumina, CA, USA) using MiSeq Reagent Kit v3 (600-cycle, #MS-102-3003), according to manufacturer’s protocol.\n\nQuality control (QC) was carried out before viral genome fasta generation, as previously described.13 Briefly, demultiplexed fastq files generated by sequencing were used as an input data for the analysis. Reads were trimmed based on quality scores with a cutoff threshold of Q30 to remove low-quality regions, in addition to adapter sequences. QC assessment for sequence reads was performed using FastQC (v0.11.9)14 and MultiQC (v1.9).15\n\nFor those reads passing the QC cutoff, we used Pangolin COVID-19 lineage assigner (v3.0.5)16 to assign SARS-CoV-2 viral lineages. Phylogenetic analysis was carried out in order to understand the evolution of this virus within the Ugandan population, including other SARS-CoV-2 genomes from Uganda that had been submitted by the Uganda Virus Research Institute (UVRI) to the GISAID database by April 22, 2021, and only complete sequences were included, totaling 328 SARS-CoV-2 genomes.\n\nMultiple sequence alignment of 328 Ugandan SARS-CoV-2 genomes and 28 from MakCHS Molecular Diagnostics Laboratory was performed using the web version of MAFFT v.7.475.17 In each alignment, the SARS-CoV-2 reference sequence (NCBI Reference Sequence: SARS-CoV-2 isolate Wuhan-Hu-1, complete genome, NC_045512.2) was included. The alignment from MAFFT was then subjected to snp-sites v2.3.318 to generate a phylip file format, which was later used to infer a maximum likelihood tree using PhyML v3.3.319 with the tool’s default parameters. The tree generated by PhyML was stored in a newick file format. The file was then uploaded to the interactive tree of life (iTOL v4.0)20 - an online tool for phylogenetic tree display and annotation for visualization (Figure 1). We then used snipit v1.0.321 to zoom into the 28 genomes from MakCHS sequencing lab to visualize their snps in reference to the SARS-CoV-2 reference genome (Figure 2).\n\n\nResults\n\nThe average age of the study participants was 40 years with an equal ratio of males to females. Their nationalities included 21 Ugandans, 3 Eritreans, 2 Indians, 1 Israeli and 1 South Sudanese. After WGS, we successfully generated a total of 28 out 30 (93%) analyzable SARS-CoV-2 genomes. It was probable that only these archived specimens had adequate viral RNA for successful genomic sequencing. Targeted SARS-CoV-2 RT-qPCR was positive for N, ORF, and S viral genes, however two samples did not have detectable Ct values for viral S gene.\n\nBoth patient demographics and summary characteristics of these genomes are shown in Table 1. We found more A strains of SARS-CoV-2 than B strains. The different quality metrics used included SARS-CoV-2 draft genome length, GC-content and average depth of coverage. The coverage of all samples was above 30X.\n\nOf the virus genomes generated, we were unable to identify any known SARS-CoV-2 variants of concern or interest from the sampled specimens when we compared the genomes from specimens collected in September 2020 to February 2021. Phylogenetic analysis of all the sequenced SARS-CoV-2 genomes by then from Uganda shows genomic relatedness as seen in Figure 1; however, this observation was of limited value given that there was inadequate epidemiologic data from the patients from whom these specimens had been collected. There was close genomic relatedness detected in MAKCHScov28 and cov10 as well as MAKCHScov5 and cov6 that had been collected from different patients on the same date. MAKCHScov26 and cov16 as well as MAKCHScov25 and cov4 were equally closely linked through phylogenetic analysis, though collected at least two months apart. Hence, this phylogenetic relatedness is likely to indicate local transmission events of SARS-CoV-2 in Kampala metropolitan area.\n\n\nDiscussion\n\nDue to the fact that 28/30 of SARS-CoV-2 genomes were successfully sequenced on the MiSeq platform, we have demonstrated a proof-of-concept project on SARS-CoV-2 WGS using archived clinical nasopharyngeal swab samples from the IBRH3AU. This study has successfully optimized and validated SARS-CoV-2 WGS using the ARTIC amplicon sequencing protocol. This has enabled us to adopt SARS-CoV-2 WGS at MakCHS Molecular Diagnostic Laboratory. As of June 8, 2021, Uganda had registered 53,961 COVID-19 cases and more than half (~26,000) of the samples are stored at the IBR3HAU biorepository.\n\nGlobally, researchers are being encouraged to sequence and share more genomes of SARS-CoV-2 via the GISAID platform, and there are currently more than 1.8 million coronavirus genome sequences from 172 countries and territories, which is a great testament to the hard work of researchers around the world during the COVID-19 global pandemic.22 Ironically, approximately 98% of these genomes have been submitted by high-income countries, underscoring the need to build a similar capacity in lower-middle-income settings (LIMC).\n\nIn this study, we estimated that the cost of WGS per SARS-CoV-2 clinical specimen in our laboratory to be $110 compared to $57.8723 in the United States using a multiplex PCR followed by sequencing on an Illumina MiSeq apparatus. In the United Arab Emirates, the cost of SARS-CoV-2 full genome sequencing was estimated to be ~$87 per specimen when sequencing 96 samples in a batch at 400× using the target enrichment method.24 It should be noted that target enrichment sequencing is still a more cost-effective approach and is scalable in many settings that handle large volumes of these samples. As of June 8, 2021, GISAID had 1,885,406 hCoV-19 genomic data with only a total of 19,065 submissions being from Africa while on June 1 2021, a total of 4,843,874 COVID-19 cases and 130,814 deaths (CFR: 2.7%) had been reported in 55 African Union (AU) member states representing 3% of all cases reportedly globally.25 However, a majority of the low-quality SARS-CoV-2 genomes submitted in this same online genome database have been submitted from sequencing facilities in Africa.\n\nAs many countries and territories globally continue to find the optimal approach in managing the health-related consequences of COVID-19, more laboratories in these settings must find the best or affordable protocols to implement WGS of SARS-CoV-2 to inform public health measures. We performed WGS of 30 samples and specifically successfully evaluated the performance of ARTIC SARS-CoV-2 sequencing protocol performance in our settings using the Illumina MiSeq platform. In this study, genomic libraries were generated using RNA samples isolated from either newly prepared nasopharyngeal swabs in AVL buffer, which is a lysis buffer intended for purifying viral nucleic acids, or previously collected and frozen nasopharyngeal swabs preserved in AVL buffer. Therefore, the findings of this study offer guidance on implementing the low-cost ARTIC SARS-CoV-2 genome sequencing protocol to study SARS-CoV-2 genomic variations in resource limited settings. Many of these settings are currently unable to perform real-time WGS of such samples either due to absence of sequencing infrastructure, which in some cases has been overcome by establishing collaborations with sequencing facilities in other countries and therefore requiring shipment of the samples, unsustainable supply of sequencing reagents, or lack of trained genomics and bioinformatics personnel. WGS of SARS-CoV-2 remains vital in elucidating COVID-19 disease26 for the unforeseeable future as researchers globally continue to identify new SARS-CoV-2 variants of concern and interests such as B.1.1.7 (Alpha), B.1351 (Beta), P.1 (Gamma), B.1.617.2 (Delta) and B.1.427/B.1.429 (Epsilon), P.2 (Zeta), B.1.525 (B.1.525), P.3 (Theta), B.1.526 (Iota), and B.1.617.1 (Kappa) respectively.27 WGS allows detection and characterization of these emerging viral variants, generating essential new information about their genomic, immunologic, virologic, epidemiologic, and clinical characteristics.\n\nIncomplete epidemiological and clinical characteristics as well as lack of COVID-19 disease severity of study participants are some of the limitations of this study. Also, the small sample size used as well as sequencing of samples that had been collected from Kampala metropolitan area may not represent the true proportion of identified SARS-CoV-2 lineages and variants in Uganda during the study period.\n\nWe recommend the establishment of more collaborative consortia between researchers, as well between National Public Health Institutions in LIMCs and developed countries to build low-cost, sustainable, functioning pathogen genome sequencing facilities to accelerate pathogen discovery and outbreak surveillance using WGS. Furthermore, these facilities can equally utilize recently developed multiplex RT-qPCR assays to screen for SARS-CoV-2 variants of concern or interest and monitor their frequencies. These variant genotyping assays not only complement WGS in such settings but also offer a cost-effective way to identify which samples can be prioritized for WGS, especially those that are unidentifiable by routine genotyping tests. Even during the vaccination phase of COVID-19, documenting incidences and prevalence of these SARS-CoV-2 re-or-emerging variants is key in identifying vaccine escape mutants. This offers the opportunity for judicious use of WGS for rapid discovery of novel SARS-CoV-2 variants.\n\n\nConclusion\n\nIn conclusion, our proof-of-concept study shows that ARTIC SARS-CoV-2 sequencing protocol on Illumina MiSeq is sensitive and accurate at higher SARS-Cov-2 template concentration (e.g., Ct value <25) in the Ugandan settings. We successfully validated the protocol and evaluated the process in mappability, genome length, GC-content, viral genome coverage, and variations in SNV calling. The result of our study provides a thorough affirmation of carrying out whole-genome sequencing for clinical SARS-CoV-2 samples in resource limited settings, thereby providing information to mitigate the impact of COVID-19 on our society. We have further contributed to SARS-CoV-2 global dataset.\n\n\nAuthor contributions\n\nMLJ conceived and designed the study. EN and RK helped the patient nasopharyngeal swab sample collection. DPK, BSB, MW, ML, JMK, EK, SK and AFK performed experiments including RNA isolation, qRT-PCR, SARS-CoV-2 WGS library construction and sequencing. GM, SM, PN, SK and EK performed bioinformatics data analyses and drafted the manuscript. EN helped coordinate the project meetings and IRB application. All authors reviewed the final manuscript.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nPresidential Scientific Initiative on Epidemics (PRESIDE)Fast Grants (Emergent Ventures at George Mason University)\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Dr. Sarah Stanley, Dr. Julia Schaletzky and Isabelle Charles from the Center for Emerging and Neglected Diseases (CEND) at the University of California, Berkeley (USA) for a generous gift of SARS-CoV-2 sequencing kits, reagents, and the ARTIC Network for their extensive support with protocols. This work was supported in part by funding from Fast Grants (part of Emergent Ventures at George Mason University) to Sarah Stanley.We also gratefully acknowledge the Authors from the Originating laboratories responsible for obtaining the specimens and the Submitting laboratories where genetic sequence data were generated and shared via the GISAID Initiative,5 on which this research is based. Submitting and the Originating laboratories of the GISAID data used in this study are listed in the Acknowledgment Table (see Extended data28).\n\n\nData availability\n\nThese samples are available in GenBank:\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov2/2020, complete genome. Accession number MZ287347; https://www.ncbi.nlm.nih.gov/nuccore/MZ287347\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov14/2020 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protei... Accession number: MZ287348; https://www.ncbi.nlm.nih.gov/nuccore/MZ287348\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov21/2021 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protei... Accession number: MZ287349; https://www.ncbi.nlm.nih.gov/nuccore/MZ287349\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov21/2021 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protei... Accession number: MZ287349 https://www.ncbi.nlm.nih.gov/nuccore/MZ287349\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov6/2020, complete genome. Accession number: MZ287350 https://www.ncbi.nlm.nih.gov/nuccore/MZ287350\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov11/2021 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protei... Accession number: MZ287351 https://www.ncbi.nlm.nih.gov/nuccore/MZ287351\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov30/2021, complete genome. Accession number: MZ287352 https://www.ncbi.nlm.nih.gov/nuccore/MZ287352\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov18/2020 ORF1ab polyprotein (ORF1ab) and ORF1a polyprotein (ORF1ab) genes, partial cds; surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycopr... Accession number: MZ287353 https://www.ncbi.nlm.nih.gov/nuccore/MZ287353\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov26/2021, complete genome. Accession number: MZ287354 https://www.ncbi.nlm.nih.gov/nuccore/MZ287354\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov20/2021 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protei... Accession number: MZ287355 https://www.ncbi.nlm.nih.gov/nuccore/MZ287355\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov13/2020 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protei... Accession number: MZ287356 https://www.ncbi.nlm.nih.gov/nuccore/MZ287356\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov29/2021 ORF1ab polyprotein (ORF1ab) and ORF1a polyprotein (ORF1ab) genes, partial cds; and surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane gly... Accession number: MZ287357 https://www.ncbi.nlm.nih.gov/nuccore/MZ287357\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov4/2020, complete genome. Accession number: MZ287358 https://www.ncbi.nlm.nih.gov/nuccore/MZ287358\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov28/2021, complete genome. Accession number: MZ287359 https://www.ncbi.nlm.nih.gov/nuccore/MZ287359\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov19/2020, complete genome. Accession number: MZ287360 https://www.ncbi.nlm.nih.gov/nuccore/MZ287360\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov7/2021, complete genome. Accession number: MZ287361 https://www.ncbi.nlm.nih.gov/nuccore/MZ287361\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov12/2021, complete genome. Accession number: MZ287362 https://www.ncbi.nlm.nih.gov/nuccore/MZ287362\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov5/2020, complete genome. Accession number: MZ287363 https://www.ncbi.nlm.nih.gov/nuccore/MZ287363\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov1/2020, complete genome. Accession number: MZ287364 https://www.ncbi.nlm.nih.gov/nuccore/MZ287364\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov15/2020 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protei... Accession number: MZ287365 https://www.ncbi.nlm.nih.gov/nuccore/MZ287365\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov3/2020 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protein... Accession number: MZ287366 https://www.ncbi.nlm.nih.gov/nuccore/MZ287366\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov9/2021, complete genome Accession number: MZ287367 https://www.ncbi.nlm.nih.gov/nuccore/MZ287367\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov16/2020, complete genome. Accession number: MZ287368 https://www.ncbi.nlm.nih.gov/nuccore/MZ287368\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov25/2021, complete genome. Accession number: MZ287369 https://www.ncbi.nlm.nih.gov/nuccore/MZ287369\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov8/2021 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protein... Accession number: MZ287370 https://www.ncbi.nlm.nih.gov/nuccore/MZ287370\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov10/2021 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycoprotein (M), ORF6 protei... Accession number: MZ287371 https://www.ncbi.nlm.nih.gov/nuccore/MZ287371\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov17/2020 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), and surface glycoprotein (S) genes, partial cds; and ORF3a protein (ORF3a), envelope protein (E), membrane gl... Accession number: MZ287333 https://www.ncbi.nlm.nih.gov/nuccore/MZ287333\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov22/2021 ORF1ab polyprotein (ORF1ab), ORF1a polyprotein (ORF1ab), and surface glycoprotein (S) genes, partial cds; ORF3a protein (ORF3a) gene, complete cds; envelope protein (... Accession number: MZ287334 https://www.ncbi.nlm.nih.gov/nuccore/MZ287334\n\nGenBank: Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/UGA/MAKCHScov23/2021 ORF1ab polyprotein (ORF1ab) and ORF1a polyprotein (ORF1ab) genes, partial cds; surface glycoprotein (S), ORF3a protein (ORF3a), envelope protein (E), membrane glycopr... Accession number: MZ287335 https://www.ncbi.nlm.nih.gov/nuccore/MZ287335\n\nData has also been deposited in the Global Initial on Sharing All Influenza Data (GISAID’s EpiCoV database: http://www.gisaid.org)\n\nZenodo: Whole-genome sequencing of SARS-CoV-2 in Uganda: implementation of the low-cost ARTIC protocol in resource-limited settings. https://doi.org/10.5281/zenodo.5055843.28\n\nThis project contains the following extended data:\n\n- Acknowledgments table for GISAID data\n\nExtended data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nCOVID-19 Map: Johns Hopkins Coronavirus Resource Center.Accessed June 12, 2021. Reference Source\n\nGenomic sequencing of SARS-CoV-2: a guide to implementation for maximum impact on public health. Accessed March 22, 2021. Reference Source\n\nUganda Coronavirus: 60,250 Cases and 423 Deaths - Worldometer. Accessed June 12, 2021. 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Publisher Full Text" }	[ { "id": "89859", "date": "09 Aug 2021", "name": "Anthony Mukwaya", "expertise": [ "Reviewer Expertise Infection biology", "functional genomics", "Applied bioinformatics", "molecular epidemiology", "immunology", "molecular biology", "diagnostic methods", "Angiogenesis", "eye diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary The article by Mboowa et al., evaluates the ARTIC protocol for Whole-genome sequencing of SARS-CoV-2 in Uganda. Authors test the protocol using previously collected qPCR positive COVID-19 samples, and they found that 93.3% of the samples generated readable genetic sequences. Of these samples, about 78% were of the A lineage, and 21% were of the B lineage. Authors show a relatedness of the isolates in the current study to other SARS-CoV-2 genomes available in GISAID. The study shows the feasibility of conducting the ARTIC protocol in a resource limited setting, thus allowing for more SARS-COV-2 WGS in the country. The idea behind the study is interesting, vital, and timely, given the role played by WGS in disease epidemiology. The article is properly written, the methods are robust, but few minor details need to be clarified. The comments below should be taken into consideration as they aim to further improve on the overall quality of the article.\nMinor corrections\nThe following statement needs to be revised for accuracy: \"Of the 131 Uganda full SARS-CoV-2 genomes analyzed in December 2020, 50 (38%)\". I suspect authors meant \"Of the 131 full SARS-CoV-2genomes analyzed in Uganda in December 2020…\"?\n\nI suggest a brief introduction of the SARS-CoV2 lineages/nomenclature be included in the introduction to guide the reader for example the \"Pango lineages\".\n\nIn the introduction, authors write \"that this situation is very similar to almost all other African countries…\" however, the \"situation\" being referred to is not properly problematized, thus needs to be explicitly defined in the preceding sentence (i.e. is it an issue with low testing rate or low/no sequencing rate or both)?\n\nA brief introduction of ARTIC including the principle behind it would be good to summaries in the introduction, while highlighting the advantages of ARTIC over the currently available SARS-CoV-2 genome sequencing platforms.\n\nUnder the subheading study settings, the exact test used for COVID-19 diagnosis should be stated in this section as well. Under ethical consideration, please clearly state that \"no patient information that can be used to identify an individual such as names and personal identification number etc. were used in the study\".\n\nAuthors should elaborate on the details of the diagnostic protocol used for testing. For example, did all the amplification curves for all the three genes have to be below Ct 25 for a sample to be declared positive? What if only two genes had Ct values below 25, and the other gene above Ct 25, how was a such a sample classified? What would a Ct value of 26 mean? This being a diagnostic PCR, Interpretation of the diagnostic data should be clarified to indicate that many factors were taken into consideration, for example the total number of cycles run, and the Ct value range for interpretation, the positive control, and that of the internal control etc.\n\nIt is not clear if the samples used for sequencing were amplicons stored from the diagnostic process or if it was aliquots of RNA previously extracted during testing, or separate samples were collected from the individual specifically for this study. Please specify this in the methods.\n\nFigures 1 and 2 need to be appropriately cited in the results main text.\n\nIn the recommendations, authors should recommend further testing of the ARTIC SARS-CoV-2 sequencing protocol using a much bigger sample size, different sample types, and preservation buffers, to draw more comprehensive conclusions.\n\nAuthors write that \"the result of our study provides a thorough affirmation of carrying out whole genome sequencing for clinical SARS-CoV-2 samples in resource limited settings, thereby providing information to mitigate the impact of COVID-19 on our society\". However, I think that it is important that the importance of sequencing on disease epidemiology be clearly stated as part of the study contribution.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "89860", "date": "19 Aug 2021", "name": "Alan Christoffels", "expertise": [ "Reviewer Expertise Microbial bioinformatics", "biobank information systems", "data standards" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMboowa et al. describes the application of the ARTIC protocol for sequencing of SARS-COV-2 genomes and subsequently analysis in preparation for submission of data to GISAID.\nA few minor comments:\n1. Abstract, Background The context of this work points to the need for more adoption of WGS in Africa and therefore increase disease surveillance. One way of ordering the background paragraph could be: Start with the last sentence of that paragraph, remove the Wuhan sentence and then follow with the first sentence then mention that to date, x % of sars-cov-2 genomes submitted to GISAID were contributed from Africa. This data includes low quality viral genomes and a need for improved protocols for implementing WGS.\n2. Introduction, last paragraph Replace UK variant and SA variant sentence with the acceptable nomenclature, \"Alpha\" and \"Beta\" variants respectively, so that this scientific record in F1000 assists in rectifying the public usage of terms.\n3. Methods section, study design Is it standard protocol to archive SARS-CoV-2 biospecimens in the local biobank (IBRH3AU)? If not then it would be good to extend the sentence to add reproducibility for others wanting to do the same.\n4. Methods, seq and bioinformatics analysis section Delete the first sentence that starts \"These samples had been...\". Edit the next sentence to read, \"Metadata associated with the randomly selected 30 COVID-19 positive patient samples included the data of sample collection...\"\n5. Discussion This article is key to accelerating adoption of methods to respond to COVID-19. This focus of the paper was to show the success with using the ARTIC protocol. You could add a sentence in the results section after the phylogenetic analysis using iTOL to point readers to the option of using Nextstrain within the analysis process and the value of richer meta data in this context.\nFigures 1 and 2 have a title but no caption/description.\nTable 1: could you order the info in this table according to one of the columns? That would make reading the table a bit easier - maybe group by lineage? Or order by GC-content?\nAny comment on the 45.96 GC-content for MAKCHScov17/2020? Curious why the spike in comparison to the other samples.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-598
https://f1000research.com/articles/10-594/v1	19 Jul 21	{ "type": "Case Report", "title": "Case Report: Duodenoscopy diagnosis of a secondary aorto-duodenal fistula: A case report and review of literature.", "authors": [ "Hanen Elloumi", "Ben Mrad Melek", "Imen Ganzoui", "Sonia Ben Hamida", "Wissem Triki", "Ilhem Mchirgui", "Makrem Ben Hmida", "Bilel Derbel", "Imed Cheikh", "Imen Ganzoui", "Sonia Ben Hamida", "Wissem Triki", "Ilhem Mchirgui", "Makrem Ben Hmida", "Bilel Derbel", "Imed Cheikh" ], "abstract": "Secondary aorto-enteric fistula (SAEF) is a rare life-threatening complication occurring in patients with previous infrarenal aortic prosthetic reconstruction. The main symptom is a gastrointestinal bleeding. Its diagnosis is challenging due to the lack of a specific clinical signs. The failure of early diagnosis and treatment of this entity can lead to fatal issue. Actually, the abdominal computed tomography angiogram represents the principal exploration to confirm the diagnosis, but it is associated with a moderate specificity and sensibility. Duodenoscopy can highlight the communication between the duodenum and the prosthetic graft, but it is often inconclusive. We report in this manuscript a case of secondary aorto-enteric fistula revealed by occult gastrointestinal bleeding in an elderly patient who is admitted for severe anemia. The SAEF diagnosis was suspected by the computed tomography scan and confirmed by the duodenoscopy showing an exceptional image of Dacron graft protruding in the third duodenum lumen. Unfortunately, the patient died from cataclysmic shock before intervention. We overview also the rare previous published case reports concerning the endoscopic images of secondary aortoenteric fistula and we contrast our findings with those reported in the literature.", "keywords": [ "Duodenoscopy", "aorto-duodenal fistula" ], "content": "Introduction\n\nSecondary aorto-enteric fistula (SAEF) is an abnormal communication between a prosthetic graft and the gastrointestinal tract that happens after an abdominal infrarenal aortic graft implant to treat aortic aneurysm or occlusive disease.1 SAEF is a rare life-threatening complication with an incidence lower than 2% after aortic prosthetic reconstruction.1 Its diagnosis is sometimes challenging and delayed because of a lack of specific signs.2 Upper endoscopy should be done primarily to eliminate another cause of digestive bleeding. In rare cases it can also confirm the diagnosis by showing the protrusion of the aortic graft in the distal duodenum.3 We report in this paper a case of secondary aorto-duodenal fistula revealed by a severe anemia and confirmed with exceptional duodenoscopy images showing the Dacron prothesis protruding into the lumen of the duodenum. It is only the second report in the medical literature where we clearly and directly see a Dacron vascular graft protruding into the duodenum.\n\n\nThe case\n\nA 75-year-old North African man was referred to our department for management and exploration of severe anemia, symptoms of dyspnea, vertigo, and asthenia. Four years previously, he had undergone an aorto-bifemoral bypass by a Dacron prosthesis for aorto-iliac Leriche occlusive disease.\n\nThe patient did not report any history of gastric ulcers or other gastro-duodenal pathologies.\n\nUpon physical examination, the patient was pale but awake and alert. The hemodynamic state was stable, his blood pressure was 130/70 mmHg and his heart rate was 92 bpm. He was febrile (temp = 38 EC). Cardiac and pulmonary examinations were normal. Neurological and otorhinolaryngological examinations were normal also.\n\nThe digital rectal examination did not show any bleeding.\n\nComplete blood count revealed microcytic hypochromic anemia with a hemoglobin level of 5,5 g/dL and hyperleukocytosis with a leukocyte count of 12.1 × 109/L. The C-reactive protein level was 17 mg/dL.\n\nThe patient received blood transfusion by four units of packed red blood cells and the hemoglobin level reached 9 g/dL.\n\nIn order to determine the cause of this severe anemia, the patient had a colonoscopy and upper gastrointestinal endoscopy under general anesthesia. Both exams were normal, not showing an obvious cause of intestinal bleeding.\n\nContrast Computed Tomography (CT scan) was performed urgently, showing a closed contact between the third duodenum and the proximal part of the aorto-bifemoral graft with air bubbles present in the aortic graft associated with a fluid collection of 8,8 × 3,9 mm in diameter in contact with the Dacron prothesis (Figure 1). At this stage the diagnosis of SAEF was strongly suspected and we decide to perform another endoscopic exploration of the duodenum.\n\nContrast Computed Tomography revealing a closed contact between the third duodenum and the proximal edge of aorto-bifemoral graft with air bubbles in the aorta (red arrow) without extravasation of the aortic contrast material into the duodenum lumen associated with a little collection of 8,8 × 3,9 mm in diameter.\n\nThe second esophagogastroduodenoscopy showed a large duodenal fold beside a white foreign body evoking a synthetic material (Figure 2). In order to better elucidate this finding, a duodenoscopy was performed, demonstrating a large parietal defect >3 cm in diameter involving the third duodenum with visualization of Dacron mesh through the mucosal defect (Figure 3).\n\nThe patient received a broad-spectrum intravenous probabilistic antibiotic therapy based on Teicoplanin, Imipenem, and Amikacin. The patient was transferred to the vascular surgery department for surgical intervention.\n\nUnfortunately, the day before his operation, the patient presented a cataclysmic hemorrhage and died from hemorrhagic shock.\n\n\nDiscussion\n\nIn 1956, Claytor reported the first case of SAEF after an aortic prosthetic graft.4 More than 75% of SAEF occur between the aorta and the 3rd or 4th part of the duodenum.5 This localization is explained by the retroperitoneal fixation and the close proximity of the duodenum to the aorta.6–8 Rarely the fistula is located in the ascending, transverse, sigmoid colon, or rectum.6–8\n\nThe SAEF pathogenesis is controversial. Several hypotheses have been postulated. The most significant mechanisms for its development are graft infection and inflammation, and mechanical factors.2,6–9 Mechanical effect is explained by the presence of a continuous pulsatile movement between the graft and intestine, leading to the erosion of the aortic prosthetic material into the adjacent digestive structure.6,9 This hypothesis is supported by the fact that most SAEFs involve, like our case, the third and the fourth duodenum.6 Another possible mechanism of SAEF is infection introduced at the time of the surgery or secondary to the prosthetic graft colonization by the bowel flora, passed through the suture line.6,9\n\nThe clinical presentation varies significantly. A patient with SAEF may present with gastrointestinal bleeding, sepsis, or weight loss. Gastrointestinal hemorrhage, as manifested by hematemesis, hematochezia, melena, hemorrhagic shock, or chronic anemia is the most frequent presentation, encountered in 18-100% of cases.5,6,10 After GI bleeding, sepsis or fever is the commonest presentation in patients with SAEF accounting to 30-87% of cases.5,6 Signs of infection such as fever and leukocytosis sometimes accompany gastrointestinal bleeding.7,9 Our patient presented an occult gastrointestinal bleeding revealed by a severe anemia associated with fever and hyperleukocytosis.\n\nThe SAEF diagnosis must be suspected first in every patient who has gastrointestinal bleeding and a history of abdominal graft. Diagnosis is aided by morphological exams such as computerized tomography (CT scan) and digestive endoscopy.6,8,9 The CT scan is the most frequent initial diagnostic test.6,8,9 It allows the visualization of the fistula conduit and its location and may aid in identification of an infection or abscess if present. Its sensibility and its specificity are around 94% and 85% respectively.11 The most specific CT scan features suggestive of SAEF are gas shadow in or around the graft (sensitivity 40%; specificity 100%) and visible graft (sensitivity 22%, specificity 100%).5,12,13 In our case, CT scan showed the presence of air bubbles around the aortic graft. Esophagogastroduodenoscopy is recommended in suspected SAEF patients presenting with upper gastrointestinal bleeding.6,9 Sometimes, the diagnosis should be suspected when we discover stigmata of arterial bleeding, adherent clot, a pulsatile blood clot, or a pulsatile structure on the lumen of the distal duodenum.14\n\nWhen the synthetic graft is visible in the intestinal lumen, the diagnosis is confirmed. The accuracy of EGD is however limited beyond the second portion of the duodenum while most of SAEFs occur in the third and fourth parts of the duodenum. This explains the scarcity of reports in the medical literature that have been able to highlight by duodenoscopy this aortoduodenal fistula.3,15–19 Compared with these reports, the duodenoscopy findings in our case are the more relevant allowing for the first time to clearly see the characteristic cutmarks of the Dacron prothesis.\n\nThe endoscopic findings of prosthetic graft within the duodenum allows the certain diagnosis and surgical treatment should be performed immediately to avoid fatal hemorrhage, as seen in our patient.19\n\nThe main objectives of SAEF treatment are maintenance of hemodynamic stability, surgical repair of the underlying defect, infection control via empiric intravenous antibiotics and perfusion of the lower limbs.3,5,6,20 The optimal SAEF repair technique is currently not well defined. The conventional vascular treatment consists of an extra-anatomic bypass grafting with aortic ligation and subsequent graft removal associated with bowel repair.7,21\n\nManagement of sepsis consists of early administration of empiric antibiotics covering the majority of identified organisms.6,22 The prognosis of patients with SAEF remains uncertain, and depends on hemodynamic status of the patient at presentation, the operative technique performed and the time of surgical exploration.6,9 A delay in surgical exploration is the main predictive factor of mortality.23\n\n\nConclusion\n\nSAEF is a rare life threatening complication. The diagnosis must be suspected in front of a patient having a gastrointestinal bleeding or severe anemia with the history of previous aortic prosthetic reconstruction. Unfortunately, the lack of specific signs is responsible for a diagnostic delay. The diagnosis depends on clinical and biological examinations and CT scan findings. In rare cases, meticulous duodenoscopy exploring the third and the fourth segment of duodenum can confirm the diagnosis by showing the prosthetic graft into the duodenal lumen. The management of SAEF must be done urgently to avoid fatal cataclysmic bleeding.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the family of the patient.", "appendix": "Acknowledgments\n\nThis paper would not have been possible without the exceptional support of our friend Jack Hukill, especially for his help in verifying English this article.\n\n\nAuthor contributions\n\nElloumi Hanen: Writing – Original Draft Preparation\n\nBen Mrad Melek: Writing – Review and Editing\n\nGanzoui Imen: Writing – Original Draft Preparation\n\nBen Hmida Sonia: Resources\n\nTriki Wissem: Writing – Original Draft Preparation\n\nMchirgui Ilhem: Review and Editing\n\nBen Hmida Makrem: Review and Editing\n\nDerbel Bilel: Supervision\n\nCheikh Imed: Validation\n\n\nReferences\n\nPeck JJ, Eidemiller LR: Aortoenteric fistulas. Arch Surg. 1992 Oct; 127(10): 1191–3; discussion 1193-4. PubMed Abstract \| Publisher Full Text\n\nSimon T, Feller E: Diverse presentation of secondary aortoenteric fistulae. Case Rep Med. 2011; 2011: 406730. Epub 2011 Dec 29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLirici MM, Tierno SM, Giudice R, et al.: Secondary aortoenteric fistula successfully treated with staged endovascular repair and duodenal resection without graft removal. Minim Invasive Ther Allied Technol. 2020 Apr; 29(2): 114–9. Epub 2019 Mar 8. PubMed Abstract \| Publisher Full Text\n\nBirch L, Cardwell ES, Claytor H, et al.: Suture-line rupture of a nylon aortic bifurcation graft into the small bowel. AMA Arch Surg. 1956 Dec; 73(6): 947–50. PubMed Abstract \| Publisher Full Text\n\nXiromeritis K, Dalainas I, Stamatakos M, et al.: Aortoenteric fistulae: present-day management. Int Surg. 2011 Jul-Sep; 96(3): 266–73. PubMed Abstract \| Publisher Full Text\n\nMalik MU, Ucbilek E, Sherwal AS: Critical gastrointestinal bleed due to secondary aortoenteric fistula. J Community Hosp Intern Med Perspect. 2015 Dec 11; 5(6): 29677. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpanos K, Kouvelos G, Karathanos C, et al.: Current status of endovascular treatment of aortoenteric fistula. Semin Vasc Surg. 2017 Jun-Sep; 30(2-3): 80–4. Epub 2017 Oct 26. PubMed Abstract \| Publisher Full Text\n\nKhan A, Ahmad E, Javaid S, et al.: An Insidious Gastrointestinal Bleeding from Secondary Aortoduodenal Fistula Leading to Septic Shock. Case Rep Gastrointest Med. 2019 May 13; 2019: 6261526. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang C, Chen X, Li J, et al.: A case report of successful treatment of secondary aortoenteric fistula complicated with gastrointestinal bleeding and retroperitoneal abscess in an elderly patient. Medicine (Baltimore). 2018 Jun; 97(24): e11055. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPipinos II, Carr JA, Haithcock BE, et al.: Secondary aortoenteric fistula. Ann Vasc Surg. 2000 Nov; 14(6): 688–96. PubMed Abstract \| Publisher Full Text\n\nLow RN, Wall SD, Jeffrey RB Jr, et al.: Aortoenteric fistula and perigraft infection: evaluation with CT. Radiology. 1990 Apr; 175(1): 157–62. PubMed Abstract \| Publisher Full Text\n\nHughes FM, Kavanagh D, Barry M, et al.: Aortoenteric fistula: a diagnostic dilemma. Abdom Imaging. 2007 May-Jun; 32(3): 398–402. PubMed Abstract \| Publisher Full Text\n\nHagspiel KD, Turba UC, Bozlar U, et al.: Diagnosis of aortoenteric fistulas with CT angiography. J Vasc Interv Radiol. 2007 Apr; 18(4): 497–504. PubMed Abstract \| Publisher Full Text\n\nBaker MS, Fisher JH, van der Reis L, et al.: The endoscopic diagnosis of an aortoduodenal fistula. Arch Surg. 1976 Mar; 111(3): 304. PubMed Abstract \| Publisher Full Text\n\nAbernethy W, Sekijima JH: Images in clinical medicine. Aortoenteric fistula. N Engl J Med. 1997 Jan 2; 336(1): 27. PubMed Abstract \| Publisher Full Text\n\nBaker BH, Baker BS, van der Reis L, et al.: Endoscopy in the diagnosis of aortoduodenal fistula. Gastrointest Endosc. 1977 Aug; 24(1): 35–7. PubMed Abstract \| Publisher Full Text\n\nBrand EJ, Sivak MV Jr, Sullivan BH Jr: Aortoduodenal fistula: endoscopic diagnosis. Dig Dis Sci. 1979 Dec; 24(12): 940–4. PubMed Abstract \| Publisher Full Text\n\nGalloro G, De Palma GD, Siciliano S, et al.: Secondary aortoduodenal fistula. Rare endoscopic finding in the course of digestive hemorrhage. Hepatogastroenterology. 2000 Nov-Dec; 47(36): 1585–7. PubMed Abstract\n\nIkeno T, Sugiyama A, Nishimaki K, et al.: Endoscopic image of secondary aortoenteric fistula. Digestive Endoscopy . 2001; 13: 65–6. Publisher Full Text\n\nAntoniou GA, Koutsias S, Antoniou SA, et al.: Outcome after endovascular stent graft repair of aortoenteric fistula: A systematic review. J Vasc Surg. 2009 Mar; 49(3): 782–9. Epub 2008 Nov 22. PubMed Abstract \| Publisher Full Text\n\nKakkos SK, Bicknell CD, Tsolakis IA, et al.: Hellenic Co-operative Group on Aortic Surgery. Editor’s Choice - Management of Secondary Aorto-enteric and Other Abdominal Arterio-enteric Fistulas: A Review and Pooled Data Analysis. Eur J Vasc Endovasc Surg. 2016 Dec; 52(6): 770–86. Epub 2016 Nov 9. PubMed Abstract \| Publisher Full Text\n\nHerdrich BJ, Fairman RM: How to manage infected aortic endografts. J Cardiovasc Surg (Torino). 2013 Oct; 54(5): 595–604. PubMed Abstract\n\nBaril DT, Carroccio A, Ellozy SH, et al.: Evolving strategies for the treatment of aortoenteric fistulas. J Vasc Surg. 2006 Aug; 44(2): 250–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "89870", "date": "02 Sep 2021", "name": "Mouna Medhioub", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting, well-written article with clear data.\nThe article highlights the major role of morphologic and iterative endoscopic examinations in the diagnosis of secondary aorto-enteric fistula.\nDiscussion:\nI recommend that the authors add a part for the prognosis and the mortality rate to emphasize the diagnostic and therapeutic emergency.\n\nFor treatment options, it is mandatory to mention endovascular surgery; an alternative to the classic extra-anatomic bypass.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "263992", "date": "09 May 2024", "name": "Khamin Chinsakchai", "expertise": [ "Reviewer Expertise Vascular surgeon" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 case report titled \"Duodenoscopy Diagnosis of a Secondary Aorto-Duodenal Fistula,\" several comments are noted:\n1. The abstract highlights the use of abdominal computed tomography angiogram for diagnosis, noting its moderate specificity and sensitivity. However, it lacks clarity on why duodenoscopy may be inconclusive. Clarifying the limitations of each diagnostic method would enhance study comprehension. 2. Include normal laboratory testing ranges (hemoglobin, leukocyte count, and C-reactive protein levels) in the case section for reader reference. 3. Inadequate discussion on the crucial clinical presentation of herald bleeding, a characteristic symptom of aortoenteric fistula. 4. Insufficient exploration of treatment options, including modalities, success rates, and potential challenges. 5. I Recommend addressing prevention strategies for this complicated post abdominal aortic procedures to enhance the manuscript's quality and convey important information to readers.\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": "98770", "date": "29 May 2024", "name": "Rahul Gupta", "expertise": [ "Reviewer Expertise Gastrointestinal surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have presented an interesting case of Dacron graft eroding the duodenum. I have the following comments regarding the manuscript:\nWhy was the finding of Dacron graft erosion missed during the first endoscopy? Please add the explanation in the manuscript.\n\nWhat was the cause of sudden hemorrhage in this patient as there was no contrast leak on CT?\n\nDid the endoscopy in any way precipitate the catastrophic bleed?\n\nPlease discuss the risk factors for development of secondary aortoenteric fistula, apart from infection and inflammation.\n\nWhat precautions should be taken during the aortofemoral bypass surgery to prevent the development of aortoenteric fistula?\n\nPlease add a table summarizing the clinical presentation, investigations, treatment and outcome of previously reported cases/series\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-594
https://f1000research.com/articles/10-381/v1	13 May 21	{ "type": "Case Report", "title": "Case Report: PsAPSASH syndrome: an alternative phenotype of syndromic hidradenitis suppurativa treated with the IL-17A inhibitor secukinumab", "authors": [ "Georgios Nikolakis", "Katja Kreibich", "Aristeidis Vaiopoulos", "Katarzyna Kaleta", "Joud Talas", "Markus Becker", "Christos C. Zouboulis", "Katja Kreibich", "Aristeidis Vaiopoulos", "Katarzyna Kaleta", "Joud Talas", "Markus Becker", "Christos C. Zouboulis" ], "abstract": "Syndromic hidradenitis suppurativa (HS) is a form of symptom constellations, which differs from the familial and genetic form and comprises predominantly osteoarticular manifestations. Many forms include pyoderma gangrenosum and acne (PASH), pyogenic arthritis (PAPASH), spondyloarthritis (PASS) and psoriatic arthritis (PsaPASH) and are categorized in the autoinflammatory syndromes. anti-TNF-α and anti-IL-1a blockade are between the therapeutic approaches that improve skin symptoms and prevent permanent osteoarticular damage. This case report refers to the successful treatment of a mixed phenotype of the aforementioned symptoms using the IL-17A inhibitor secukinumab after initial treatment with adalimumab. The therapy improved both cutaneous and reported osteoarticular symptoms. Different approaches for these recalcitrant HS syndromes are essential in order to achieve long-term remission for those patients.", "keywords": [ "PASH", "PAPASH", "PASS", "\"hidradenitis suppurativa\"", "SAPHO", "PAPA", "\"acne inversa\"", "hidradenitis", "secukinumab", "syndrome", "arthritis", "autoinflammatory", "pustulosis" ], "content": "Introduction\n\nHidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disorder of the terminal hair follicle characterized by the presence of nodules, abscesses, tunnels and extensive scarring in the apocrine gland-rich areas of the body.1 Immune dysregulation has been implicated in HS, with a wide range of cytokines identified. Significant increase of proinflammatory cytokines IL-1β, TNF-α, IL-17 and the antiinflammatory cytokine IL-10 has been detected in lesional and perilesional skin.2,3 Apart from sporadic cases, there is a genetic background for certain HS patients, correlating with mutations in the γ-secretase genes nicastrin, presenilin enhancer 2 and presenilin.4 HS has been described in association with several clinical syndromes that include comorbid disorders, such as pyogenic arthritis (PA), pyoderma gangrenosum (PG), acne, ulcerative colitis (UC) and psoriatic arthritis. Such syndromes include the triad of PG, acne and HS (PASH) alone or in combination with PA (PAPASH) or psoriatic arthritis (PsAPASH). In addition, HS can also be a feature of other syndromes such as the SAPHO syndrome,5 which appear recalcitrant to treatment, even after the use of various biologics, such as anti-TNF and anti-IL-1. Here we describe a patient with a novel phenotypic variant of the syndromes described, who responded to treatment with the IL-17A inhibitor secukinumab.\n\nManifestations of HS localized in the inguinofemoral region before (1c) and after (1d) treatment. Pyoderma gangrenosum two months after adalimumab discontinuation (1e), clinical image 4 months after secukinumab treatment (1f) and post-prednisolone i.v pulse therapy (1g). Plantar pustulosis before (1h) and after secukinumab treatment (1i).\n\n(2c): histopathologic evaluation of palmar pustular eruptions revealed characteristic microabscesses of subcorneal neutrophilic accumulation. (2d): Histology of the lower-leg ulcerations demonstrated diffuse neutrophilic inflammation as a hallmark of pyoderma gangrenosum.\n\n\nCase\n\nA 50-year-old female, Caucasian, unemployed patient was admitted to our departments suffering from HS. The patient reported a disease onset of 25 years, describing exacerbations with relapsing nodules, abscesses, and draining sinus tracts. Among the risk factors correlated with the disease, the patient was obese (BMI = 36) and a smoker (34-pack years). Moreover, she had a positive familial history, with her maternal grandmother having had severe refractory HS. Sequencing of the γ-secretase gene complex did not reveal any relevant mutations. The patient reported to having up to 10-15 stools daily. A colonoscopy was performed three years ago, which excluded inflammatory bowel disease. Twelve years after being diagnosed with HS, the patient developed acne conglobata (AC), which was treated without systemic therapy. In addition, she underwent numerous incisions and radical excisions, antibiotic treatment with doxycycline, and the combination of clindamycin and rifampicin over 3 months, according to the HS treatment guidelines,1,2 without sustained remission of the lesions. A previous 1-year therapy with isotretinoin did not improve the HS lesions. The patient was included in a clinical trial combining weekly administration of 40 mg adalimumab s.c. vs. placebo for 3 months, followed by continuation of adalimumab treatment in the same dose over 15 months. After this period, the patient was lost to follow-up. She described an improvement of the HS lesions and reduction of flares under adalimumab. During this time, her general practitioner discontinued adalimumab treatment, judging that the treatment lacked efficacy.\n\nOne month after discontinuation of adalimumab, the patient developed confluent erythematous pustules on the palms (Figure 1a) and soles (Figure 1h) with psoriasis-like scaling of the lesions followed by intermittent shoulders and knee pain and swelling. We performed a bone scan, which showed intense radiopharmaceutical accumulation of the left knee (Figure 2a) and the wrist joints showed signs of arthritis and synovitis (Figure 2b), with no typical pattern of psoriatic arthritis. The pattern of the bull’s head sign (Figure 2a, red arrow) was detected. The patient did not admit having a pain or recurrent swelling of the costoclavicular region. No typical signs of osteitis were detected. Dermatohistology of the palmoplantar lesions revealed characteristic neutrophilic abscesses, compatible with pustular psoriasis (Figure 2c).\n\nTwo months after therapy discontinuation, single, disseminated, painful pustules appeared on both thighs and lower legs (Figure 1e), which progressed to painful ulcers (Figure 1f) with elevated violaceous margins. The histopathological evaluation confirmed a PG (Figure 2d). Based on the disease pathophysiology, we initiated therapy with secukinumab 300 mg s.c weekly for the first month and then monthly thereafter. Four months later, the patient demonstrated a significant remission of her HS lesions (Figure 1d) (ΔIHS4 9 and ΔDLQI 11), joint pains and pustular psoriasis (Figure 1b, Figure 1i), with only moderate improvement of her PG lesions. An epithelization was not observed. The PG showed no improvement and 100 mg prednisolone daily i.v. over three days was added to the treatment, which was subsequently tapered over one month. The treatment was followed by PG improvement (Figure 1g).\n\n\nDiscussion\n\nHS can also be a main or secondary element of certain syndromes highlighted through their unique phenotypes,5 known as autoinflammatory diseases (AID). AID manifests with recurrent sterile inflammation, while high autoantibody serum levels or antigen-specific lymphocytes are lacking.6–9 These syndromes combine dermatological manifestations (HS, PPP, AC, PG), musculoskeletal disorders (arthritis, synovitis, hyperostosis, osteitis) and gastroenterological manifestations (ulcerative colitis, M. Crohn). Combinations lead to already described syndromes such as PASH,10,11 PASS,8 PAPASH,10 PsAPASH12 and SAPHO.13 These disorders are characterized by aberrant release of IL-1β, which mediates the increase of tumor necrosis factor α (TNF-α), interferon γ (IFN-γ) and other chemokines, which are responsible for neutrophilic recruitment and might promote an anti-apoptotic microenvironment.14–17 IL-17 also promotes neutrophilic recruitment and activation and has a synergistic effect with TNF-α.18 An imbalance of the Th17/Treg lymphocyte ratio is believed to aggravate autoinflammation and was reported both in PG and HS independently.19,20\n\nWe describe a new syndromic HS-related phenotype (pustular Psoriasis, Arthritis, PG, Synovitis, Acne, Suppurative Hidradenitis). The pathophysiology based on the dysregulation of IL-17 production provided the rationale for the treatment with the IL-17A inhibitor secukinumab. Moreover, this case underlines the role of the dermatologist in diagnosing such symptoms and leading a multidisciplinary approach for these patients. Dermatological manifestations can precede the osteoarticular or other organ symptoms and timely treatment initiation might avoid irreversible complications.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nThe patient has provided written consent for the use of all photos provided in the manuscript. The consent included potential use of the material in lecture(s) and/or publication(s).", "appendix": "References\n\nZouboulis CC, Desai N, Emtestam L, et al.: European S1 guideline for the treatment of hidradenitis suppurativa/acne inversa. J Eur Acad Dermatology Venereol. 2015; 29(4): 619–44. PubMed Abstract \| Publisher Full Text\n\nZouboulis CC, Bechara FG, Fritz K, et al.: S1-Leitlinie zur Therapie der Hidradenitis suppurativa/Acne inversa * (ICD-10 Ziffer: L73.2). JDDG - J Ger Soc Dermatol. 2012; 10(SUPPL. 5). PubMed Abstract \| Publisher Full Text\n\nKurzen H, Kurokawa I, Jemec GBE, et al.: What causes hidradenitis suppurativa? Exp Dermatol. 2008; 17(5): 455–6.\n\nZouboulis CC, Tzellos T, Kyrgidis A, et al.: Development and validation of the International Hidradenitis Suppurativa Severity Score System (IHS4), a novel dynamic scoring system to assess HS severity. Br J Dermatol. 2017 Nov; 177(5): 1401–9. PubMed Abstract \| Publisher Full Text\n\nGasparic J, Theut Riis P, Jemec GB: Recognizing syndromic hidradenitis suppurativa: a review of the literature. J Eur Acad Dermatology Venereol. 2017 Nov; 31(11): 1809–16. PubMed Abstract \| Publisher Full Text\n\nMcDermott MF, Aksentijevich I, Galon J, et al.: Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell. 1999 Apr; 97(1): 133–44. PubMed Abstract \| Publisher Full Text\n\nCiccarelli F, Martinis M, Ginaldi L: An Update on Autoinflammatory Diseases. Curr Med Chem. 2013 Dec; 21(3): 261–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeuenberger M, Berner J, Di Lucca J, et al.: PASS Syndrome: An IL-1-Driven Autoinflammatory Disease. Dermatology. 2016 Apr; 232(2): 254–8. PubMed Abstract \| Publisher Full Text\n\nContassot E, Beer HD, French LE: Interleukin-1, inflammasomes, autoinflammation and the skin. Swiss Med Wkly. 2012 May; 142(MAY): w13590. PubMed Abstract \| Publisher Full Text\n\nSonbol H, Duchatelet S, Miskinyte S, et al.: PASH syndrome (pyoderma gangrenosum, acne and hidradenitis suppurativa): a disease with genetic heterogeneity. Br J Dermatol. 2018; 178(1): e17–8. PubMed Abstract \| Publisher Full Text\n\nBraun-Falco M, Kovnerystyy O, Lohse P, et al.: Pyoderma gangrenosum, acne, and suppurative hidradenitis (PASH)-a new autoinflammatory syndrome distinct from PAPA syndrome. J Am Acad Dermatol. 2012 Mar; 66(3): 409–15. PubMed Abstract \| Publisher Full Text\n\nSaraceno R, Babino G, Chiricozzi A, et al.: A new syndrome associated with hidradenitis suppurativa with response to tumor necrosis factor inhibition. J Am. Dermatol. 2015; 72: e42–4. PubMed Abstract \| Publisher Full Text\n\nChamot AM, Benhamou CL, Kahn MF, et al.: Acne-pustulosis-hyperostosis-osteitis syndrome. Results of a national survey. 85 cases. Rev Rhum Mal Osteoartic. 1987; 54(3): 187–96. PubMed Abstract\n\nMarzano AV, Fanoni D, Antiga E, et al.: Expression of cytokines, chemokines and other effector molecules in two prototypic autoinflammatory skin diseases, pyoderma gangrenosum and Sweet’s syndrome. Clin Exp Immunol. 2014 Oct; 178(1): 48–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMankan AK, Dau T, Jenne D, et al.: The NLRP3/ASC/Caspase-1 axis regulates IL-1β processing in neutrophils. Eur J Immunol. 2012 Mar; 42(3): 710–5. PubMed Abstract \| Publisher Full Text\n\nGuma M, Ronacher L, Liu-Bryan R, et al.: Caspase 1-independent activation of interleukin-1β in neutrophil-predominant inflammation. Arthritis Rheum. 2009 Dec; 60(12): 3642–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitroulis I, Kourtzelis I, Kambas K, et al.: Regulation of the autophagic machinery in human neutrophils. Eur J Immunol. 2010 May; 40(5): 1461–72. PubMed Abstract \| Publisher Full Text\n\nDonetti E, Cornaghi L, Gualerzi A, et al.: An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17. Cytokine. 2014 Jul; 68(1): 1–8. PubMed Abstract \| Publisher Full Text\n\nMoran B, Sweeney CM, Hughes R, et al.: Hidradenitis Suppurativa Is Characterized by Dysregulation of the Th17:Treg Cell Axis, Which Is Corrected by Anti-TNF Therapy. J Invest Dermatol. 2017 Nov; 137(11): 2389–95. PubMed Abstract \| Publisher Full Text\n\nNegus D, Ahn C, Huang W: An update on the pathogenesis of hidradenitis suppurativa: implications for therapy. Expert Rev Clin Immunol. 2018 Apr; 14(4): 275–83. PubMed Abstract \| Publisher Full Text" }	[ { "id": "85309", "date": "26 May 2021", "name": "Mathilde Daxhelet", "expertise": [ "Reviewer Expertise Hidradenitis Suppurativa" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me the opportunity to review this really interesting case.\nThis case report describes an mixed syndrome encountered in a patient initially suffering from HS and treated with secukinumab.\nThe clinical, scintigraphic and histopathological images provided are of high quality.\nHowever I would like to add some details to the case description:\nThe locations of the HS lesions on the patient's skin.\n\nWhy is she unemployed? Because of HS?\n\nI would also add the initial severity assessment scores (IHS4 and DLQI) before secukinumab to better understand the improvement of symptoms with treatment.\n\nI would specify that the bull's head sign is typical of SAPHO syndrome to facilitate understanding of the case as not all doctors are experts in interpreting bone scans.\nFinally, I would add to the discussion that the initial response to secukinumab is promising but long-term follow-up of the patient is needed to assess long-term remission.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6784", "date": "16 Jul 2021", "name": "Nikolakis Georgios", "role": "Author Response", "response": "We would like to thank Dr. Daxhelet for their comments and their valuable contribution to improve the manuscript We have mentioned the initial HS scores. Unemployment was not correlated with HS. The localization of the lesions was highlighted. We added the comment about the bull's head sign and its correlation with SAPHO syndrome. A sentence was added in the discussion, underlining the importance of long-term follow-up also in combination with the other reviewer's remarks" } ] }, { "id": "85308", "date": "07 Jun 2021", "name": "Simone Garcovich", "expertise": [ "Reviewer Expertise Hidradenitis suppurativa" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 report on a exemplary case of syndromic hidradenitis suppurativa, requiring a complex systemic treatment. While potentially interesting, there are some minor points to be addressed by the authors, to further improve an already well-written case report:\n1. Regarding joint manifestations: it seems there is some minor uptake of radiotracer localized to the sacroiliac joints. Osteoarticular involvement strongly reminds the SAPHO pattern. Did the patient refer any back pain? Differential diagnosis with SAPHO should be discussed by the authors.\n\n2. The authors propose a novel acronym for the reported case. In my opinion, the conclusion section should be more conservative, as there is no experimental (genetic, molecular, cytokine data etc.) supporting this concept. Furthermore, this is a heavily pre-treated patient with a long disease history of HS. Could be there be an association between the previous exposure to multiple drugs (retinoids, anti-TNFs, etc.) and the development of \"paradoxical\" autoinflammatory extracutaneous involvement (such as recalcitrant PG)? This could be a more interesting take on the case? Please include some additional references.\n3. Treatment: the authors should better describe the take-home messages for the therapeutic management of such complex/severe disease phenotypes. Maybe, a combination treatment strategy for complex autoinflammatory disease phenotypes is more effective than monotherapy (i.e. secukinumab alone)?\nPlease include some additional references on this such as:\n\nGarcovich et al. (2017)1. Jin et al. (2019)2. Tan et al. (2021)3.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6785", "date": "16 Jul 2021", "name": "Nikolakis Georgios", "role": "Author Response", "response": "We would like to thank Dr. Garcovich for approving our manuscript, their careful evaluation of the case and their contribution to its improvement. The important differential diagnosis of the case being a variant of SAPHO syndrome including HS and a paradoxical reaction to the switch of the biologic(s) or to the previous adalimumab treatment were also addressed. An extensive opinion on combination of treatments (IL17/TNFα) or IL-1β was included but because of the lack of controlled studies - as Dr Garcovich mentioned in their article - it cannot have a strong recommendation level." } ] } ]	1	https://f1000research.com/articles/10-381
https://f1000research.com/articles/10-235/v1	24 Mar 21	{ "type": "Study Protocol", "title": "The evidence for interventions in early childhood allergy prevention – towards a living systematic review: protocol", "authors": [ "Uwe Matterne", "Christina Tischer", "Jiancong Wang", "Helge Knüttel", "Jon Genuneit", "Michael Perkin", "Christian Apfelbacher", "Christina Tischer", "Jiancong Wang", "Helge Knüttel", "Jon Genuneit", "Michael Perkin", "Christian Apfelbacher" ], "abstract": "Background: Research in early childhood allergy prevention (ECAP) is flourishing and new intervention strategies have proven to be promising. Due to the dynamic nature of ECAP, gaps between what is known and how guidelines inform practice are likely. A living systematic review (LSR) can narrow this gap by incorporating new evidence as it becomes available. No efficacy comparisons across various ECAP interventions for similar outcomes have been carried out. Networks of randomised clinical trials can be evaluated in the context of a network meta-analysis (NMA). We aim to establish a LSR on the efficacy and safety of any intervention investigated in randomised controlled trials (RCT) to prevent the occurrence of allergic sensitisation (AS), symptoms or diagnoses of allergic diseases in infancy and early childhood (0-3 years). Methods: A baseline SR will synthesise the evidence from existing SRs of RCTs as well as RCTs not yet considered in these. After completion of the baseline SR we propose to conduct a LSR. Using this methodology, we aim to undertake constant evidence surveillance, three-monthly search updates, and review updates every three months, should new evidence emerge. Conclusions: The ECAP evidence landscape has undergone dramatic transformations and this process is likely to continue. As a response to this, a LSR offers the potential to allow more timely synthesis of new evidence as it emerges. Long gaps between updates of SRs makes it harder for guidelines and recommendations to be up to date. Users of information, such as parents, may be confused if they encounter new evidence that is not part of a trusted guideline. A LSR approach allows us to continuously search the literature and update the evidence-base of existing ECAP interventions resulting in a decreased timespan from evidence accrual to informing clinical practice.", "keywords": [ "Early childhood allergy prevention", "randomised controlled trial", "living systematic review" ], "content": "Introduction\n\nAllergy in children is common. Frequent food allergies (FA) in children include hen’s egg, cow’s milk and peanut.1 Around 10% of children are affected by FA and the incidence is still rising in developing countries.1 Food allergy impacts quality of life.2–4 Allergic diseases (eczema,5–7 asthma and hay fever/allergic rhinitis)8,9 are also highly prevalent and associated with decreased health-related quality of life (HRQOL).10\n\nTo counteract the large number of allergies and reduce their burden, a major shift from merely managing manifest allergy to preventing its occurrence has taken place. Previous prevention efforts revolved around avoidance of potential allergens (particularly in at risk individuals), while more recently a new paradigm has been embraced whose focus is on early allergen exposure to induce immune tolerance. This new paradigm has been informing study designs for food allergy prevention. So far it has been established that oral tolerance induction is allergen specific and efficacious in single introduction trials of peanut and egg.11\n\nThere is also research activity revolving around environmentally derived prevention paradigms (i.e. exposure to rural environments, cowshed pill, or unpasteurised milk).12 However, one trial found no evidence that an orally applied bacterial lysate affected allergy development in at risk infants.13 It is however, expected that more research in this field will emerge soon.\n\nIn general, the design of preventive strategies has been informed by several hypotheses regarding the aetiology of allergy and allergic disease. The original hygiene hypothesis states that lack of exposure to common infections causes allergy.14 However, not all infections protect from allergy, resulting in criticism of the hypothesis.15 Further research has led to modifications of this hypothesis, suggesting that exposure to specific pathogens, commensals and symbionts protects against development of allergy.16,17 The dual-allergen-exposure hypothesis proposes that exposure to food allergens through the skin leads to allergy, while early consumption of these foods induces tolerance.18 While in the past it was advocated that avoidance of allergenic food would reduce the onset of allergy19 several recent trials have lent support to the dual-allergen-exposure hypothesis.20–22 The vitamin D hypothesis suggests that low vitamin D levels increase the risk of developing food allergy.23,24 There are, however, conflicting data on the relationship between vitamin D and the development of food allergy.25\n\nResearch in early childhood allergy prevention (ECAP) is flourishing and more than 50 systematic reviews (SR) that exclusively or partly reviewed RCTs26–81 have been published, some of which provide useful insights. However, not all include a standardised evaluation of the quality of the evidence (e.g. Grading of Recommendations Assessment, Development and Evaluation (GRADE))82 for randomised controlled trials (RCT).83 Risk of bias (RoB) assessment was done by a variety of different tools. Some used the National Institute for Health and Care Excellence (NICE) methodological checklist84 or the Strength of Recommendation Taxonomy (SORT)85 criteria. Thus, a homogenous RoB assessment and consistent evidence grading approach for each study type would be desirable.\n\nAs intervention effects may vary depending on whether at risk, not at-risk or children with manifest allergy are investigated, studies will be grouped accordingly. ECAP in the latter would still be considered primary prevention, rather than secondary or tertiary prevention, since we would look only at the prevention of new allergies.\n\nMany of the above SRs included observational studies as well as clinical trials. Studies examining the effect of breastfeeding versus no breastfeeding on allergy development cannot use an RCT design; however, for most other ECAP strategies, there are no ethical or other constraints that prevent the use of the gold standard RCT. Hence, we will only consider RCTs in our SR.\n\nNot all ECAP findings may yet be part of allergy prevention guidelines. As outlined above, due to the wealth of studies and new ECAP paradigms, more than 50 SRs have attempted to synthesise the evidence accumulated within the various approaches to ECAP. It is likely that there are gaps between what we know from the best available research and what happens in healthcare practice due to the dynamic nature of ECAP research. The incorporation of new findings into existing reviews or their updates is time-consuming and not always feasible. Hence, adapting prevention guidelines in light of new findings and providing health care providers and other information users with up-to-date evidence may be impeded. Recent advances in the presentation of systematically reviewed (qualitative and meta-analytic) data have led to the concept of a living systematic review (LSR). A LSR is defined as “a systematic review that is continually updated, incorporating relevant new evidence as it becomes available”.86\n\nFurthermore, no efficacy comparisons across various ECAP strategies for similar outcomes have been carried out. Networks of randomised clinical trials can be evaluated in the context of a network meta-analysis (NMA). NMA refers to a procedure that allows inferences about the comparative effectiveness of interventions that may or may not have been evaluated directly against each other.87–89\n\nWe aim to establish an LSR on the efficacy and safety of any intervention to prevent the occurrence of allergic sensitisation (AS), symptoms or diagnoses of allergic diseases in infancy and early childhood (0-3 years).\n\nThe specific objectives are:\n\n1. To identify all individual-level interventions using the oral, skin, environmental or pharmaceutical route for the prevention of allergy in allergy-free children or the prevention of new allergies in children with manifest allergy\n\n2. To identify all community-level interventions (such as community programmes promoting dietary and environmental diversity in early life) which have thus far been investigated in RCTs to prevent the occurrence of allergy in infancy.\n\n3. To summarise the evidence regarding the effects (efficacy and safety) of these interventions in preventing the occurrence of allergy in infancy and early childhood.\n\n4. To judge the quality of this evidence.\n\n5. To provide a corresponding plain language summary (PLS) accessible for consumers.\n\n6. To develop a workflow for an LSR, which ensures that the evidence synthesis is continuously kept up-to-date.\n\n\nMethods\n\nThe review will be undertaken according to the methods outlined in the Cochrane Handbook for Systematic Reviews of Interventions,90 PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses)91 and its extension for application to NMA.92 A protocol registration will be made at PROSPERO.93 Any updates to the protocol will be made through PROSPERO. The protocol was developed closely considering the Prisma-P94 checklist.95 A baseline SR will synthesise the evidence available so far and then transformed into a LSR by regular updates.\n\nStudies will be eligible for inclusion in the review should they meet the following (PICO) criteria:\n\n- Date of publication: 1980 onward\n\n- Types of study: Randomised controlled trials,\n\n- Population: expectant and/or breastfeeding mothers of and/or children 0 - 3 years (at time of intervention), at risk children (0 - 3 years) (at least one parent with known allergic disease), not at risk children (0-3 years) (no parental allergic disease), children (0-3 years) with manifest allergy (only if intervention aims at preventing a new allergy or study reports this outcome)\n\n- Interventions: any aimed at the individual or community level at preventing the onset of new allergy or allergic disease or allergic sensitisation\n\no Oral route (supplements (e.g. vitamins, minerals, pro-, pre-, symbiotic, gut bacteria), time and presence of allergenic food introduction (e.g. peanut, egg protein, cow’s milk, fish), variation in condition (raw, cooked, pasteurised, fermented) and/or amount of complimentary food introduction, avoidance of potential allergens\n\no Skin route (e.g. emollients, treated water for washing)\n\no Environmental interventions (exposure to natural outdoor environments, green space, outdoor spaces, exposure to farm environments (cow- and other animal-shed bacteria, farming associated bacteria and microbes, bacterial lysate, acinetobacter, microbiome, mucous membrane, microbiota), avoidance of chemicals or allergens (e.g. mattress protector (mites))\n\no Pharmaceutical prevention (e.g. allergen-immuno-therapy (AIT), Bacillus Calmette-Guérin (BCG) vaccination))\n\n- Comparator: inactive comparator such as placebo, no intervention or usual care\n\n- Primary outcomes:\n\no Physician-diagnosed or parent-reported incidence of allergic asthma (AA), allergic rhinitis (AR), atopic eczema (AE), food allergy (FA)\n\no Physician-diagnosed or parent-reported recurrent symptoms of sneeze, wheeze, cough, itch, flexural eczema, or FA\n\n- Secondary outcomes:\n\no incidence of AS measured by in-vivo tests such as skin-prick test (SPT)\n\no incidence of AS measured by in-vitro tests such as fluorescently labelled anti-IgE antibody, enzyme-linked immunosorbent assay (ELISA), or Radio-Allergo-Sorbent-Test (RAST)\n\no adverse events (AE), severe adverse events (SAE), withdrawals\n\nECAP has revolved around antenatal and postnatal strategies targeting the mother and strategies targeting the child after birth (often during a critical period). Strategies that have been explored in RCTs are illustrated in Table 1.\n\nAny observational research (cross-sectional, case-control, case-series, prospective/retrospective cohort studies) or quasi-experimental studies (matched controlled designs) will be excluded. We will not consider interventions aimed at treating allergy unless the intervention is also hypothesised to reduce the onset of a new allergic manifestation.\n\nWe will use five approaches for the identification of studies for the baseline SR:\n\n\n\n1) Topic-based searches in databases and registries\n\nWe will search for all relevant RCTs regardless of publication status (published, unpublished, in press, or ongoing) in the following bibliographic databases: MEDLINE (Ovid), Embase (Ovid), CENTRAL (Cochrane Library), Science Citation Index Expanded & Social Sciences Citation Index (Web of Science), Cochrane Skin Group Specialized Register, GREAT (The Global Resource of EczemA Trials, Centre of Evidence Based Dermatology), in clinical trial registries (U.S. National Institutes of Health ClinicalTrials.gov, ISRCTN registry, Australian New Zealand Clinical Trials Registry, World Health Organization International Clinical Trials Registry Platform, EU Clinical Trials Register).\n\nWe will search the conference proceedings (European Academy of Dermatology and Venereology (EADV), European Academy of Allergy and Clinical Immunology (EAACI), American Academy of Dermatology (AAD), American Academy of Allergy, Asthma & Immunology (AAAI), Asia Pacific Association of Allergy, Asthma and Clinical Immunology (APAAACI) and Asia Pacific Association of Pediatric Allergy, Respirology & Immunology (APAPARI), World Allergy Organisation (WAO)) and Sociedad Latinoamericana de Alergia, Asma e Inmunología, Asunción (SLAAI). Additionally, we will review published documents from health technology assessment agencies (e.g. NICE, IQWIG). No restriction on status or year of publication will be applied. The search will be restricted to publications in English, German, French, Italian or Spanish. An initial draft search strategy for MEDLINE was developed by a medical librarian experienced in comprehensive searches for systematic reviews (Box 1). The performance of his strategy will be checked against the growing set of known relevant records. The final search strategy will be peer reviewed. The search strategy is composed of the components Population, Intervention, Outcome and Study filter that will be intersected with the Boolean AND operator. For each of the components relevant terms from text fields and controlled vocabulary were used in order to achieve high sensitivity. This strategy will be adapted to the other databases as appropriate.\n\nBox 1: Initial draft search strategy for MEDLINE\n\n\n\n1 exp infant/ or Child, Preschool/ or (child or children).ti,ab,kf. or (pre-school$ or preschool$).ti,ab,kf. or Nurseries/ or (nursery or nurseries).ti,ab,kf. or exp Parents/ or (parent or parents or mother or mothers).ti,ab,kf. or (infant or infants).ti,ab,kf. or infancy.ti,ab,kf. or toddler?.ti,ab,kf. or (baby or babies).ti,ab,kf. or newborn$.ti,ab,kf. or neonat$.ti,ab,kf. or Pediatrics/ or (pediatric$ or paediatric$).ti,ab,kf. or early childhood.ti,ab,kf. or (Pregnant Women/ or Pregnancy/ or Prenatal Nutritional Physiological Phenomena/) or pregnan$.ti,ab,kf. or Prenatal Exposure Delayed Effects/or Maternal Exposure/or ((maternal or prenatal) adj1 exposure$).ti,ab,kf. or (fetus or fetuses or fetal or foetus or foetuses or foetal).ti,ab,kf. or Fetus/ (Population)\n\n2 exp Preventive Health Services/ or Preventive Medicine/or \"prevention control\".fs. or prevent$.ti,ab,kf. or prophyla$.ti,ab,kf. or Infant Formula/or (formula or supplement$).ti,ab,kf. or ((risk or protect$ or development or avoidance or exposure or introduction) adj6 (allerg$ or hypersensitivit$ or atopy or atopic or dermatitis or neurodermatitis or asthma)).ti,ab,kf. (Intervention)\n\n3 exp Hypersensitivity/ or Allergens/ or allerg$.ti,ab,kf. or hypersensitivit$.ti,ab,kf. or prick test$.ti,ab,kf. or exp asthma/ or Dyspnea/or (asthma$ or dyspnea or wheezing).ti,ab,kf. or (difficult$ adj1 breathing).ti,ab,kf. or rhinoconjunctivitis.ti,ab,kf. or (atopic adj1 (dermatit$ or neurodermatit$ or eczema or disease)).ti,ab,kf. or Diaper Rash/or ((infant or infantile or diaper) adj1 (rash or rashes or eczema or dermatit$)).ti,ab,kf. or Disseminated Neurodermat$.ti,ab,kf. (Outcome)\n\n4 ((randomized controlled trial or controlled clinical trial).pt. or randomized.ab. or placebo.ab. or drug therapy.fs. or randomly.ab. or trial.ab. or groups.ab.) not (exp animals/not humans.sh.) (Cochrane Highly Sensitive Search Strategy for identifying randomized trials in MEDLINE: sensitivity-maximizing version (2008 revision))\n\n5 1 and 2 and 3 (Combined concepts: Patients AND Intervention AND Outcome)\n\n6 5 and 4 (Combined concepts: Patients AND Intervention AND Outcome AND RCTs)\n\n2) Searches by trial registry numbers\n\nRegistry numbers of eligible trials will be collected to be used in follow-up searches with the aim to identify additional trial reports.\n\n3) Trials included in relevant SRs\n\nWe will search for relevant SRs in order to identify additional trials in these SRs.\n\n4) Reference lists of included trials\n\nWe will screen the reference lists of all included study reports. To aid in this process the reference lists will be downloaded from Web of Science when available.\n\n5) Existing systematic reviews\n\nFor the creation of the baseline SR previous SRs on ECAP will be systematically searched for.\n\nDatabase search results will be imported into EppiReviewer (version 4.11.5.3)96 for deduplication and study selection. Assessment of eligibility, data extraction, risk of bias (RoB) evaluation and quality of the evidence assessment will be carried out by at least two researchers independently. The latter will be done according to the GRADE82 recommendations.\n\nThe following data will be extracted:\n\nStudy characteristics: Author, year of publication, geographical region, study design, type of intervention, type of control, number of participants in intervention group and control group, study duration, time points of assessment, follow-up period, type of primary and secondary outcomes and safety indicators.\n\nParticipant characteristics: Person in whom intervention took place (mother (prenatal, postnatal), child, both), age, sex, ethnicity, allergic risk status, parental atopy, presence of allergic sensitisation and/or condition.\n\nStudy outcomes: Efficacy outcomes (unadjusted/adjusted): Incidence of allergic sensitization and/or allergic disease; safety outcomes: proportions of AEs, SAEs, withdrawal due to AEs\n\nExisting systematic reviews will be incorporated into the baseline SR. Assessment of the quality of these will be carried out by A MeaSurement Tool to Assess systematic Reviews-2 (AMSTAR-2).97 AMSTAR-2 is a critical appraisal tool for SRs that include randomised or non-randomised studies of healthcare interventions, or both.97 Findings of SRs whose quality has been judged adequate will be summarised and studies of the same intervention and outcome not considered in these SR will be individually assessed for RoB and quality of the evidence.\n\nIf no obvious qualitative heterogeneity within studies exists, we will perform meta-analyses across similar applications of interventions. Pair-wise meta-analyses between two intervention conditions (provided at least two eligible studies exist that are not too heterogeneous) for each different endpoint/outcome (FA, AS, AE, asthma, AR) will be conducted. These are to be updated in the LSR as relevant new studies emerge. To guard against potential type I error inflation and occurrence of type II errors which are a function of the number of analyses done with the same data (as new data is incorporated with each update) we will follow the recommendations outlined by Simmonds et al.98\n\nPairwise meta-analysis is a statistical technique for quantitatively synthesising similar studies in a systematic review. Useful in its own right, it is, however, limited in that it can only compare two interventions simultaneously, and only those evaluated directly in head-to-head trials.89 Pairwise meta-analysis allows for comparisons between pairs of interventions (an experimental intervention and a comparator intervention) for a specific outcome in a particular population or setting. It is, however, often the case that a variety of different interventions are available for any given condition. A single SR that includes all relevant interventions and presents their comparative effectiveness and potential for harm would help people to decide between alternative interventions. NMA affords an analysis option for such a review.88 A network of interventions consists of any set of studies that links three or more interventions via direct comparisons. Within a network, there can be numerous ways to make indirect comparisons between interventions. They refer to comparisons that have not been made directly within studies. Mathematical combinations of the available direct intervention effect sizes are used to estimate indirect effects. In NMA direct and indirect estimates across a network of interventions are combined in a single analysis.88\n\nWe also aim to conduct NMA to compare different interventions for the same endpoint/outcome (PO, SO and AE), should assumptions for NMA be met. We will also perform a full evaluation of the confidence in the results from NMA by using the web application CINeMA (Confidence in Network Meta-Analysis).99 This web application simplifies the evaluation of confidence in the findings from NMA and has evolved out of the GRADE approach. The GRADE82 approach offers an assessment of the confidence in the results from systematic reviews and meta-analyses. Many organisations, for example the World Health Organisation, have adopted the GRADE approach.100 Based on GRADE, two systems have been proposed to evaluate the credibility of results from NMAs,101,102 but the complexity of the methods and lack of suitable software have limited their wide adoption.103 CINeMA is based on six domains: within-study bias (referring to the impact of RoB in the included studies), across-studies bias (publication or reporting bias), indirectness (relevance to the research question and transitivity), imprecision (comparing the range of treatment included in the 95% confidence interval with the range of equivalence), heterogeneity (predictive intervals), and incoherence (if estimates from direct and indirect evidence disagree).111 Judgements across the six domains are then summarised to obtain four levels of confidence for each relative treatment, corresponding to the usual GRADE82 approach: very low, low, moderate or high.\n\nIf possible, we will also perform subgroup analysis (sex, atopic predisposition as a marker of high risk to develop allergic sensitization or allergic disease).\n\nThis review will continuously evaluate the role of interventions for the prevention in early childhood (ECAP). A living systematic review is a cumulative synthesis that is updated regularly as new evidence becomes available.86 According to Cochrane’s Living Evidence Network104 transformation into a LSR is justified when the review question is a particular priority for decision-making, there is an important level of uncertainty in the existing evidence and there is likely to be emerging evidence that will affect the conclusions of the LSR. The review question is of particular priority for decision making because one SR on ECAP105 is among the Cochrane Priority Reviews and the rate of publications on ECAP interventions has substantially increased over the past years (Figure 1) and is expected to continue so. The level of uncertainty remains an issue. A recent SR on interventions for pregnant or breastfeeding women and/or infants concluded that while dietary avoidance of food allergens, vitamin supplements, fish oil, probiotics, prebiotics, synbiotics, and emollients may have little to no effect on preventing food allergy, the evidence was judged as very uncertain.43 New evidence is expected to emerge on variations of the induction of tolerance paradigm as paved by previous trials such as EAT20 or LEAP.22,106\n\nAn artificial intelligence algorithm for automated searches will be developed in collaboration with information specialists and software engineers. We will gradually incorporate more and more intelligent software tools for eligibility tests, data extraction, and analysis and synthesis presentation as they prove to be reliable in the subsequent update processes.\n\nThis evolving search algorithm is to be run at three-monthly intervals and will be set up to notify the author team about studies with a high likelihood of being eligible. At the same interval manual searches will be conducted until the automated search algorithm is sufficiently reliable. The author team will review the search results, decide upon inclusion, and update the living SR’s web version after a new evidence synthesis is deemed necessary every three months. At the same time references of included studies and the corresponding tables and figures will be updated. Every three months the date of each subsequent search, the number of new included studies, and new summary of findings tables will be published on the LSR’s website along with an updated plain language summary.\n\nThe LSR will be maintained in its ‘living’ form until no new evidence is likely to arise and/or so long as the certainty of the evidence for particular ECAP interventions remains unsettled. The necessary future funding will be sought.\n\nThe baseline SR will be published in a peer-reviewed journal and indexed publication platform that allows the linkage of review updates through versioning of the review publication. A plain language summary will be provided on a project website. This website is intended to also contain graphical and other information for health care providers and the public.\n\nFor the LSR, we will consider resubmission to the journal in which the baseline SR is published should the direction of the effect on the critical outcomes change or a substantial modification to the evidence’s certainty occur.\n\nAt submission of this protocol the final search strategy is being peer-reviewed.\n\n\nDiscussion\n\nThe prevention of allergy in early childhood is important given the high personal burden and societal costs of allergic diseases. The ECAP evidence landscape has undergone dramatic transformations and this process is likely to continue. As a response to this, a LSR offers the potential to undertake a more timely synthesise of new evidence as it emerges. Long gaps in between updates of SRs may make it harder for guidelines and recommendations to be current and up to date. Users of information, such as parents may be confused if they encounter new evidence that is not part of a trusted guideline. A LSR approach allows us to continuously search the literature and update the evidence-base of existing ECAP interventions resulting in a decreased timespan from evidence accrual to informing clinical practice.\n\nIt is also crucially important to assess whether an ECAP intervention is associated with harm. For instance, there is evidence suggesting that early egg white powder introduction at 4 to 6 months of age for the prevention of egg allergy in children from the general population may increase the occurrence of allergy.108\n\nFurther, a consistent assessment of the certainty of the evidence in line with the GRADE recommendation will be carried out across all included studies. This will allow a high level of concordance between the certainty of the evidence and future guideline recommendations.\n\nWhilst our approach of including all interventions for the prevention of all allergies in early childhood may seem ambitious, none of the individual systematic reviews being undertaken at present (e.g.33,34) will be able to conduct a comparison of multiple interventions (network meta-analysis). The latter provides information regarding the relative treatment effect and the ranking order for multiple treatments for a particular outcome irrespective of whether they were conducted in the same or different trials.\n\nThe baseline and subsequent LSR will identify research gaps needing to be addressed by future research. The clinical implications of the LSR will be the provision of up-to-date information that can dynamically inform national, international clinical and public health guidelines and influence the practice of allergists, paediatricians, ENT specialists, other primary care providers and public health authorities in terms of evidence-based ECAP strategies. The provision of a regularly updated plain language summary will benefit parents as they can easily access a widely visible website with information provided in lay terms. The LSR will have include research evidence from across the world and we hope will have a multinational benefit.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nFigshare: PRISMA-P checklist for “The evidence for interventions in early childhood allergy prevention – towards a living systematic review: protocol” https://doi.org/10.6084/m9.figshare.14135450.v1.95\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nLoh W, Tang MLK: The Epidemiology of Food Allergy in the Global Context. Int J Environ Res Public Health. 2018; 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDunn Galvin A, Dubois AEJ, Flokstra-de Blok BMJ, et al.: The effects of food allergy on quality of life. Chem Immunol Allergy. 2015; 101: 235–52. 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Reference Source\n\nWells G, Shea B, O’Connell D, et al.: The Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomised studies in metaanalyses.Cited 8 Jul 2020. Reference Source\n\nNational Institute for Health and Care Excellence: Developing NICE guidelines: the manual.Cited 8 Jul 2020. Reference Source\n\nEbell MH, Siwek J, Weiss BD, et al.: Strength of recommendation taxonomy (SORT): a patient-centered approach to grading evidence in the medical literature. Am Fam Physician. 2004; 69: 548–56. PubMed Abstract\n\nElliott JH, Synnot A, Turner T, et al.: Living systematic review: 1. Introduction-the why, what, when, and how. J Clin Epidemiol. 2017; 91: 23–30. PubMed Abstract \| Publisher Full Text\n\nMills EJ, Thorlund K, Ioannidis JPA: Demystifying trial networks and network meta-analysis. BMJ. 2013; 346: f2914. PubMed Abstract \| Publisher Full Text\n\nChaimani A, Caldwell DM, Li T, et al.: Chapter 11: Undertaking network meta-analyses. In: Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, Welch VA (editors). Cochrane Handbook for Systematic Reviews of Interventions version 6.1 (updated September 2020). Cochrane; 2020 Cited 20 Jan 2021. Reference Source\n\nRouse B, Chaimani A, Li T: Network meta-analysis: an introduction for clinicians. Intern Emerg Med. 2017; 12: 103–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins JPT, Thomas J, Chandler J, et al.; Cochrane Handbook for Systematic Reviews of Interventions. 2nd ed. Chichester (UK): John Wiley & Sons; 2019.\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Journal of clinical epidemiology. 2009; 62: 1006–12. 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Reference Source\n\nThomas J, Graziosi S, Brunton J, et al.: EPPI-Reviewer: advanced software for systematic reviews, maps and evidence synthesis: EPPI-Centre Software.UCL Social Research Institute; 2020.\n\nShea BJ, Reeves BC, Wells G, et al.: AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. BMJ. 2017; 358: j4008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimmonds M, Salanti G, McKenzie J, et al.: Living systematic reviews: 3. Statistical methods for updating meta-analyses. J Clin Epidemiol. 2017; 91: 38–46. PubMed Abstract \| Publisher Full Text\n\nInstitute of Social and Preventive Medicine, University of Bern: CINeMA: Confidence in Network Meta-Analysis [Software].2017. Reference Source\n\nWorld Health Organisation: WHO handbook for guideline development.2012 Cited 20 Jan 2021. Reference Source\n\nPuhan MA, Schünemann HJ, Murad MH, et al.: A GRADE Working Group approach for rating the quality of treatment effect estimates from network meta-analysis. BMJ. 2014; 349: g5630. PubMed Abstract \| Publisher Full Text\n\nSalanti G, Del Giovane C, Chaimani A, et al.: Evaluating the quality of evidence from a network meta-analysis. PLoS One. 2014; 9: e99682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNikolakopoulou A, Higgins JPT, Papakonstantinou T, et al.: CINeMA: An approach for assessing confidence in the results of a network meta-analysis. PLoS Med. 2020; 17: e1003082. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrooker J, Synnot A, McDonald S, et al.: Guidance for the production and publication of Cochrane living systematic reviews: Cochrane Reviews in living mode.Version December 2019 Cited 25 Feb 2021. Reference Source\n\nBest CM, Xu J, Patchen BK, et al.: Vitamin D supplementation in pregnant or breastfeeding women or young children for preventing asthma. Cochrane Database Syst Rev. 2019. Publisher Full Text \| Free Full Text\n\nDu Toit G, Sayre PH, Roberts G, et al.: Allergen specificity of early peanut consumption and effect on development of allergic disease in the Learning Early About Peanut Allergy study cohort. J Allergy Clin Immunol. 2018; 141: 1343–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmidt L, Olorisade BK, McGuinness LA, et al.: Data extraction methods for systematic review (semi)automation: A living review protocol. F1000Res 2020; 9: 210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellach J, Schwarz V, Ahrens B, et al.: Randomized placebo-controlled trial of hen's egg consumption for primary prevention in infants. J Allergy Clin Immunol. 2017; 139: 1591–1599. e2. PubMed Abstract \| Publisher Full Text\n\nLitonjua AA, Carey VJ, Laranjo N, et al.: Effect of Prenatal Supplementation With Vitamin D on Asthma or Recurrent Wheezing in Offspring by Age 3 Years: The VDAART Randomized Clinical Trial. JAMA. 2016; 315: 362–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCabana MD, McKean M, Caughey AB, et al.: Early Probiotic Supplementation for Eczema and Asthma Prevention: A Randomized Controlled Trial. Pediatrics. 2017; 140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGore C, Custovic A, Tannock GW, et al.: Treatment and secondary prevention effects of the probiotics Lactobacillus paracasei or Bifidobacterium lactis on early infant eczema: randomized controlled trial with follow-up until age 3 years. Clin Exp Allergy. 2012; 42: 112–22. PubMed Abstract \| Publisher Full Text\n\nZeiger RS, Heller S: The development and prediction of atopy in high-risk children: follow-up at age seven years in a prospective randomized study of combined maternal and infant food allergen avoidance. J Allergy Clin Immunol. 1995; 95: 1179–90. PubMed Abstract \| Publisher Full Text\n\nTachimoto H, Imanari E, Mezawa H, et al.: Effect of Avoiding Cow's Milk Formula at Birth on Prevention of Asthma or Recurrent Wheeze Among Young Children: Extended Follow-up From the ABC Randomized Clinical Trial. JAMA Netw Open. 2020; 3: e2018534. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamamoto-Hanada K, Kobayashi T, Williams HC, et al.: Early aggressive intervention for infantile atopic dermatitis to prevent development of food allergy: a multicenter, investigator-blinded, randomized, parallel group controlled trial (PACI Study)-protocol for a randomized controlled trial. Clin Transl Allergy. 2018; 8: 47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZolkipli Z, Roberts G, Cornelius V, et al.: Randomized controlled trial of primary prevention of atopy using house dust mite allergen oral immunotherapy in early childhood. J Allergy Clin Immunol 2015; 136: 1541–1547. e11. PubMed Abstract \| Publisher Full Text\n\nValovirta E, Petersen TH, Piotrowska T, et al.: Results from the 5-year SQ grass sublingual immunotherapy tablet asthma prevention (GAP) trial in children with grass pollen allergy. J Allergy Clin Immunol. 2018; 141: 529–538. e13. PubMed Abstract \| Publisher Full Text\n\nSteenhuis TJ, van Aalderen WMC, Bloksma N, et al.: Bacille-Calmette-Guerin vaccination and the development of allergic disease in children: a randomized, prospective, single-blind study. Clin Exp Allergy. 2008; 38: 79–85. PubMed Abstract \| Publisher Full Text\n\nThøstesen LM, Kjaer HF, Pihl GT, et al.: Neonatal BCG has no effect on allergic sensitization and suspected food allergy until 13 months. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "84078", "date": "02 Jun 2021", "name": "Carlo Caffarelli", "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 present manuscript describes a project on future systematic reviews for interventions in early childhood allergy prevention. This is an interesting issue. However, some important points need to be clarified.\nIntroduction:\nThe rationale of the study is focused on dual-allergen-exposure hypothesis. It should be underlined that several strategies have been proposed to reduce the burden of allergic diseases as depicted in Table 1. For example, a multifaceted intervention that reduces exposure to allergens has been shown to be effective in preventing allergy onset. On the other hand, the list of “more than 50 systematic reviews” on early childhood allergy prevention is incomplete. For example see Mastrorilli et al. (20201).\n\nStudy selection:\nIt seems that systematic reviews can only be useful for retrieving papers that have not been identified with other means. It is therefore difficult to understand why you should incorporate and assess existing systematic reviews. This point needs to be discussed and it should be providing possible explanations since it is not an objective of the current project.\n\nMeta-analysis:\nIt should be clarified whether a meta-analysis will be done when it results that heterogeneous studies with different design and outcome have been included. It seems that sometimes, this frequent limitation is not considered by meta-analysis. So, a statement on this issue is warranted.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6889", "date": "09 Jul 2021", "name": "Uwe Matterne", "role": "Author Response", "response": "We thank the reviewer very much for having taken the time to critically review our manuscript. We have revised the manuscript taking your suggestions into account and hope to have improved the manuscript sufficiently. Introduction: The rationale of the study is focused on dual-allergen-exposure hypothesis. It should be underlined that several strategies have been proposed to reduce the burden of allergic diseases as depicted in Table 1. For example, a multifaceted intervention that reduces exposure to allergens has been shown to be effective in preventing allergy onset. On the other hand, the list of “more than 50 systematic reviews” on early childhood allergy prevention is incomplete. For example see Mastrorilli et al. (20201). Thank you for this valuable comment. We agree with the reviewer, that there are several strategies within each route of administration. Table 1 lists the main routes of administration along with one illustrative example. Table 1 is not meant to be fully comprehensive of all strategies but instead meant to provide an indication of the hitherto used routes of intervention with an accompanying example. We would like to highlight, though, that for the purposes of the manuscript we had not intended to do a systematic search of all potentially relevant reviews. Thus, there may still be other reviews having examined randomised controlled trials of ECAP that are not listed in the manuscript. The suggested example by Mastrorilli et al., though a review, is not a systematic review. Study selection: It seems that systematic reviews can only be useful for retrieving papers that have not been identified with other means. It is therefore difficult to understand why you should incorporate and assess existing systematic reviews. This point needs to be discussed and it should be providing possible explanations since it is not an objective of the current project. We appreciate the reviewer’s comment very much. The respective section was revised to more fully convey the methodological approach to using existing SRs in the production of the baseline review. Meta-analysis: It should be clarified whether a meta-analysis will be done when it results that heterogeneous studies with different design and outcome have been included. It seems that sometimes, this frequent limitation is not considered by meta-analysis. So, a statement on this issue is warranted. We are grateful for the reviewer’s comment and would like to stress that in the ‘Meta-analyses and network-meta-analysis’ section it is said, that only similar interventions (tested in RCTs only) and the same outcome will be considered in meta-analyses. We added another sentence at the end of this section to clarify that we intend to examine the sources of heterogeneity and the impact of inclusion of studies at various risk of bias in meta-analyses." } ] }, { "id": "86682", "date": "16 Jun 2021", "name": "Mirjana Turkalj", "expertise": [ "Reviewer Expertise allergen immunotherapy", "environment and allergy development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the study \"The evidence for interventions in early childhood allergy prevention - towards a living systematic review: protocol\" the authors propose a new, better and more objective intervention concept in early childhood allergy prevention (ECAP). The authors present concrete and objective methodology and metrics. In fact, living systematic review (LSR) requires regular and continuous updates with the implementation of new knowledge in guidelines and clinical practice. - However, there are several drawbacks to this concept; confusing and contradictory messages for the clinician in practice, as well as for patients (parents of children), giving guidelines for treatment that may not be applicable to every patient.\nIt is therefore important to emphasize that evidence for interventions in early childhood allergy prevention, in fact, only \"current guidelines\" that can be changed or modified, and that they are not applicable to every patient. It is important for the authors to emphasize that personalized approach is still crucial in prevention strategy.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-235
https://f1000research.com/articles/10-575/v1	15 Jul 21	{ "type": "Research Article", "title": "Residues of tetracyclines and β-lactams antibiotics induce carbonylation of chicken breast ", "authors": [ "Johana Marquez", "Albeiro Marrugo-Padilla", "Darío Méndez Cuadro", "Erika Rodríguez Cavallo", "Johana Marquez", "Albeiro Marrugo-Padilla", "Darío Méndez Cuadro" ], "abstract": "Background: Worldwide, chicken meat is widely consumed due to its low cost, high nutritional value and non-interference with religious or cultural beliefs. However, during animal husbandry chickens are exposed to many chemical substances, including tetracyclines and β-lactams, which are used to prevent and cure several infections. Some residues of these compounds may bioaccumulate and be present in chicken meat after slaughtering, promoting oxidative reactions. Methods: In order to evaluate in vitro carbonylation induced by tetracyclines and β-lactams residues, a proteomic approach was used. For this, chicken muscle was individually contaminated with tetracyclines (tetracycline, chlortetracycline, oxytetracycline, and doxycycline) and β-lactams (ampicillin, benzathine penicillin, dicloxacillin and oxacillin) at 0.5, 1.0 and 1.5 times their maximum residue level (MRL). Then, sarcoplasmic, myofibrillar and insoluble proteins were extracted and their content were measured using the Bradford method. Protein carbonylation was measured using the 2,4-Dinitrophenylhydrazine alkaline method. Results: Residues of tetracyclines and β-lactams induced in vitro carbonylation on sarcoplasmic, myofibrillar and insoluble proteins even at 0.5MRL concentrations (p<0.05). When comparing the carbonylation induced by both antibiotics no differences were found (p>0.05). Variables such as the partition coefficient (log P) and the concentration of these antibiotics showed a high correlation with the oxidative capacity of tetracyclines and β-lactams on chicken breast proteins. Conclusions: This study shows that the presence of tetracyclines and β-lactams residues at MRLs concentrations promotes in vitro carbonylation on chicken breast proteins. Our results provide important insights about the impact of antibiotics on the integrity of meat proteins intended for human consumption.", "keywords": [ "Tetracyclines", "β-lactams", "carbonylation", "proteins", "meat" ], "content": "Introduction\n\nTetracyclines (TCs) and β-lactams (β-Lts) are widely used antibiotics in veterinary medicine for the treatment and prophylaxis of bacterial diseases affecting animals.1 In poultry, these antibiotics are employed to treat diseases caused by gram-positive and gram-negative bacteria, and in some cases are also administered at sub-therapeutic concentrations in the feed to promote the animal’s growth and ensure productive performance.2,3 In this context, this extensive and often indiscriminate use of TCs and β-LTs in poultry can induce the presence of their residues in poultry products such as chicken breast and eggs; these residues can reach and be ingested by consumers, and promote negative effects on their health, mainly through antimicrobial resistance.1\n\nIn order to protect to consumer, the European Commission and the Codex Alimentarius have stablished the maximum residue level (MRL) for antibiotics in products of animal origin. The MRL is defined as the highest level of residue of a pesticide or antibiotic that is legally tolerated in food and is not expected to cause harm to humans. Despite the regulation, several investigations have reported levels of TCs and β-LTs greater than their MRLs in chicken meat and other products derived from poultry farms, such as eggs; this could promote negative effects on consumers stated above.4,5\n\nDespite the recurring presence of antibiotic residues in chicken meat and its derivatives, no investigations evaluating the effects of TCs and β-LTs at their MRLs on chicken meat components, including proteins, are being carried out, even though proteins are one of its most abundant constituents. In previous studies we have demonstrated that residues of these antibiotics at their MRLs are capable of inducing oxidative stress on biological models such as beef6 and milk,7 which could have a negative impact on food safety and quality. Protein carbonylation is currently recognized as one of the main biomarkers for oxidative stress in food,8 and several publications have demonstrated its impact in chicken meat and the alteration of its functional properties such as solubility and digestibility.9,10 In addition, the effects promoted by the consumption of foods with carbonylated proteins, such as metabolic disorders and inflammatory processes at the intestinal level and even the promotion of carcinogenic processes, have also been demonstrated.11,12\n\nChicken meat is the most common product consumed in the word due its affordability, although chicken meat proteins are susceptible to suffer oxidative reactions which can be promoted by external factors as animal feed, slaughter, processing and storage. Thus, the objective of this study is to evaluate the in vitro carbonylation induced by tetracyclines and β-lactams residues on chicken muscle proteins.\n\n\nMethods\n\nPrimary standards of Ampicillin trihydrate (99.0%, AMP), Benzathine penicillin G tretrahydrate (97.2%, PNG), Dicloxacillin sodium hydrate (99.0%, DCL), Oxacillin salt hydrate (99.0%, OXA), Tetracycline hydrochloride (97.7%, TC), Doxycycline hyclate (98.7%, DXC), Chlortetracycline hydrochloride (94.6%, CTC) and Oxytetracycline hydrochloride (95.0%, OTC) were supplied by Dr. Ehrenstofer GmbH (Germany). The 2,4-Dinitrophenylhydrazine, potassium phosphate, sodium azide, hydrochloric acid, sodium bicarbonate and calcium chloride were purchased from PanReac (Barcelona, Spain). Analytical grade methanol, sodium hydroxide EMSURE (99%), biotechnological grade sodium chloride, 2-mercaptoethanol, and coomassie blue brilliant G250 (CBB) were purchased from Merck (Darmstadt, Germany). Bovine serum albumin (BSA) free of fatty acids (98%) was supplied from Sigma Aldrich (San Luis, USA). Water was purified with a Milli-Q system (Millipore, Bedford, MA, USA).\n\nIndividual stock solutions of tetracyclines and β-lactams (100 μg·mL-1) were prepared with milli-Q water and methanol, respectively. For the sample’s contamination with the antibiotics of interest, serial dilutions of the stocks were made until obtaining an antibiotic concentration of 10 μg·mL−1 (working solution), in order to avoid sample protein precipitation during the sample preparation process.13\n\nChicken breast samples were purchased from a local market in Cartagena, Colombia. They were stored in plastic polyethylene bags, transported to the laboratory maintaining cold chain (4°C), cut and cleaned to remove visible connective tissue and then, mechanically homogenized using a blender (Powergen by Fisher). Then, one-gram replicates of homogenized samples were collected in falcon tubes and stored at -20°C until further assays. This procedure was described by Marquez et al. (2020).13\n\nChicken breast aliquots were randomly divided in control samples and samples to contaminate with tetracyclines and β-lactams. Then, samples were contaminated individually with 200 μL of each antibiotic working solutions until reaching final concentrations of 0.5, 1.0 and 1.5 MRL (Table 1), followed by vortexing for 30s and then incubation for 1 h at room temperature in the dark.14 All assays were carried out with three replicates.\n\nTo obtain the sarcoplasmic, myofibrillar and insoluble proteins, the method developed by Marquez et al. (2020) was employed. Briefly, homogenized samples were mixed with 10 mL of low ionic strength buffer (0.05 M K3PO4, 1 mM NaN3, 2 mM EDTA, pH 7.3), then, vortexing at 3,000 rpm for 3.5 minutes and centrifugation at 11,150 xg 10 minutes and 1 °C were carried out. The supernatant (sarcoplasmic proteins) was collected and refrigerated, and the pellet was resuspended with 5 mL of low ionic strength buffer, centrifuged and the supernatant was unified with the sarcoplasmic proteins fraction initially obtained. The remaining pellet was resuspended with 5 mL of high ionic strength buffer (0.55 M KCl, 0.05 M K3PO4, 1mM NaN3, 2 mM EDTA, pH 7.3) and the same vortexing and centrifugation steps were realized.). The new supernatant (myofibrillar proteins) was collected and also kept refrigerated at 4°C. The final pellet was resuspended in 0.15 M KCl to obtain the insoluble proteins.13 Protein concentration was determined by the Bradford method (Coomassie blue brilliant/ethanol/phosphoric acid). For this, a calibration curve was realized using standard solutions of bovine serum albumin at concentrations between 0.0625 and 1.0 mg·L−1, which were put in microplates with the samples and the Braford reactive. Then, the absorbance was measured at 595 nm.15\n\n* The concentration are expressed as μg. Kg-1 chicken breast.\n\nProtein carbonyl content was measured according to Mesquita et al. (2014) with some modifications described by Marquez et al. (2020).13,16 For this, 300 μL of 2,4-dinitrophenylhydrazine (DNPH, 10 mM in 0.5M H3PO4) was added to the same volume of protein solution (150 μg) and incubated in the dark for 10 minutes. Next, 160 μL of this solution was placed in a 96-well plate and 40 μL of 6M NaOH were added and incubation for 10 minutes was carried out. The change of colour was measured at 450 nm (FLUOstar Omega spectrophotometer, BMG-Lab Tech). The protein carbonyl content was calculated using the DNPH molar extinction coefficient corrected for microplates (ε = 11154 μM-1.cm-1). Protein carbonylation was expressed as nmol of carbonyls·mg-1 of protein.\n\nValues are reported as mean ± SEM of three independent determinations. To assess the effect of antibiotics concentration on protein carbonylation, a unifactorial ANOVA with three levels and blank at 95% confidence was performed (GraphPad Prism V5.01). Multiple comparisons of the means were made using the Tukey adjustment when the ANOVA was significant (p < 0.05) for control and contaminated samples.\n\n\nResults\n\nThe oxidative capability of the assayed antibiotics was evaluated by determination of the carbonylation degree of exposed sarcoplasmic, myofibrillar and insoluble chicken breast proteins, and was expressed as the carbonyl index (CI) in nmol of carbonyls·mg-1 of proteins. CIs found in the control samples were on average 7.40 ± 0.85; 8.67 ± 1.03 and 12.79 ± 0.5178 nmol of carbonyls·mg-1 of proteins for sarcoplasmic, myofibrillar and insoluble, respectively. When those values were compared with the treated samples, it was observed that 16 of the 24 treatments assayed (66.7%) significantly increased their carbonyl groups, as shown in Figure 1.\n\nThe colors of the violins allude to the type of protein fraction: Green: sarcoplasmic proteins; Blue: myofibrillar proteins and Purple: insoluble proteins. The medians and quartiles are showed as black and red lines in the violin boxes, respectively. Different letters on the plots expressed significant differences in protein carbonylation between samples.\n\nThe CIs induced by antibiotics on protein fractions were between 3.92 – 31.38; 6.72 – 50.07 and 11.77–61.49 nmol of carbonyls·mg-1 of proteins for sarcoplasmic, myofibrillar and insoluble, respectively. In addition, the developed Kruskal-Wallis test among the CIs from the three proteins groups showed that there were significant differences between their medians (p<0.05), the insoluble proteins being the ones that showed the highest CIs (28.04 ± 1.61 nmol of carbonyls·mg-1 of proteins), followed by myofibrillar and sarcoplasmic proteins with mean values of 23.07 ± 1.42 and 17.28 ± 0.89 nmol of carbonyls·mg-1 of proteins, respectively. Next, we described the behavior of induced carbonylation by each group of antibiotics.\n\nTCs’ oxidative capability is shown in Figure 2. In sarcoplasmic proteins, all assayed concentrations of CTC, OTC and DXC induced a significant carbonylation compared to the control (p < 0.05); in contrast, the samples contaminated with TC, only showed a significant increase at 0.5 and 1.5 MRL (p < 0.05). The maximum oxidant power of TC, CTC and DXC was observed at 1.5 MRL, while OTC was set at 0.5 MRL. Contrarily, in myofibrillar proteins, TC, CTC and OTC promoted a significantly higher carbonylation compare to control (p < 0.05) at all concentrations assayed. DXC only induced a significant carbonylation at 1.0 MRL regarding to the control (p < 0.05). Unlike to sarcoplasmic proteins, the maximum oxidant power of TC, OTC and DXC was observed at 1.0 MRL, while CTC’s continued at 1.5 MRL (p < 0.05).\n\nThe columns in the graph are grouped as: a) TC and DXC treatment´s and their blanks b) CTC and OTC and their blanks. Mean values ± SEM. a-j Different letters on the bars denote statistically significant differences. Means having different superscripts differ between blank and tetracyclines concentration (p < 0.05). **p < 0.0001, p < 0.005, p < 0.05 denote statistically significant differences among treatment and their respective control.\n\nFinally, for insoluble proteins, all concentrations of tetracyclines induced significant carbonylation with respect to control (p < 0.05). The maximum carbonylation promoted by TC and CTC was observed at 1.0 and 1.5 MRL (p > 0.05), while OTC and DXC were at 0.5 and 1.5 MRL, respectively.\n\nβ-Lts-induced carbonylation is shown in Figure 3. In sarcoplasmic proteins, all the assayed concentrations induced significant carbonylation on contaminated samples compared to control (p < 0.05). For samples contaminated with AMP and PNG, maximum carbonylation was observed at 1.5 MRL, while in samples contaminated with DCL, maximum carbonylation was set at 1.0 MRL;for OXA the values were set at 0.5 and 1.0 MRL. There were no statistical differences between their CIs (p > 0.05). In contrast, in myofibrillar proteins, DCL and OXA induced the same carbonylation in treated samples at all assayed concentrations (p > 0.05), while AMP only induced a significant carbonylation at 0.5 and 1.5 MRL with respect to control (p < 0.05), and PNG a 1.5 MRL carbonylation. Finally, for insoluble proteins, PNG, DCL and OXA promoted a significantly higher carbonylation than control at all concentrations assayed (p < 0.05), while AMP only led to a 1.5 MRL carbonylation. For AMP and OXA, the maximum oxidant power was induced at 1.5 MRL, whereas that of DCL was similar for all three concentrations (p > 0.05).\n\nMean values ± SEM. Letters a-j above the bars denote statistically significant differences in carbonylation compared to blank. Means having different superscripts differ between blank and tetracyclines concentration (p < 0.05). *p < 0.0001, p < 0.005, *p < 0.05 denote statistically significant differences among treatment and their respective control.\n\nFigure 4 shows the oxidant power of TCs and β-LTs on sarcoplasmic, myofibrillar and insoluble proteins from chicken breast; oxidant power was calculated using the ratio between the CIs from treated samples and from the controls (Carbonyl index of protein fraction from contaminated samples/Carbonyl index of blank samples). As showed, among all antibiotics assayed, CTC and OTC promoted the highest carbonylation in myofibrillar proteins at all assayed concentrations (with average ratios of 7.9 and 5.4, respectively); these were followed by DCL in the insoluble (average ratio of 5.4) and myofibrillar fractions (average ratio of 4.0) and OXA in the sarcoplasmic and myofibrillar fractions (average ratios of 3.7 and 3 respectively). Oppositely, DXC, AMP and PNG induced the lowest carbonylation, mainly in myofibrillar (average ratios of 0.4, 0.5 and 0.5, respectively) and insoluble fractions (average ratios of 1.4, 0.7 and 1.4, respectively).\n\nThe results of the comparative analysis between the carbonylation induced by TCs and β-Lts according to the type of fraction is described in the first item (Figure 1). On the other hand, the medians of the carbonylation indices (CIs) promoted by the two groups of antibiotics evaluated through the Mann-Whitney test were compared; results showed that there were no significant differences between the CIs in the samples exposed to both groups of antibiotics (p = 0.3714), Figure 5.\n\nThe medians and quartiles are showed as black and red lines in the violin boxes, respectively.\n\nAccording to the carbonylation variability induced by the assayed antibiotics, we decided to assess individual and multiple correlations among CIs and independent variables assessed in the study: protein fraction, type of antibiotic, evaluated concentration of each antibiotic, and octanol-water partition coefficient (LogP). This last variable was included given that the antibiotics evaluated, by belonging to the same group, have similar chemical structures (pharmacophore) differing only in their radicals; therefore one of the most used parameters to evaluate differences in their toxicological behaviors in in vitro models are their solubility variations (LogP).17\n\nSimple regression analysis results between CIs induced by antibiotics at their respective concentration, and LogP revealed a significantly positive relationship for both variables combinations (R2 = 0.0327, p = 0.0044 for CIs and antibiotics concentrations, and R2 = 0.1235, p = 0.0019, for CIs and LogP). This indicates that they represent important parameters in the oxidant properties of antibiotics (Figure 6a and 6b). When comparing adjusted determination coefficients obtained in correlation analyses, we were able to establish a hierarchy in the effect exercised by the evaluated independent variables (concentration and LogP) on the antibiotics-induced carbonylation, showing the following increasing order: LogP > concentration. Nevertheless, in order to evaluate the joint correlation level of independent variables assayed (concentration and LogP) vs the antibiotic-induced carbonylation, a multiple linear regression analysis was performed. Thus, the comparison between adjusted R2 obtained in the simple and multiple linear regression analysis showed that R2 in combined variables (14.48%) was higher than that of individual ones (3.27% and 12.35% for concentration and LogP, respectively), thus attributing the antibiotic-induced carbonylation effect to the synergic effect of the evaluated independent variables (p < 0.0001).\n\nThese parameters were used to evaluate the variables’ relation level were the correlation and determination coefficients.\n\n\nDiscussion\n\nThe results described before show the capability of TCs and β-LTs to promote the carbonylation of chicken breast proteins even at concentrations under their MRLs.\n\nIn addition, when comparing the carbonylation induced by tetracyclines in three different protein fractions, it can be observed that sarcoplasmic and myofibrillar proteins were the most oxidized ones (Figure 4). This behavior could be associated to accessibility of proteins in muscle cells: sarcoplasmic being more available than myofibrillar, which are more available than insoluble proteins; this is due to sarcoplasmic proteins being soluble in sarcolemma, while myofibrillar and insoluble proteins are stored in myofibrilla and connective tissue, respectively.13,18\n\nFinally, carbonylation of chicken breast proteins provides evidence for the ability of tetracyclines to promote oxidative stress, which is consistent with assays carried out with several biological models.19–24 According to Wen et al. (2012), CTC induced oxidative stress in maize root through the production of hydroxyl radicals19; Pes et al. (2018) reported a decrease of antioxidant enzymes in fish when these were exposed to OTC; Hang et al. (2019) showed that exposure to TC induced oxidative stress in ryegrass seedlings mediated by the increase of lipid oxidation and decrease of antioxidants enzymes.9 However, according to the literature, DXC has antioxidant properties, which is contradictory to our results.21–23 This fact is interesting since the concentrations assayed in this study were lower than the ones used in studies where the protecting effect against oxidation was observed (16 times approximately)21–23; maybe this behavior could be associated to endocrine-disrupting effects.\n\nIn a similar way as tetracyclines, these results show the capacity of β-lactams to induce oxidation in chicken breast proteins even at levels under their MRLs, and therefore their ability to induce oxidative stress. However, these results are contradictory to the literature, because β-lactams have been attributed antioxidant properties: Berczyński et al. (2017) demonstrated the antioxidant capacity of β-lactams in the following descending order: ampicillin, penicillin, dicloxacillin and finally oxacillin, against reactive oxygen species.24 Although Dwyer et al. (2014), observed that some antibiotics, suchas ampicillin induced oxidative stress to increase their antibiotic lethality. Thus, β-lactam behavior could be bivalent (oxidant/antioxidant), explaining the lower levels of carbonylation induced of chicken breast proteins (Figure 4). While comparing the carbonylation on three protein fractions, only DCL and OXA promoted greater oxidative damage on these (Figure 4), probably mediated by a largest number of molecules of DCL and OXA available in the muscle cell, these amounting to 6 times the concentrations of PGN and AMP.4,13\n\nAccording to the literature, the carbonylation induced by residues of tetracyclines and β-lactams at MRL concentrations are similar to those promoted by fluroquinolones MRLs on beef proteins, which was reported by Marquez et al. (2020). In this study, fluoroquinolones also induced carbonylation even at concentrations under MRL values, and related to solubility loss and decreased protein digestibility.13 Therefore, it would be expected that these types of changes can occur in chicken muscle proteins as well and influence the meat’ nutritional value.14\n\nIn conclusion, our results showed the oxidizing effect of tetracycline and β -lactam residues on chicken breast proteins, even at concentrations considered safe. This causes concern because carbonylation continues to occur even after the animal is slaughtered, which means that under in vivo conditions protein oxidation may be greater. This could also affect the nutritional value of meat proteins, since carbonylation causes solubility loss and decreased digestibility in proteins, as well as loss of protein functionality.\n\n\nData availability\n\nFigshare: Raw data of chicken breast proteins exposure to antibiotics, https://doi.org/10.6084/m9.figshare.14799147.v3\n\nThis project contains the following underlying data:\n\n• Data set 1: Raw data from carbonyl assay of tetracyclines.\n\n• Data set 2: Raw data from carbonyl assay of β-lactams.\n\n• Raw data Figures.\n\n• Raw data Figures from GraphPad Prism.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors express their gratitude to the University of Cartagena for grants 078 and 095/2019 and Corporación Universitaria Rafael Núñez.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nA. Marrugo-Padilla thanks Colciencias for the scholarships National Doctorates No 785-1047457972. The authors would like to thank Judith Rodriguez Cavallo for her technical assistance and María Alejandra Mendez for editing.\n\n\nReferences\n\nSarker YA, Hasan MM, Paul TK, et al.: Screening of Antibiotic Residues in Chicken Meat in Bangladesh by Thin Layer Chromatography. J Adv Vet Anim Res. 2018; 5(2): 140–145. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCornejo J, Pokrant E, Figueroa F, et al.: Assessing Antibiotic Residues in Poultry Eggs from Backyard Production Systems in Chile, First Approach to a Non-Addressed Issue in Farm Animals. Animals. 2020, 10 (6), 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMárquez-Lázaro JP, Mora L, Méndez-Cuadro D, et al.: In Vitro Oxidation Promoted by Chlorpyrifos Residues on Myosin and Chicken Breast Proteins. Food Chem. 2020; 326: 126922. PubMed Abstract \| Publisher Full Text\n\nPadilla AM, Cuadro DM, Cavallo ER: Combined Tetracycline and Pyrethroid Residues Increases Protein Carbonylation in Bovine Milk. Int Dairy J. 2020: 104708. Publisher Full Text\n\nRodríguez-García A, García-Vicente R, Morales ML, et al.: Protein Carbonylation and Lipid Peroxidation in Hematological Malignancies. Antioxidants . 2020; 9(12): 1–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKorzeniowska M, Króliczewska B, Kopeć W: Carbonyl and Sulfhydryl Groups of Chicken Meat Proteins after Dietary Modulation with Selenium. Open Chem. 2015; 13(1): 1293–1302. Publisher Full Text\n\nEstévez M: Oxidative Damage to Poultry: From Farm to Fork. Poult. Sci. 2015; 94(6): 1368–1378. PubMed Abstract \| Publisher Full Text\n\nQuaik S, Embrandiri A, Ravindran B, et al.: Veterinary antibiotics in animal manure and manure laden soil: Scenario and challenges in Asian countries. J King Saud University - Sci. Elsevier B.V.2020; 32: 1300–1305. Publisher Full Text\n\nAmelin VG, Bol’shakov DS, Podkolzin IV: Rapid Screening and Determination of Residual Amounts of β-Lactam Antibiotics in Foods by Ultrahigh-Performance Liquid Chromatography–High-Resolution Quadrupole Time-of-Flight Mass Spectrometry. J Anal Chem [Internet]. 2020 Sep 3; 75(9): 1177–88. Reference Source\n\nPalisoc S, De Leon PG, Alzona A, et al.: Highly sensitive determination of tetracycline in chicken meat and eggs using AuNP/MWCNT-modified glassy carbon electrodes. Heliyon. 2019 Jul 1 [cited 2021 May 3]; 5(7): e02147. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nEstévez M, Luna C: Dietary protein oxidation: A silent threat to human health? Crit Rev Food Sci Nutr. [cited 2021 Jun 24]; 2017; 57(17): 3781–93. PubMed Abstract \| Publisher Full Text Reference Source\n\nMuñoz S, Méndez L, Dasilva G, et al.: Targeting Hepatic Protein Carbonylation and Oxidative Stress Occurring on Diet-Induced Metabolic Diseases through the Supplementation with Fish Oils. Mar Drugs. 2018 [cited 2021 Jun 24]; 16(10): 353. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nMárquez-Lázaro J, Méndez-Cuadro D, Rodríguez-Cavallo E: Residues of Fluoroquinolone Antibiotics Induce Carbonylation and Reduce in vitro Digestion of Sarcoplasmic and Myofibrillar Beef Proteins. Foods. 2020 Feb 11; 9(2): 170. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nMárquez-Lázaro JP, Mora L, Méndez-Cuadro D, et al.: In vitro oxidation promoted by chlorpyrifos residues on myosin and chicken breast proteins. Food Chem. 2020 Oct 1 [cited 2021 Apr 7]; 326: 126922. PubMed Abstract \| Publisher Full Text Reference Source\n\nBradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7 [cited 2021 Apr 7]; 72(1–2): 248–54. PubMed Abstract \| Publisher Full Text Reference Source\n\nMesquita CS, Oliveira R, Bento F, et al.: Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins. Anal Biochem . 2014 Aug 1 [cited 2021 Apr 7]; 458: 69–71. PubMed Abstract \| Publisher Full Text Reference Source\n\nBennion BJ, Be NA, McNerney MW, et al.: Predicting a Drug’s Membrane Permeability: A Computational Model Validated With in vitro Permeability Assay Data. J Phys Chem B. 2017 May 25 [cited 2021 May 20]; 121(20): 5228–5237. Reference Source\n\nGuo W, Greaser ML: Muscle Structure, Proteins, and Meat Quality. In: New Aspects of Meat Quality. Elsevier; 2017 [cited 2021 May 5]. p. 13–31. Reference Source\n\nWen B, Liu Y, Wang P, et al.: Toxic effects of chlortetracycline on maize growth, reactive oxygen species generation and the antioxidant response. J Environ Sci. 2012 Jun 1 [cited 2021 May 5]; 24(6): 1099–1105. PubMed Abstract \| Publisher Full Text Reference Source\n\nPês TS, Saccol EMH, Londero ÉP, et al.: Protective effect of quercetin against oxidative stress induced by oxytetracycline in muscle of silver catfish. Aquaculture. 2018 Feb 1 [cited 2021 May 5]; 484: 120–5. Reference Source\n\nYeh Y-C, Lai H-C, Ting C-T, et al.: Protection by doxycycline against doxorubicin-induced oxidative stress and apoptosis in mouse testes. Biochem Pharmacol. 2007 Oct 1 [cited 2021 May 5]; 74(7): 969–980. PubMed Abstract \| Publisher Full Text Reference Source\n\nLai H-C, Yeh Y-C, Ting C-T, et al.: Doxycycline suppresses doxorubicin-induced oxidative stress and cellular apoptosis in mouse hearts. Eur J Pharmacol. 2010 Oct 10 [cited 2021 May 5]; 644(1–3): 176–187. PubMed Abstract \| Publisher Full Text Reference Source\n\nCastro MM, Rizzi E, Ceron CS, et al.: Doxycycline ameliorates 2K-1C hypertension-induced vascular dysfunction in rats by attenuating oxidative stress and improving nitric oxide bioavailability. Nitric Oxide. 2012 Mar 31 [cited 2021 May 5]; 26(3): 162–8. PubMed Abstract \| Publisher Full Text Reference Source\n\nBerczyński P, Kładna A, Kruk I, et al.: Radical-scavenging activity of penicillin G, ampicillin, oxacillin, and dicloxacillin. Luminescence. 2017 May; 32(3): 434–442. PubMed Abstract \| Publisher Full Text" }	[ { "id": "102491", "date": "18 Feb 2022", "name": "Małgorzata Korzeniowska", "expertise": [ "Reviewer Expertise Poultry meat quality" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written, based on a well-designed, reliable, and reproducible experiment. The subject of the study is interesting for readers and relevant to the discipline.\n\nThe abstract is informative and clearly states the most relevant findings. The introduction shows the main findings in the area and impact on chicken meat and possible influence on humans. The objective is short and straight to the point. The methodology is known and already established. It would be beneficial to have data on the respective antibiotic concentrations in the used material which was purchased in the local market. The lack of this information (analysis) is the main gap in the study. It would be great to have chicken meat collected directly from the farm to have the knowledge about real residues of the antibiotics in meat and to be able to draw a conclusion about the mechanism of action. Results and discussion are well prepared, leading to the conclusion.\n\nThe manuscript can be indexed in its current form.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "121739", "date": "11 Mar 2022", "name": "Chenliang Li", "expertise": [ "Reviewer Expertise Meat science", "protein oxidation", "peptide chemistry", "omics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe main results of this study show that the presence of tetracyclines and β-lactams residues at MRLs concentrations promotes in vitro carbonylation on chicken breast proteins. The authors give an interesting insight into the impact of antibiotics on the integrity of meat proteins towards human consumption. This manuscript has been well-written and discussed in terms of the topic which has good novelty. This reviewer would thereby recommend it for indexing as it is.\nSome suggestions:\nThe third paragraph of the Introduction section: 'Despite...are one of its most abundant constituents' This sentence was too long, suggest to isolate it into two individual ones.\n\nIn the section entitled 'Preparation of antibiotics solutions':change '...until obtaining an antibiotic concentration...' into '...to obtain an antibiotic concentration...'; change '...in order to avoid sample protein precipitation...' into '...to avoid protein precipitation...'\n\nIn section entitled with 'Behavior of TCs-induced carbonylation': change '...higher carbonylation compare to control (p < 0.05) at all concentrations assayed' into '...higher carbonylation compare to control (p < 0.05) regardless of concentrations assayed'\n\nIn the section entitled 'Comparison between carbonylation induced by TCs and β-LTs': change 'Thus, the comparison between adjusted R2 obtained in the simple and multiple linear regression analysis showed that...' into 'As a result, the comparison between...'\n\nIn the Discussion section: change '...; maybe this behavior could be associated to endocrine-disrupting effects.' into '..., which sheds light in endocrine-disrupting effects.'\n\nIn the Discussion section: 'In this study, fluoroquinolones also induced carbonylation even at concentrations under MRL values, and related to solubility loss and decreased protein digestibility'. Please specify clearer which concentrations (with values) you mentioned.\n\nIn the Discussion section: 'This could also affect the nutritional value of meat proteins since carbonylation causes solubility loss and decreased digestibility in proteins, as well as loss of protein functionality.' Change '...loss of protein functionality' into 'an alteration in protein (bio)availability and/or functionality'.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/10-575
https://f1000research.com/articles/10-84/v1	08 Feb 21	{ "type": "Brief Report", "title": "SWAT 76 evaluation: randomised evaluation of sending pre-notification cards to trial participants before a face-to-face primary outcome measurement to increase attendance", "authors": [ "Shaun Treweek", "Stephanie Gallant", "Annie S. Anderson", "Stephanie Gallant", "Annie S. Anderson" ], "abstract": "Background: Retention is considered the second highest trial methods priority in the UK after recruitment. Methods: This Study Within A Trial (SWAT) evaluated whether sending a pre-notification card around one month before a face-to-face primary outcome measurement visit compared to not sending the card increased trial retention. The SWAT was a two-arm, parallel randomised (1:1 allocation ratio), stratified by centre, study. It was embedded within the ActWELL host trial, which evaluated whether women receiving lifestyle change counselling from volunteer coaches improved outcomes including weight and physical activity. The text on the card was not developed using formal behavioural change theory but did target factors thought to influence attendance. The SWAT primary outcome was the difference in the proportion of participants attending the host trial primary outcome measurement visit. The secondary outcome was the direct cost of sending cards. Host trial participants and research staff at the primary outcome visit were blind to the SWAT. Analysis was intention-to-treat. GRADE was used the assess the certainty of evidence. Results: 558 host trial participants took part in the SWAT and were included in the analysis. Sending a pre-notification card may result in a slight increase in attendance at a face-to-face primary outcome measurement visit: risk difference = 3.3% (95% confidence interval = -3.0% to 9.6%). This is GRADE low certainty evidence. A recording error meant it was unclear whether 17 participants allocated to the card were actually sent one but a sensitivity analysis did not change the overall result or conclusion. The direct cost of producing and sending the cards was £192 GBP (€213 EUR; $260 USD). Discussion: Trialists could consider using pre-notification as they may gain a slight increase in retention to face-to-face trial measurement visits but further evaluations are needed.", "keywords": [ "SWAT", "retention", "pre-notification cards", "randomised trial" ], "content": "Introduction\n\nRetention is considered the second highest trial methods priority in the UK after recruitment1. A recent UK study found that the median loss-to-follow-up in a sample of 151 trials was 11%2. Reminders are generally an effective way of increasing response rates to questionnaires and there is evidence that pre-notification (contacting participants to say that they will soon be sent a questionnaire) is beneficial, although it is not high certainty evidence3.\n\nThere is no clear evidence from the Cochrane systematic review of trial retention interventions that pre-notification is effective for trial retention for face-to-face visits4. However, at the time the review was published, an ongoing Study Within A Trial (SWAT) in a trial involving women aged between 70 and 84 years at high risk of osteoporotic fractures did find that sending a newsletter to participants approximately six weeks before a trial questionnaire increased retention by around 1%5.\n\nDoes sending a pre-notification card around one month before a face-to-face primary outcome measurement visit compared to not sending the card increase trial retention?\n\n\nMethods\n\nThis SWAT is registered on the SWAT repository as SWAT 76. See: http://www.qub.ac.uk/sites/TheNorthernIrelandNetworkforTrialsMethodologyResearch/FileStore/Filetoupload,864300,en.pdf\n\nThis SWAT evaluation was embedded in the ActWELL trial (ISRCTN11057518)6. ActWELL evaluates whether women who receive two, face-to-face lifestyle change sessions from volunteer coaches followed by up to nine telephone calls over a year, compared to no counselling, improves a range of lifestyle outcomes. The two primary outcomes were weight change and change in physical activity at 12-months. Women were invited to take part in ActWELL when they attended their routine mammography appointment (all women aged 50 - 70 in Scotland receive an offer of mammography every three years) at one of four Scottish National Health Service Breast Screening centres. A total of 560 women were randomised into the ActWELL trial.\n\nAll host trial participants were eligible.\n\nThe intervention is a pre-notification card sent around one month before the face-to-face primary outcome measurement visit. The text on the card was not developed using formal behavioural change theory but did target factors thought to influence attendance. Women were thanked to make them feel valued, were told their data were valuable regardless of how things had gone in the trial and the number of other women in the trial was highlighted. The card was signed by the Chief Investigator of the host trial and the Chief Executive of Breast Cancer Now, the charity involved in delivering the host trial intervention. The card is shown in Figure 1.\n\nNo pre-notification card.\n\nPrimary outcome: the difference in the proportion of participants attending the host trial primary outcome measurement visit (i.e., retention).\n\nSecondary outcome: the direct cost of sending pre-notification cards.\n\nThe sample size was determined by the host trial so no sample size calculation was done. See Trial Forge Guidance 1 for more information about SWAT sample size calculation7.\n\nTwo-arm, parallel randomised with a 1:1 allocation ratio, stratified by centre. One of the authors (ST) prepared a central randomisation list for each centre for up to 150 participants using https://www.random.org/sequences/. This was then passed to the trial manager and trial administrator who sent out the pre-notification cards.\n\nWomen in the host trial had no knowledge of the SWAT. Host trial primary outcome visits were organised and done by research nurses, who had no knowledge of the SWAT or host trial allocation. The SWAT primary outcome, retention, was objective.\n\nThe study was approved by East of Scotland Research Ethics Service REC 1 as part of the host ActWELL trial (17/ES/0073). The low risk nature of the SWAT evaluation meant that individual informed consent from host trial participants to be involved was not required by the ethics committee, in line with most SWATs in the UK.\n\nThe difference in the proportion of attended visits between groups was calculated using Comprehensive Meta-Analysis Version 3 (https://www.meta-analysis.com/).\n\nGRADE was used to assess the certainty of the evidence8. In addition to the numerical result, the result is summarised as an informative statement as per GRADE Guidelines 269.\n\n\nResults\n\nTwo host trial participants withdrew before the 12-month host trial primary outcome measurement meaning 558 were included in the SWAT, which ran between March 2018 and July 2019 (Figure 2; Table 1). One host trial centre recruited 151 participants, which was beyond its recruitment target and one participant beyond the randomisation list for that centre. The extra participant was manually allocated to the comparator arm. A discrepancy between the randomisation log (which indicated who should get a card) and the host trial's tracker system (which confirmed that a card had been sent to a participant) meant that we could not confirm whether 17 participants who should have been sent the pre-notification card were actually sent one. Three further participants who should have received a card are known to have not been sent a card because the participant was called in for a host trial measurement visit before the card could be sent.\n\nThe summary statement below and the primary analysis in Table 1 are intention-to-treat as per the randomisation schedule. A sensitivity analysis done using the tracker data is also shown.\n\nSummary statement of result: Sending a pre-notification card may result in a slight increase in attendance at a face-to-face primary outcome measurement visit. Risk difference = 3.3% (95% confidence interval = -3.0% to 9.6%). GRADE = low certainty evidence.\n\nA sensitivity analysis was done to explore the impact of a record-keeping error (see main text).\n\nThe direct costs of printing the cards was £72 GBP. Design work was extremely modest, bundled with other host trial design work and not charged separately. Second class (i.e., delivery within two days) postage costs were run through the University of Dundee mailroom at an estimated cost of £120 GBP. The total direct cost was therefore £192 GBP (€212 EUR; $259 USD).\n\n\nDiscussion\n\nSending a simple card about one month prior to a face-to-face primary outcome measurement visit may result in a slight improvement in attendance. This is GRADE low certainty evidence because there is just this single evaluation and it is imprecise.\n\nWe are not aware of other pre-notification interventions that target face-to-face trial visits. An upcoming 2021 update of the Cochrane retention review4 found no additional pre-notification studies (ST is a co-author of this update). Mitchell and colleagues5 added their evaluation to a meta-analysis of pre-notification evidence done outside trials and healthcare. As might be expected, there was substantial heterogeneity but the overall direction of effect was also in favour of pre-notification.\n\nWe had a record-keeping error, which means we cannot say with confidence that all participants who should have been sent a pre-notification card were sent one. However, the number of participants affected was relatively small and our overall results and conclusion remain the same regardless of whether we analyse according to the randomisation schedule or the tracker system. There are currently only two evaluations of pre-notification in trials, our own of cards aimed at face-to-face visits and that of Mitchell and colleagues of a newsletter to increase questionnaire response5. This does not provide a broad range of contexts for this evidence base. Both evaluations were done in the UK and in women only.\n\nTrialists could consider using pre-notification as they may gain a slight increase in retention to face-to-face trial measurement visits.\n\nLooking at the existing evidence and referring to Trial Forge Guidance 2 as to whether further SWATs evaluating this intervention are required10, we conclude that more evaluations are needed because the GRADE certainty in the evidence is not high, there is only a single evaluation meaning cumulative meta-analysis cannot converge and few host trial contexts are included.\n\nFurther evaluations of pre-notification in trials could target either face-to-face or questionnaires but should aim to add new host trial contexts. Future host trials should involve men. Formal approaches to developing intervention content may increase effect sizes.\n\n\nData availability\n\nOpen Science Framework: SWAT 76 data for host trial ActWELL, https://doi.org/10.17605/OSF.IO/N64HU11\n\nThis project contains the following underlying data:\n\nPrimary analysis 8-1-2021 (public).csv\n\nSensitivity analysis 8-1-2021 (public).csv\n\nOpen Science Framework: CONSORT checklist for ‘SWAT 76 evaluation: randomised evaluation of sending pre-notification cards to trial participants before a face-to-face primary outcome measurement to increase attendance’, https://doi.org/10.17605/OSF.IO/B78JT12\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nSmith CT, Hickey H, Clarke M, et al.: The trials methodological research agenda: results from a priority setting exercise. Trials 2014; 15: 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalters SJ, Bonacho dos Anjos Henriques-Cadby I, Bortolami O, et al.: Recruitment and retention of participants in randomised controlled trials: a review of trials funded and published by the United Kingdom Health Technology Assessment Programme. BMJ Open 2017; 7(3): e015276. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdwards PJ, Roberts I, Clarke MJ, et al.: Methods to increase response to postal and electronic questionnaires. Cochrane Database Syst Rev 2009; Issue 3. Art. No.: MR000008. PubMed Abstract \| Publisher Full Text\n\nBrueton VC, Tierney J, Stenning S, et al.: Strategies to improve retention in randomised trials Cochrane Database Syst Rev 2013 Dec 3; 12: MR000032. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitchell N, Hewitt CE, Lenaghan E, et al.: Prior notification of trial participants by newsletter increased response rates: a randomized controlled trial. J Clin Epidemiol 2012; 65: 1348–52. PubMed Abstract \| Publisher Full Text\n\nAnderson AS, Craigie AM, Gallant S, et al.: Randomised controlled trial to assess the impact of a lifestyle intervention (ActWELL) in women invited to NHS breast screening. BMJ Open 2018; 8: e024136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreweek S, Bevan S, Bower P, et al.: Trial Forge Guidance 1: What is a Study Within A Trial (SWAT)? Trials 2018; 19: 139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuyatt GH, Oxman AD, Kunz R, et al.: Going from evidence to recommendations. BMJ 2008; 336: 1049–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSantesso N, Glenton C, Dahm P, et al.: GRADE Guidelines 26: informative statements to communicate the findings of systematic reviews of interventions. J Clin Epidemiol 2020; 119: 126–35. PubMed Abstract \| Publisher Full Text\n\nTreweek S, Bevan S, Bower P, et al.: Trial Forge Guidance 2: How to decide if a further Study Within A Trial (SWAT) is needed. Trials 2020; 21: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreweek S: SWAT 76 data for host trial ActWELL.2021, January 14. Publisher Full Text\n\nTreweek S: SWAT 76 data for host trial ActWELL.2021, January 14. Publisher Full Text" }	[ { "id": "79342", "date": "01 Mar 2021", "name": "Christopher Sutton", "expertise": [ "Reviewer Expertise Clinical trial methodology", "particularly around trial conduct and retention methods." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this is an excellent manuscript and an extremely welcome and useful addition to the literature on trial retention. The work provides a clear citation of the relevant literature.\nThe methodology is sound and the trial performed well; it has significant academic merit. There are, however, aspects of the methods and analysis which are a little under-specified and unclear, and suggestions for clarification are detailed below. Likewise, the statistical and economic analysis is appropriate, although I do suggest additional analyses and results be presented to enhance interpretation and generalisability.\n\nThe source data underlying the results are available. The conclusions are drawn adequately and supported by the results, with an appropriate sensitivity analysis performed to investigate the potential impact of some degree of failure to definitively send the pre-notification card to 20 participants allocated to receive the card.\nSpecific comments:\nIntroduction: clearly state that the previous SWAT of the impact of a newsletter on retention was statistically significant (p<0.05 … just!).\n\nI would query the statement that “All trial participants were eligible.” From my perspective, the eligibility criteria were (a) Trial participant of the ActWELL trial (b) remaining in ActWELL at 1 month prior to the 12-month final follow-up.\n\nThe primary outcome would be better specified as a binary outcome measure (i.e. at the participant level e.g. “attendance at the host trial primary outcome measurement visit”).\n\nStatistical analysis: the method of analysis should be stated, in addition to the software used.\n\nIn line with CONSORT 2010, for binary outcome measures both absolute and relative measures of effect are recommended. Across different trial populations, interventions, duration of follow-up etc., it is likely that there are different ‘control’ rates of retention and the impact of any intervention is probably more likely to be on a relative rather than an absolute scale. For example, the newsletter trial referenced in the Introduction (Mitchell et al., (2012)) had a control group retention rate of 94.6% (and an absolute increase of 1.6% in the intervention group), compared with this trial which had a control group retention rate of 81.0% (and an absolute increase of 3.3%). It is therefore recommended that the results are also presented as a Risk Ratio (Relative Risk) or Odds Ratio.\n\nI would like to see more information on the economic analysis. For example, how much did the intervention cost per participant retained?\n\nIn the Discussion and Strengths and Limitations, the authors state that there are no additional pre-notification studies; it would be better to stated that there are no additional completed pre-notification studies (there are at least two other pre-notification trials registered on the SWAT Repository Store (SWAT77 and SWAT86).\n\nReference list: references 11 and 12 re identical (Reference 12 should refer to the CONSORT checklist for this SWAT).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6878", "date": "15 Jul 2021", "name": "Shaun P. Treweek", "role": "Author Response", "response": "Response to comments on F1KR00CDE F1R-VER53982-A 5/7/2021 Thanks for the reviewers’ comments on our manuscript. Our responses are below. We have made the necessary changes to the manuscript using track changes so that they can be easily seen. We have also submitted a clean version with the changes accepted. Reviewer #1 1. Introduction: clearly state that the previous SWAT of the impact of a newsletter on retention was statistically significant (p<0.05 … just!) Done. We have also added the exact difference of 1.6% rather than saying ‘around 1%’. 2. I would query the statement that “All trial participants were eligible.” We have changed our text to: ‘All host trial participants who had not withdrawn were eligible.’ 3. The primary outcome would be better specified as a binary outcome measure (i.e. at the participant level e.g. “attendance at the host trial primary outcome measurement visit”). We have thought about this but since the primary result we report is the difference in attendance between those who got the card and those who did not (i.e. 3.3%) and not the actual attendance for each group (84% and 81%), we think the way we describe the primary is correct. 4. Statistical analysis: the method of analysis should be stated, in addition to the software used We have added that we used a fixed effects model. 5. Present relative relative measures of effect as well. Good idea, apologies for not doing it originally. Done. 6. I would like to see more information on the economic analysis. For example, how much did the intervention cost per participant retained? We only collected direct costs so our economic analysis is very limited. However, we liked the suggestion to calculate a cost per (extra) participant retained, which means we have added the text below to the cost information. We have also added the cost per additional participant retained to the abstract. ‘Cost per additional participant retained If the 274 participants who received the card had attended at the same rate as those who did not, a total of 222 participants would have attended the next visit. In fact, 231 attended, meaning an extra nine participants were retained. The cost per additional participant retained was £192/9, or £21.33 (€23.55; $28.77).’ 7. In the Discussion and Strengths and Limitations, the authors state that there are no additional pre-notification studies; it would be better to stated that there are no additional completed pre-notification studies We have now amended the Discussion text to: ‘The 2021 update of the Cochrane retention review [4] found no additional completed pre-notification studies (ST is a co-author of this update). There are at least two pre-notification protocols (SWAT 77 and SWAT 86) registered on the SWAT repository (https://www.qub.ac.uk/sites/TheNorthernIrelandNetworkforTrialsMethodologyResearch/SWATSWARInformation/Repositories/SWATStore/) in addition to our own SWAT 76, meaning there may be additional studies underway but not yet complete.’ 8. Reference list: references 11 and 12 re identical (Reference 12 should refer to the CONSORT checklist for this SWAT). References 11 and 12 were added by the publisher, not us, but they do work as they should in the version I downloaded yesterday (8/6/2021). There may perhaps have been an error in the version the reviewer was sent that has now been corrected by the publisher. Reviewer #2 1. Results – To help the reader understand the impact on the trial, I recommend the authors first report the total number of women who attended their primary outcome follow-up visit, then report the proportion by group. We have added this number to the header row of Table 1 and we have done this for both the intention-to-treat and sensitivity analyses. 2. Results – I initially found Table 1 confusing. Since those who did not attend the visit is the complement of those who did attend a visit, I suggest reporting only those who attended the visit. The risk difference represents the difference between those who attended the visit. I also suggest adding the 95% CI around the estimates, and report 1 decimal point for consistency with the risk difference. We have thought about the reviewer’s comment re. only showing the number who attended a visit but have decided that we would prefer to be explicit and give both the number who did attend and the number who did not. The main reason is that it avoids the reader having to do a calculation and all the raw numbers are there for readers to do other calculations should they wish. There is no confidence interval for the proportions attending a visit: the numbers are not an estimate but the actual number attending. The risk difference (and now relative risk in response to Reviewer 1, Comment #5) does have a confidence interval around it. We have changed our presentation so that we now consistently report to 1 decimal place as requested by the reviewer. 3. Can the authors report whether patients recalled receiving the pre-notification card before their outcomes visit? Participants were not asked whether they recalled getting a card so we cannot unfortunately report on this. Both the participants and the research nurses who took measurements at the primary outcome visits did not know that we were running this study (i.e. they were blind) and to maintain the blind, we did not ask about the cards. It is possible that some participants mentioned this to the nurse but we have no record of this. 4. Implications for trial practice – I suggest adding text to further contextualize the implications of the relative cost of this intervention, the overall investment costs in the enrolled patients to-date, and the importance of ascertaining the primary outcome. Even a 3% improvement in primary outcome ascertainment could affect trial results. Good point. We have re-written the Implications for trial practice section: ‘Given the paucity of evidence to support retention decisions [Reference 4], trialists could consider using pre-notification as they may gain a slight increase in retention to face-to-face trial measurement visits. Trials are expensive and in that context pre-notification cards are a very cheap intervention that may provide a small increase in the proportion of primary outcome data a trial team obtains. We had no negative reaction to them from participants (i.e. there were no complaints) and our cost of around £21 per additional retained participant is likely to be substantially cheaper that recruiting an additional participant to replace these lost primary outcome data.’ 5. Abstract – I suggest reporting absolute values in the intervention and control groups, then the risk difference. Done. 6. Introduction – I suggest adding a statement regarding the importance of the primary outcome in a trial and the need for interventions to optimize ascertainment. We have added the following text to the Introduction: ‘This reduces the amount of trial data available for analysis, which is especially problematic for the trial’s most important outcome– the primary outcome– because this is the outcome that will be used to judge whether the trial intervention is effective. Ensuring that retention is high is therefore of great importance to trialists.’ 7. Intervention – I suggest moving the text regarding behavioural change theory in the development of the pre-notification card from the methods to the limitations section of the manuscript. Done. 8. Results – Data error for 17 participants (3%) – suggest also reporting % of total group to help the reader easily evaluate the small number. Interestingly, the risk difference was also 3%. The authors conducted a sensitivity analysis, and the results were similar. I suggest this is also a strength of the study. We have added the proportion (3%) to the Results and also mentioned that the similarity of the sensitivity analysis results to the intention-to-treat results shows the results are robust in the face of this recording error. 9. Implications for trial practice. I suggest framing the study findings specific to 1-year follow-up measures. We have made the change suggested and also added ‘at 1-year’ to our evidence summary statement in the Abstract and Results." } ] }, { "id": "79341", "date": "27 Apr 2021", "name": "Michelle E Kho", "expertise": [ "Reviewer Expertise Critical care clinical trials", "health research methodology", "rehabilitation", "physiotherapy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTreweek et al. conducted a study within a trial to evaluate whether a post card sent 1 month prior to an in-person primary outcome research visit improved retention within the multicentre ActWELL randomized trial of lifestyle coaching. 558 participants from the host trial were sent a pre-notification card. Of this cohort, 84% who were allocated a pre-notification card attended the primary outcome visit, compared to 81% who were not allocated a pre-notification card. This is a well-written, well-designed, and well-reported study. It will make important contributions to further understanding ways to improve retention to clinical trials. I have some considerations for the authors.\nModerate issues:\nResults – To help the reader understand the impact on the trial, I recommend the authors first report the total number of women who attended their primary outcome follow-up visit, then report the proportion by group.\n\nResults – I initially found Table 1 confusing. Since those who did not attend the visit is the complement of those who did attend a visit, I suggest reporting only those who attended the visit. The risk difference represents the difference between those who attended the visit. I also suggest adding the 95% CI around the estimates, and report 1 decimal point for consistency with the risk difference.\n\nCan the authors report whether patients recalled receiving the pre-notification card before their outcomes visit?\n\nImplications for trial practice – I suggest adding text to further contextualize the implications of the relative cost of this intervention, the overall investment costs in the enrolled patients to-date, and the importance of ascertaining the primary outcome. Even a 3% improvement in primary outcome ascertainment could affect trial results.\nMinor issues:\nAbstract – I suggest reporting absolute values in the intervention and control groups, then the risk difference.\n\nIntroduction – I suggest adding a statement regarding the importance of the primary outcome in a trial and the need for interventions to optimize ascertainment.\n\nIntervention – I suggest moving the text regarding behavioural change theory in the development of the pre-notification card from the methods to the limitations section of the manuscript.\n\nResults – Data error for 17 participants (3%) – suggest also reporting % of total group to help the reader easily evaluate the small number. Interestingly, the risk difference was also 3%. The authors conducted a sensitivity analysis, and the results were similar. I suggest this is also a strength of the study.\n\nImplications for trial practice. I suggest framing the study findings specific to 1-year follow-up measures.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6879", "date": "15 Jul 2021", "name": "Shaun P. Treweek", "role": "Author Response", "response": "Response to comments on F1KR00CDE F1R-VER53982-A 5/7/2021 Thanks for the reviewers’ comments on our manuscript. Our responses are below. We have made the necessary changes to the manuscript using track changes so that they can be easily seen. We have also submitted a clean version with the changes accepted. Reviewer #1 1. Introduction: clearly state that the previous SWAT of the impact of a newsletter on retention was statistically significant (p<0.05 … just!) Done. We have also added the exact difference of 1.6% rather than saying ‘around 1%’. 2. I would query the statement that “All trial participants were eligible.” We have changed our text to: ‘All host trial participants who had not withdrawn were eligible.’ 3. The primary outcome would be better specified as a binary outcome measure (i.e. at the participant level e.g. “attendance at the host trial primary outcome measurement visit”). We have thought about this but since the primary result we report is the difference in attendance between those who got the card and those who did not (i.e. 3.3%) and not the actual attendance for each group (84% and 81%), we think the way we describe the primary is correct. 4. Statistical analysis: the method of analysis should be stated, in addition to the software used We have added that we used a fixed effects model. 5. Present relative relative measures of effect as well. Good idea, apologies for not doing it originally. Done. 6. I would like to see more information on the economic analysis. For example, how much did the intervention cost per participant retained? We only collected direct costs so our economic analysis is very limited. However, we liked the suggestion to calculate a cost per (extra) participant retained, which means we have added the text below to the cost information. We have also added the cost per additional participant retained to the abstract. ‘Cost per additional participant retained If the 274 participants who received the card had attended at the same rate as those who did not, a total of 222 participants would have attended the next visit. In fact, 231 attended, meaning an extra nine participants were retained. The cost per additional participant retained was £192/9, or £21.33 (€23.55; $28.77).’ 7. In the Discussion and Strengths and Limitations, the authors state that there are no additional pre-notification studies; it would be better to stated that there are no additional completed pre-notification studies We have now amended the Discussion text to: ‘The 2021 update of the Cochrane retention review [4] found no additional completed pre-notification studies (ST is a co-author of this update). There are at least two pre-notification protocols (SWAT 77 and SWAT 86) registered on the SWAT repository (https://www.qub.ac.uk/sites/TheNorthernIrelandNetworkforTrialsMethodologyResearch/SWATSWARInformation/Repositories/SWATStore/) in addition to our own SWAT 76, meaning there may be additional studies underway but not yet complete.’ 8. Reference list: references 11 and 12 re identical (Reference 12 should refer to the CONSORT checklist for this SWAT). References 11 and 12 were added by the publisher, not us, but they do work as they should in the version I downloaded yesterday (8/6/2021). There may perhaps have been an error in the version the reviewer was sent that has now been corrected by the publisher. Reviewer #2 1. Results – To help the reader understand the impact on the trial, I recommend the authors first report the total number of women who attended their primary outcome follow-up visit, then report the proportion by group. We have added this number to the header row of Table 1 and we have done this for both the intention-to-treat and sensitivity analyses. 2. Results – I initially found Table 1 confusing. Since those who did not attend the visit is the complement of those who did attend a visit, I suggest reporting only those who attended the visit. The risk difference represents the difference between those who attended the visit. I also suggest adding the 95% CI around the estimates, and report 1 decimal point for consistency with the risk difference. We have thought about the reviewer’s comment re. only showing the number who attended a visit but have decided that we would prefer to be explicit and give both the number who did attend and the number who did not. The main reason is that it avoids the reader having to do a calculation and all the raw numbers are there for readers to do other calculations should they wish. There is no confidence interval for the proportions attending a visit: the numbers are not an estimate but the actual number attending. The risk difference (and now relative risk in response to Reviewer 1, Comment #5) does have a confidence interval around it. We have changed our presentation so that we now consistently report to 1 decimal place as requested by the reviewer. 3. Can the authors report whether patients recalled receiving the pre-notification card before their outcomes visit? Participants were not asked whether they recalled getting a card so we cannot unfortunately report on this. Both the participants and the research nurses who took measurements at the primary outcome visits did not know that we were running this study (i.e. they were blind) and to maintain the blind, we did not ask about the cards. It is possible that some participants mentioned this to the nurse but we have no record of this. 4. Implications for trial practice – I suggest adding text to further contextualize the implications of the relative cost of this intervention, the overall investment costs in the enrolled patients to-date, and the importance of ascertaining the primary outcome. Even a 3% improvement in primary outcome ascertainment could affect trial results. Good point. We have re-written the Implications for trial practice section: ‘Given the paucity of evidence to support retention decisions [Reference 4], trialists could consider using pre-notification as they may gain a slight increase in retention to face-to-face trial measurement visits. Trials are expensive and in that context pre-notification cards are a very cheap intervention that may provide a small increase in the proportion of primary outcome data a trial team obtains. We had no negative reaction to them from participants (i.e. there were no complaints) and our cost of around £21 per additional retained participant is likely to be substantially cheaper that recruiting an additional participant to replace these lost primary outcome data.’ 5. Abstract – I suggest reporting absolute values in the intervention and control groups, then the risk difference. Done. 6. Introduction – I suggest adding a statement regarding the importance of the primary outcome in a trial and the need for interventions to optimize ascertainment. We have added the following text to the Introduction: ‘This reduces the amount of trial data available for analysis, which is especially problematic for the trial’s most important outcome– the primary outcome– because this is the outcome that will be used to judge whether the trial intervention is effective. Ensuring that retention is high is therefore of great importance to trialists.’ 7. Intervention – I suggest moving the text regarding behavioural change theory in the development of the pre-notification card from the methods to the limitations section of the manuscript. Done. 8. Results – Data error for 17 participants (3%) – suggest also reporting % of total group to help the reader easily evaluate the small number. Interestingly, the risk difference was also 3%. The authors conducted a sensitivity analysis, and the results were similar. I suggest this is also a strength of the study. We have added the proportion (3%) to the Results and also mentioned that the similarity of the sensitivity analysis results to the intention-to-treat results shows the results are robust in the face of this recording error. 9. Implications for trial practice. I suggest framing the study findings specific to 1-year follow-up measures. We have made the change suggested and also added ‘at 1-year’ to our evidence summary statement in the Abstract and Results." } ] } ]	1	https://f1000research.com/articles/10-84
https://f1000research.com/articles/10-572/v1	15 Jul 21	{ "type": "Case Report", "title": "Case Report: Reinfection of COVID-19, with second infection less severe", "authors": [ "Nawar Jasim Alsalih", "Zeayd Fadhil Saeed", "Hazim Talib Thwiny", "Ali Mosa Rashid Al-Yasari", "Ahmed Waleed Dheyab Alnassar", "James P. Hobkirk", "Mohenned A. Alsaadawi", "Nawar Jasim Alsalih", "Zeayd Fadhil Saeed", "Hazim Talib Thwiny", "Ali Mosa Rashid Al-Yasari", "Ahmed Waleed Dheyab Alnassar", "James P. Hobkirk" ], "abstract": "There is concern that an individual may contract COVID-19 twice, either as a result of being a viral carrier that was not entirely cleared from the body in the first instance or as a result of reinfection. The recurrent infection may be qRT-PCR positive, which must be distinguished from post-COVID-19 symptoms that are qRT-PCR negative. Although it is known that recovered patients of viral diseases can be immune for the next infection, recurrent infections of COVID-19 have been recorded in Brazilian healthcare workers. We report a case of recurrent COVID-19 infection in a 34-year-old man working in the Gynecology and Children Hospital in Al-Muthanna Province, south of Iraq. The patient suffered from a sharp and noticeable rise in the body temperature at 39 ºC and cough on the 16th of July 2020. Then, the patient was symptomized with another course of COVID-19 on the 27th of August 2020, which was contracted from the patient’s workmate. Nose swab PCR test and CT scan were performed to confirm the second infection. The clinical signs of repeated infection with coronavirus were obviously less than the first infection of the same patient. It is clear that the first infection symptoms of COVID-19 are more severe than the signs of recurrent disease.", "keywords": [ "COVID-19", "reinfection", "PCR", "CT scan", "Real-time PCR" ], "content": "Introduction\n\nThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the cause of the global COVID-19 pandemic, which began in Wuhan, China, in December 2019 and has since spread throughout the world.1 The virus may infect a wide range of systems, including the respiratory, gastrointestinal, hepatic, and central neurological systems, with severe symptoms that may need hospitalization.2 According to recent studies, around 4–5 days is the typical incubation time from SARS-CoV-2 exposure to the beginning of symptoms, with 97.5% of symptomatic individuals showing symptoms within 11.5 days.3 Although most cases are asymptomatic or moderate, advanced age, cardiovascular disorders, chronic lung illness, systemic arterial hypertension, diabetes, obesity, and Black and minority ethnicity are the main risky factors of the pandemic.4 At a time when millions of first-wave infections have occurred and many communities are suffering from a second wave, there is worry over whether the sickness may continue, whether by the recurrence of a viral reservoir that was not completely removed from the body in the first place or by reinfection.2 The common symptom of recurrent CVID-19 syndrome occurs again after the first appearance of the initial pandemic infection and can be qRT-PCR positive, which needs to be differentiated from the post-COVID-19 symptoms, typically negative on qRT-PCR.4 Recurrent infection is far from the more serious “chronic post-viral syndrome”, or long-term symptoms that persist despite no viral activity. While certain viral sequences can be identified by sequencing, and thus whether a patient has a new infection can be determined, there are other circumstances in which a patient is known to have an infection, but we do not have information on their exact viral sequence. To account for these situations, the phrase ‘recurrence’ instead of ‘persistence’ should be used.5–8 The importance of considering recurrent infection relates to the immune interactions and the ability of the immune system to stop subsequent viral infections. It is known that during the viral infections, immunity can be developed after the first infection and provides a strong defense against subsequent SARS-Cov-2 exposure.9\n\nIn this case report, we present a patient with recurrence of COVID-19 symptoms, qRT-PCR positive, whom was diagnosed as having COVID-19 recurrence.\n\n\nCase description\n\nA 34-year-old man working in the Gynecology and Children Hospital in Al-Muthanna Province, south Iraq, suffered from a sharp and noticeable rise in body temperature of 39°C and cough on 16th of July 2020. The patient also suffered from congestion in the tonsils and pharynx, difficulty swallowing, severe fatigue, emaciation, joint pain, persistent sweating, pain along the spinal cord, in addition to choking and pain in the chest area, and difficulty breathing. On 19th July, the patient visited a doctor who ran various laboratory tests and a complete blood count (Tables 1 and 2). Due to suspicion of COVID-19, the patient was on mandatory vacation for a month. One week later, the patient lost his sense of smell for two days. These symptoms continued for eight days, after which the patient recovered from his symptoms and returned to normal.\n\nNasopharyngeal swabs, which had been collected on 23rd of July 2020 to detect the nucleic acid of SARS-CoV2, were tested by reverse transcriptase-polymerase chain reaction in real time, with positive results to IgM of COVID-19. On 26th July 2020, complete blood count was returned as normal. The patient was given a COVID-19 diagnosis.\n\nThe patient underwent the recommended treatment involved using bronchium syrup (three times per day), TAVANIC tablets 500MG, co-codamol tablets 500MG (three times per a day), Heparin (two times per a day) at dose of 18 units/kg/hr IV for five days, and desloratadine tablets 10mg (one time per a day.\n\nThe PCR test of COVID-19 was done again on 19th of August 2020, which showed a negative result. Other tests performed on the same date included C-reactive protein (CRP), D-dimer and serum ferritin were 0.42 mg/L, 181 ng/ml and 97.5 ng/ml, respectively. These tests showed that the patient was completely recovered.\n\nThe patient resumed his work on 19th of August 2020 in the microbiology laboratory of the hospital. Five days later (23rd August), the patient's workmate tested positive to COVID-19 after PCR test and CT scan. Our patient was suspected to be COVID-19 positive on COVID-19 on 27th of August 2020, due to the following symptoms: intermittent fever, pain in tonsil and pharynx, and intermittent diarrhea. Nasopharyngeal swab was taken again on the same day and was confirmed as COVID-19 positive by PCR. A CT scan was performed to confirm the second infection and this showed bilateral consolidation and linear opacities (Figure 1), indicative of COVID-19 infection.\n\nAfter five days of second appearance of symptoms in the patient, the patient’s cough was continuous and dry without sputum with suffocation (SpO2 dropped to 85%), which continued for two days. The patient started to use medicinal oxygen to compensate the lost oxygen. Treatment provided to the patient was as follows: Heparin (twice per a day) at dose of 18 units/kg/hr IV for five days; Livofloxacin 500 mg once daily; Panadol tablets 500 mg thrice daily; Desloratadine 10 ml (5 mg) oral solution once a day; Dexamethasone 4 mg used only in case of severe hypoxia (the patient used it twice); and multivitamins (C, D, and zinc) once daily. After two days, the patient reported feeling more normal. A week later, the patient’s symptoms had disappeared except coughing and pharyngitis.\n\nA complete blood count taken on 3rd September showed that there was a smaller number of lymphocytes than normal and higher than normal number of neutrophils (Table 3). CRP was positive (42 mg/dl) while D-dimer and serum ferritin were normal (181 ng/ml and 97.5 ng/ml, respectively). The status of the patient became more stable and he gradually started to recover. However, the patient still reports feeling tired and not having the ability to do normal activities compared to his abilities before the recurrent infection.\n\n\nDiscussion\n\nOur patient suffered from a rapid increase in body temperature, which reached a peak of 39°C with congestions of the tonsils and pharynx, difficulty swallowing, and coughing and discomfort in the chest. Following these symptoms, the patient’s sense of smell was lost. Rapid test showed that the patient was IgG positive to COVID-19. Blood work showed that the patient had a small elevation in D-Dimer. In COVID-19 patients, higher values of D-Dimer could occur as a sequalae of SARS-CoV-2 or a secondary complication of a systemic inflammatory response syndrome which can be considered as an indicator for incidence of acute pulmonary embolism.10 CRP was also elevated, which is expected to increase due to pulmonary injuries during SARS-CoV-2 infection. Increased of CRP might indicate the seriousness of the disease.11 In addition, serum ferritin was moderately increased. Ferritin can be considered as a biomarker for COVID-19 prognosis.12 It is recommended to use these three parameters during hospitalization to track the prognosis of COVID-19 patients.13\n\nThe role of acquired immunity and SARS-CoV-2 antibodies in patient protection against further COVID-19 infection is still under debate.14,15 It was reported that acquired immunity could help in preventing further infection.16–19 However, Bentivegna et al.11 presented a case of a recovered positive serological pneumonia COVID-19 patient, accompanied by six negative PCR tests. A month later, and after exposure to the virus, another positive PCR test and seroconversion of IgM were performed.\n\nThe coronavirus infection might cause a thrombosis that can induce severe symptoms that could lead to death. Complete blood testing in our patient showed a significant elevation in the mean platelet volume, which could increase the probability of thrombosis.21,22\n\nA previous COVID-19 positive patient was shown to have a high level of peptide precursor of calcitonin hormone [procalcitonin (PCT)].23,24 In our case, both blood tests of the patient showed an increase in serum levels of PCT, which could indicate serious infection with pathological bacteria associated with bronchiolitis.25 Another increased marker in both of our patient’s blood tests was lymphopenia, which declined in the first and second COVID-19 infection. The low level of lymphocytes in the blood stream is usually associated with COVID-19 patients, resulting in immune suppression.26 An increase in neutrophils (as seen in our patient) can happen during a severe infection or exposure to stress, seen in COVID-19 infection.27\n\nDuring our patient’s second infection, a CT scan was ordered to confirm the second infection. The scan results showed consolidation and opacities in different sites of both lungs, indicating that the patient has a second COVID-19 infection, although he was infected before.\n\nIn conclusion, all the above tests strongly indicate that the patient had a recurrent infection with SARS-CoV-2 virus. However, the symptoms and severity of COVID-19 were less during the second infection than the first one.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of the 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": "Acknowledgements\n\nThe authors thank all the medical staff members involved in treating this patient.\n\n\nReferences\n\nZhu N, Zhang D, Wang W, et al.: A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Y, Liu Q, Guo D: Emerging coronaviruses: genome structure, replication, and pathogenesis. J Med Virol. 2020; 92(4): 418–423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGandhi RT, Lynch JB, Del Rio C: Mild or moderate Covid-19. N Engl J Med. 2020; 383(18): 1757–1766. PubMed Abstract \| Publisher Full Text\n\nDos Santos LA, de Góis Filho PG, Silva AMF, et al.: Recurrent COVID-19 including evidence of reinfection and enhanced severity in thirty Brazilian healthcare workers. J Infect. 2021; 82(3): 399–406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzam M, Sulistiana R, Ratnawati M, et al.: Recurrent SARS-CoV-2 RNA positivity after COVID-19: a systematic review and meta-analysis. Sci Rep. 2020; 10(1): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBongiovanni M, Vignati M, Giuliani G, et al.: The dilemma of COVID-19 recurrence after clinical recovery. J Infect. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang R, Deng W, He J, et al.: Case Report: Recurrence of Positive SARS-CoV-2 Results in Patients Recovered From COVID-19. Front Med. 2020; 7: 882. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrington D, Kele B, Pereira S, et al.: Confirmed Reinfection with SARS-CoV-2 Variant VOC-202012/01. Clin Infect Dis. 2021. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSekine T, Perez-Potti A, Rivera-Ballesteros O, et al.: Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19. Cell. 2020; 183(1): 158–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeonard-Lorant I, Delabranche X, Severac F, et al.: Acute pulmonary embolism in COVID-19 patients on CT angiography and relationship to D-dimer levels. Radiology. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLing W: C-reactive protein levels in the early stage of COVID-19. Med Mal Infect. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKappert K, Jahić A, Tauber R: Assessment of serum ferritin as a biomarker in COVID-19: bystander or participant? Insights by comparison with other infectious and non-infectious diseases. Biomarkers. 2020 Jul 23; 0(ja): 1–36. PubMed Abstract \| Publisher Full Text\n\nHenry BM, de Oliveira MHS, Benoit S, et al.: Hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (COVID-19): a meta-analysis. Clin Chem Lab Med CCLM. 2020 Jun 25; 58(7): 1021–1028. PubMed Abstract \| Publisher Full Text\n\nKirkcaldy RD, King BA, Brooks JT: COVID-19 and Postinfection Immunity: Limited Evidence, Many Remaining Questions. Jama. 2020; 323(22): 2245–2246. PubMed Abstract \| Publisher Full Text\n\nRoy S: COVID-19 Reinfection: Myth or Truth? SN Compr Clin Med. 2020: 1–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChandrashekar A, Liu J, Martinot AJ, et al.: SARS-CoV-2 infection protects against rechallenge in rhesus macaques. Science. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLong Q-X, Liu B-Z, Deng H-J, et al.: Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med. 2020: 1–4. PubMed Abstract \| Publisher Full Text\n\nOta M: Will we see protection or reinfection in COVID-19? Nature Publishing Group . 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatson J, Whiting PF, Brush JE: Interpreting a covid-19 test result. Bmj. 2020; 369. PubMed Abstract \| Publisher Full Text\n\nBentivegna E, Sentimentale A, Luciani M, et al.: New IgM seroconversion and positive RT-PCR test after exposure to the virus in recovered COVID-19 patient. J Med Virol. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConnors JM, Levy JH: COVID-19 and its implications for thrombosis and anticoagulation. Blood. 2020 Jun 4; 135(23): 2033–2040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhai Z, Li C, Chen Y, et al.: Prevention and Treatment of Venous Thromboembolism Associated with Coronavirus Disease 2019 Infection: A Consensus Statement before Guidelines. Thromb Haemost. 2020 Jun; 120(6): 937–948. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhesi Z, Enne VI, O‘Grady J, et al.: Rapid and Point-of-Care Testing in Respiratory Tract Infections: An Antibiotic Guardian? ACS Pharmacol Transl Sci. 2020 Jun 12; 3(3): 401–417. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaitao T, Vermunt JV, Abeykoon J, et al.: COVID-19 and Sex Differences: Mechanisms and Biomarkers. Mayo Clin Proc. 2020 Oct 1; 95(10): 2189–2203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlejandre C, Guitart C, Balaguer M, et al.: Use of procalcitonin and C-reactive protein in the diagnosis of bacterial infection in infants with severe bronchiolitis. Eur J Pediatr. 2020 Sep 14 [cited 2020 Oct 15]. PubMed Abstract \| Publisher Full Text\n\nTan L, Wang Q, Zhang D, et al.: Lymphopenia predicts disease severity of COVID-19: a descriptive and predictive study. Signal Transduct Target Ther. 2020 Mar 27; 5(1): 1–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Li Q, Yin Y, et al.: Excessive Neutrophils and Neutrophil Extracellular Traps in COVID-19. Front Immunol. 2020 [cited 2020 Oct 12]; 11. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source" }	[ { "id": "89733", "date": "17 Aug 2021", "name": "Aida B. Allawe", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research is greatly needed in order to get a better understanding of this new pandemic. The details of the clinical signs and case history are clearly explained. In addition, the clinical diagnostic steps and treatment are both properly illustrated.\nHowever, more research is needed to better understand the disease process, diagnosis, and treatment since it is a new pandemic and scientists have not fully understood it. For instance, the effect of age, sex, other illnesses, and the environment on the susceptibility of infection. Additionally, there is no evidence that refers that the virus persists after infection.\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/10-572
https://f1000research.com/articles/10-569/v1	14 Jul 21	{ "type": "Research Article", "title": "Semen biomarker TEX101 predicts sperm retrieval success for men with testicular failure", "authors": [ "Keith A Jarvi", "Peter Schlegel", "Christina Schiza", "Andrei Drabovich", "Susan Lau", "Antoninus Soosaipillai", "Dimitrios Korbakis", "Davor Brinc", "Brendan Mullen", "Eleftherios Diamandis", "Peter Schlegel", "Christina Schiza", "Andrei Drabovich", "Susan Lau", "Antoninus Soosaipillai", "Dimitrios Korbakis", "Davor Brinc", "Brendan Mullen", "Eleftherios Diamandis" ], "abstract": "Background: Azoospermia could be due to either obstruction (obstructive azoospermia: OA) or spermatogenic failure (non-obstructive azoospermia: NOA). Close to 50% of men with NOA have small pockets of sperm in the testis which could be retrieved surgically and then injected into oocytes in a program of intra-cytoplasmic sperm insertion. Presently, there are no accepted non-invasive tests allowing clinicians to predict the success rates of sperm retrieval. Previously, we have identified a germ cell-specific protein TEX101 in semen found in the primary spermatocytes and more mature sperm forms, but not in spermatogonia, Sertoli or Leydig cells. We hypothesized that the semen concentration of TEX101 could be used to predict sperm production in men with NOA. Methods:\n\nThis was a prospective cohort study on men with NOA being treated at a male infertility centre.\n\nMen with NOA planning sperm retrieval provided 1–3 semen samples prior to surgery. Semen TEX101 concentrations were measured by an in-house-developed ELISA assay and compared with the results of the surgery to retrieve sperm.\n\nResults: 20/60 karyotypically normal men with NOA had semen TEX101 < LOD (<0.2ng/mL). Of these, 0% had successful sperm retrieval(0-17%: 95% CI) . In contrast, of the 40 men with TEX101> LOD, sperm was found in 50% (34-66%: 95% CI, sig diff. Fisher’s exact test, p<0.05). Conclusions: Undetectable (<0.2 ng/mL) semen TEX101 is highly predictive of sperm retrieval failure for karyotypically normal men with NOA and is the single strongest non-invasive predictor of sperm retrieval failure reported so far. Semen TEX101 concentration will help couples decide their individual chances of successful sperm retrieval.", "keywords": [ "Azoospermia", "germ cell-specific protein", "sperm production", "sperm retrieval", "TEX101" ], "content": "Introduction\n\nOf men with infertility, ~ 20% have azoospermia or no sperm in the ejaculate, with the majority (49–93%) of these men having spermatogenic failure (non-obstructive azoospermia (NOA)). While the term “failure” would seem to indicate complete absence of spermatogenesis, men with testicular failure have either reduced spermatogenesis (hypo-spermatogenesis), maturation arrest at either an early or late stage of spermatogenesis or a complete lack of spermatogenic cells noted with Sertoli-cell only syndrome.1–5\n\nUntil approximately 25 years ago, there were no available therapies that allowed men with NOA to have biologically related children. The advent of the era of intra-cytoplasmic sperm insertion (ICSI) with the capacity to inject a single sperm into an oocyte opened the door for successful therapies for men with very low sperm counts. This revolutionary therapy for couples with infertility was then extended to treat men with azoospermia, with sperm retrieved from the testis/epididymis/vas deferens being successfully used for ICSI.6–10 Testicular sperm extraction for men with NOA is now used world-wide with reported successful sperm retrieval rates of 30–70% and ICSI pregnancy rates between 19–50%.8,10–16\n\nNon-invasive markers to predict the chances of a successful testicular sperm extraction technique are urgently needed. Several candidate clinical markers intended to predict the presence of sperm within the testis have been proposed including testis size, FSH, serum testosterone, inhibin B, genetic profiles (karyotype, Y-microdeletions) and blood flow patterns within the testis on Doppler studies.15–19 Unfortunately, none have demonstrated consistent clinical reliability compared with an in-depth testicular tissue analysis and sampling using the surgical microscope. Even the pathology on a previous diagnostic testicular biopsy (usually a randomly sampled tissue biopsy) does not definitively predict whether sperm are present or absent throughout the remaining of testis among men with non-obstructive azoospermia.7,8,11,13,14,18,20,21\n\nIn previous work from our laboratory, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified two seminal proteins, epididymis-specific protein ECM1 and germ cell-specific protein TEX101 that were able to differentiate obstructive versus non-obstructive azoospermia with 100% specificity and 81% sensitivity.22–27 Immunohistochemical staining of testicular tissue revealed that TEX101 is found in the primary spermatocytes and more mature sperm forms, but not in spermatogonia, Sertoli or Leydig cells.24,27 In addition, according to Human Protein Atlas, TEX101 protein is expressed in germ cells only, and not in any other of 75 tissues and cell types studied (http://www.proteinatlas.org/ENSG00000131126).\n\nThis background information suggests that TEX101 concentrations should be non-detectable in the semen of men with diffuse Sertoli-Cell-only syndrome and maturation arrest at the spermatogonial stage of development (early maturation arrest) (Figure 1). Non-detectable TEX101 could be useful to predict sperm retrieval failure.\n\nIn our previous work, we identified TEX101 as a potential marker for sperm production in men with NOA.23,24 This work used mass spectrometry (MS) to measure the abundance of TEX101 in the semen of men with NOA but was limited due to fairly high limit of detection (~5 ng/mL) and the cost of MS, making routine use for diagnostic testing impractical.\n\nWe have produced several monoclonal antibodies (mAbs) against human TEX101 and paired two mAbs to develop a highly sensitive, reproducible and low-cost ELISA assay with detection limit of < 0.2 ng/mL.27–29 With our recent development of a highly sensitive, reproducible and inexpensive ELISA assay for TEX101, we are now able to better study how accurately semen TEX101 levels predict sperm retrieval for men with NOA.\n\n\nMethods\n\nThis study was approved by the research ethics boards of both the Sinai Health System and the Weill Cornell Medical Center and all participants provided written informed consent for the use and publication of the patient’s data.\n\nBetween Jan 2016 to Jan 2019, patients were recruited prospectively from the specialist male infertility clinics at the University of Toronto and Weill–Cornell University. Semen samples were collected prior to surgery with the last samples being collected in June 2019. Men who were eligible for the study (presumed NOA with a minimum of two semen assays confirming azoospermia/cryptozoospermia and clinical parameters compatible with NOA) who were planning on proceeding with sperm retrieval were approached to enter the study. Since no additional semen samples were required and the surgery offered to the men was not changed by participation in this study, non-participation in the study was uncommon. To avoid bias, all potential patients were recruited.\n\nThe semen specimens were collected by masturbation into a sterile collection cup either at home or at the clinic. Following liquefaction (~1 hour at room temperature) a semen analysis was performed, then the semen samples were immediately centrifuged at 4oC and the supernatant (seminal plasma, SP) was frozen at −80oC. Whenever possible, semen samples were provided monthly for up to 3 months.\n\nThe production and selection of mouse monoclonal anti-TEX101 antibodies has been previously described.29,30 For the immunoassay development, white 96-well ELISA plates were coated with 400 ng/well of mouse monoclonal anti-TEX101 antibody 34-ED-629.3 in 50 mM Tris buffer, pH 7.8. Plates were washed (2×) with 0.05% Tween 20 in 20 mM Tris, 150 mM NaCl, pH 7.4). TEX101 calibrators and diluted samples in assay buffer containing 60 g/L BSA, 20 mg/L Mouse IgG, 100 mg/L goat IgG,1 g/L Bovine IgG and 0.25 M KCl in 5 0mM Tris with 0.05% Tween 20, pH 7.8, were added into each well (100 μL/well) and incubated for 2 h with gentle shaking. Plates were then washed (3×) with the washing buffer. A biotinylated mouse monoclonal anti-TEX101 antibody 34-ED-470.3, diluted in a solution containing 60 g/L BSA, 20 mg/L mouse IgG, 100 mg/L goat IgG, and 1 g/L bovine IgG in 50 mM Tris with 0.05% Tween 20, pH 7.8, were added (25 ng of antibody per 100 μL of solution per well) and incubated for 1 h. Plates were washed (6×) and alkaline phosphatase-conjugated streptavidin was added in the wells (100 μL per well). Incubation was for 20 min at RT with gentle shaking, followed by a final wash (6×). Diflunisal phosphate (DFP) solution was prepared in substrate buffer (0.15 M NaCl, 1 mM MgCl2 in 0.1 M Tris, pH 9.1), added on the plate (100 μL per well) and incubated for 10 min at RT with gentle shaking. Subsequently, the developing solution (1 M Tris, 0.15 M NaOH, 2 mM TbCl3, and 3 mM EDTA) was added on top and mixed for 1 min. Time-resolved fluorescence was measured with the Wallac EnVision 2103 Multilabel Reader (Perkin Elmer).\n\nThe calibrators were prepared by pooling several seminal plasma samples. TEX101 concentration in the pool was calculated using a quantitative SRM method, described elsewhere.30 Aliquots (volume of 20 μL each) of the pool were prepared and stored at −20 °C.\n\nTo assess the assay’s linearity, serial dilutions of the calibrator sample were prepared in a two-fold dilution step (ranging from ~7 ng/mL to ~0.05 ng/mL) and added on ELISA plate. Each dilution was analyzed in four replicates. For within-run and total precision, three levels of TEX101 concentration in the calibrator sample were used (mean 0.0770 ng/mL, 0.322 ng/mL, and 3.70 ng/mL). Each concentration was analyzed in eight replicates per run (plate), in two runs per day, over three days.\n\nTo assess assay’s linearity and within-run and total precision, DataInnovations software was used. The open-source software HYPERLINK “http://www.gnumeric.org/” HYPERLINK “http://www.gnumeric.org/” Gnumeric (Gnumeric, RRID:SCR_018462) may also be used in place of EP Evaluator to preform similar analyses.\n\nTEX101 ELISA assay limit of detection (LOD) is 0.015 ng/mL. Since seminal plasma samples are diluted 10-fold before analysis, the LOD for patient samples is 0.15 ng/mL; rounded to 0.2 ng/mL.\n\nTEX101 concentration is stable in SP samples stored at 4°C and 22°C over a period of at least 7 days. Samples were run in triplicate.\n\nSperm retrieval was performed using a standard technique as described by Schlegel in 1999.8 If any sperm was identified during or after the procedure, this was considered positive sperm retrieval.\n\nFrom previous studies on semen TEX101, we had shown that men having a Sertoli Cell Only pattern (lacking germ cells) would have undetectable TEX101 in the semen, while those with complete spermatogenesis would have detectable TEX101 levels.23,24 Since successful sperm retrieval rates for men with NOA are reported to be between 30-70%, we calculated the sample size required of 54 based on an estimated successful sperm retrieval rate of 50% in those with detectable TEX101 in the semen vs 15% for those with undetectable TEX101 in the semen (power of 0.8 and alpha of 0.05).8,10–16 We aimed to recruit an additional 25% to account for drop-outs and incomplete information.\n\nComparison of binomial results was performed using Fisher’s exact testing using Microsoft Excel (RRID:SCR_016137; An open access alternative is Google Sheets (RRID:SCR_017679)), and 95% CI were estimated using an online calculator.\n\n\nResults\n\nTwo mouse monoclonal antibodies (34-ED629.3 and 34-ED-470.3) targeting different epitopes of native TEX101 were used to develop a sensitive immunofluorometric assay. The new assay allowed a significant increase of ELISA signal with no sample pre-treatment required.30\n\nThe limit of quantification (LOQ) of the immunoassay was set at the TEX101 concentration showing CV≤20%, which was 0.015 ng/mL. The limit of detection (LOD) of the assay was defined as LOD=LOQ/3, which was 0.05 ng/mL. The linear range of the assay spanned from 0.015 ng/mL to 7.2 ng/mL (slope 0.966; intercept −0.0010) (Figure 3). Within-run (n=3 different levels, each in eight replicates) and between-run (n=3 different levels, each in eight replicates per run) precision assessed over 3 days were <6 and 9%, respectively (Table 2).\n\nRecovery was assessed by spiking a seminal plasma pool (of TEX101 concentration: 0.27 ng/mL and 1.1 ng/mL) into four seminal plasma samples with known TEX101 concentration (measured by a quantitative SRM method). Recovery of TEX101by this assay ranged from 93 to 104%.\n\nA total of 69 men (mean age 35±4.6 years) with NOA, considering undergoing surgical sperm retrieval, were enrolled in this study. Karyotype analysis revealed non-mosaic Klinefelter’s syndrome in four out of 69, a deletion of Y(q11.22) in one out of 69 and 64 out of 69 with normal karyotypes.\n\nAll of the 69 men enrolled in the study provided at least one semen sample for the TEX101 assay, with 37 providing more than one sample for analysis at least one month following the initial testing. Surgical sperm retrieval was performed in 65 out of 69 men, of whom 60 had normal karyotypes.\n\nSemen TEX101 concentration was less than the limit of detection (LOD <0.2 ng/mL) for 25 out of 69 initial semen specimens tested, with the median concentration of the remaining 44 semen samples being 0.98 ng/mL (IQR 0.35–126 ng/mL: range 0.2–23,102 ng/mL).\n\nA total of 37 out of 69 men had more than one semen sample tested. Of these 37 men, eight had consistent TEX101<LOD, 20 had consistent TEX101>LOD and nine had variable TEX101 (above and below LOD).\n\nThe results of the surgical sperm retrieval procedures and the semen TEX101 levels were available for a total of 65 men. Overall, sperm was found in 23/65 sperm retrieval procedures.\n\nWhile there were only four men with Klinefelter’s syndrome, TEX101 semen levels were low in this group with 3/4 having semen TEX101 <0.2ng/mL but sperm retrieval rates were high (75%).\n\nOut of the 65 men above, a total of 60 men had a normal karyotype. Of these 60 men, zero out of 20 (0%) of those with semen TEX101 <0.2 ng/mL (TEX101 measure on the semen sample provided closest to the date of the surgical sperm retrieval if more than one sample provided) had sperm identified during the surgical sperm retrieval procedure, while 20 out of 40 (50%) of those with semen TEX101 >0.2 ng/mL had sperm retrieved with surgery (significantly different, Fisher’s exact test, p < 0.05) (Table 1).\n\n* Significantly higher than TEX101 < 0.2 ng/mL, Fisher’s exact test, p < 0.05.\n\n1 Standard deviation.\n\n\nConclusion/discussion\n\nThis new study documents that TEX101 semen concentrations predict sperm retrieval outcomes for men with NOA and normal karyotypes. None of the men with TEX101 semen concentrations below the assay’s LOD had sperm found, while sperm was found in 50% of those with semen TEX101 levels above the assay’s LOD.\n\nWe have previously shown that the concentration of two semen protein markers, TEX101 and ECM1 differentiate with high specificity and sensitivity NOA from OA.24 ECM1 is an epididymal mono-specific marker which was below detection limits for men with OA, but present in the semen in detectable amounts in men with NOA. TEX101 is a germ cell mono-specific marker found on sperm, round spermatids, and spermatocytes, but not in spermatogonia, Sertoli cells or Leydig cells.24,27–29 Since TEX101 is germ cell mono-specific, it follows that semen TEX101 levels will be non- detectable in men with obstructive azoospermia and should also be non-detectable in men with Sertoli cell-Only syndrome and those with maturation arrest at the spermatogonia stage.24\n\nIt is important to note that the majority of the small number of studied men with Klinefelter’s syndrome had undetectable TEX101 semen levels but sperm retrieval rates remained high. The lack of detection of TEX101 of men with Klinefelter syndrome may reflect the focal nature of spermatogenesis that is commonly present in these men and/or sclerosis of the tubular lumen.\n\nThese results should have important implications in how we test and manage men with azoospermia. The information from the semen concentrations of ECM1 and TEX101 will differentiate OA from NOA with high specificity and sensitivity (Figure 1).27 For those with NOA, normal karyotype and undetectable semen TEX101, the test predicts that these men are very likely to have uniform Sertoli cell-only Syndrome or early maturation arrest with low/zero success rates with sperm retrieval (0%: 95% CI 0–17%). We have proposed an algorithm to diagnose the category of azoospermia and the expected outcomes of the sperm retrieval procedure based upon this study and our previously published work (Figure 2). This information should help clinicians counsel couples about their expected success rates as they manage azoospermia.\n\nScatter plot illustrating the assigned (theoretical) and measured TEX101 concentration in serial dilutions of seminal plasma calibrator sample analyzed in four replicates (left); Residual plot showing the assigned (theoretical) TEX101 concentration and the residual concentration (the difference between the measured and the assigned TEX101 concentration) (right).\n\nInterestingly, we did notice variability of semen TEX101 levels, with some men varying between detectable and undetectable TEX101 levels. It is possible that this variability indicates some underlying changes in spermatogenesis (similar in concept to the known variability in sperm counts in men with sperm in the ejaculate), so potentially TEX101 levels may be used in the future to time surgical sperm retrievals for men with NOA to optimize success rates.\n\nThere are two important caveats to consider with the results of this study. The observed sperm retrieval failure rates for those with semen TEX101 concentration <LOD was 100%, with a 95% CI of 83–100%. This study should not be used to conclude that men with TEX101 < 0.2 ng/mL universally have no sperm production, but should suggest that the prognosis for sperm retrieval is very poor for this group of men. Further studies on more men from different centers are required to provide a more accurate estimate of the negative predictive value of this test, as techniques for, and success of, sperm retrieval may vary at different centers.\n\nSecondly, the men seen and the type of surgery performed at the Toronto and Weill–Cornell centers may not be representative of the general male infertility practice. A different population of men with NOA will change the mean TEX101 concentration and fraction with TEX101 <LOD, while the type of surgery alters the success rates of the sperm retrieval. Further studies on men with NOA from different male infertility clinics are needed.\n\n\nAuthor contribution\n\nKeith A. Jarvi had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\n\nData availability\n\nUniversity of Toronto Dataverse: Underlying data for ‘Semen biomarker TEX101 predicts sperm retrieval success for men with testicular failure’. https://doi.org/10.5683/SP2/DQYDQW.31\n\nThe project contains the following underlying data:\n\n• TEX 101_Cornell-Jun 1 2021.tab (Cornell sample with their TEX101 concentration and if sperm was identified during mTESE)\n\n• TEX101_Sinai-Mar 11, 2019.xlsx (Sinai sample with their karyotype, TEX101 concentration and if sperm was identified during mTESE)\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\nFogle RH, Steiner AZ, Marshall FE, et al.: Etiology of azoospermia in a large nonreferral inner-city population. Fertil Steril. 2006; 86: 197–199. PubMed Abstract \| Publisher Full Text\n\nJarow JP, Espeland MA, Lipshultz LI: Evaluation of the azoospermic patient. J Urol. 1989; 142: 62–65. PubMed Abstract \| Publisher Full Text\n\nFedder J, Cruger D, Oestergaard B, et al.: Etiology of azoospermia in 100 consecutive nonvasectomized men. Fertil Steril. 2004; 82: 1463–1465. PubMed Abstract \| Publisher Full Text\n\nWang C, So SY, Wong KK, et al.: Chronic sinopulmonary disease in Chinese patients with obstructive azoospermia. J Androl. 1987; 8: 225–229. PubMed Abstract \| Publisher Full Text\n\nMatsumiya K, et al.: Clinical study of azoospermia. Int J Androl. 1994; 17: 140–142. PubMed Abstract \| Publisher Full Text\n\nVan Perperstraten AM, Proctor ML, Phillipson G, et al.: Techniques for surgical retrieval of sperm prior to ICSI for azoospermia. Cochrane Database Syst Rev. 2001; CD002807. PubMed Abstract \| Publisher Full Text\n\nSu LM, et al.: Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia: testicular histology can predict success of sperm retrieval. J Urol. 1999; 161: 112–116. PubMed Abstract\n\nSchlegel PN: Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod. 1999; 14: 131–135. PubMed Abstract \| Publisher Full Text\n\nGil-Salom M, et al.: Testicular sperm extraction and intracytoplasmic sperm injection: a chance of fertility in nonobstructive azoospermia. J Urol. 1998; 160: 2063–2067. PubMed Abstract\n\nSchlegel PN, et al.: Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia. Urology. 1997; 49: 435–440. PubMed Abstract \| Publisher Full Text\n\nColpi GM, et al.: Sperm retrieval for intra-cytoplasmic sperm injection in non-obstructive azoospermia. Minerva Urol Nefrol. 2005; 57: 99–107. PubMed Abstract\n\nSchlegel PN: Causes of azoospermia and their management. Reprod Fertil Dev. 2004; 16, 561–572. PubMed Abstract \| Publisher Full Text\n\nRaman JD, Schlegel PN: Testicular sperm extraction with intracytoplasmic sperm injection is successful for the treatment of nonobstructive azoospermia associated with cryptorchidism. J Urol. 2003; 170: 1287–1290. PubMed Abstract \| Publisher Full Text\n\nSchiff JD, et al.: Success of testicular sperm extraction [corrected] and intracytoplasmic sperm injection in men with Klinefelter syndrome. J Clin Endocrinol Metab. 2005; 90: 6263–6267. PubMed Abstract \| Publisher Full Text\n\nTsujimura A, et al.: Prediction of successful outcome of microdissection testicular sperm extraction in men with idiopathic nonobstructive azoospermia. J Urol. 2004; 172: 1944–1947. PubMed Abstract \| Publisher Full Text\n\nBohring C, Schroeder-Printzen I, Weidner W, et al.: Serum levels of inhibin B and follicle-stimulating hormone may predict successful sperm retrieval in men with azoospermia who are undergoing testicular sperm extraction. Fertil Steril. 2002; 78: 1195–1198. PubMed Abstract \| Publisher Full Text\n\nVernaeve V, et al.: Serum inhibin B cannot predict testicular sperm retrieval in patients with non-obstructive azoospermia. Hum Reprod. 2002; 17: 971–976. PubMed Abstract \| Publisher Full Text\n\nSouza CA, Cunha-Filho JS, Fagundes P, et al.: Sperm recovery prediction in azoospermic patients using Doppler ultrasonography. Int Urol Nephrol. 2005; 37: 535–540. PubMed Abstract \| Publisher Full Text\n\nBernie AM, Ramasamy R, Schlegel PN: Predictive factors of successful microdissection testicular sperm extraction. Basic and clinical andrology. 2013; 23: 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamasamy R, Schlegel PN: Microdissection testicular sperm extraction: effect of prior biopsy on success of sperm retrieval. J Urol. 2007; 177: 1447–1449. PubMed Abstract \| Publisher Full Text\n\nTunc L, et al.: Power Doppler ultrasound mapping in nonobstructive azoospermic patients prior to testicular sperm extraction. Arch Androl. 2005; 51: 277–283. PubMed Abstract \| Publisher Full Text\n\nDrabovich AP, Jarvi K, Diamandis EP: Verification of male infertility biomarkers in seminal plasma by multiplex selected reaction monitoring assay. Mol Cell Proteomics. 2011; 10: M110 004127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrabovich AP, Saraon P, Jarvi K, et al.: Seminal plasma as a diagnostic fluid for male reproductive system disorders. Nat Rev Urol. 2014; 11: 278–288. PubMed Abstract \| Publisher Full Text\n\nDrabovich AP, et al.: Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma. Sci Transl Med. 2013; 5: 212ra160. PubMed Abstract \| Publisher Full Text\n\nBatruch I, et al.: Analysis of seminal plasma from patients with non-obstructive azoospermia and identification of candidate biomarkers of male infertility. J Proteome Res. 2012; 11: 1503–1511. PubMed Abstract \| Publisher Full Text\n\nBatruch I, et al.: Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system. J Proteome Res. 2011; 10: 941–953. PubMed Abstract \| Publisher Full Text\n\nKorbakis D, et al.: Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility. BMC Medicine. 2017; 15: 60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchiza C, Korbakis D, Jarvi K, et al.: Identification of TEX101-associated Proteins Through Proteomic Measurement of Human Spermatozoa Homozygous for the Missense Variant rs35033974. Mol Cell Proteomics. 2019; 18: 338–351. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchiza C, et al.: Discovery of a Human Testis-specific Protein Complex TEX101-DPEP3 and Selection of Its Disrupting Antibodies. Mol Cell Proteomics. 2018; 17: 2480–2495. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKorbakis D, et al.: Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids. Mol Cell Proteomics. 2015; 14: 1517–1526. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJarvi K: TEX101_Sinai_Cornell. Scholars Portal Dataverse. 2021; V1. Publisher Full Text" }	[ { "id": "90893", "date": "04 Aug 2021", "name": "Kristian Almstrup", "expertise": [ "Reviewer Expertise Male reproduction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Jarvi et al. uses an ELISA assay to detect TEX101 concentrations in seminal plasma of men with non-obstructive azoospermia and correlate TEX101 levels with the success of sperm retrieval by TESE.\nI found the manuscript clear and well-written, the experimental procedures solid, and the scientific context interesting. However, as listed below, a range of issues should be resolved by the authors.\nIt is unclear to what extent Figure 1 represent novel data or reuse from earlier publications. This reviewer was able to find identical images in ref 241 by the same authors. Furthermore, it is unclear if samples/measurements of TEX101 are reused from the same publication1. This needs to be clear.\n\nPlease define better the “clinical parameters compatible with NOA” that were used as inclusion parameters.\n\nSome information is lacking regarding the stability of the measurements. The author just writes that TEX101 concentrations are stable in seminal plasma for 7 days, without providing any data. Similarly, the analysed seminal plasma samples were stored at -80C for ~5 years and it has not been investigated if this storage affects TEX101 measurements.\n\nFigure 3 and Table 2 are mentioned in the text before Figure 2 and Table 1, respectively.\n\nInstead of just stating how many were above and below the LOD, the authors should be able to provide more information about the biological variability from the men that delivered repeated samples. What was the coefficient of variation? A plot of the detected levels in repeated samples could also be informative. It is particularly important to report if men could be LOD in another.\n\nA Fisher’s exact test is insufficient to describe the diagnostic performance of the assay. The authors should include a receiver operating characteristic curve and provide the area under the curve. In addition, the authors need to report the sensitivity and specificity of the TEX101 assay in predicting successful sperm retrieval clearer. Presently, it is unclear what the numbers listed in Figure 2 cover.\n\nThis reviewer does not think the authors can conclude that their assay “ is the single strongest non-invasive predictor of sperm retrieval failure reported so far” without benchmarking their assay to other available methods.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "93559", "date": "30 Sep 2021", "name": "Mohamed Mostafa Arafa", "expertise": [ "Reviewer Expertise More clinical data and correlations are needed as discussed before" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 very important research providing a potential marker for sperm retrieval in the difficult group of non-obstructive azoospermia (NOA). The manuscript is well written and the methodology is solid. I have a few comments:\nWhat is the clinical data of the patients? Including testicular size, hormonal levels (FSH, LH, and Testosterone)\n\nWas there any correlation between TEX101 and these clinical parameters?\n\nCan the authors divide the NOA patients into high FSH and Normal FSH and find out if the findings will remain the same or better they can do a predictive model containing more than one parameter e.g. FSH, Testosterone, TEX101 that may increase the reproducibility of the study in other sites\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-569
https://f1000research.com/articles/9-1325/v1	13 Nov 20	{ "type": "Systematic Review", "title": "Aerobic, resistance, and mind-body exercise are equivalent to mitigate symptoms of depression in older adults: A systematic review and network meta-analysis of randomised controlled trials", "authors": [ "Kyle J. Miller", "Pinyadapat Areerob", "Declan Hennessy", "Daniela C. Gonçalves-Bradley", "Christopher Mesagno", "Fergal Grace", "Pinyadapat Areerob", "Declan Hennessy", "Daniela C. Gonçalves-Bradley", "Christopher Mesagno", "Fergal Grace" ], "abstract": "Background: Exercise has been identified as an allied health strategy that can support the management of depression in older adults, yet the relative effectiveness for different exercise modalities is unknown. To meet this gap in knowledge, we present a systematic review and network meta-analysis of randomised controlled trials (RCTs) to examine the head-to-head effectiveness of aerobic, resistance, and mind-body exercise to mitigate depressive symptoms in adults aged ≥ 65 years. Methods: A PRISMA-NMA compliant review was undertaken on RCTs from inception to September 12th, 2019. PubMed, Web of Science, CINAHL, Health Source: Nursing/Academic Edition, PsycARTICLES, PsycINFO, and SPORTDiscus were systematically searched for eligible RCTs enrolling adults with a mean age ≥ 65 years, comparing one or more exercise intervention arms, and which used valid measures of depressive symptomology. Comparative effectiveness was evaluated using network meta-analysis to combine direct and indirect evidence, controlling for inherent variation in trial control groups. Results: The systematic review included 81 RCTs, with 69 meeting eligibility for the network meta-analysis (n = 5,379 participants). Pooled analysis found each exercise type to be effective compared with controls (Hedges’ g = -0.27 to -0.51). Relative head-to-head comparisons were statistically comparable between exercise types: resistance versus aerobic (Hedges’ g = -0.06, PrI = -0.91, 0.79), mind-body versus aerobic (Hedges’ g = -0.12, PrI = -0.95, 0.72), mind-body versus resistance (Hedges’ g = -0.06, PrI = -0.90, 0.79). High levels of compliance were demonstrated for each exercise treatment. Conclusions: Aerobic, resistance, and mind-body exercise demonstrate equivalence to mitigate symptoms of depression in older adults aged ≥ 65 years, with comparably encouraging levels of compliance to exercise treatment. These findings coalesce with previous findings in clinically depressed older adults to encourage personal preference when prescribing exercise for depressive symptoms in older adults, irrespective of severity. Registration: PROSPERO CRD42018115866 (23/11/2018).", "keywords": [ "Older adults", "elderly", "seniors", "exercise", "physical activity", "depression", "randomised controlled trial", "RCT" ], "content": "Introduction\n\nAt the close of this decade, the last remaining ‘baby boomers’ will transition to an expanding peer demographic aged ≥ 65 years projected to constitute more than one billion older adults, worldwide1. Physical exercise is proposed as a low-risk adjunctive mitigant of age-associated functional deterioration in mental health, including for dementia2 and depression3,4. In light of impending demographic shifts, and with the burden of age-associated depression estimated to affect ~20% of older adults5–7, 2030 may confer a burden of 200 million adults aged ≥ 65 years presenting with clinical depression. In preparation for inevitable future demands on primary care systems8, there is prevailing opportunity for concerted efforts to support primary and allied health personnel with informed preventative strategies.\n\nInternational public health consortia are in concert with the antidepressant effects of exercise as a low-risk adjunct for optimal mental health9–11. While we do not yet have the answers for low uptake of exercise in older adults12, it may in some way be due to nuanced regimen design, which in turn, may similarly impact compliance in exercise prescription by primary and stakeholders in aged care. By exemplar, ‘Exercise is Medicine’ is a global initiative promulgated through 40 member countries by the American College of Sports Medicine with a platform to promote and encourage routine physical exercise for general health and a broad range of medical conditions. This manifesto encourages primary care physicians and health care providers to refer patients to qualified exercise personnel when prescribing treatment plans. However, despite conventional agreement for exercise as a prophylactic for geriatric depression, contemporary literature is yet to quantify the antidepressant treatment effectiveness for individual exercise types.\n\nPerhaps a lesser appreciated obstacle to optimising exercise prescription for mental health in older adults lies with the ‘catch all’ characterisation of exercise. During the past four decades, widely different metabolic, social, and environmental demands between exercise modalities (i.e., running vs. weightlifting vs. Tai Chi) have been well-characterised. Given that there is variation between exercise regimens, and these variations are not merely semantics, one may be surprised to discover that only a few randomised controlled trials (RCTs) have deliberately compared the antidepressant effects of different exercise regimens in older adults13,14. This begs a meaningful question, ‘are all exercise types equal?’.\n\nIn attempting to quantify the magnitude of potential antidepressant effects of exercise, researchers have deployed conventional pairwise meta-analysis4,15,16 during recent years. In departure from the value offered by pooling conventional treatments during pairwise meta-analysis, this amalgamation is inherently prone to overgeneralisation and concomitant overestimation of treatment effectiveness17. More specifically in mental health literature, pooling individual trial effect comparisons during the pairwise meta-analytical process precludes any opportunity for head-to-head comparison between different exercise types. In the same vein, a further nuance of pairwise meta-analysis is that effect-sensitivity is compromised by foregoing potentially relevant characterisation of control arms (i.e., wait-list, usual care, attention-control) of included trials18. In this respect, one must acknowledge that pairwise meta-analysis has intrinsic restrictive boundaries in circumstances where head-to-head comparison of different exercise treatments are required.\n\nIn circumstances where relative effectiveness of multiple treatment comparisons are required, network meta-analysis offers a methodological solution to provide more precise pooled head-to-head effect estimates than may otherwise be achieved19,20. Performed correctly, network meta-analysis can allow comparative effectiveness of exercise treatment regimens (aerobic, resistance, and mind-body), avoiding the assumption of treatment homogeneity, controlling for bias from small study effects, and likewise, controlling for characteristically different control arms (wait-list, usual care, and attention-control).\n\nExercise regimens are broadly categorized into either aerobic, resistance, or mind-body exercise types21,22, and any individual exercise program may consist of multiple combinations of these activities. Extended description of these three exercise types may be found as Extended data. It is well established that independent regimens of either aerobic or resistance exercise elicit uniquely different metabolic and phenotypic adaptations, whereas mind-body exercise is unique as a low impact form of exercise. Therefore, it is reasonable to theorise comparative differences in the capacity to elicit any antidepressant effect. Until recently, investigation of head-to-head treatment effects of aerobic, resistance, and mind-body exercise had not been undertaken and their comparative ability to moderate symptoms of depression in older adults without pre-existing clinical depression is unknown.\n\nRecently, Miller and colleagues23 quantitatively compared head-to-head effectiveness of aerobic, resistance, and mind-body exercise in older adults with pre-existing clinical depression. In realisation of their study hypothesis, Miller et al.23 excluded studies of older adults which lacked participant-level diagnosis of clinical depression. In doing so, there is an assumption that older adults with diagnosis of clinical depression should not be pooled with similarly aged counterparts without diagnosis. These differences are not merely semantics, and while beyond the broad scope of the present study, are worthy of summary.\n\nPresent day phenotypic characterisation of clinically depression in older adults is supported by the coalition of more than six decades of cross-disciplinary research into major depressive disorders. Limbic brain regions, monoamine neurotransmitters, and the hypothalamic-pituitary-adrenal (HPA) axis contribute to the pathophysiology of depression24. In addition, hypothalamic oversight of pulsatile stress hormone perception from brain regions is central for homeostatic regulation of human stress response and maintenance of systemic feedback loop physiology in concert with the pituitary and adrenal cortex. Similarly, it is well-established that stress hormones have broad infiltration through large areas of the brain via neuronal supply originating from midbrain and brainstem regions. Indeed, these monoaminergic systems have been widely recognised by neuroscientists, pharmacologists, and clinicians alike as key determinants (thus targets) of a person’s mood, cognition, sleep quality, appetite, and reward systems; each of which is known to be affected by physical exercise and the cornerstone of many pharmacological treatments for major depressive disorders.\n\nLong-term prospective data25 has demonstrated qualitatively distinct populations within patients with major depressive disorder, and a more recent systematic review26 of 67,318 participants enrolled to longitudinal cohort studies identified that people with subthreshold depression had an elevated risk of developing major depressive disorder. However, the existence for a continuum of depressive symptomology is inconclusive27 and subsyndromal symptoms are not yet classified as a continuum28. Taken together, it remains prudent to respect the binary threshold separating clinical diagnosis with subclinical depressive symptomology, and it stands to reason that clinical and mentally healthy categories should not be ‘merged’ into the same network in order to compare head-to-head effectiveness of different exercise treatments.\n\nWith these aspects in mind, the purpose of this systematic review and network meta-analysis was to quantitively assess the best evidence from RCTs to establish relative (head-to-head) effectiveness of resistance, aerobic, and mind-body exercise in adults aged ≥ 65 years below the clinical threshold for diagnosed depression. More specifically, we investigated whether (i) resistance, aerobic, and mind-body exercise training can induce substantive treatment effect on depressive symptoms in older adults, (ii) while considering relative treatment compliance to each exercise regimen, and further, (iii) to juxtapose the optimal exercise treatment for all adults aged ≥ 65 years irrespective of depressive symptomology.\n\n\nMethods\n\nThis review was prospectively registered with PROSPERO (registration number: CRD42018115866, 23/11/2018). The network meta-analysis extension for the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA-NMA) guidelines29 and the Cochrane Intervention Review that Compares Multiple Interventions30 each provided further support in guiding this review. Guidelines specific for geriatric meta-analyses31 were consulted to identify baseline characteristics, equity considerations, inclusion/exclusion criteria, known confounders, and potentially important effect modifiers. Extended data for this review can be found online32.\n\nStudies were eligible for inclusion if they (i) followed an RCT protocol, (ii) used a wait-list, usual care, or attention-control group, (iii) included one or more aerobic, resistance, or mind-body exercise intervention arms, (iv) reported depressive symptoms as an outcome at baseline and during follow-up, (v) used one or more psychometrically validated depression questionnaires, (vi) recruited participants with a minimum mean sample age of 65 years. Studies were excluded when (i) the intervention condition used a multicomponent treatment including non-exercise components with the exercise condition, or (ii) the participants were diagnosed with clinical depression, defined by DSM or ICD criteria, or a clinical threshold on a questionnaire validated against a structured diagnostic interview prior to study enrollment. Eligibility was not restricted to specific years, languages, or publication status.\n\nStudies were identified from computerised searches of the following databases: PubMed, Web of Science, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Health Source: Nursing/Academic Edition, PsycARTICLES, PsycINFO, and SPORTDiscus. Databases were searched for key terms pertaining to four main concepts: older adults, exercise, depressive symptoms, and RCTs. Search terms were identified using text mining procedures and an example of a search strategy is presented in detail as Extended data. Published studies, systematic reviews, and meta-analyses4,15,16,33–36 were also screened for additional articles. Databases were searched up to and including September 12th, 2019.\n\nAll articles collated from the systematic search initially went through a title and abstract screening, followed by a full-text screening. Eligibility criteria were used to determine whether each article should be included or excluded. The screening process was performed concurrently for both the current review and a parallel review23. Once the screening process was complete, the remaining studies were included in the systematic review and descriptively reported. Studies were only included in the quantitative analysis if they contained sufficient outcome data.\n\nDetailed data extraction was undertaken independently by a minimum of two researchers (KJM, PA, and/or DH) in compliance with a data extraction form (see Extended data), with any inconsistencies being arbitrated by another researcher (CM). Study characteristics are presented in Table 1. Methodological characteristics of each study were used to evaluate the quality of evidence, which included publication status, intention-to-treat principle, use of a cluster design, and validated measure(s) of depressive symptoms used.\n\nNote. N/R = not reported; n = Total participants included in intention-to-treat or post-treatment data analysis; HRmax = Heart rate maximum; VO2max = Maximal oxygen uptake; 1Rmax = One-repetition maximum; RPE = Rating of perceived exertion; BDI = Beck Depression Inventory; CESD = Center for Epidemiological Studies Depression Scale; CSDD = Cornell Scale for Depression in Dementia; DASS-D = Depression, Anxiety, and Stress Scale (depression subscale); GADS-D = Goldberg Anxiety and Depression Scale (depression subscale); GDS = Geriatric Depression Scale; GHQ-D = General Health Questionnaire (depression subscale); HADS-D = Hospital Anxiety and Depression Scale (depression subscale); HRSD = Hamilton Rating Scale for Depression; IDS-C = Inventory of Depression Symptomatology; MADRS = Montgomery-Asberg Depression Rating Scale; POMS-D = Profile of Mood States (depression subscale); PROMIS-EDD SF-8a = Patient Reported Outcome Measurement Information System - Emotional Distress and Depression Short Form-8a; TDQ = Taiwanese Depression Questionnaire.\n\nOutcome measure used in network meta-analysis.\n\n*Additional unsupervised exercise component.\n\nControl groups within each individual study were further categorised as either wait-list, usual care, or attention-control. Participants undergoing wait-list conditions received the exercise intervention following trial completion. Participants randomised to usual care were those in sole receipt of conventional treatment during the trial. Participants randomised to attention-control conditions (also known as attention placebo control or active control) received activities completely unrelated to physical activity and/or exercise during their respective trials (e.g., social activities, educational programs, etc.).\n\nImportant participant and intervention characteristics were used to verify whether potential effect modifiers were similarly distributed across comparisons within the network. Participant characteristics were coded according to sample size, age (mean and standard deviation), percentage of females, and place of residence (community-dwelling, residential care, clinical setting). Relevant inclusion criteria (e.g., sedentary, dementia, etc.) were further used to assess the risk of bias from equity considerations116.\n\nIntervention characteristics were appropriately coded. Exercise types were categorised as aerobic, resistance, or mind-body exercise. Length of interventions were coded in weeks or months. Exercise intensities were coded according to ratings of perceived exertion117, heart rate maximum (HRmax; low = <50%, moderate = 50-70%, high = >70%), maximal oxygen uptake (VO2max; low = <40%, moderate = 40-60%, high = >60%) or one-repetition maximum (1Rmax; low = <50%, moderate = 50-74%, high = >74%)118, or where unavailable, this was estimated according to the assessment of the original author(s). Frequency was coded as total sessions per week. Duration was coded as the average number of minutes engaging in exercise per session. Format of program was dichotomously coded as exercise in a group or individual setting. Format of supervision was dichotomously coded as supervised or unsupervised exercise. Agreement between the three researchers was 91.3%.\n\nRisk of bias of the included RCTs was assessed using the Cochrane Collaboration’s Tool for Assessing Risk of Bias119, which were independently conducted by a minimum of two researchers (KJM, PA, and/or DH). Discrepancies were arbitrated by another co-author (CM). Appraisal for ‘other sources of bias’ were evaluated with consideration for small sample size (n < 15), low adherence (less than 80%), cluster randomisation, and inequity in the selection of the sample.\n\nOutcome statistics including means (M), standard deviations (SD), and sample sizes (n) for depressive symptoms were used to calculate the mean change in the primary outcome. Test statistics (i.e., t-, F-, and p-values) were used to estimate effect sizes when descriptive statistics were unavailable. Pairwise relative (head-to-head) treatment effects for depressive symptoms were estimated using Hedges’ g120, which corrects for overestimation biases due to small sample sizes121. Hedges’ g coefficients were interpreted according to Cohen’s conversions122, whereby effects were considered small (0.2), medium (0.5), and large (0.8). Independent subgroups (e.g., males vs. females) within studies were treated as independent effect size estimates123. When individual studies reported more than one post-treatment depression score for the same group of participants, only the initial post-treatment time-point was used. When studies reported depression scores on multiple outcome measures, the included depression measure was selected in compliance with clinical applicability124,125.\n\nSecondary outcome data were also extracted for study attrition, treatment adherence, and adverse events. Pairwise relative (head-to-head) treatment effects for study attrition were reported as odds ratios based on the pre-treatment sample size versus post-treatment dropout in the treatment and comparison conditions. Treatment adherence were reported as a percentage of total attendance in, or compliance to, the treatment condition. Adverse events were qualitatively reported according to the descriptive information provided in the transcript.\n\nOne notable assumption of network meta-analysis relates to comparison group estimates, which are derived from pooling studies with homogenous between-study effect modifiers126. If the distribution of an effect modifier is heterogeneous across studies within a specific comparison group, the assumption of transitivity can be violated. In the context of this network meta-analysis, we separate exercise conditions (i.e., aerobic, resistance, mind-body) and control conditions (i.e., wait-list, usual care, attention-control) to account for the between-study variation of these effect modifiers. Given the intricacies of depression severity, a forest plot was generated to depict the juxtaposition of treatment effects between (i) participants in the present network meta-analysis with depressive symptomology but not clinically diagnosed and (ii) participants in a previous network meta-analysis with clinical depression23.\n\nRisk of bias assessment identified the potential for unit-of-analysis error within studies using a cluster design for treatment allocation. Thus, sample sizes were recalculated to account for error in cluster RCTs by determining a design effect127 with a conservative intracluster correlation coefficient of 0.05, in accordance with past studies45,128. The certainty of evidence contributing to network estimates was assessed according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach129.\n\nData were analysed and figures were generated using STATA/SE 15.1130. Comparison-adjusted funnel plots were used to evaluate publication bias and small-study effects. Random-effects meta-regression was used to investigate modifying effects of age, gender, source of participants, length of intervention, exercise intensity, frequency, duration, and format of program, which have been identified as potential risks to transitivity in geriatric meta-analyses4,35,131.\n\nA multivariate random-effects meta-analysis was conducted using the ‘mvmeta’ command132. A random-effects model assumes variance both within and between studies, explaining the heterogeneity of treatment effects133. A common heterogeneity parameter was assumed across comparisons. Heterogeneity was evaluated using tau-squared (τ2), which estimates the deviation in effect sizes across the population of studies123. The 95% prediction interval (PrI) was also used to estimate the true dispersion of effect within two standard deviations of the mean effect size123. The significance level was p < 0.05 for all analyses.\n\n\nResults\n\nThe initial systematic search yielded a total of 3,704 citations. When duplicate studies were removed (n = 1,395), 2,309 eligible records were identified. Titles and abstracts were screened by two independent researchers (KJM and DCGB) with an agreement of 91.9%. Subsequently, 356 full-text articles were assessed for compliance with inclusion criteria and outcome data. Full-text screening was performed independently by two researchers (KJM and PA) with an agreement of 81.8%, where discrepancies were arbitrated and resolved by consensus with a third researcher (CM). Studies using duplicate sample sets (n = 12) were identified, where the most informative publication with complete data being included in the quantitative analysis. Finally, 81 studies fulfilled all inclusion criteria to be included in the systematic review. In cases of missing data, authors were emailed by the first author (KJM). If authors failed to respond following two contact efforts, and there were insufficient data to calculate effect size estimates, the study was excluded from the quantitative analysis (n = 9). Additionally, three studies60–62 fulfilled all criteria but necessitated exclusion from the quantitative analysis due to control conditions being insufficiently defined, and the authors being unobtainable. Figure 1 outlines study selection and additional exclusion criteria.\n\nData from a total of 5,379 participants (2,815 treatment and 2,564 control) across 69 studies were pooled in the quantitative analysis. Each study reported pre- and post-treatment measures of depression symptoms, and data from 60 of the 69 studies were obtained for post-treatment attrition. Figure 2 illustrates the network of pairwise comparisons across all studies. Aerobic, resistance, and mind-body conditions had one or more direct comparison with each of the five comparison conditions. Direct comparisons for depressive symptoms were robustly characterised between intervention and corresponding control conditions (aerobic = 28, resistance = 22, mind-body = 32), with proportions being similar for study attrition (aerobic = 24, resistance = 21, mind-body = 25).\n\nLine width is proportional to the number of pairwise effect size estimates and node size is proportional to the number of participants.\n\nFrom the 69 included studies, 43 studies enrolled young-old samples (65–75 years) and 26 studies enrolled old-old samples (>75 years). Participants were identified as either community-dwelling (n = 46), living in residential care (n = 18), clinical (n = 4), or uncategorised (n = 1). Twelve studies enrolled participants with sedentary lifestyles, while the remainder enrolled participants with a range of active and sedentary lifestyles. Some studies recruited older adults that were either frail (n = 5), wheelchair bound (n = 3), or identified as presenting with a risk of falling (n = 2). Participants with age-associated comorbidities were also recruited, including cardiovascular problems (n = 6), dementia (n = 3), cognitive impairment (n = 3), cancer (n = 2), and other chronic illness (n = 6).\n\nFrom 79 independent exercise interventions, the majority were supervised in group exercise classes (n = 64). The remainder were either individually supervised (n = 5), unsupervised in an individual format (n = 2), supervised in an undisclosed format (n = 1), or entirely undisclosed (n = 1). The average exercise program length (weeks) was shorter for mind-body (M = 13.67, SD = 6.77) than aerobic (M = 19.76, SD = 14.44) or resistance (M = 18.00, SD = 9.07). Average weekly exercise minutes (calculated as frequency multiplied by duration) were calculated for aerobic (M = 107.78, SD = 40.88), resistance (M = 126.90, SD = 50.28), and mind-body exercise (M = 132.75, SD = 76.07). Treatment adherence (n = 44) was comparable between aerobic (M = 81.01, SD = 13.72), resistance (M = 81.17, SD = 9.00), and mind-body exercise (M = 79.34, SD = 14.73). Adverse events were observed during several of the included trials, which are presented in Table 2.\n\nRisk of bias for each study is presented comprehensively as Extended data (see Figure S1). Both blinding of participants and personnel to treatment was implausible due to the implicit nature of exercise training interventions, and thus, the remaining six criteria were used to assess the overall risk of bias within each study. Methodological quality of included studies can be considered low-to-moderate (M = 4.08/6, where low = 1, unclear, = 0.5, high = 0). Assessment of random sequence generation (selection bias), incomplete outcome data (attrition bias), and selective outcome reporting (reporting bias) may be considered adequate for most studies, whereas allocation concealment (selection bias) and blinding of outcome assessment (detection bias) were diverse. High ‘other sources of bias’ was due to low study adherence (n = 14), small sample sizes (n = 8), or both (n = 1). See Figure 3 for a summary of the risk of bias in the included studies.\n\nInconsistency network models were used to test the global consistency of direct and indirect effects of pairwise and multi-arm comparisons. Assumption of consistency was satisfied for each treatment (p > 0.05). Tests for inconsistency between direct and indirect estimates were not significant (p > 0.05), thus indirect and direct estimates were not different to direct evidence. Inconsistency tables can be found as Extended data (see Tables S1 and S2).\n\nLoop-specific heterogeneity was explored using inconsistency plots (see Extended data, Figure S7 and S8). Within the depressive symptoms network, inconsistency factors (IF) did not indicate high inconsistency (IF = 0.00 to 0.65) or loop-specific heterogeneity (τ2 = 0.05 to 0.28). The AER-MB-UC loop departed from the minimum lower-bound confidence interval (CI), yet fell short of presenting risk for heterogeneity (IF = 0.54, τ2 = 0.05, CI = 0.04, 1.03). Within the attrition network, the ratio of two odds ratios (RoR) indicated a high degree of inconsistency for the AER-RES-UC (RoR = 1.87) and MB-RES-UC (RoR = 1.77) loops. Each were interpreted as presenting elevated risk for heterogeneity, which were subsequently downgraded during GRADE assessment. All remaining loops satisfied assumption of consistency.\n\nComparison-adjusted funnel plots were used to detect publication bias and small-study effects. Funnel plots were roughly symmetrical for both depressive symptoms and attrition, indicative of low risk of publication bias and no presence of small-study effects. See Figure 4 for the depressive symptoms network and Extended data for the attrition network (see Figure S9).\n\nThe red vertical line represents the null hypothesis that independent effect size estimates are not different to comparison-specific pooled estimates. AC = attention-control; AER = aerobic; MB = mind-body; RES = resistance; UC = usual care; WL = wait-list.\n\nIn order to test transitivity across networks, potential effect modifiers were tested with meta-regression sensitivity analyses for the entire pool of studies and separately for each exercise comparison (aerobic vs. resistance vs. mind-body). No significant modifying effects were observed for the pool of studies or any separate exercise comparison for age, gender, source of participants, length of intervention, format of program, exercise intensity, frequency, duration, adherence, year of study, risk of bias, publication status, intention-to-treat analysis, nor cluster design, indicating that the assumption of transitivity was upheld. Full analyses are presented as Extended data (see Tables S6–S8).\n\nData pooled from 69 eligible studies provided a total of 88 individual comparisons for depressive symptoms and 76 individual comparisons for study attrition. Table 3 presents the network meta-analysis of depressive symptoms and attrition. Network estimates were calculated to establish relative effectiveness between pairs of comparisons.\n\nNote. Depressive symptoms (upper) are reported as Hedges’ g and 95% confidence intervals. Negative scores indicate a greater decrease in depressive symptoms for the column group. Attrition (lower) is reported as odds ratios and 95% confidence intervals. An odds ratio lower than 1 indicate a greater attrition for the column-defining comparison.\n\nEach exercise type effectively reduced depressive symptoms compared with control conditions (see Figure 5). Ranking of treatments for depressive symptoms are presented on SUCRA plots of ranked mean values, which can be found as Extended data (see Figure S11 and Table S3). Ranked order of quantitative values determined mind-body exercise to be the most effective type of exercise to mitigate depressive symptoms, followed closely by resistance and aerobic exercise, respectively. The magnitude of study effect did not reach statistical threshold to favour any individual exercise treatment.\n\nBlack diamonds represent the difference in the effect size estimate (Hedges’ g). Narrow horizontal lines represent the confidence intervals (CI) and the wider horizontal lines represent the prediction intervals (PrI). The blue vertical line represents the null hypothesis (Hedges’ g = 0). Negative scores indicate a greater decrease in depressive symptoms for the comparison (left) group. AC = attention-control; AER = aerobic; MB = mind-body; RES = resistance; UC = usual care; WL = wait-list.\n\nResistance exercise demonstrated the highest study compliance compared with each of the other comparison groups, but the dispersion of effect estimates presented a level of heterogeneity that confounded any substantive differences. Comprehensive reporting of study attrition can be found as Extended data (see Figure S10) in addition to SUCRA plots and ranked order (see Figure S12 and Table S4).\n\nA two-dimensional clustered ranking plot was employed to illustrate the average reduction in depressive symptoms for each comparison, relative to average attrition rate. Figure 6 presents the ranking of the exercise conditions with respective control conditions in conjunction with SUCRA values for depressive symptoms (effectiveness) and attrition. The three exercise conditions amalgamated a single cluster and were more effective than control conditions.\n\nEach colour represents a group of treatments that belong to the same cluster. Treatments in the upper right corner represent effective treatments with low attrition.\n\nThe certainty of evidence was assessed with the GRADE approach129. All control comparisons in the depressive symptoms network were rated as high or moderate certainty, which were downgraded due to small sample size or potential risks of bias (i.e., attrition bias, detection bias, or bias resulting from low adherence). Comparisons between exercise interventions were moderate-to-low certainty, resulting from imprecision in confidence intervals and small sample sizes. Detailed summary of the depressive symptoms network is presented in Table 4. Estimates of the attrition network were downgraded due to inconsistency and imprecision, which reflect moderate to very low confidence this outcome (see Extended data, Table S5).\n\naPotential attrition bias due to high number of studies with incomplete outcome data.\n\nbSmall sample size.\n\ncPotential risk of bias due to high number of studies with low adherence.\n\ndPotential detection bias due to high number of studies without blinding of outcome assessment.\n\neConfidence intervals include values favouring either treatment.\n\nFigure 7 presents collective representation for all adults aged ≥ 65 years. This comparison employs scaled distribution (Hedges’ g) for the present data and that of clinically depressed older adults23.\n\nCircles represent the difference in the effect size estimate (Hedges’ g) for those with depressive symptomology but not diagnosed with clinical depression (n = 5,379). Triangles represent the difference in the effect size estimate (Hedges’ g) for those with clinical depression (n = 596; effect sizes adapted from Miller et al., 2020). Horizontal lines represent the confidence intervals (CI). The dotted vertical line represents the null hypothesis (Hedges’ g = 0). Negative scores indicate a greater decrease in depressive symptoms for the left group, and vice versa.\n\n\nDiscussion\n\nThe present review offers new information for general exercise prescription to support mental health outcomes for adults aged ≥ 65 years. Specifically, (i) aerobic, resistance, and mind-body each demonstrate equivalent benefit to mitigate symptoms of depression in adults aged ≥ 65 years, (ii) compliance to exercise treatment is notably encouraging for each exercise types, and (iii) when combined with the pool of data from clinically depressed older adults23, the effectiveness of aerobic, resistance, and mind-body exercise is comparably consistent for all adults aged ≥ 65 years, irrespective of depression severity. These findings should provide reassurance for personnel and stakeholders in healthy ageing to encourage exercise prescription from a point of pragmatism and in collaboration with patient preference.\n\nBehavioural and physiological research24,25,27,28 coalesce to provide ample reasoning to separate participant cohorts with existing clinical depression from those without diagnosis. This study is no different. In agreement with findings from previous research13,14,35, aerobic and resistance exercise demonstrated similar treatment effectiveness in older samples aged ≥ 65 years (Hedges’ g = -0.06, PrI = -0.91, 0.79). Exercise characteristics (i.e., intensity, frequency, duration, etc.) are often similar between aerobic and resistance exercise, representing two sides of the same coin. Meta-analytical data15 on older adults with existing clinical depression observed that exercise programs incorporating a combination of aerobic and resistance training were most beneficial. Although the synergistic effects of combined exercise types were beyond the scope of the current review, it is conceivable that aerobic and resistance exercise may complement one another in an exercise intervention.\n\nPooled direct and indirect estimates marginally favoured treatment with mind-body exercise over either aerobic (Hedges’ g = -0.12, PrI = -0.95, 0.72) or resistance exercise (Hedges’ g = -0.06, PrI = -0.90, 0.79). However, it must be noted that the magnitude of effect falls short of being statistically different between groups and should be considered equivalent. Certainty of evidence is moderate, due to the dispersion in effect size estimates resulting in imprecision. Direct comparisons from multi-arm RCTs have offered mind-body exercise to be more effective than aerobic91 or resistance44 exercise. Moreover, subgroup analyses16 have indicated that clinically depressed older adults respond more favourably to mind-body exercise, but this hypothesis has not be substantiated4.\n\nSince mind-body exercise engages low intensity muscular activity (i.e., yoga, Tai Chi, qigong), the novel evidence in the current systematic analysis challenge the idea that intensity is the primary mechanism for the antidepressive effect of exercise. Rather, mind-body exercise combines the mental and physical aspects of exercise, which may result in similar antidepressive effects to higher intensity exercise134,135. Critical to these mental aspects is interoceptive sensations such as an internally directed focus on breathing and proprioception, which have previously been linked to the resilience of depressive states136,137. Thus, it is plausible that mind-body exercise allows older adults to regulate negative mood states, which is not normally possible during aerobic and resistance activities.\n\nOther important determinants of successful programming include study attrition, adherence rate, and adverse events. Here, we hypothesised that each exercise condition would demonstrate lower compliance to treatment than wait-list, usual care, and attention-control comparisons. Contrary to expectations, pooled direct and indirect estimates indicated that study attrition was comparable for all comparisons apart from resistance exercise, which offered a higher degree of compliance. However, on deeper scrutiny of absolute sample size, any differences observed in attrition become abrogated due to relatively smaller participant numbers within the resistance exercise studies (n = 705) compared with aerobic (n = 1,143) or mind-body (n = 1,005). Thus, in consideration of the wide dispersion in effect size estimates and a moderate risk of bias in individual studies, there are limitations in the certainty to confidently conclude substantive difference in study attrition between any comparisons.\n\nEach of the three types of exercise had similar adherence rates and time spent per week (calculated as frequency multiplied by duration). Interestingly, mind-body exercise had relatively shorter intervention length than either aerobic or resistance exercise (Mdiff = 6.09 and 4.33 weeks, respectively) despite having a greater reduction in depressive symptoms. This could be explained by (i) mind-body exercise having the same antidepressive effects in a shorter time than aerobic and resistance exercise, or (ii) a potential plateau effect whereby the antidepressive effects reach a maximum threshold during the first 10–15 weeks of an exercise program and are then maintained during the remaining weeks. Either way, it seems plausible that mind-body exercise provides a slightly more effective intervention against depressive symptoms in older populations without clinical depression, but that these treatment effects are not substantive enough to constitute statistical difference.\n\nWith consideration to projected population estimates over the next decade and the consequential demand on healthcare services1,8, the findings from this network meta-analysis offer a message of support for exercise prescription to promote mentally healthy ageing. When considering the collective findings of the present review in conjunction with the recent network meta-analysis in clinically depressed adults aged ≥ 65 years23, stakeholders in healthy ageing and exercise prescription have encouraging pooled RCT evidence for the antidepressant effects of either aerobic, resistance, or mind-body exercise for older adults across the mental health continuum.\n\nTreatment safety is a matter of ongoing importance in gerontological health, and exercise treatment programs are no different. Systematic scrutiny of the included RCTs found that study participants reported no major adverse events and only a few minor somatic complaints (n = 28). Taken together, this provides encouraging support for personnel wanting to safely prescribe exercise-based intervention programs in older populations. Of course, there is always a possibility of underreporting adverse events in clinical trials, and the present review was no exception. Therefore, the importance of reporting event outcomes, adverse or otherwise, cannot be understated. In fact, there is a known phenomenon in geriatric exercise research whereby adverse events are often underreported because authors do not consider minor adverse events to be noteworthy and/or essential to the primary purpose of the trial138, giving rise to an ongoing issue that will not be corrected until all studies routinely report event outcomes.\n\nNevertheless, participants engaging in aerobic exercise reported the least adverse events (n = 3), including minor medical attention and hip pain. Amongst studies included in this meta-analysis, aerobic exercise predominantly involved walking and stationary cycling, which may reflect a safe and natural form of exercise for older adults. Resistance exercise was typically associated with participants experiencing mild muscular pain and falls (n = 16), which may be explained by the progressive overloading of resistance-based training. Notably, incidents of falling were reported in an unsupervised exercise program. Finally, mind-body exercise was typically associated with different types of muscular pain and body strain (n = 9). It is speculated that the higher rates of injury in mind-body exercise are predominantly because it incorporates flexibility, balance, and stability movements, which may be unique to older bodies. In general, exercise seems to be a relatively safe intervention for older adults living in both the local community and residential aged care, although intensity and supervision, particularly for resistance training, should be monitored to ensure falls and injury do not occur.\n\nThe present review has some notable advantages above a traditional pairwise meta-analysis. RCTs with considerable non-exercising components, such as those using a multicomponent exercise intervention, were excluded because they may have overestimated the magnitude of the true effect in past reviews. Specifically, it is likely that multicomponent exercise interventions such as laughter therapy139, depression awareness training128, or self-efficacy training140 may have introduced a risk of bias by inflating the observed effectiveness of the exercise program on depressive symptoms through a secondary, complementary treatment effect. Pairwise meta-analysis also assumes that all control groups are the same, which is not always the case18. To manage heterogeneity from this assumption, control groups were separated into individual network comparisons. Taken together, the current findings provide a more accurate estimate of the true effects of exercise on depressive symptoms in adults aged ≥ 65 years.\n\nThe present network meta-analysis is not without limitations. As study participants and personnel cannot be successfully blinded, there is an inherent risk of performance bias. It is also believed that many exercise-based interventions have a small number of participants, shorter follow-up, and do not adequately conceal randomisation141, which are all likely to reduce the quality of RCTs and increase the risk of bias. However, we mitigated the impact of this by comparing relative effects with multiple control groups in order to increase reliability and specificity. This, combined with the relatively low risk of bias in individual RCTs, were extremely important in minimising overall risk of bias and achieving accurate effect comparisons in the present review.\n\nSince most RCTs did not explicitly describe the exclusion of participants with ongoing diagnosis of clinical depression, there was potential contamination with data from participants with existing clinical diagnosis and medical treatment that went unreported. This was primarily managed by separating (i) participants with depressive symptomology but not clinically diagnosed in the present review from (ii) participants with clinical depression in a previous network meta-analysis23. Within this review, we further mitigated this effect modifier by only including RCTs, where this risk would be balanced by control participants. We recommended that ageing researchers encourage the reporting of all ongoing pharmacological regimens in trials recruiting older participants.\n\nAlthough modifying effects were explored using meta-regression, potential compounding effects from exercise modifiers (e.g., fitness improvements, length of program, session frequency and duration, exercise intensity, supervision, group format) were outside the scope of our network meta-analysis. There has been a modicum of such exploration in subgroup and meta-regression analyses of previous reviews4,16,35, providing researchers with an encouraging opportunity in their planning of future similar work. Future meta-analyses with extensive subgroup analyses should explain the heterogeneity of effect sizes between similar exercise intervention studies in older persons.\n\n\nConclusions\n\nPooled RCT evidence highlights that each individual exercise mode (aerobic, resistance, and mind-body) demonstrate equivalence to mitigate symptoms of depression in older adults, irrespective of depression severity. As each exercise treatment demonstrated encouraging levels of treatment compliance, we endorse personnel and stakeholders in healthy ageing to encourage individual/patient preference when prescribing exercise to older adults ≥ 65 years presenting with depressive symptomology.\n\n\nData availability\n\nFigshare: Aerobic, resistance, and mind-body exercise are equivalent to mitigate symptoms of depression in older adults: A systematic review and network meta-analysis of randomised controlled trials (extended data). https://doi.org/10.6084/m9.figshare.12998549.v232.\n\nThis project contains the following underlying data:\n\n- Data File D1 (PDF file containing raw outcome data)\n\n- STATA/SE 15.1 files (DTA files containing raw data)\n\nFigshare: Aerobic, resistance, and mind-body exercise are equivalent to mitigate symptoms of depression in older adults: A systematic review and network meta-analysis of randomised controlled trials (extended data). https://doi.org/10.6084/m9.figshare.12998549.v232.\n\nThis project contains the following extended data:\n\n- Supplementary File S1 (PDF file containing additional information, tables, and figures not in the main manuscript)\n\nFigshare: PRISMA-NMA checklist for “Aerobic, resistance, and mind-body exercise are equivalent to mitigate symptoms of depression in older adults: A systematic review and network meta-analysis of randomised controlled trials (extended data). https://doi.org/10.6084/m9.figshare.12998549.v232\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 graciously acknowledge the skilled contributions of Dr. Beyon Miloyan for his technical guidance, thoughtful discussions, and expert critique of the present work.\n\n\nReferences\n\nUnited Nations DoEaSA, Population Division: World Population Prospects 2019. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "80099", "date": "08 Mar 2021", "name": "Walid Kamal Abdelbasset", "expertise": [ "Reviewer Expertise Exercise Intervention", "Geriatric Rehabilitation", "Rehabilitation 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\nThis study aims to examine the head-to-head effectiveness of aerobic, resistance, and mind-body exercise to mitigate depressive symptoms in adults aged ≥ 65 years. The topic is interesting, however, some corrections should be addressed.\nGeneral comments:\nThe English presentation needs to be improved throughout the manuscript.\n\nThis study is a systematic review. Therefore, transfer kindly the PRISMA checklist, which can help clarify the missed information.\nSpecific comments: Introduction:\nDefine depression in detail. Determine the relationship between exercise and depression status in older adults. I suggest the authors expose the following points in the introduction: What is known about depression and exercise training in older adults? What is not known? Why the study was done? The hypothesis is also missed in this section.\n\nDiscussion:\nThe main findings should be presented in the introductory paragraph with respect to the study objectives and hypothesis.\n\nInconsistency models need more details in the discussion section.\n\nPlease, re-frame the discussion along the following lines: Main findings of the present study Comparison with other studies Implication and explanation of findings Strengths and limitations Conclusion, recommendation, and future direction.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6849", "date": "14 Jul 2021", "name": "Kyle Miller", "role": "Author Response", "response": "This study aims to examine the head-to-head effectiveness of aerobic, resistance, and mind-body exercise to mitigate depressive symptoms in adults aged ≥ 65 years. The topic is interesting, however, some corrections should be addressed. We extend our gratitude to the Reviewer for their critique and offering an opportunity to improve our article. The author team have taken the time to reflect on each comment and reached consensus, which are provided in detail below. General comments: The English presentation needs to be improved throughout the manuscript. The majority of the authorship team are native English language speakers and while each of us acknowledge that we are in continual development, we are experienced in communicating scientific writing in our native language. However, we do acknowledge this criticism in the spirt of improvement that it is meant. Thus, we have taken time to consider literary communication and addressed to the best of our collective ability. This study is a systematic review. Therefore, transfer kindly the PRISMA checklist, which can help clarify the missed information. Should we be accurate in the interpretation of this comment, the Reviewer considers this article to be (i) a systematic review. This is correct. However, it is a systematic review combined with (ii) network meta-analysis, which requires compliance with a specific set of reporting guidelines that are an extension of the noted PRISMA checklist. This article is presented in acknowledgement of a complimentary extension to PRISMA guidelines where we have adhered to the ‘PRISMA-NMA guidelines’, where ‘NMA’ accounts for a quantitative addition within this ‘network meta-analysis’. To support our response of confirmatory compliance, we direct the Reviewer to the Methodology section within this article which specifies a formal statement of compliance with PRISMA-NMA guidelines. To further offer confirmatory compliance with PRISMA-NMA guidelines, we direct this Reviewer to supplementary materials within the online repository, found here: ‘Data Availability’ à ‘Reporting Guidelines’ à ‘PRISMA-NMA Checklist’ https://doi.org/10.6084/m9.figshare.12998549.v3 Specific comments: Introduction: Define depression in detail. We thank the Reviewer for identifying potentially important rationale for conducting this systematic review and network meta-analysis. In order to provide a comprehensive response to the query, it is necessary to offer two components of ‘depression’: Definition of depression – This is an interesting request, and the authors believe it to be adequately rationalised within the Introduction section. In compliance PRISMA-NMA criteria, we have specified the generalised term ‘depression’ as having a characteristic binary format for this work where explicitly (i) ‘clinically defined depression’ is divergent with (ii) ‘depressive symptoms’ which fall short of the threshold for the former. The present article focuses on the latter, where we offer contextual dialogue of literature within the domain of ‘clinical depression’ and articulate within the Discussion section of this article. Operational definition for this article – The discrete definition for depressive symptoms used in this review are further detailed within the methodology, where RCTs were deemed eligible where valid determination of depressive symptoms was achieved using accepted psychometrically valid tools. Determine the relationship between exercise and depression status in older adults. I suggest the authors expose the following points in the introduction: What is known about depression and exercise training in older adults? What is not known? We direct the Reviewer to Paragraphs 2 of the Introduction section: “International public health consortia are in concert with the antidepressant effects of exercise as a low-risk adjunct for optimal mental health (American Psychiatric Association, 2010; Stubbs et al., 2018; World Health Organization, 2010). While we do not yet have the answers for low uptake of exercise in older adults (Knowles et al., 2015), it may in some way be due to nuanced regimen design, which in turn, may similarly impact compliance in exercise prescription by primary and stakeholders in aged care.” Similarly, Paragraph 3 of the same Introduction section: “During the past four decades, widely different metabolic, social, and environmental demands between exercise modalities (i.e., running vs. weightlifting vs. Tai Chi) have been well-characterised. Given that there is variation between exercise regimens, and these variations are not merely semantics, one may be surprised to discover that only a few randomised controlled trials (RCTs) have deliberately compared the antidepressant effects of different exercise regimens in older adults (Martins et al., 2011; Penninx et al., 2002).” The authors acknowledge that may not be ideal for the Reviewer and are optimistic that the body of referenced material in coalition with the short steps of logical reasoning, offer mitigatory satisfaction of this Reviewer’s exposed points. Why the study was done? We agree that this is an important attribute which must be present within empirically sound research efforts. Accordingly, we direct the Reviewer to view the final paragraph within the Introduction section, which reads: “The purpose of this systematic review and network meta-analysis was to quantitively assess the best evidence from RCTs to establish relative (head-to-head) effectiveness of resistance, aerobic, and mind-body exercise in adults aged ≥ 65 years, and below the clinical threshold for diagnosed depression. More specifically, we investigated whether (i) resistance, aerobic, and mind-body exercise training can induce substantive treatment effect on depressive symptoms in older adults, (ii) while considering relative treatment compliance to each exercise regimen, and further, (iii) to juxtapose the optimal exercise treatment for all adults aged ≥ 65 years irrespective of depressive symptomology.” The hypothesis is also missed in this section. We are thankful to the Reviewer for addressing this important consideration. PRIMA-NMA guidelines stipulate that those wishing to conduct meta-analytical review should “provide an explicit statement of questions being addressed, with reference to participants, interventions, comparisons, outcomes, and study design (PICOS).” There is opportunity to investigate this further, where we direct the Reviewer the emergent work from Bruce Charlton titled ‘The used and abuses of meta-analysis’, wherein it is prescribed that “meta-analysis is not a hypothesis-testing activity” and where Charlton further offers that meta-analysis “cannot legitimately be used to establish the reality of a putative hazard or therapy” (Charlton, 1996). In this respect, we prefer to present the research questions in its current active format. More generally, we offer that the majority of the author team are experimental scientists where research is beset on the presentation of formal hypotheses. We agree that it could be a route to detail a specific question of focus. However, as (for example) the present work relies upon and leverages the efforts from many RCT authors to satisfy our independent question, we must also acknowledge that their individual nuanced differences become amplified to induce collective heterogeneity towards reaching a resolution to our research question. For reasons such as this, we selected to side with formal guidelines (in this case, PRISMA-NMA) to achieve the outcomes detailed within the present article. Discussion: The main findings should be presented in the introductory paragraph with respect to the study objectives and hypothesis. The authors offer some satisfaction to resolving the ‘hypothesis’ component of the Reviewer’s suggestion in the prior response. In terms of addressing the substantive part of this Reviewer’s query, we offer the main findings from this review to be presented at the beginning of the Discussion section, and specifically, in the inaugural paragraph, which reads: “The present review offers new information for general exercise prescription to support mental health outcomes for adults aged ≥ 65 years. Specifically, (i) aerobic, resistance, and mind-body each demonstrate equivalent benefit to mitigate symptoms of depression in adults aged ≥ 65 years, (ii) compliance to exercise treatment is notably encouraging for each exercise types, and (iii) when combined with the pool of data from clinically depressed older adults the effectiveness of aerobic, resistance, and mind-body exercise is comparably consistent for all adults aged ≥ 65 years, irrespective of depression severity. These findings should provide reassurance for personnel and stakeholders in healthy ageing to encourage exercise prescription from a point of pragmatism and in collaboration with patient preference.” Inconsistency models need more details in the discussion section. Within the present article, inconsistency models are detailed within the Results section (see ‘Assessment of Inconsistency’). Our generalised ambition in delivering coherence within the Discussion section was to integrate our findings from inconsistency modelling and to communicate in a more active voice in appreciative context with other available literature. As it turned out, our tests of inconsistency became largely satisfied with generalised assumption of consistency for (i) global and (ii) loop-specific heterogeneity for our main (depressive symptom) network which limited an expanded discussion of inconsistency. To support this Reviewer’s query and in our compliance with Cochrane Review guidelines (Higgins et al., 2021), network inconsistency was further interrogated using the GRADE assessment tool (Salanti et al., 2014). In doing this, any important inconsistencies are dialogued in context to the primary objective, which in turn, are dialogued within the Discussion section and benchmarked against our primary outcomes. For instance, Paragraph 5 of the Discussion section reads: “Certainty of evidence is moderate, due to the dispersion in effect size estimates resulting in imprecision.” Please, re-frame the discussion along the following lines: Main findings of the present study Comparison with other studies Implication and explanation of findings Strengths and limitations Conclusion, recommendation, and future direction. The authors have partially satisfied this request (RE: ‘Specific Comments - Discussion’), with requisite response above. In compliance with previously identified guidelines, we have attempted to provide a coherent Discussion section in this article which is presented in the following generalised flow: Paragraph 1: Summary of main findings. Paragraph 2-6: Theoretical implications and comparisons with past studies. Paragraph 7-10: Practical considerations and recommendations. Paragraph 11-13: Strengths, limitations, and future directions. Paragraph 14: Conclusion and final recommendation. We thank this Reviewer for constructive critique and the authors are satisfied that we have an improved article with support from Reviewer commentary. References American Psychiatric Association: Practice guideline for the treatment of patients with Major Depressive Disorder. 3 ed. Washington, DC: American Psychiatric Association;2010. Charlton BG: The uses and abuses of meta-analysis. Family Practice. 1996;13(4): 397–401. 8872100 10.1093/fampra/13.4.397 Higgins JPT, Thomas J, Chandler J, et al.: Cochrane handbook for systematic reviews of interventions version 6.2 (updated February 2021). Cochrane, 2021. Available from www.training.cochrane.org/handbook Knowles AM, Herbert P, Easton C, et al.: Impact of low-volume, high-intensity interval training on maximal aerobic capacity, health-related quality of life and motivation to exercise in ageing men. Age (Dordr). 2015;37(2):25. 25773069 10.1007/s11357-015-9763-3 4359174 Martins R, Silva MCE, Pindus D, et al.: Effects of strength and aerobic-based training on functional fitness, mood and the relationship between fatness and mood in older adults. J Sports Med Phys Fitness. 2011;51(3):489–96. 21904289 Penninx BWJH, Rejeski WJ, Pandya J, et al.: Exercise and depressive symptoms: A comparison of aerobic and resistance exercise effects on emotional and physical function in older persons with high and low depressive symptomatology. J Gerontol B Psychol Sci Soc Sci. 2002;57(2):P124–32. 11867660 10.1093/geronb/57.2.p124 Salanti G, Del Giovane C, Chaimani A, et al.: Evaluating the quality of evidence from a network meta-analysis. PLoS One. 2014;9(7):e99682. 24992266 10.1371/journal.pone.0099682 4084629 Stubbs B, Vancampfort D, Hallgren M, et al.: EPA guidance on physical activity as a treatment for severe mental illness: A meta-review of the evidence and Position Statement from the European Psychiatric Association (EPA), supported by the International Organization of Physical Therapists in Mental Health (IOPTMH). Eur Psychiatry. 2018;54:124–44. 30257806 10.1016/j.eurpsy.2018.07.004 World Health Organization: Global recommendations on physical activity for health. Geneva, Switzerland: World Health Organization;2010." } ] }, { "id": "83980", "date": "24 May 2021", "name": "Andrea Camaz Deslandes", "expertise": [ "Reviewer Expertise Physical exercise and mental health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nObjective: The study aimed to investigate the head-to-head effectiveness of aerobic, resistance, and mind-body exercise in ameliorating depressive symptoms in older adults. The study is relevant and original, considering the increased prevalence of depression in older adults and the need for treatment through complementary and alternative practices, such as exercise. This topic has indeed received little attention in literature until now. The method used is adequate to the purpose of the study, and the results and the discussion were developed appropriately. However, some minor adjustments are necessary for a better understanding of the manuscript. Please find below some of the specific items regarding the issues.\nIntroduction: I suggest a presentation of the concepts of physical activity and exercise, as well as a discussion about divergences in the classification of Yoga and other mind-body practices as physical exercise.\nMethods: The inclusion of older adults with Dementia (n=3), Cognitive Decline (n=3), Parkinson's Disease (n=1), and Cognitive frailty (n=1) can be a possible limitation of the present study, considering the differences related to comorbidity. I suggest the author exclude these studies. Neuropsychiatric symptoms in MCI and Dementia are related to several different mechanisms, as well as the psychological and physiological effects of physical exercise in these populations. The characteristics of Depression in these cases can be very specific, with causes, neurophysiological changes and treatments that differ from depressive symptoms in healthy older adults. The inclusion of these studies can contribute of shrinking the effect size of exercise. For example, the effectiveness of pharmacological approaches to managing depressive symptoms in Dementia and MCI is less effective and the antidepressant effect of exercise in these populations is still controversial. If the authors decide to include these population (MCI, Parkinson, Dementia), several other studies that investigated the effect of exercise on neuropsychiatric symptoms (such as depression) in patients with MCI and Dementia should be included in the present meta-analysis, For more details, see Liang 2018 and Leng et al., 2018.1 Moreover, this issue should be included in the limitation section.\n\nFactors related to the prescription of exercise (frequency, intensity, duration, length, supervision, cognitive engagement) should be more explored in analyses and results.\nDiscussion: The author should discuss the possible different mechanisms related to the antidepressant effect of mind-body practices, aerobic and resistance training in older adults, considering both psychological and physiological hypotheses. Please consider more recent neurobiological mechanisms related to the effect of exercise on depressive symptoms (e.g., myokines, neurotrophins, inflammatory cytokines, etc.).\nConclusion: The authors concluded: “Pooled RCT evidence highlights that each individual exercise mode (aerobic, resistance, and mind-body) can effectively mitigate symptoms of depression in older adults, irrespective of depression severity”. I suggest excluding the phrase “irrespective of depression severity”, since the authors did not run a specific analysis to evaluate this issue.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6850", "date": "14 Jul 2021", "name": "Kyle Miller", "role": "Author Response", "response": "Objective: The study aimed to investigate the head-to-head effectiveness of aerobic, resistance, and mind-body exercise in ameliorating depressive symptoms in older adults. The study is relevant and original, considering the increased prevalence of depression in older adults and the need for treatment through complementary and alternative practices, such as exercise. This topic has indeed received little attention in literature until now. The method used is adequate to the purpose of the study, and the results and the discussion were developed appropriately. However, some minor adjustments are necessary for a better understanding of the manuscript. Please find below some of the specific items regarding the issues. Firstly, the authors are extremely grateful for the considered commentary from this Reviewer. We take this opportunity to extend our gratitude for being provided an opportunity to improve our article. The author team have taken time to deliberate on each point and have reached consensus within our responses below. Thank you. Introduction: I suggest a presentation of the concepts of physical activity and exercise, as well as a discussion about divergences in the classification of Yoga and other mind-body practices as physical exercise. We acknowledge this as an important point, and one which would require detailed expansion and integration of broad knowledge of the different forms of exercise classification and their nuanced differences. In order to preserve the flow of rationale within the Introduction section, while similarly being complete with exercise characteristics of aerobic, resistance, and mind-body regimens, we have provided supplementary reading to satisfy the broader readership of exercise specialists, including background material for readers out-with this specialist topic. We have taken this opportunity to include further statement within the Introduction section to highlight the differences between types of exercise: “We direct the reader to the Extended data of this article for detailed description of the exercise classifications included in this review.” ‘Extended data’ for publication in F1000 is repository for non-essential information or data. To diligently tackle important divergences in classifications in exercise type required 750 words, which the authors deemed as an important point with an appropriate home within the online repository, which can be found here: ‘Data Availability’ > ‘Extended data’ > ‘Supplementary File S1’ https://doi.org/10.6084/m9.figshare.12998549.v3 Methods: The inclusion of older adults with Dementia (n=3), Cognitive Decline (n=3), Parkinson's Disease (n=1), and Cognitive frailty (n=1) can be a possible limitation of the present study, considering the differences related to comorbidity. I suggest the author exclude these studies. Neuropsychiatric symptoms in MCI and Dementia are related to several different mechanisms, as well as the psychological and physiological effects of physical exercise in these populations. The characteristics of Depression in these cases can be very specific, with causes, neurophysiological changes and treatments that differ from depressive symptoms in healthy older adults. The inclusion of these studies can contribute of shrinking the effect size of exercise. For example, the effectiveness of pharmacological approaches to managing depressive symptoms in Dementia and MCI is less effective and the antidepressant effect of exercise in these populations is still controversial. We agree with the Reviewers comments relating to the antidepressant effects of exercise, and in particular that evidence in these populations remains controversial. Before undertaking this review, specific inclusion criteria for this work were considered greatly, wherein, the authors remained agnostic to any particular effect size resulting from exercise participation on depressive symptoms in older adults. The purpose of the present review was to provide point-specific information that is generalisable to all older adults aged >65 years without diagnosis of clinical depression. The binary approach taken within this review was (a) without diagnosis of clinical depression, thus excluding (b) those diagnosed with clinical depression. This review included RCTs limited to the former, which became the defining characteristic. Specifically, we cannot bias the inclusion criteria based on ubiquitous conditions, and as such, any given adult aged >65 years is by definition a primary candidate for each of the suggested limiting covariates. We are genuinely grateful to this Reviewer for generating some important points relating to exercise gerontology which remain unresolved. Until such time as epidemiological controversy such as nuanced and unresolved characteristics amongst ageing cohorts is evidenced, it is our belief that we must accept perceived imperfections within the literature, which is a notable limitation of meta-analytical investigation. This Reviewer does imply an important fact, and we do not offer this article as being definitive as such potential limitations are unresolved. However, we do believe that it is best practice to present in the article’s current form, but importantly, accept and acknowledge the value of the Reviewer’s observation by noting this as a generalised limitation within the limitations section. By doing this, there is opportunity for researchers to improve on this research, should new evidence add confirmatory knowledge. Supplementary sensitivity analysis specific to this can be found in the online repository, located here: ‘Data Availability’ > ‘Extended data’ > ‘Additional Sensitivity Analysis’ https://doi.org/10.6084/m9.figshare.12998549.v3 If the authors decide to include these population (MCI, Parkinson, Dementia), several other studies that investigated the effect of exercise on neuropsychiatric symptoms (such as depression) in patients with MCI and Dementia should be included in the present meta-analysis, For more details, see Liang 2018 and Leng et al., 2018.1 Moreover, this issue should be included in the limitation section. We are further grateful to this Reviewer for providing a list of potential references and have applied article inclusion criteria in review of each. From this list, one study (i.e., Abd El-Kader & Al-Jiffri, 2016) met the inclusion criteria for the systematic review but was not eligible for inclusion in network meta-analytics. Thus, the Systematic Review component of this article has been updated, yet the network meta-analysis remains unchanged. In response to the Reviewer’s recommendation, we have carefully examined the review conducted by Leng et al. (2018). We have applied the present article inclusion criteria in review of each study and further included detailed reasoning for inclusion/exclusion for each which may be in the online repository, each of which may be observed at: ‘Data Availability’ > ‘Extended data’ > ‘Suggested Articles Provided During Peer Review’ https://doi.org/10.6084/m9.figshare.12998549.v3 Factors related to the prescription of exercise (frequency, intensity, duration, length, supervision, cognitive engagement) should be more explored in analyses and results. This is an important point to consider (and partially addressed in the previous comment). One primary difference between network meta-analysis and its more widely used counterpart, the ‘meta-analysis’, relates to the limited ability for network meta-analysis to address significant covariates by sub-group analytics and meta-regression of known moderators of the primary outcome. However, such important factors have a place to address this within network meta-analysis. Although not overtly observable, we have done so within the present article. More specifically, “in order to test transitivity across networks, potential effect modifiers were tested with meta-regression sensitivity analyses for the entire pool of studies and separately for each exercise comparison (aerobic vs. resistance vs. mind-body). No significant modifying effects were observed for the pool of studies or any separate exercise comparison for age, gender, source of participants, length of intervention, format of program, exercise intensity, frequency, duration, adherence, year of study, risk of bias, publication status, intention-to-treat analysis, nor cluster design, indicating that the assumption of transitivity was upheld.” Further information can also be found in the online repository, found here: ‘Data Availability’ > ‘Extended data’ > ‘Supplementary File S1’ https://doi.org/10.6084/m9.figshare.12998549.v3 Discussion: The author should discuss the possible different mechanisms related to the antidepressant effect of mind-body practices, aerobic and resistance training in older adults, considering both psychological and physiological hypotheses. Please consider more recent neurobiological mechanisms related to the effect of exercise on depressive symptoms (e.g., myokines, neurotrophins, inflammatory cytokines, etc.). Possible different mechanisms, and unique characteristics relating to the antidepressant potential of different forms of exercise, remain to be fully defined. The main purpose of meta-analytical review of randomised controlled trials (RCTs) is to leverage the work of other researchers, and by doing so improve statistical power and point estimation for a specific question. In this respect, the present review cannot offer psychological or physiological hypotheses to a degree greater than any one specific RCT. However, we do accept this Reviewer’s guidance and further accept that it is important to contextualise contemporary mechanisms relating to the antidepressant effect of exercise. In light of this, we have included a separate extended piece of 784 words within the online repository to provide an extension of this discussion, found here: ‘Data Availability’ > ‘Extended data’ > ‘Supplementary File S1’ https://doi.org/10.6084/m9.figshare.12998549.v3 Additionally, the authors have added the following text “we direct the reader to the Extended data for further discussion relating to potential mechanisms for the antidepressant effects of exercise in older adults”, to add value to this overall body of work and contemporary mechanistic information for the interested reader. Conclusion: The authors concluded: “Pooled RCT evidence highlights that each individual exercise mode (aerobic, resistance, and mind-body) can effectively mitigate symptoms of depression in older adults, irrespective of depression severity”. I suggest excluding the phrase “irrespective of depression severity”, since the authors did not run a specific analysis to evaluate this issue. Thank you for drawing our attention to this critical oversight from the authors. The authors agree and this has now been removed for reasons of specific accuracy. Specifically, this has been moderated and subsequently amended within the Abstract, Discussion, and Conclusion sections. References Abd El-Kader SM, Al-Jiffri OH: Aerobic exercise improves quality of life, psychological well-being and systemic inflammation in subjects with Alzheimer’s disease. Afri Health Sci. 2016;16(4): 1045-55. 28479898 10.4314/ahs.v16i4.22 Leng MA, Liang B, Zhou H, et al.: Effects of physical exercise on depressive symptoms in patients with cognitive impairment: A systematic review and meta-analysis. J Nerv Ment Dis. 2018;206: 809–23. 30273278 10.1097/NMD.0000000000000887" } ] }, { "id": "83975", "date": "09 Jun 2021", "name": "Christian Imboden", "expertise": [ "Reviewer Expertise Depression", "physical activity for 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\nGeneral The review and network meta-analysis are very well written and conclusive. I especially appreciate the aim of the authors to analyze studies of different modalities of physical activity (PA) on depressive symptoms in older adults without clinical depression and comparing them to their already published network-MA on the effects of PA in clinically depressed older adults. The methods are described thoroughly. The authors analyze RCTs evaluating the effects of aerobic, resistance or mind-body exercise on depressive symptoms separately. In addition, they also group control groups by waitlist, attention control and care as usual which yields a very detailed analysis. They found significant positive effects on depressive symptoms for all three modalities of PA versus control interventions with no specific modality yielding significantly stronger effects than the other. They also analyzed attrition to the study protocol, an important variable that adds additional weight to the analysis. Considering attrition, the authors found comparable values in all comparisons. The results are displayed in appropriate ways by graphical and numerical means. And thus, provides a good overview of the various results. The statistical methods and interpretation seem absolutely appropriate to me, but since I am not a specialist for network meta-analysis I can not provide an in-depth evaluation.\nI have some minor comments to the authors:\nP 24, Results of the network-MA: Please spell out “SUCRA” at its first mentioning\nFigure 7: In the notes, the confidence interval is not specified. Is it 95% CI? Please specify. I also don’t understand why the horizontal lines (95%? CI) are different from Figure 5: i.e., the comparison AER vs. WL has a 95% CI of -0.63 to -0.14. So why does the horizontal line depicting the CI cross the vertical zero-line in Figure 7? Please explain.\nDiscussion: p. 27: you described that mind-body exercise has additional mental effects such as interoceptive sensations. Often mind-body exercise also incorporates aspects of mindfulness. Therefore, I believe it would be appropriate to insert a short paragraph discussing the effects of mindfulness (such as meditation and MBSR) on depressive symptoms. A suggested reference might be: Li et al. (2019) DOI: 10.1111/inm.125681\nOn the same page, you discuss compliance to treatment. You state, that you hypothesized that compliance would be lower in exercise treatments. However, the meta-analysis on dropout rates of Stubbs et al. (2016), DOI: 10.1016/j.jad.2015.10.0192 found similar dropout rates for exercise and control conditions but analyzed samples of adult patients (not specifically elderly). Nonetheless, you might discuss this in a sentence.\nConclusions: You state that exercise mitigates symptoms of depression regardless of depression severity. If interpreted strictly, according to the current paper this only applies to subclinical severity of symptoms. If you incorporate your earlier paper (network MA of exercise in clinically depressed elderly) into the conclusion, I believe this should be mentioned accordingly.\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? 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": "6851", "date": "14 Jul 2021", "name": "Kyle Miller", "role": "Author Response", "response": "General The review and network meta-analysis are very well written and conclusive. I especially appreciate the aim of the authors to analyze studies of different modalities of physical activity (PA) on depressive symptoms in older adults without clinical depression and comparing them to their already published network-MA on the effects of PA in clinically depressed older adults. The methods are described thoroughly. The authors analyze RCTs evaluating the effects of aerobic, resistance or mind-body exercise on depressive symptoms separately. In addition, they also group control groups by waitlist, attention control and care as usual which yields a very detailed analysis. They found significant positive effects on depressive symptoms for all three modalities of PA versus control interventions with no specific modality yielding significantly stronger effects than the other. They also analyzed attrition to the study protocol, an important variable that adds additional weight to the analysis. Considering attrition, the authors found comparable values in all comparisons. The results are displayed in appropriate ways by graphical and numerical means. And thus, provides a good overview of the various results. The statistical methods and interpretation seem absolutely appropriate to me, but since I am not a specialist for network meta-analysis I can not provide an in-depth evaluation. We wish to firstly thank the Reviewer for their thoughtful commentary. We have taken time to consider all feedback and make the necessary revisions to the article. I have some minor comments to the authors: P 24, Results of the network-MA: Please spell out “SUCRA” at its first mentioning We have included “surface under the cumulative ranking curve” before the first mention of the acronym. Figure 7: In the notes, the confidence interval is not specified. Is it 95% CI? Please specify. We thankful to the Reviewer for picking up this clerical error, on our behalf. This has now been rectified to state the “95% CI” in notes for Figures 5 and 7. I also don’t understand why the horizontal lines (95%? CI) are different from Figure 5: i.e., the comparison AER vs. WL has a 95% CI of -0.63 to -0.14. So why does the horizontal line depicting the CI cross the vertical zero-line in Figure 7? Please explain. We are grateful to the Reviewer for highlighting a lack of consistency in the article, specifically for intervals between Figure 5 and 7. Version 1 of the article as read by Reviewers contained both confidence and prediction intervals for Figures 5 and 7, respectively. In light of this, Figure 7 is now converted to 95% confidence intervals in order to maintain observational consistency with Figure 5. We have also proofed consistency of data (and presentation thereof) within article figures. Discussion: p. 27: you described that mind-body exercise has additional mental effects such as interoceptive sensations. Often mind-body exercise also incorporates aspects of mindfulness. Therefore, I believe it would be appropriate to insert a short paragraph discussing the effects of mindfulness (such as meditation and MBSR) on depressive symptoms. A suggested reference might be: Li et al. (2019) DOI: 10.1111/inm.125681 This is a very reasonable query relating to the potential for ‘mindfulness’ being an adjunct to potential improvement from mind-body exercise. The authors have taken the opportunity to record these findings within the article, with enhanced detail within the online supplementary material. This supplementary material extends upon the current article discussion to include potential discrete mechanisms that may underpin the psycho-adaptive response to exercise in older adults. This can be found within the online repository which accompanies this article: ‘Data Availability’ > ‘Extended data’ > ‘Supplementary File S1’ https://doi.org/10.6084/m9.figshare.12998549.v3 On the same page, you discuss compliance to treatment. You state, that you hypothesized that compliance would be lower in exercise treatments. However, the meta-analysis on dropout rates of Stubbs et al. (2016), DOI: 10.1016/j.jad.2015.10.0192 found similar dropout rates for exercise and control conditions but analyzed samples of adult patients (not specifically elderly). Nonetheless, you might discuss this in a sentence. This is an interesting point and one which we credited with consideration during the planning of this review. As the Reviewer will be aware, every epidemiological study conducted to date identify older adults to be the least active and least compliant with exercise compared with younger demographics. For reasons of being conservative and containing the potential for observer bias, this was handled in the negative unless data demonstrated otherwise. However, on reflection we deem the use of ‘hypothesised’ as potentially inappropriate compared with alternatives such as ‘anticipated’. This has been amended in the Discussion section to read: “Here, we anticipated that each exercise condition would demonstrate lower compliance to treatment than wait-list, usual care, and attention-control comparisons.” Conclusions: You state that exercise mitigates symptoms of depression regardless of depression severity. If interpreted strictly, according to the current paper this only applies to subclinical severity of symptoms. If you incorporate your earlier paper (network MA of exercise in clinically depressed elderly) into the conclusion, I believe this should be mentioned accordingly. We extend our gratitude to this Reviewer, who in coalition with Reviewer 2, identified this very same point. On reflection, this was clearly an overreach by the authors. We fully accept the requirement for amendment, which resulted in amendments to the Abstract, Discussion, and Conclusion sections." } ] } ]	1	https://f1000research.com/articles/9-1325
https://f1000research.com/articles/8-214/v1	24 Feb 19	{ "type": "Research Article", "title": "Potential predictors of adoption of the Tobacco Heating System by U.S. adult smokers: An actual use study", "authors": [ "Steve Roulet", "Christelle Chrea", "Claudia Kanitscheider", "Gerd Kallischnigg", "Pierpaolo Magnani", "Rolf Weitkunat", "Steve Roulet", "Claudia Kanitscheider", "Gerd Kallischnigg", "Pierpaolo Magnani", "Rolf Weitkunat" ], "abstract": "Background: This was a pre-market actual use study with the Tobacco Heating System (THS), a candidate modified risk tobacco product, conducted with adult smokers in eight cities in the United States. The main goal of the study was to describe THS adoption in a real-world setting. The aim of this analysis was to identify potential predictors for adoption of THS using stepwise logistic regression method. Methods: This actual use study was an observational study assessing self-reported stick-by-stick consumption of the THS product compared with the use of commercial cigarettes over six weeks. The study aimed at replicating the usage of THS in real-world conditions with participants being able to consume cigarettes, THS, and any other nicotine-containing products (e.g., e-cigarettes, cigars, etc.) ad libitum. Results: 14.6% of participants adopted THS, which comprised 70% or more of their total tobacco consumption by the end of the observational period (in Week 6). The main predictors of adoption were the liking of the smell, taste, aftertaste, and ease of use of THS. The proportion of adoption was higher in participants aged 44 years and older and in Hispanic or Latino adult smokers. Additionally, adoption of THS was more likely in participants who had never attempted to quit smoking and in participants who smoked up to 10 cigarettes per day. Finally, the adoption of THS was higher in participants who consumed both regular and menthol THS compared with those who consumed only one THS variant. Conclusions: The main predictors of THS adoption were positive sensory assessment and the ease of use. Socio-demographic characteristics and smoking habits appeared much less important. Post-marketing studies will provide further insights on the impact of the THS at the individual and the overall population level.", "keywords": [ "Harm Reduction", "Heat-Not-Burn", "Modified Risk Tobacco Product", "Actual Use", "Product Adoption" ], "content": "Abbreviations\n\nAIC: Akaike Information Criterion; CDC: U.S. Centers for Disease Control and Prevention; CRF: case report form; FDA: U.S. Food and Drug Administration; MRTP: modified risk tobacco product; THS: tobacco heating system\n\n\nIntroduction\n\nCigarette smoking causes pulmonary, cardiovascular, and other serious diseases and is responsible for the largest number of preventable deaths in the United States (U.S.)1,2. It is widely known that the best way to avoid these risks is to never start smoking. For smokers, the best way to reduce the risks and adverse health consequences of smoking is to quit3. However, as smoking is addictive, smoking cessation has proven difficult to achieve. Despite a decline in the smoking prevalence in the U.S. from 21% to 16% over the last decade, an estimated 40 million people in the U.S. smoked cigarettes in 2015, with around 30% of them smoking menthol cigarettes4.\n\nThe U.S. Food and Drug Administration (FDA) and other international health authorities have recognized that in order to more rapidly reduce the burden of death and disease from tobacco use, current tobacco control measures should be enriched and complemented by tobacco harm reduction strategies1,5,6. The latter focus on pragmatic goals that aim to provide smokers who do not want to stop nicotine use alternative, noncombustible tobacco and nicotine-containing products or nicotine delivery systems that eliminate exposure to smoked tobacco and thus substantially reduce harm compared with smoking combustible products7–14.\n\nIn the U.S., this has given rise to a regulatory framework for manufacturers to market modified risk tobacco products (MRTP), defined as “any tobacco product that is sold or distributed for use to reduce harm or the risk of tobacco-related disease associated with commercially marketed tobacco products”15.\n\nMRTPs aim to avoid imposing increased risks of chronic disease, morbidity, and mortality at levels caused by smoking cigarettes on their users, and their risk profile is an essential factor in estimating the public health effects of these products16. One approach to harm reduction involves e-cigarettes, which various authorities (Public Health England, 2018; Royal College of Physicians, 2016; U.S. Department of Health and Human Services, 2014) have concluded are likely to be substantially safer than cigarettes. A more recent approach consists of products that heat tobacco rather than burning it, thus producing far lower quantities of harmful and potentially harmful constituents (HPHC) than are found in cigarette smoke13. While it has been acknowledged that more research on the relative risk of heated tobacco products compared with that of combustible tobacco is needed, the available evidence suggests that heated tobacco products may be considerably less harmful than cigarettes17,18. Currently, the most widely-available heat not burn product is the Tobacco Heating System (THS) developed by Philip Morris International (PMI), sold under the IQOS® brand name. IQOS was launched in 2014 in Italy and Japan and is currently available in more than 30 countries. In May 2017, PMI MRTP Applications for IQOS with three THS variants were filed for scientific review by the FDA in the U.S.19.\n\nTHS uses a precisely controlled heating system into which the THS Tobacco Stick is inserted to generate an aerosol without combusting tobacco. The device heats tobacco to significantly lower temperatures (no more than 350°C) than cigarettes, thereby significantly reducing or eliminating HPHCs from the inhaled aerosol compared with cigarette smoke. The substantial reduction in toxic emission and subsequent body exposure have been established by the THS manufacturer (PMI) and competitors20–35. Though a few studies have brought contradictory evidence36,37, the weight of evidence produced by independent studies, including FDA laboratory tests, confirms PMI’s findings on the substantial reduction of major carcinogens17,29,38–42. While prevalence data are still sparse, evidence from Japan, where IQOS was first launched, suggest a steady increase in awareness and use of IQOS between 2015 and 201743,44. Analysis of predictors of IQOS current use (use in the previous 30 days) in 2017 showed that current Japanese smokers with intention to quit had higher odds to use IQOS than that of those with no intention to quit (13.3 vs. 6.7), while women aged 60 years or more showed significantly lower odds than reference categories. Ever-use of e-cigarettes was associated with greater odds of using IQOS. These findings suggest that the large majority of IQOS users in Japan switched from cigarettes to IQOS and that there is minimal uptake from nonsmokers. However, they provide limited information on how IQOS would impact public health in countries other than Japan. More specifically, in the context of an MRTP application, the FDA recommends assessment of the public health impact of candidate MRTPs under close to real-world conditions to understand how U.S. adult consumers actually use the product45, thus requiring actual use evidence for a product which is not yet commercialized in the U.S. The U.S. Institute of Medicine recommended studies that provide real-world evidence, including ad libitum use of MRTPs alone and in combination with cigarettes7.\n\nAlthough real-world evidence is generally gathered from observational studies in a post-market setting, as with over-the-counter drugs, where consumers are provided with the product together with labeled directions for use46–48, most of the actual use data that have been collected on potential MRTPs have been done in an artificial setting where the MRTP is provided for free, as opposed to what happens for other commercialized tobacco products in real-life conditions49–51.\n\nThe present study reports the findings of a pre-market actual use study performed in the context of IQOS MRTP application to the FDA19. The goal of the study was to measure change of use patterns in U.S. adult daily cigarette smokers and to assess THS product acceptance.\n\nTo mimic real-life situations as closely as possible, adult daily smokers had access to THS regular and menthol flavor products and were free to consume cigarettes, THS, and any other nicotine-containing products ad libitum.\n\nThe present analysis aims at identifying the potential predictors (i.e., socio-demographics, smoking habits, sensory assessment, and ease of use) of THS adoption in adult cigarette smokers. The effect of THS product flavor (i.e., regular or menthol) was also investigated.\n\n\nMethods\n\nThe actual use study consisted of one-week baseline period, a six-week observational period, and a one-week close-out period (see Figure 1)19. During the baseline period, participants recorded their regular cigarette consumption. During the subsequent observational period, participants recorded their consumption of both cigarettes and THS. Throughout the entire observational period, all participants were free to consume cigarettes, THS, and any other nicotine-containing products ad libitum. The observational period served to assess the development of THS use patterns. A close-out period was implemented for safety surveillance.\n\n* During the baseline and the observational periods, participants recorded their stick-by-stick consumption of cigarettes and/or THS into an electronic diary (e-diary). Participants were able to call the toll-free telephone hotline to raise queries related to the study, resolve issues related to the e-diary or THS, and report product quality complaints and adverse health events associated with the use of THS.\n\nThe study was conducted between 21 September 2015 and 7 January 2016 in eight cities located across the U.S. (Asheville, NC; Charlotte, NC; Denver, CO; Detroit, MI; Las Vegas, NV; Miami, FL; Oklahoma City, OK; Tampa, FL). All study materials were reviewed and approved on 28 August 2015 by Sterling Institutional Review Board (ID: 5149-001) before actual study implementation. This study was performed in accordance with Good Epidemiological Practice.\n\nStudy participants were recruited from the C&C Market Research databases. C&C’s databases consist of approximately 400,000 individuals nationwide who are recruited to join the site database via mall intercept, word of mouth, or by visiting the C&C Market Research website. The sampling was designed using quotas in terms of sex (male (56%); female (44%)), age (18–24 years old (34%); 25–44 years old (34%); 45+ years old (32%)), race (white (70%); black or African American (30%)), and income (low (48%); moderate/high (52%))1.\n\nBased on information available for each person (e.g., age, gender, smoker/nonsmoker, etc.) within the database, individuals employed by C&C Market Research randomly contacted potential study participants via telephone. No specific method or particular order was utilized for the selection of study participants beyond ensuring that the quotas were met. Individuals who met the following inclusion criteria were eligible for the study: (a) 18 years of age or above according to the minimum legal age), (b) currently living in the U.S., (c) current daily smokers of regular and/or menthol cigarettes with no intention of quitting within the next 30 days, (d) interest in participating in an eight-week study and providing informed consent. The following individuals were excluded from the study: (a) women who, based on self-report, were either pregnant, breastfeeding, or of childbearing potential and not using adequate means of contraception and (b) individuals who had started smoking within the last 30 days. Eligible individuals were then invited to a study site, where they were rescreened for eligibility based on their ID document for proof of age and were asked their intention to use THS based on their reading of a multipage information brochure on THS (Extended data52). Only participants with a positive intention (i.e., “somewhat likely”, “very likely”, “definitely” using a bipolar six-point scale ranging from “definitely not” to “definitely”) were enrolled in the study.\n\nSample size calculation was based on a precision-based approach (accuracy in parameter estimation) based on predetermined tightness of the confidence intervals. Given a precision of ± 5% for 95% confidence intervals of prevalence estimates and assuming a proportion of 50% of participants passing a consumption threshold of 100 THS products and 40% attrition, the study aimed to recruit 1,300 participants.\n\nThe THS is made up of three distinct components20: (1) a Tobacco Stick, specifically designed for use at low temperatures and containing specially processed crimped tobacco, (2) a holder for the Tobacco Stick that electronically heats the tobacco and controls the temperature, and (3) a charger for recharging the holder after each use. THS was provided by PMI. Products available to participants during the observational period had a neutral design with study identification elements to ensure confidentiality of the THS material, given the pre-market nature of the actual use study. U.S. Surgeon General’s warnings were present on each THS pack in a rotating fashion.\n\nAt enrollment in the study, participants completed an informed consent form and were interviewed in person by trained staff from the C&C Market Research study site in order to provide information on the purpose and goal of the study and instructions on how to use an electronic diary to report tobacco consumption. Questionnaires were also administered to collect demographic information, such as sex, age, race, ethnicity, education, occupation, and income as well as information on smoking habits, including the average number of cigarettes smoked per day, type of cigarette (menthol, regular), current usage of e-cigarettes, current usage of nicotine replacement therapy products, attempts to quit smoking, and the likelihood to use THS regularly as well as the reasons for this.\n\nDuring the one-week baseline period, participants were requested to make an entry into an electronic diary every time they consumed a cigarette. Upon completion of the one-week baseline period, participants returned to the site to receive THS and choose between THS regular, menthol, or a combination of the two products, according to their taste preference. Participants were provided with a maximum of 100 THS products at the start of the observational period. This supply ensured that all participants had access to THS on the initial days of participation in the observational period. During the remaining study period, participants could request additional THS products. Excessive ordering of additional THS was prevented by fixing an individual maximum number, based on self-reported cigarette consumption assessed at enrollment and then applying an “inflation factor” of three to allow for potential increase of use of THS.\n\nDuring the six-week observational period, participants were requested to make an entry into the electronic diary every time they consumed a THS or a cigarette. If no entries were made until a predefined time point per day, the e-diary sent an acoustic signal and displayed a reminder to record consumption. E-diary data were transferred automatically to a central database each night. In addition, participants were interviewed every two weeks to assess the taste, smell, aftertaste, and ease of use of THS (telephone interviews at Weeks 3 and 5 and personal interview at Week 7). Taste, smell, and aftertaste were assessed using a seven-point Likert scale ranging from one to seven, where one represented “I don’t like it at all”, and seven represented “I like it very much”. Similarly, ease of use was measured using a seven-point Likert scale ranging from one to seven, where one was “not easy to use at all”, and seven was “very easy to use”.\n\nParticipants were able to call the toll-free telephone hotline to raise queries related to the study, resolve issues related to the e-diary or THS, and report product quality complaints and adverse health events associated with the use of THS. At the end of the observational period, participants were asked to return all study materials.\n\nStudy participation was voluntary, and participants were free to withdraw at any time. Compensation in the study was based on participants’ level of participation and on compliance with the study procedures (maximum of $440) and paid via check at the end of the study.\n\nThe main outcome measure was self-reported consumption of cigarettes and THS during the observational period. This measure was used to derive a variable describing the percentage of THS use on a weekly basis by dividing the number of THS products by the number of total tobacco products used (THS products plus cigarettes). In order to facilitate meaningful description and interpretation of THS use patterns and future comparison across various studies53, this product use variable was then trichotomized into the following predefined symmetrical usage categories: (1) THS use (≥ 70% of total tobacco product used being THS[70–100]% THS), (2) combined use (> 30% to < 70% of total tobacco product used being THS ]30–70[% THS), and (3) cigarette use (≤ 30% of total tobacco product used being THS [0–30]% THS). In addition, “Adoption of THS” at Week 6 was defined as ≥ 70% of THS products in a participant’s combined consumption of tobacco products during Week 6.\n\nThe following variables were evaluated as potential predictors of THS adoption (Table 1):\n\n1 n = 965, excluding three participants without any reported Tobacco Stick or cigarette use within Week 6. Only nonmissing data are shown in the table.\n\n2 Categories recorded in the case report form (CRF) were condensed in order to reduce the number of estimators and balance the number of subjects per category: Persons in household in categories: 1 person, > 1 person\n\nChildren in household in categories: None; 1 or more children. Information on children in household was missing for two participants.\n\nMarital status: Relationship (CRF categories: Living with someone / Married), No relationship (CRF categories: Never married / Legally separated / Divorced / Widowed)\n\nOccupational status: At work (CRF category: working now), Not at work (CRF categories: Only temporarily laid off, sick leave or maternity leave / Looking for work, unemployed / Retired /Disabled, permanently or temporarily / Homemaker, keep housing / Student / Other). Information on occupational status was missing for one participant.\n\nEducational attainment: Low (CRF category: less than high school diploma) / moderate (CRF category: high school diploma), High (CRF categories: some university training or university degree). Information on educational attainment was missing for one participant.\n\nIncome levels: Low (CRF categories: Less than $30,000), moderate (CRF categories: $30,000 to less than $60,000), High (CRF categories: $60,000 and more). Information on income level was missing for 41 participants.\n\nSocio-economic status is derived as a combination of income levels and educational attainment: Low (low income and low education), Moderate (low income and moderate education, low income and high education, moderate income and low education, and high income and low education), and High (moderate income and moderate education, moderate income and high education, high income and moderate education, and high income and high education). Information on socio-economic status was missing for 42 participants.\n\nRace: White, Black or African American/Other (CRF categories: American Indian or Alaska Native, Asian, Native Hawaiian or Other Pacific Islander). Information on race was missing for two participants.\n\nLast attempt to quit smoking: Some time in the past (CRF categories: less than 6 months ago, more than 6 months ago), Never.\n\n3 The taste, smell, and aftertaste of the product were assessed using a seven-point scale ranging from 1 = “I don’t like it at all” to 7 = “I like it very much”. For the scale assessments, Cronbach’s alpha was calculated as measure of internal consistency among the scales. Because of an alpha of 0.89 (above the threshold value of 0.8), a combined construct of sensory acceptance was calculated using the mean scale assessments over taste, smell, and aftertaste. Four categories were created based on the quartiles of the distribution of these mean scale assessments. Information on sensory assessment was missing for 28 participants.\n\n4 Ease of use of the product was assessed using a seven-point scale ranging from 1 = “not easy to use at all” to 7 = “very easy to use”. Information on ease of use was missing for 28 participants.\n\nDemographics. From the demographic collection at enrollment, the following variables were derived: sex, age (18–24 years, 25–44 years, above 44 years), race (white, black or African American/Other), ethnicity (Hispanic or Latino, not Hispanic or Latino), income (low, moderate, high), number of persons (1 person, > 1 person) and children (none, 1 or more children) in household, marital status (no relationship, relationship), occupational status (at work, not at work), educational attainment (low/moderate, high), socio-economic status (low/moderate, high). In addition, study site location (eight cities) was also considered as a potential demographic predictor.\n\nSmoking behavior. From the smoking habits questionnaire at enrollment, the following variables were derived: average number of cigarettes per day (1–10 cigarettes, 11–20 cigarettes, ≥ 21 cigarettes), usage of e-cigarettes (yes, no), intention to quit smoking within the next six months (no or don’t know, yes), last attempt to quit smoking (some time in the past, never). In addition, the type of THS products ordered through the study observational period was also considered as a predictor of THS adoption (only regular, only menthol, both types).\n\nProduct assessment. Taste, smell, and aftertaste assessment collected at the end of the study (Week 7) were aggregated to quantify sensory assessment into four quartiles. Ease of use assessment was aggregated into three categories (not easy to use, quite easy to use, easy to use).\n\nThe study population for analysis included all participants who (1) fulfilled all eligibility criteria, (2) had at least one documented consumption of a cigarette during the baseline period, and (3) had at least one documented consumption of a THS product during the observational period.\n\nPotential predictors of THS adoption underwent bivariate screening using the Chi-squared test (see Table 1). Predictors with a p-value < 0.2 were subsequently subjected to stepwise logistic regression, with sex, age, and THS product types ordered being forced-in variables. Backward selection was applied to identify the final model, with p < 0.05 as the selection threshold to retain variables. The resulting model was compared with that identified by forward selection using the same variables. In case of a difference between the models, the better model based on the Akaike Information Criterion (AIC) was chosen54.\n\nAdditionally, the process was repeated using two-way interaction terms between THS product types ordered and each independent variable with p-value < 0.2 from the bivariate screening with simple logistic regressions. The two resulting multiple logistic regression models with and without interaction terms were compared using the AIC.\n\nAnalysis was conducted using SAS, version 9.4 (SAS Institute Inc. Cary, NC, USA). All analyses were descriptive and exploratory. No imputation of missing data was applied. Percentages were calculated as proportion of each category based on all nonmissing values.\n\n\nResults\n\nOut of the database managed by C&C Market Research, 8,858 members were contacted via telephone. Of these, 1,860 refused to continue the telephone conversation, 5,630 did not meet the eligibility criteria, and the remaining 1,368 were invited to the closest study site and rescreened against inclusion/exclusion criteria to verify eligibility. Of the 1,336 participants who were enrolled into the study, 1,106 participants self-reported at least one cigarette during the baseline period and at least one THS product during the observational period. At the end of the observational period (Week 6), 968 participants had reported data in e-diaries. Of these, three participants reported use of zero THS products or cigarettes. Thus, the analysis population consisted of 965 participants.\n\nThe proportion of male participants (49%) in the analysis population was very similar to the proportion of female participants (51%). More than 75% of the participants were 25+ years old, about two thirds (68%) were white, and slightly more than half (56%) had a yearly household income below $45,000 (Table 1).\n\nOf the analysis population (965 participants), 424 participants (43.9%) ordered only menthol THS products, 365 participants (37.8%) ordered only regular THS products, and 172 participants (17.8%) ordered both types.\n\nThe proportion of participants with THS use decreased between Week 1 (19.4%) and Week 6 (14.6%). Usage patterns of THS products were relatively stable in Weeks 4, 5, and 6 of the observational period.\n\nThe proportion of participants with combined use (> 30% and < 70% THS) decreased from 41.5% at Week 1 to 22.4% at Week 6, while the proportion of participants with cigarette use (≤ 30% THS) increased from 39.0% at Week 1 to 62.7% at Week 6.\n\nThe number of tobacco products (THS products and cigarettes) consumed per day during the observational period was lower than the number of cigarettes consumed per day during the baseline period across all participant groups at Week 6. The mean (± standard deviation) number of tobacco products decreased from 9.0 ± 5.89 to 8.1 ± 5.37 in participants with THS use, from 9.3 ± 6.34 to 8.9 ± 6.21 in participants with combined use, and from 10.9 ± 7.69 to 9.9 ± 6.75 in participants with cigarette use (Table 2).\n\n1 Definitions: THS use: ≥ 70% of total tobacco product used being THS, (2) combined use: > 30% to < 70% of total tobacco product used being THS, and (3) cigarette use: ≤ 30% of total tobacco product used being THS.\n\nAt the end of the observational period (Week 6), 14.6% of the analysis population had adopted THS use (Table 1). The proportion of participants adopting THS was higher in males (17.1% vs. 12.2%), in participants aged more than 25 years (25 to 44 years: 16.3%, above 44 years: 15.3% vs. 18 to 24 years: 10.8%), in one person households (17.6% vs. 13.8%), in participants with no relationship (15.6% vs. 11.4%), in black or African Americans (17.1% vs. 13.5%), and in Hispanic or Latino participants (23.5% vs. 13.4%).\n\nWith regard to smoking habits, the proportion of participants adopting THS was higher in participants smoking from one to 10 cigarettes per day (17.8% vs. 11 to 20 cigarettes per day: 13.2% and ≥ 21 cigarettes per day: 9.1%), e-cigarette users (23.1% vs. 14.1%), and in participants who never attempted to quit smoking (17.1% vs. 11.0%). The proportion of participants who adopted THS was higher in those who ordered both THS products (22.7% vs. 13.9% for menthol only vs. 11.8% for regular only).\n\nThe proportion of participants adopting THS was higher in participants who liked the taste, smell, and aftertaste of THS (increasing from 5.8% in the first quartile to 29.3% in the fourth quartile for sensory assessment scores) and in participants who found THS easy to use (increasing from 6.0% in participant who found THS not easy to use to 24.2% in participants who ound THS easy to use).\n\nStepwise main effects logistic regression analysis resulted in the same model, max-rescaled R-square of 0.1968 and 76.2% of concordant pairs, regardless of the selection method (i.e., forward or backward). The predictors of adoption of THS at the end of the observational period are summarized in Figure 2.\n\nThe vertical line shows the value where chances of adopting are equal in both the reference and the comparator group. Horizontal lines show the confidence intervals. The size of the diamonds is proportional to the number of participants in the comparator group.\n\n1 The taste, smell, and aftertaste of the product were assessed using seven-point scales ranging from 1 = “I don’t like it at all” to 7 = “I like it very much”. For the scale assessments, Cronbach’s alpha was calculated as measure of internal consistency among the scales. Because of an alpha of 0.89 (above the threshold value of 0.8), a combined construct of sensory acceptance was calculated using the mean scale assessments over taste, smell, and aftertaste. Four categories were created based on the quartiles of the distribution of these mean scale assessments.\n\n2Ease of use of the product was assessed using a seven-point scale ranging from 1 = “not easy to use at all” to 7 = “very easy to use”.\n\n3Average number of cigarettes/day at enrollment.\n\nNo influence of sex (OR = 0.71 [95% CI: 0.48–1.06]) was found, but adoption of THS was more likely in participants aged more than 44 years (OR = 2.01 [95% CI: 1.13–3.58]) and in participants who ordered both THS product types (OR = 1.86 [95% CI: 1.10–3.14]).\n\nSensory assessment and ease of use were the main predictors for THS adoption. The odds of adopting THS were more than four times higher in participants who liked the smell, taste, and aftertaste of THS (≥ 5.0 points on a seven-point scale) (OR = 4.44 [95% CI: 2.26–8.73]). Similarly, the odds to adopt THS were more than three times higher in participants who found THS easy to use (OR = 3.39 [95% CI 1.89–6.07]).\n\nParticipants who had never attempt to quit smoking had a higher chance of adopting THS compared with those who attempted to quit at some time in the past (OR = 1.73 [95% CI 1.14–2.63]).\n\nParticipants who smoked on average ≥ 21 cigarettes/day had a lower chance of adopting THS compared with those who smoked on average 1–10 cigarettes/day (OR = 0.44 [95% CI 0.21–0.89]), and the same applied for non-Hispanic or Latino participants compared with Hispanic or Latino participants (OR = 0.57 [95% CI 0.33–0.99]) (Figure 2). Interaction terms with the consumed THS product type did not improve the overall model fit.\n\n\nDiscussion\n\nThe main goal was to describe THS adoption in a real-world setting and to identify potential predictors for adoption of THS. This actual use study was conducted in U.S. adult daily smokers and included 1,106 participants self-reporting their consumption of cigarettes and/or THS products using an electronic diary. The study was conducted in eight cities spread across the U.S. to recruit a sufficiently large and diverse number of U.S. adult daily smokers. Quota sampling in terms of sex, age, race, and income was applied.\n\nThe proportion of participants with THS use was stable from Week 4 onwards. By Week 6, almost 15% of the participants had adopted THS, suggesting that THS is a viable alternative to cigarettes for adult smokers. The results do not indicate an increase of tobacco consumption over the observational period. Therefore, even though dual use is likely to happen in the first weeks of THS use, it is unlikely to lead to higher abuse liability and increase exposure to tobacco and nicotine products.\n\nThe adoption of THS was higher in participants ordering both THS types compared with participants ordering only regular or only menthol THS, suggesting that the availability of several variants of THS, including menthol, might result in a higher proportion of U.S. adult smokers substituting cigarettes with THS. Similar findings have been reported in studies with electronic cigarettes and noncombustible nicotine products55–58. Some of these studies also indicated that the use of menthol can facilitate the transition from cigarettes to reduced-risk products (RRP)55–58. “RRPs” is the term that PMI uses to refer to products that present, are likely to present, or have the potential to present less risk of harm to smokers who switch to these products versus continued smoking.\n\nParticipants who liked the smell, taste, and aftertaste of THS and participants who found THS easy to use were more likely to adopt THS, compared with participants who did not like THS smell, taste, and aftertaste or did not find THS easy to use. This finding supports results from previous studies that found that one of the main reasons that people stop using e-cigarettes after trying them is that they do not like the taste58–61.\n\nParticipants smoking 1–10 cigarettes per day were more likely to adopt THS than participants smoking more than 21 cigarettes/day. A similar outcome has been reported for e-cigarettes, as indicated by the prevalence of regular use of e-cigarettes being higher among adult smokers who smoke a lower number of cigarettes per day62.\n\nParticipants who had previously attempted to quit smoking were less likely to adopt THS than participants who never attempted to quit smoking in the past. This finding suggests that the availability of THS is unlikely to prevent those willing to quit tobacco from doing so. This is further confirmed by the fact that the intention to quit smoking within the next six months was not associated with THS adoption.\n\nThe proportion of THS adoption was higher in participants aged 44 years and older compared with participants aged between 18 and 24 years old. Hispanic or Latino participants had a slightly higher likelihood of adopting THS than not-Hispanic or Latino participants.\n\nOther demographic characteristics, such as sex, household size, educational attainment, income levels, or race, were not associated with THS adoption.\n\nOverall, these findings show that the socio-demographic characteristics of smokers who are more likely to adopt THS tend to differ from what has been recently reported on e-cigarettes, particularly in terms of age, ethnicity, and previous quit attempts62. This suggests that THS may be seen as an acceptable substitute for cigarettes to a different category of smokers than those who are currently using e-cigarettes. This is corroborated by the fact that current e cigarette use was not associated with THS adoption.\n\nImportantly, the study findings highlight the importance of offering alternatives that are close to cigarettes from a sensory experience for the adoption of RRPs58–61, with product liking and ease of use being more important predictors for adoption of THS than socio-demographic characteristics and smoking habits.\n\nThe key strengths of this actual use study included (1) the high ecological validity due to the near to real-world setting of the study, (2) the broad regional coverage, (3) the large sample size, and (4) the duration of the observational period of six weeks (which is slightly longer than in previous studies of alternative tobacco products63,64).\n\nLimitations include the fact that due to the study having been conducted in a pre-market setting, the study participants did not pay for the THS products, while they continued to pay for their cigarettes, which may have overestimated the level of THS adoption in this study. Also, the sample was not representative of the U.S. adult smoker population, which should be considered when interpreting the results. Finally, no biochemical verification of tobacco consumption, such as CO monitoring, was used, as the method of data collection relied exclusively on self-reported tobacco consumption. With regard to this point, it should be noted that validation studies have shown that self-reported tobacco consumption behaviors among adults are consistent and reliable50,65.\n\nFactors that were not measured may have influenced THS adoption (e.g., repeated exposure to product communication, peer-to-peer information sharing, risk perception [the product possibly being perceived as possible risk-reduced], familiarity, and acceptability of alternative tobacco usage behavior, as it may develop once the product has been marketed for some time)66.\n\nIn view of the above limitations, post-market studies are needed to provide actual levels of THS adoption and use patterns once THS is commercially marketed in the U.S. More studies are also needed to further understand what the drivers of THS adoption are. Consistent with several theoretical frameworks that have been used to understand the impact of intervention or prevention policies67,68, research should not only look at factors intrinsic to the users or to the product to explain use behavior but also take into consideration the influence of social (e.g., family background, peer influence) and societal/environmental factors (e.g., media influence, public health policy).\n\n\nConclusions\n\nThis actual use study showed that after a six week period of ad libitum use of THS provided at no expense, almost 15% of U.S. daily adult smokers substituted cigarettes with THS.\n\nIn this context, potential predictors for adoption of THS were a positive opinion of the taste, smell, and aftertaste and the ease of use, while socio-demographic characteristics and smoking habits were less important. Adoption of THS was higher in smokers aged over 44 years and in Hispanic or Latino smokers. For other demographic characteristics, such as sex, household size, educational attainment, income levels, or race, an influence on the adoption of THS was not found. Moreover, the adoption of THS was higher in smokers who used both menthol THS and regular THS products.\n\nThe results suggest that the introduction of THS in the U.S. has the potential to result in adoption by adult smokers who would otherwise continue to smoke cigarettes. On the basis of this adoption rate, this could benefit public health by having a positive impact on this particular population of adult smokers69. In particular, the results indicate that the adoption of THS is unlikely to result in an increase of tobacco consumption. Epidemiologic and post-marketing studies can provide further insights on the impact of the THS at the individual and the overall population level.\n\n\nData availability\n\nOpen Science Framework: Potential predictors of adoption of the Tobacco Heating System (THS) by U.S. adult smokers. https://doi.org/10.17605/OSF.IO/SBDXG52.\n\nThis project contains the following underlying data files:\n\n- Raw dataset.sas\n\n- Variable Coding Book.pdf\n\nOpen Science Framework: Potential predictors of adoption of the Tobacco Heating System (THS) by U.S. adult smokers. https://doi.org/10.17605/OSF.IO/SBDXG69.\n\nThis project contains the following extended data files:\n\n- Brochure.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": "Grant information\n\nPhilip Morris International is the sole source of funding and sponsor of this project.\n\n\nAcknowledgements\n\nThe authors would like to thank Kantar Health for assistance in data analysis and medical writing support.\n\n\nFootnotes\n\n1Low income (annual household ≤ $44,999); moderate/high income (annual household ≥ $45,000).\n\n\nReferences\n\nNational Center for Chronic Disease Prevention, Health Promotion Office on Smoking and Health: The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoulakova JN, Hartman AM, Liu B, et al.: Reliability of adult self-reported smoking history: data from the tobacco use supplement to the current population survey 2002-2003 cohort. Nicotine Tob Res. 2012; 14(8): 952–960. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPepper JK, Emery SL, Ribisl KM, et al.: How risky is it to use e-cigarettes? Smokers' beliefs about their health risks from using novel and traditional tobacco products. J Behav Med. 2015; 38(2): 318–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFong GT, Cummings KM, Borland R, et al.: The conceptual framework of the International Tobacco Control (ITC) Policy Evaluation Project. Tob Control. 2006; 15 Suppl 3: iii3–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHyland A, Ambrose BK, Conway KP, et al.: Design and methods of the Population Assessment of Tobacco and Health (PATH) Study. Tob Control. 2017; 26(4): 371–378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDjurdjevic S, Lee PN, Weitkunat R, et al.: Modeling the Population Health Impact of Introducing a Modified Risk Tobacco Product into the U.S. Market. Healthcare (Basel). 2018; 6(2): pii: E47. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "51842", "date": "28 Aug 2019", "name": "Kenneth A. Mundt", "expertise": [ "Reviewer Expertise Epidemiology with a primary focus on concepts and methods as used to evaluate occupational", "environmental and consumer product exposures." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\nThis article presents the methods and findings of a study in which a heated (not combusted) tobacco product (referred to as the Tobacco Heating System, or THS) was made available to current cigarette smokers who volunteered to participate and to maintain detailed diaries of their tobacco consumption, including cigarettes, THS, e-cigarettes, and any other nicotine-containing products. The study consisted of a one-week baseline period, six-weeks of observation, and one week ‘close-out’ period.\nParticipants were invited from a large market research database. Approximately 1300 participants enrolled, of which 1100 met the minimal cigarette and THS use criteria (one each) and by the end of the study 965 had completed diaries and were included in the statistical analyses. In addition to data on tobacco product consumption, participants provided information on socio-demographics (sex, age, race/ethnicity, household information, marital status, educational attainment, occupation, income, etc.); smoking and tobacco use (number of cigarettes or THS or other products, intention to quit); and THS product assessment (taste, smell, aftertaste and ease of use).\nPatterns of tobacco product use and changes over the six-week observation period were reported. Logistic regression with stepwise inclusion/elimination of variables (and first order interaction terms) was used to identify predictors of THS adoption (defined as >70% THS use). Participants of older age, selecting both THS flavors, users of and more favorable product assessment (taste and ease of use factors) and those never having attempted to quit were significantly more likely to adopt THS. Gender (not statistically significant) and ethnicity (statistically significant) contributed less.\n\nStudy strengths and weaknesses are well presented. The main strength was that this study closely monitored actual use of cigarettes and THS (and other nicotine products) over six weeks. One unavoidable limitation is that THS was provided to participants at no charge, and whether the observed patterns would be different if THS users were required to purchase their supplies.\n\nGeneral comments\nThis study generated several interesting and potentially helpful insights regarding real-world selection and use of THS among current conventional cigarette smokers. It appears to be the first study to provide such preliminary insights regarding how THS might be received in the United States.\n\nThe study methods, data collected, statistical analyses and results all are clearly presented. However, the discussion remains somewhat thin, i.e., individual analytical findings are addressed in succession, but the overall conclusion and integrated findings have not been fully explored. Interpretations of study findings should be compared more broadly with the published literature on other non-combusted tobacco or nicotine-containing products, as well as the possible impact of THS adoption among current smokers in the US. While important insights were generated regarding the group of smokers who volunteered for the study, no inference can be drawn with regard to non-tobacco users or users of other nicotine-containing products. The need for post-marketing studies is noted, but suggestions for their objectives (or specific research questions) are not offered. What specific additional studies would build on the initial findings reported here?\nA more minor point: the text would benefit from careful editing to correct some grammatical errors and primarily to make it more idiomatic.\nSpecific comments\nAbstract\nThe abstract needs improvement in content as well as structure.\nBackground: The final sentence presents the study aim; this largely is restated (with more detail) under the methods section. The reference to logistic regression reflects part of the study methods and should be moved.\nMethods: The only information provided is that tobacco users were observed over six weeks. A brief but more informative overview of the study design would be helpful. Results: This appears to be reasonably complete. Perhaps response rates (which were remarkably good) should be stated.\nConclusions: This section largely repeats results (and only some) and recommends post-marketing studies, but provides no interpretations, synthesis or policy implications based on this study.\n\nIntroduction\nThe introduction is informative and well presented.\nIs it possible to provide some examples of MRTPs available in the US? Perhaps it would be helpful to clarify the status of e-cigarettes with respect to MRTPs.\nMethods\nThe methods are clearly described. A few suggestions:\nSetting: How or why were these study locations chosen? The reference to “Good Epidemiological Practice” should carry a citation, and perhaps be moved under Study Design.\nProducts: THS is described in greater detail here, and does not reflect any methods. Perhaps the description of the three components of the THS should be moved to the introduction where the THS is first described.\nData collection and measurements: There is no mention of what took place during the final “close-out” week (it may not belong here, however).\nAnalysis: the logistic regression approach is reasonably clear, i.e., stepwise selection and (presumably) backward elimination to remove parameters with p<0.05. However, the choice of p<0.05 should be more clearly justified - what is the impact of eliminated parameters on the coefficients of those retained (i.e., is there evidence of confounding and does confounding increase when these terms are eliminated)?\nResults\nStudy participants: response/participation rates (which are remarkably good) should be presented.\nPotential predictors of adoption of THS: Adoption of THS by men and women differed by nearly 30% but was not statistically significant. Should this be presented as “no influence of sex”?\nDiscussion\nHow might the statistically significant reduced adoption of THS among heavier smokers and among non-Latino smokers be interpreted? Did these groups increase their conventional cigarette use or simply fail to adopt THS as much as other groups? Might the results suggest that these smokers are more habituated or committed to smoking conventional cigarettes?\n\nIt seems intuitive that adoption of THS would be preferentially higher among those who found it easy to use and more enjoyable. Is there any alternative interpretation? Similarly, might study participants selecting both THS types reflect populations more interested in variety than single product loyalty? How might these observations be used to predict what might happen in the US if THS were broadly available to current conventional cigarette smokers? Can some quantitative range of projection(s) be made regarding what proportion of cigarette smokers might adopt THS, i.e., quit conventional cigarette smoking?\nI noticed that the two study locations in a US state (i.e., NC) where tobacco is an important crop – and cigarettes are produced – were the least likely to adopt THS. This is interesting and might be explored further.\nJapanese studies demonstrated higher rates of THS adoption among smokers intending to quit smoking combusted cigarettes. In contrast, this study demonstrated higher adoption of THS among those who never attempted to quit. How might this be explained? What are the characteristics of smokers not intending to quit but adopting THS (vs. those not adopting, or those intending to quit)?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6820", "date": "14 Jul 2021", "name": "Christelle Chrea", "role": "Author Response", "response": "General comments Discussion – General comments Several parts of the Discussion section have been reworked to address the Reviewers comments on the Discussion. In particular, the Reviewer commented that, while important insights were generated regarding the group of adult smokers who volunteered for the study, no inference can be drawn with regard to non-tobacco users or users of other nicotine-containing products. We have reflected this comment in the Discussion as follows: “The sample was not representative of the U.S. current adult smoker population, which should be considered when interpreting the results.” Regarding the need for post-market studies, we outline in the Discussion that objectives of such research is to provide actual levels of THS adoption and use patterns once THS is commercially marketed in a given market (in the context of this manuscript, in the U.S.) and what are the drivers of adoption in the population. Specific comments Abstract The Abstract has been slightly edited according to the Reviewer’s comments on its content and structure, with no alteration to the message presented (i.e. removed repetitious sentences, as well as addition, restructuration and move of sentences to a different part of the abstract for clarity, etc…). As also recommended by the Reviewer, the overall text of the manuscript has been further edited to correct any grammatical errors and further improve the flow. Introduction As suggested by the Reviewer, the Introduction was edited to present how other regulatory authorities are considering different types of products, including e-cigarettes, and to provide examples of the MRTPs available in the U.S. (including MRTP applications accepted for review). We also updated in the Introduction the status of the PMI MRTP Applications for IQOS to indicate that, in July 2020, the FDA authorized the following claim “AVAILABLE EVIDENCE TO DATE: The IQOS system heats tobacco but does not burn it. This significantly reduces the production of harmful and potentially harmful chemicals. Scientific studies have shown that switching completely from conventional cigarettes to the IQOS system significantly reduces your body’s exposure to harmful or potentially harmful chemicals.”, together with the appropriate citation. In addition, we specified that “As of March 31, 2021 IQOS was available in 66 countries.” Methods The Methods section was edited to outline why the study locations were chosen (“The study locations were chosen to recruit a sufficiently large and diverse number of current U.S. adult daily smokers. In each city, C&C Market Research was operating a dedicated booth within a mall, which was used as study site.”). A citation has been added to reference “Good Epidemiological Practice” (Hoffmann, 2019), and this study characteristic remains indicated under Setting sub-section of the Methods, along with the information related to the Institutional Review Board. THS description has been moved under Introduction section as suggested. Regarding the final “close-out” week, the Data collection and measurements sub-section of the Methods has been edited as follows: “During the \"close-out\" week period participants were not required to record any data, however, they were able to call the toll-free telephone hotline for the continued surveillance of potential adverse events (AEs).” For the logistic regression described under Analysis sub-section of the Methods, we think that there is a misunderstanding. The backwards elimination was performed with a criteria p<0.05 as a selection threshold to retain the variable in the model, not to remove it. In addition, it was tested if the model could be improved using this procedure. Results The Reviewer recommended to present the response/participation rates. The aim of this analysis was to assess the potential predictors of adoption of the Tobacco Heating System (THS) by current U.S. adult smokers after six weeks of THS product use. With this in mind, the response/participation rates are, therefore, of limited value. This being said, the response/participations rates can be found as part of the full study report, which is publicly available as part of Philip Morris Products S.A. Modified Risk Tobacco Product (MRTP) Applications and can be accessed through the following link: https://www.fda.gov/tobacco-products/advertising-and-promotion/philip-morris-products-sa-modified-risk-tobacco-product-mrtp-applications. Regarding potential predictors of adoption of THS, the Reviewer suggested that, because our results showed that “Adoption of THS by men and women differed by nearly 30% but was not statistically significant,” it should be indicated as “no influence of sex”. We agree with this comment and we confirm that the Results section include the following wording: “No influence of sex was found” (after Figure 2). Discussion – Specific comments The Reviewer requested how the statistically significant reduced adoption of THS among heavier smokers and among non-Latino smokers could be interpreted. Indeed, heavy current adult smokers might need more time to adopt THS than current adult smokers smoking fewer cigarettes per day. Actually, based on the literature, Latino smokers tend to consume fewer cigarettes per day than non-Latino smokers. Therefore, the results of non-Latino smokers in our sample are likely related to the findings on heavy smokers who have a reduced adoption of THS. The Reviewer asked whether some quantitative range of projection(s) could be made regarding what proportion of cigarette smokers might adopt THS. We think that a quantitative projection would go beyond the objective of the study and would be difficult to justify. The Reviewer noticed that the two study locations in a U.S. state (i.e., NC) where tobacco is an important crop – and cigarettes are produced – were the least likely to adopt THS, and suggested to further explore this aspect. The study locations (eight cities spread across the U.S.) were chosen to recruit a sufficiently large and diverse number of U.S. adult daily smokers. The study was aimed at assessing differences by demographic characteristics and smoking habits rather than by study location. The Reviewer asked our perspective on Japanese studies data, which reported higher rates of THS adoption among current adult smokers intending to quit smoking combusted cigarettes, compared to our study, which, in contrast, demonstrated higher adoption of THS among those who never attempted to quit. Comparing the data from Japan and the U.S. is difficult, because we would compare pre-market data (U.S. data) with post-market data (Japan data) and different cultural environment. When it comes to the characteristics of the current adult smokers not intending to quit but adopting THS, we would not expect a difference in profile." } ] }, { "id": "52578", "date": "02 Sep 2019", "name": "Riccardo Polosa", "expertise": [ "Reviewer Expertise Tobacco research (including ECIG and THP)" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 6-weeks observational study, self-reported stick-by-stick consumption of IQOS was assessed and potential predictors of product adoption identified. Understanding predictors of e-cig/THP adoption is important as it may lead to improved smoking cessation/reduction rates. I have the following comments:\n\nMajor Comments\nRecruiting method is a potential (and probable) source of bias. As noted by the Authors, market panel members are not a representative sample of the population of US smokers. The participant remuneration was significantly high ($440); this - together with the fact that IQOS and consumables were given for free - would encourage participation and spin study findings. All the above casts doubts on whether the study is really conducted under real-world setting conditions and jeopardize the main goal of the study, which was to describe IQOS adoption in a real-world setting.\n\nThe study should be better reported.\nNo report of refusal to participate.\n\nNo report of partial compliance with diary reporting.\n\nNo report of the number non-compliant with interview schedule.\n\nNo report of the number not turning in study materials (used for measurement, so significant).\n\nThe only report is the number of participants who made no diary entries, and that alone is significant as 28% made no entries. If the authors conducted imputations for missing data (and certainly there were some missing data points!) this should be reported.\n\nI have concerns about the Analyses.\nE-cig users made up 12 of the 141 adopters, but represented only 5.4% of the population - clearly skewing the results. E-cig use should have been analyzed as a confounder.\n\nThe combined use categories make little sense to me.\n\nThe effectiveness for Hispanics/Latinos barely reached significance.\n\nWide CIs indicate that there are insufficient numbers for subgroup analysis.\nCan you clarify which version of the product was provided in this study; as far as I am aware the manufacturer has rolled out the third generation/evolution of IQOS. This is important for the interpretation of study findings (I was told that newer generations perform substantially better than earlier generations).\n\nFindings are products specific. It would have been interesting to have another comparator (e-cigs?) in the study in order to have a better understanding in terms of predictors of adoption of these new technologies. This should be discussed.\n\nThere’s lack of information about complete abstinence from tobacco cigarettes and this should be provided.\nMinor Comments\nIntroduction, Page 3. “These findings suggest that the large majority of IQOS users in Japan switched from cigarettes to IQOS and that there is minimal uptake from nonsmokers”. Please qualify these statements with appropriate numbers/percentages (and references).\n\nThe authors state that the study supplied a hotline for information and to collect reports of adverse effects. How many calls did the hotline receive? What adverse effects were reported? This is critical information and should be provided in the paper.\nStudy design. Is there a psychological behavioural pharmacological theory/rationale for the chosen length of study periods (i.e. 1 week for baseline period; 6 weeks for observation period)?\n\nI note that a validated psycho-diagnostic tool was used to measure participants’ intention to quit. Please specify which one.\n\nMore information on the structure and validity of the questionnaires used are need and the questionnaires should be included in the appendix.\n\nIt would have been equally important to evaluate the construct of the intention to switch to low-risk products.\n\nAn important predictor for IQOS adoption could have been the participants own cigarette brand.\n\nI was sorry to see in the analysis that heavy smokers (21+/day) were less likely to adopt than light smokers. This may reflect high level of inefficiency of (currently marketed) IQOS to adequately reproduce the experience in cigarette smoking. This should be discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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? Partly", "responses": [ { "c_id": "6821", "date": "14 Jul 2021", "name": "Christelle Chrea", "role": "Author Response", "response": "Major comments The Reviewer questioned the recruitment method and the extent to which our study should be considered to have been conducted under real-world setting conditions. From our perspective, an Actual Use Study represents the most appropriate study design to approximate real-world conditions in a premarket environment (i.e. in a condition in which the investigational product has not been authorized to be marketed, and therefore, is not commercially available to the public yet). Therefore, such study attempts to replicate to the maximum possible extent within the boundaries determined by the pre-market conditions and the limits imposed by feasibility. With respect to the recruiting method, we would like to note that the rationale for the selection of a non-probabilistic sample was to ensure the feasibility of the study. The sample was large as more than 1,300 participants were enrolled in the study and the results were robust as the study was conducted in eight different sites spread across the U.S. We chose these eight geographical locations to: i) recruit a diverse population of current U.S. adult daily smokers study participants; ii) allow distribution of the product, considering the various state requirements (e.g., retail license, tax stamping by a licensed distributer, pre-notification to fire Marshall); and iii) to ensure a good control over the investigational product. Although results are not generalizable to the U.S. smoking population, they provide a robust description of the patterns of use. Concerning the participants remuneration, we believe that participants were reimbursed adequately for their time and effort. The compensation was based on the study duration and the numerous tasks that they were asked to perform, e.g., complete an e-diary on a stick-by-stick frequency, answer to CATI interviews, go to the study site to get additional product, keep used and unused product, etc. Additionally, compensation in the study was up to $440, depending on the length of time in the study and return of the THS, used and unused HeatSticks, and the e-diary device. Assuming a full participation, this means $55 per week. To minimize the impact that reimbursement could have on participants’ tobacco consumption, each participant was reimbursed only after the completion of the study. The amount of the compensation was approved by the Institutional Review Board (Sterling IRB) and it was in line with the consumer research standards in the U.S. Finally, THS and HeatSticks were given for free, which is normal practice in consumer research, as stated by the ESOMAR code. We also note that other different features might have discouraged product use compared to a full real-world setting, e.g., lack of repeated communication, absence of a real customer care and presence of other adult users using the product. Having said that, we recognize that some of the points made by the Reviewer are the limitations of the study, which were inherent to the pre-market setting, and have been stated as such in the manuscript. The Reviewer recommended to further report the overall disposition of subjects. The aim of this analysis was to assess the potential predictors of adoption of the Tobacco Heating System (THS) by current U.S. adult smokers after six weeks of THS product use. The data from this last week of the observational period (i.e. week 6) was therefore appropriate. In terms of the overall disposition of subjects, at pre-screening, 8,858 subjects, spread over eight sites (ranging from 599 in Denver to 2,050 in Detroit), were contacted via telephone. Of these, 21% initially refused to continue the telephone conversation. A certain proportion of the remaining subjects did not fulfill pre-specified criteria. The most common of these unfulfilled criteria were as follows: (i) participation in a consumer or clinical research study in the past three months (32%), (ii) being a non-daily adult smoker (18%), (iii) not willing to participate in a consumer research study that could last up to eight weeks (10%), (iv) started smoking regularly within the past 30 days (10%), (v) and demographic criteria above quota (10%). After this pre-screening phase, subjects were invited to one of the eight sites. At the eight sites 1,368 subjects were screened in person. Of the 1,368 screened subjects, 1,336 participants were enrolled, while 32 participants were not enrolled because of violation of inclusion or exclusion criteria. The number of participants screened was overall similar for each of the eight sites (ranging from 162 in Oklahoma City to 179 in Miami). The Full Analysis Set (FAS) comprised 1,106 participants, of which 119 prematurely discontinued and 987 completed the study. Overall, 230 subjects enrolled were not included in the FAS because they did not self-report in the e-diary “at least one documented consumption of a cigarette during the one-week baseline period” or “at least one documented consumption of a HeatStick during the six-week observational period.” Moreover, no imputation of missing information was applied as part of the study. Those details can be found as part of the full study report, which is publicly available as part of Philip Morris Products S.A. Modified Risk Tobacco Product (MRTP) Applications and can be accessed through the following link: https://www.fda.gov/tobacco-products/advertising-and-promotion/philip-morris-products-sa-modified-risk-tobacco-product-mrtp-applications. The Reviewer formulated concerns about the analyses. The number of e-cigarette adult users is too small from our perspective to put into the logistic model (i.e. as a potential confounder) and the estimation of an effect would lead to wide Confidence Intervals (CIs). Regarding the effectiveness for Hispanics/Latinos, this is a true statement coming from the analysis. This means that this subgroup is no more likely than other subgroups to use THS; the reporting of the results was performed in line with the multivariate analysis, presented in Figure 2. The Reviewer outlined that wide CIs indicate that there are insufficient numbers for subgroup analysis. This is indeed correct as the planning of the sample size was not done for subgroup analysis. Considering the potential inclusion of another comparator such as e-cigarettes, this suggestion from the Reviewer is interesting and we believe it merits potential exploration in studies designed and executed with a different purpose than a Modified Risk Tobacco Product Application. We have noticed that the Reviewer has designed such a study to compare changes in cigarette consumption and adoption rates among smokers randomized to either IQOS or e-cigarettes: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962654/. Regarding information about complete switching from cigarettes (i.e. 100% THS use), indeed, this information was not intended to be part of the manuscript, however, it has been provided during the Tobacco Products Scientific Advisory Committee review of Philip Morris Products S.A. Modified Risk Tobacco Product (MRTP) Applications and can be accessed through the following link: https://www.fda.gov/media/110734/download. The percentage of 100% THS use (i.e. completely stopped using combustible cigarettes) was 6% at Week 6. Under the Variables sub-section of the Methods, we provide the definition of the combined use category. To the Reviewer’s comment about this category, we would like to outline that we have defined the product use categories with the objective of having a symmetrical classification, which allowed for practical and meaningful operationalized categorization, description and analysis. We have provided the raw data to allow scientists to create further categories. Regarding the version of the product provided for the study, the Products sub-section of the Methods has been edited to indicate that the investigation product as part of this actual use study was the Tobacco Heating System Version 2.2. Thus, the product used in this study was an earlier version of the currently commercialized THS product. Minor Comments In the Introduction, the Reviewer suggested to qualify our statement “These findings suggest that the large majority of IQOS users in Japan switched from cigarettes to IQOS and that there is minimal uptake from nonsmokers” with numbers/percentages (and references). The findings come from several studies that are cited earlier in the text including their respective references (Tabuchi, 2017 and Tabuchi, 2016). Therefore, stating one particular percentage does not seem adequate as the statement is based on the overall weight of evidence rather than on particular percentage(s). We have added the references again along the statement. We also clarified that the preceding citation (“Analysis of predictors of IQOS current use (use in the previous 30 days) in 2017…”) is linked to citation Tabuchi (2016) specifically. The Reviewer indicated that data related to the hotline and adverse events (AEs) should be provided in the manuscript. However, safety was not a primary or secondary objective of this study. AEs spontaneously reported by study participants were collected using a passive safety surveillance methodology. The safety population exposed to the product use included the 1,158 participants who used at least one HeatStick and their data were valid for safety analysis. Overall, 121 AEs (102 non-serious and 19 serious) were reported in 48 individual cases. The overall reporting rate was 4.14%. The most frequent reported AEs by preferred terms (PTs) were Headache (n=13), Malaise (n=7), Nausea (n=6), Dizziness (n=4), Abdominal discomfort (n=3), Oral discomfort (n=3), Chest pain (n=3), Cough (n=3), and Throat irritation (n=3). In total, the most frequent PTs represent 37.19% (45/121) of the total number of AEs reported during the study for all THS product variants. Headache was the most frequent reported AE in both product variants investigated in the study (reported in 13 out of 48 cases), with a reporting rate of 10.74% from all AEs received. Other frequent reported AEs were Malaise, Nausea and Dizziness (overall reporting rates from 3.30% to 5.78%). The severity of the leading event was reported as Mild in 5 cases (10.40%), Moderate in 7 cases (14.58%), Severe in 6 cases (12.50%), and Unknown in 6 cases (12.50%). The severity was not reported in half of the total number of cases (24 cases, 50.0%). Those details can be found as part of the full study report which is publicly available as part of Philip Morris Products S.A. Modified Risk Tobacco Product (MRTP) Applications and can be accessed through following link: https://www.fda.gov/tobacco-products/advertising-and-promotion/philip-morris-products-sa-modified-risk-tobacco-product-mrtp-applications. Regarding the study design and the chosen length of the observational period, the duration of six weeks was based on the assumption that participants would take between two and four weeks to either establish a regular pattern or to stop using HeatSticks altogether. The remaining weeks of the observational period would serve to confirm that HeatSticks usage patterns of participants have indeed stabilized. This study duration was also implemented by 22nd Century Group Inc. Modified Risk Tobacco Product (MRTP) Applications when conducting their actual use study pre-market. More information about 22nd Century Group Inc. Modified Risk Tobacco Product (MRTP) Applications can be accessed through the following link: https://www.fda.gov/tobacco-products/advertising-and-promotion/22nd-century-group-inc-modified-risk-tobacco-product-mrtp-applications. The Reviewer recommended to provide further details on the structure and validity of the questionnaires used, including which measure was implemented to capture participants’ intention to quit. The goal of our study was measure THS use patterns in current U.S. adult daily cigarette smokers and to assess THS product acceptance. Socio-demographics, smoking habits, sensory assessment, and ease of use were measured with ad-hoc questions. Consumption of cigarettes and THS were collected on self-reported stick-by-stick entry in an e-diary. To capture intention to quit smoking, participants were asked to complete the Prochaska Stages of Change measure. Using a dichotomous response scale (‘yes’, ‘no’), participants were first asked if they were seriously considering quitting smoking within the next six months. For those participants who indicated ‘yes’ for intention to quit smoking within the next six months, they were further asked if they were planning to quit smoking in the next 30 days. The Prochaska Stages of Change is based on the Prochaska and DiClemente stages of change concept, which is part of the Transtheoretical Model. The instrument assesses smokers’ mental state for intention to quit smoking and enables categorization of smokers into the following three stages: 1) precontemplation (not thinking of quitting smoking), 2) contemplation (thinking of quitting smoking within the next six months), and 3) preparation (thinking of quitting smoking within the next 30 days). Of note, the measure of intention to quit smoking within the next 30 days was applied for the corresponding exclusion criteria. This is because THS is aimed at current adult smokers who would otherwise continue to smoke combustible cigarettes. We also assessed intention to use THS as part of the inclusion criteria, however, measuring intention to switch to low-risk products in general was not performed. The full questionnaire (Case Report Form) can be found as part of the study report, which is publicly available as part of Philip Morris Products S.A. Modified Risk Tobacco Product (MRTP) Applications and can be accessed through the following link: https://www.fda.gov/tobacco-products/advertising-and-promotion/philip-morris-products-sa-modified-risk-tobacco-product-mrtp-applications. The Reviewer commented that the participants own cigarette brand could have been an important predictor for IQOS adoption. We did not collect the participants’ own cigarette brand as part of the survey, so we could not include this variable in the analysis. The Reviewer recommended to further discuss the results of the analyses for heavy smokers, i.e. the reduced adoption of THS among heavier smokers, and whether this may reflect high level of inefficiency of the product to adequately reproduce the experience in cigarette smoking. From our perspective, heavy adult smokers might simply need more time to adopt THS than current adult smokers who smoke fewer number of cigarettes per day." } ] }, { "id": "52579", "date": "11 Sep 2019", "name": "Fabrizio Messina", "expertise": [ "Reviewer Expertise I have several years’ experience in tobacco and nicotine products use behaviour. As part of my current role I look after all human studies from mouth level exposure", "puffing topography", "consumer risk perception and post market surveillance." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper reports the outcome from an ‘actual use’ study designed to determine if users are likely to use the product in a manner that reduces their individual health risks or exposures as compared to using other commercially marketed tobacco products. Actual use studies should allow consumers to interact freely with the product in real world conditions.\n\nThe study design, recruitment, methodology, data collection, analysis and results are well presented. However, some of the conclusions drawn are broad and statistical procedures used are unconventional, leading to interpretation based on variable selection criteria. Given the recruitment limitation of not having recruited non-tobacco users, the conclusions drawn should be limited to THS adoption among current smokers in the US.\n\nAbstract The objective of the study is stated twice in the background. Observational studies by their very nature observe individuals without manipulation or intervention. In this study the participants are asked to use the test products instead of their regular products, therefore, should be referred to as actual use study.\nThere is little if any data regarding the use of e-cigarettes, cigars etc. in the body of the paper. Number and type of participants and brief inclusion/exclusion criteria would make the methods section more informative.\nWhilst I understand that it is not for the paper to make policy implications, it would be useful to draw on the adoption of THS by vulnerable groups in conclusions.\n\nIntroduction Generally, the introduction is well presented with relevant references from the literature. Reference to actual use studies with similar tobacco heating system would enhance the articles and provide basis for comparison in discussion.\nDespite acknowledging that most actual use studies for cMRTP have been conducted in artificial setting and provided free, the products were provided free in this study.\n\nMethods The methods used are clearly presented with reference to sample size calculation, inclusion/exclusion criteria, products, data collection and analysis.\nRationale for some of the criteria for example, exclusion of individuals who had started smoking with in the last 30 days would have helped.\nSample Size Calculation Based on the reference in the paper (Dhand, N. K., & Khatkar, M. S. (2014)). Calculating the sample size n ≥ (1.96/0.05)^2 0.5 × 0.5 = 384.16 and allowing for an attrition rate of 40%, I arrive at 384.16(1/(1 - 0.4))= 641. In this paper they have recruited 1300 which is almost double as 641.\nPre-screening of the regressors I understand the practice of pre-screening the regressors to understand the relation between the dependent and the independent variables, however, excluding regressors based on an arbitrary rule (p-value <=0.2), it’s rather unconventional. According to “Five myths about variable selection” by Georg Heinze & Daniela Dunkler1: “While it is true that regression coefficients are often larger in univariable models than in multivariable ones, also the opposite may occur, if some variables (with all positive effects on the outcome) are negatively correlated. Moreover, univariable prefiltering, sometimes also referred to as “bivariable analysis,” does not add stability to the selection process as it is based on stochastic quantities and can lead to overlooking important adjustment variables needed for control in an etiologic model. Although univariable prefiltering is traceable and easy to do with standard software, one should better completely forget about it as it is neither a prerequisite nor providing any benefits when building multivariable models (Sung et al 1996)2.”\nIf the authors have not already run the logistic regression including all the regressors, it is worth re-running to see if we observe different potential predictors.\nThe study participants were asked to report the current use of NRT products – despite selecting smokers who had no intention to quit. What was reasons for them using NRT?\n\nThe main outcome measure was self-reported consumption of cigarettes and THS, does not take into account e-cig or NRT use - this would likely influence the number of THS sticks used and therefore inflate the ratio?\nResults\nIs the decrease in the number of tobacco products (Table 2) significant? Given the large SDs I suggest they are not different.\nDiscussion\nCan you say that THS is a viable alternative if 85% of users rejected the offer even when given the product for free?\n\n\"availability of THS is unlikely to prevent those willing to quit tobacco from doing so\". Can you really make this statement from the data provided? 'Previous quit attempts' is different from 'intention to quit'.\n\n\"almost 15% of U.S. daily adult smokers substituted cigarettes with THS\". While this is technically true, they didn't substitute completely which may be incorrectly inferred from this 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?\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": "6822", "date": "14 Jul 2021", "name": "Christelle Chrea", "role": "Author Response", "response": "Abstract The Abstract has been slightly edited according to the Reviewers’ comment on its structure, with no alteration to the message presented. (i.e. removed repetitious sentences, as well as addition, restructuration and move of sentences to a different part of the abstract for clarity, etc…). As suggested, the number of participants together with the description of the participants (i.e. current adult smokers) is now included in the Abstract. The full description of inclusion/exclusion criteria remains included under Participants subsection of the Methods. The Reviewers indicated that because the participants are asked to use the test products instead of their regular products, it should be referred to as an actual use study in the Abstract. We fully agree with this statement and, actually, the manuscript title \"Potential predictors of adoption of the Tobacco Heating System by U.S. adult smokers: An actual use study\" clearly reflects that it is an actual use study. The terminology of \"observational study\" is also being used to refer to the fact that, there was no intervention with the study population beyond providing the THS product. Moreover, study participants were not asked to use the THS product during the entire study periods (i.e. baseline and observational periods); they were only requested \"to make an entry into an e-diary every time they consumed a cigarette during the 1-week baseline period and a HeatStick or a cigarette during the 6-week observational period of the study. Participants were also requested to indicate on a daily basis using the e-diary whether or not they used other products containing nicotine (i.e. e-cigarettes, nicotine replacement therapy products, and other tobacco products such as cigars, cigarillos and smokeless tobacco products).\" The Reviewers suggested to further expand our conclusions and draw on the adoption of THS by vulnerable groups. THS is aimed at current adult smokers who would otherwise continue to smoke combustible cigarettes and represent the study population of this actual use study. Therefore, we believe that drawing any conclusion from this study regarding vulnerable groups is beyond the scope of this publication. The Reviewer noted that the data about the use of e-cigarettes, cigars and other tobacco products than cigarettes, is not extensively covered in our manuscript. This is because the Tobacco Heating System is aimed at current adult smokers who would otherwise continue to smoke cigarettes and hence the focus of this publication is on THS and cigarettes. Data about e-cigarette use has been captured during the rescreening phase (yes/no) and show that \"the proportion of participants adopting THS was higher among e-cigarette adult users (23.1% vs. 14.1%)\". Data about e-cigarette use, as well as nicotine replacement therapy products, and other tobacco products such as cigars, cigarillos, and smokeless tobacco products, has also been captured during the baseline and the observational periods. The data show that between the baseline and the end of the observational period, the usage of nicotine replacement therapy products remained stable, while the usage of e-cigarettes increased and the usage of other tobacco products such as cigars, cigarillos and smokeless tobacco products decreased. Introduction The Reviewers outlined that reference to actual use studies with a similar tobacco heating system (THS) would enhance the articles and provide basis for comparison in discussion. While we agree with this comment, we are not aware of any other premarket actual use study regarding another heated tobacco product (i.e. beyond the THS). Regarding the study settings, the studies referenced in Introduction which collected data on potential MRTPs were performed in an artificial setting because there was an intervention (i.e. participants being instructed to adopt a given behavior), which was not the case for our study. We slightly edited the related sentence to clarify that for these studies, the MRTP was also provided for free, as it was also the case for our pre-market study. This aspect of the study design has been stated as a study limitation in the Discussion section of the manuscript, which states, \"Limitations include the fact that due to the study having been conducted in a pre-market setting, the study participants did not pay for the THS products, while they continued to pay for their cigarettes, which may have overestimated the level of THS adoption in this study.\" Methods The Reviewer requested further insights regarding the rationale for some of the inclusion/exclusion criteria, in particular concerning the exclusion of individuals who had started smoking within the last 30 days. This exclusion criteria was applied to ensure that participants involved in the study have been smoking cigarettes for a certain period of time, and thus, have a relatively established product use pattern (of using cigarettes, in this particular case). The Reviewers provided a correct sample size calculation (i.e. 641 participants). Additionally to the number of 641, we assumed that we will have 50% \"starters\" (\"starters\" are those subjects who used 100 or more HeatSticks during the observational period) in the sample in addition to the 40% related to the attrition rate, which doubles the calculated number. Thus, we recruited 1300 participants. To the Reviewers comments on pre-screening of the regressors, we saw that different authors have favored different variable selection strategies. We have chosen this procedure to have sufficient degree of freedoms to estimate the effects. It seems unlikely that the variables in our dataset with p>0.2 in the bivariate analysis will have an additional influence. The Reviewers formulated questions regarding reasons for participants using NRT and whether number of HeatSticks used could have been influenced by use of e-cigarettes and NRT. The main data for this manuscript was the number of cigarettes and the number of HeatSticks used by study participants over a 6-week period. Information related to the use of e-cigarettes, cigars etc. can be found as part of the study report, which is publicly available as part of Philip Morris Products S.A. Modified Risk Tobacco Product (MRTP) Applications and can be accessed through the following link: https://www.fda.gov/tobacco-products/advertising-and-promotion/philip-morris-products-sa-modified-risk-tobacco-product-mrtp-applications. The percentage of NRT use was 0.1% at rescreening, which indicates that it is highly unlikely to have any impact on the number of HeatSticks used. The percentage of e-cigarette use was 5.3% at rescreening so relatively small, and hence, unlikely to have any significant impact on the number of HeatSticks used. Results The Reviewers commented on the potential significance of the decrease in the number of tobacco products presented in Table 2. Actually, the results are reported in means without any statistical test. Discussion The Reviewers asked whether stating that THS is a viable alternative was appropriate with 85% of users rejecting the offer even when given the product for free. We believe that THS is a viable alternative as about 15% of current adult smokers’ participants have replaced 70% or more of their tobacco consumption (cigarettes + THS) by THS. Further analysis has shown that about 6% (n = 968) of adult smokers’ participants completely stopped smoking combustible cigarette (i.e. THS constituted 100% of their tobacco consumption). As per the Reviewers comment regarding previous quit attempts and intention to quit, the manuscript has been amended to address your comment as follows: the old text \"Participants who had previously attempted to quit smoking were less likely to adopt THS than participants who never attempted to quit smoking in the past. This finding suggests that the availability of THS is unlikely to prevent those willing to quit tobacco from doing so. This is further confirmed by the fact that the intention to quit smoking within the next six months was not associated with THS adoption,\" was replaced with “Participants who never attempted to quit smoking in the past were more likely to adopt THS than participants who had previously attempted to quit smoking. Intention to quit smoking within the next six months was not associated with THS adoption suggesting that the availability of THS is unlikely to interfere with intention to quit smoking.\" When it comes to cigarettes substituted with THS for study participants, the manuscript has been amended as follows: the old text \"Almost 15% of U.S. daily adult smokers substituted cigarettes with THS\" was replaced with \"almost 15% of U.S. daily adult smokers replaced 70% or more of their tobacco consumption with THS\"." } ] } ]	1	https://f1000research.com/articles/8-214
https://f1000research.com/articles/10-566/v1	14 Jul 21	{ "type": "Research Article", "title": "Perception and awareness of COVID-19 among health science students and staff of Kuwait University: An online cross-sectional study", "authors": [ "Walid Alali", "Wadha AlFouzan", "Dhuha Alajmi", "Haya Al-Tawalah", "Khalid Kheirallah", "Getnet Yimer", "Wadha AlFouzan", "Dhuha Alajmi", "Haya Al-Tawalah", "Khalid Kheirallah", "Getnet Yimer" ], "abstract": "Background: Coronavirus disease 2019 (COVID-19) pandemic is unprecedented. Health science students are the future frontliners to fight pandemics. Awareness and perception toward COVID-19 among health science students and staff at Kuwait University was assessed. Methods: Between June and July 2020, an online questionnaire was distributed to all students and staff at HCS. The questionnaire was divided into six sections: socio-demography, risk and awareness, preparedness and prevention, source of information, policies, and social stigma. Results: A total of 592 students and 162 staff completed the questionnaire. The prevalence of self-reported chronic condition among students and staff was 14.0% and 19.1%, respectively. Moreover, self-reported COVID-19 prevalence among students and staff was 2.7% and 1.2%, respectively. Interestingly, 54% of students and 38.3% of staff reported that they knew someone within their immediate social environment who have been/are infected with SARS-CoV-2. Among students, 92.4% wore face mask in indoor places (outside of their home) ‘often/all the time’ compared to wearing it outdoors (69.3%); whereas, for staff, it was more common to wear it outdoor than in indoor places (75.9% vs. 81.5%). Willingness to take COVID-19 vaccine was indicated by 50% of students ‘strongly agreed’ and an additional 25.8% agreed to taking it. Interest vaccine uptake was lower among staff (28.4% and 34.6% strongly agreed or agreed, respectively). Participants strongly agreed or agreed (72.5% and 19.6% of students as well as 68.5% and 22.2% of staff) that wearing face mask in public should be obligatory. More than 18% of students and staff indicated that they would avoid contact with COVID-19 infected people. Conclusions: Responses of students and staff were mostly similar and showed that they follow precautionary measures to control spread of COVID-19, understand the viral transmission risk, and willing to raise awareness to reduce social stigma.", "keywords": [ "COVID-19", "SARS-CoV-2", "awareness", "Middle East", "Kuwait" ], "content": "Introduction\n\nThe coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a global public health threat.1 While COVID-19 vaccines have been available since November 2020, there is currently a global shortage which may prolong the pandemic.\n\nThe global health crisis of COVID-19 has grabbed the attention of the mass media around the world.2 Most of the public health messages have been focused on the control of transmission risk. Many of these messages are related to social distancing, frequent and proper hand washing, stay at home, and avoid crowded places. However, the pandemic of information has caused a great deal of anxiety, fear, excessive worry, and confusion among people worldwide. Additionally, the rapidly evolving and changing knowledge about the disease have created a cloud of misinformation and miscommunication among people in different communities.3–5\n\nKuwait went through a period of partial and full lockdowns between May and August 2020 following the large increase in cases and deaths in March and April 2020.6 School closure (including universities) was one of the first restrictions applied at the beginning of the epidemic (March 2020) in Kuwait and continued during the study period. Many countries worldwide have gone through similar school closure and continue to have it in effect.7\n\nThe COVID-19 pandemic is unprecedented, and no other disease has dominated the human population for the past 100 years. Human behavior is partially driven by the knowledge and perception gained through exposure to information from various sources such as formal education, media, family, friends and colleagues.8 In Kuwait, people receive information about COVID-19 from different sources including the Ministry of Health and official state TV programs, World health organization, expert and non-expert opinions on social media, as well as friends and colleagues. Therefore, this study was focused on assessing perception and awareness on matters related to transmission, control and prevention of COVID-19; sources of information; and social stigma. We believe the study is important for two main reasons: 1) health education and raising awareness about the disease among university students and staff as well as the larger community, and 2) findings from this survey can aid the policy makers in their decision making to control the epidemic in Kuwait and apply social policies in relation to this population.\n\nThere are number of published studies that assessed the epidemiology, pathogenesis, virology, and treatment of COVID-19. However, there are limited studies assessing the COVID-19 perception, attitude, and awareness in the academic community.9 The academic community is an important segment of the society and represent students and staff in higher education institutions. Students in the health science fields (e.g., medicine, dentistry, pharmacy and public health) will be the next generation of health leaders; hence, assessing their perception in relation to the current pandemic is expected to yield useful outcomes.\n\nKuwait University is the state national university with a student population of about 35,000 students. The Health Science Center (HSC) of Kuwait University is composed of five faculties including Medicine, Dentistry, Pharmacy, Public Health, and Allied Health. They represent a more uniformed population compared to the rest of the university’s population. Furthermore, many of the HSC staff and students have been involved with COVID-19 related activities including volunteering in health facilities, raising awareness about the disease and pursue research opportunities. The goal of this study was to describe the awareness and perception on matters related to control and prevention of COVID-19, source of information and social stigma among students and staff of HSC at Kuwait University.\n\n\nMethods\n\nThis manuscript is reported according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) protocol.10\n\nA cross-sectional survey study was conducted between June and July 2020 on students and staff at HCS of Kuwait University. Our target populations were: all undergraduate and postgraduate students enrolled in the five faculties (Medicine, Dentistry, Pharmacy, Public Health and Allied Health) of HSC and all the staff (faculty members, teaching assistants, and supporting staff) employed by HSC.\n\nData collection was conducted via online questionnaire distributed to students and staff. During the study period, the university was closed, and no classes were held on campus nor through distance learning.\n\nThe questionnaire was developed based on validated survey tools provided by World Health Organization11 and that used by Khasawneh et al.12 The WHO survey tool provides general guidelines and questionnaire standard tool to conduct behavioral insights studies related to COVID-19. The survey by Khasawneh et al. is specific for medical students’ knowledge and attitude in relation to COVID-19. Their survey tool included sections on socio-demographics, sources of information, knowledge, and attitudes regarding COVID-19, and precautionary measures. Nonetheless, the questionnaire used in this study was adapted to our target population. Non-relevant or redundant questions were excluded from both survey tools and those that met the objectives of this study were included. Some of the questions taken from the previous survey tools were modified such as changes in the number of multiple-choice answers per question as well as rewording the questions to improve clarity.\n\nThe questionnaire was divided into six sections: socio-demography, risk and awareness, preparedness and prevention, source of information, policies, and social stigma. Socio-demography questions included questions about gender, academic year, faculty, and presence of chronic illness. Additional questions for staff included job classification and age. Risk and awareness section included questions about history of COVID-19 infection, risk of infection in the future and infection severity, awareness of consequences of infection as well as awareness of availability of treatment or vaccine for COVID-19. Preparedness and prevention section included questions about probability of avoiding infection with SARS-CoV-2, prevention measures taken to avoid infection measures as frequency based on the participants response to one of the following options: most times, rarely or sometimes, and never. Additional question on participants’ opinion on attending classes or working on-campus vs. online learning methods. The source of information section had questions about main sources of acquiring information. These included television news stations, family and friends, healthcare workers, online search, social media, official government press releases, and celebrities. Policies section included questions about COVID-19 vaccine timeline and willingness to uptake the vaccine and treatment. Furthermore, opinion of participants regarding the following: mandatory quarantine of COVID-19 contacts, wearing face mask in public places, legal punishment of who is COVID-19 infected and knowingly socialize with others and those spread share/spread falsified information about COVID-19. Social stigma section included questions about: participants’ reaction to knowing someone COVID-19 positive, blaming the person of who got the infection, what information the participants would like to know about those infected, community reaction to those infected (such as being socially rejected, denied medical care, job loss), and ways to reduce social stigma.\n\nTwo questionnaires were developed; one administered to students and another to the staff. Both questionnaires were very similar with only differences in the first section (i.e., socio-demography). The questionnaires were provided in English since the students and staff are familiar with this language.\n\nThe questionnaires were distributed and administered to the participants online via Google Forms. The questionnaires were sent via an email to all students and staff within HSC through Listserv. Additionally, student representatives at each faculty distributed the questionnaire to class groups via WhatsApp. Each of the six sections was composed of several questions (the copy of the questionnaires can be found in the Extended data). The questionnaires were validated and pilot tested on 10 students and 10 staff prior to administration to all participants. Based on the pilot testing, the survey tool was improved for grammatic errors and sentence structure to provide more clarity. Multiple reminders were sent to all participants to increase response rate.\n\nThe study has two main potential biases that are also common in cross-sectional survey studies. First, selection bias due to non-probability sampling method used in the study (i.e., convenience sampling) that could lead to non-representative sample of the target populations. We used this type of sampling since no sampling frame (a list of the students and staff) was available to conduct a probability-based sampling method. Second, a recall bias could occur if the certain participants can recall certain information because of their previous exposure to COVID-19 and differentially than those who were not exposed. Our data stratification shows those who have been infected or were exposed due to interaction with COVID-19 positive cases.\n\nData were exported from Google Forms to an Excel spreadsheet for data management. The analyses were conducted in STATA statistical software ver. 15.1 (College Station, Texas). A descriptive analysis using cross tabulation of frequency and percentage of variables was conducted. Students were categorized into two groups: younger students (freshmen and junior student [years 1-3]) and older students (senior [years 4-7] and graduate students) for the purpose of the univariate (chi-square) analysis in STATA. Alpha of 0.05 was used as the significance level.\n\nAll participants were provided with informed consent statement before starting the questionnaire. Upon agreement, they ticked the ‘I consent’ box and proceeded with the questionnaire. Agreeing to informed consent prior to starting the questionnaire was mandatory. Moreover, the participants were informed that they have the option to stop answering the questions through the process of completing the questionnaire. The research study was approved by the Ethical Committee of the Health Science Center at Kuwait University (2020/0622). Confidentiality of information was also respected by not asking names and the data was stored with limited access.\n\n\nResults\n\nAt the time of the study, there was approximately 2,000 eligible undergraduate students and about 110 graduate students (Master and PhD programs). In addition, there was approximately 450 eligible staff employed by HSC at the time of the study. All of the eligible participants were invited and reminded (as described the methods) to participate in the study. There were 592 students and 162 staff completed the questionnaire with a response rate of 28.1% and 36%, respectively. We did not collect information on reasons for non-participation.\n\nThe demographics of students (n = 592) and staff (n = 162) are shown in Table 1. Most the participants were females (for both students and staff). Female students account for 75% to 90% of the student population depending on the academic year and the faculty. Participants were proportionally distributed to the population of their faculties with the largest faculties being Medicine and Allied Health. Only Medicine and Dentistry faculties have a seven-year academic program; whereas Pharmacy is five-year program and both Allied Health and Public Health are four-year programs. While the undergraduate students’ age ranged between 19 and 26 years; graduate students’ age was between 25 and 45 years. About 50% of the staff participants were faculty members. Most of the staff were older than 30 years with 24% of them over the age of 50 years. Those who participated in the study completed the full questionnaire tool before submitting their responses. Therefore, we did not have missing responses.\n\nOn chronic condition reporting, the prevalence of self-reported having any chronic condition among students and staff was 14.0% and 19.1%, respectively. The highest chronic condition among students was asthma (42.2%) and among the staff was hypertension (36.6%).\n\nThe participants answered 10 questions related to SARS-CoV-2 risk of infection, risk of transmission and their information awareness about the virus. Based on participants’ self-reported SARS-CoV-2 infection (based on PCR confirmation test), the prevalence among students and staff was 2.7% and 1.2%, respectively. However, 85.0% and 86.4% of students and staff, respectively, reported that they have not been infected with the virus. Interestingly, 54.0% of students and 38.3% of the staff reported that they knew someone in their immediate social environment, such as relatives or friends, who have been/are infected with SARS-CoV-2.\n\nWhen we asked the participants about their probability of getting infected with the virus in the near future, 3.9% of students responded as it was extremely unlikely; 23.3% indicated it is unlikely, but 5.5% responded it was likely. As for the staff, 5.6%, 21%, and 20.4% considered the probability to be extremely unlikely, unlikely, and likely, respectively. There was no significant (P = 0.374) relationship between the infection probability among staff participants and their age groups. Many of the students expressed that if they get infected with the virus, the disease will ‘not be severe’ (27.7%) or ‘slightly severe’, (24.6%). Similarly, many of the staff (20.4% and 21.6%) thought that if they get infected with the virus, the disease will ‘not be severe’ or slightly severe’, respectively.\n\nThe participant’s responses to questions regarding awareness related to proportion of COVID-19 patients that are likely to be admitted to hospital; if admitted to the hospital are likely to be admitted to the intensive care unit (ICU); and if admitted to ICU will eventually die is shown in Figure 1. Most of the participants either strongly disagreed or disagreed with the following statements: 1) majority of COVID-19 patients need hospitalization; 2) those hospitalized require ICU admission; and 3) those admitted to ICU will eventually die (Figure 1). Older students were significantly (P < 0.001) more likely to strongly disagree or disagree than younger students with the first two statements. No significant difference (P = 0.576) was detected between both student groups to the third statement. The faculty members were significantly more likely (P < 0.001) to strongly disagree or disagree compared to teaching assistant and non-academic staff. No significant difference (P = 0.583) detected between staff job groups by the response to those admitted to ICU will eventually die.\n\n* Infected to hospital admission is based on the question: Do you agree or disagree that majority of SARS-CoV-2 infected people need admission to a hospital; Hospital to admission to ICU is based on the question: Do you agree or disagree that majority of SARS-CoV-2 patients who are admitted to the hospital require receiving care in the intensive care unit (ICU); and ICU to death is based on the question: Do you agree or disagree that majority of SARS-CoV-2 patients who are admitted to the ICU will eventually die. A: students and B: staff.\n\nWe asked the participants about their reaction to suspicion of being infected; nearly 52% of the students and 50% of staff indicated that they would immediately get tested for the virus and follow government isolation instructions (if tested positive); whereas, 28.2% and 36.4% of students and staff, respectively, would stay at home and taking precautionary measures so they do not spread the virus to others, and 19.6% and 13% of students and staff, respectively, would wait for few days until symptoms worsen before they get tested and/or seek medical care. On the topic of treatment or vaccine for COVID-19, most of the participants (80.6% of students and 85.8% of staff) indicated that, at the time of this study, there is no treatment or vaccine.\n\nOn the question of self-rate perception on how to prevent SARS-CoV-2 spread, most students rated themselves as having either very good knowledge (30.4%) or good knowledge (45.7%) with significantly (P < 0.001) higher proportion among older students than younger students. On the same question, 43.8% and 37.7% of staff rated themselves as having very good knowledge and good knowledge, respectively, with significantly (P = 0.02) higher proportion among faculty members compared to other groups. In term of misconceptions, students either strongly disagreed (19.4%) or disagreed (21.8%) that SARS-CoV-2 was made in a laboratory then accidentally released. Similarly, 14.2% and 17.3% of staff either strongly disagrees or disagreed, respectively. Moreover, while most of students (30.2% strongly disagreed or 40.6% disagreed) that hot summer slowdown/stop the transmission of the virus, significantly (P < 0.001) fewer staff strongly disagreed and disagreed (14.2% and 17.3%, respectively) with that statement.\n\nThe participants answered 15 questions about precautionary measures for SARS-CoV-2 infection (Table 2). Among students, 92.4% wore face mask in indoor places (outside of their home) ‘often/all the time’ compared to wearing it outdoors (69.3%); whereas, for staff, it was more common to wear it outdoor than in indoor places (75.9% vs. 81.5%). Regular hand washing was more common than using disinfectants among both participant’s groups (Table 2). Avoiding crowded places, avoiding shaking hands, avoiding kissing, staying home as much as possible and following social distancing guidance was practiced ‘often/all the time’ by more than 80% of the participants. There was no significant difference (P > 0.05) between staff than students with regard to these percentages. Nearly 50% of students did often clean/disinfect their phone, avoided eating outside of home, got sufficient sleep, and persuade people around them to follow precautionary guidance. This percentage was between 60–70% among staff (Table 2). Additionally, we asked the participants a question on how difficult/easy it is to avoid infection with the virus, 27.8% of students indicated that it is easy to avoid infection while 21.1% thought it would be difficult. Similarly, 30.2% of staff thought it would be easy to avoid infection while 20.4% of them thought it would be difficult.\n\nWe assessed the level of trust in COVID-19 information sources among the participants using 5 Likert scale (very little trust, little trust, neutral, moderate, and great deal of trust) against 8 sources of information (Table 3). The greatest deal of trust was in official government press release such as that from the local Ministry of Health (58.8% and 53.7% among student and staff participants, respectively), and consultation with healthcare workers (49.7% and 38.9% among student and staff participants, respectively). Moderate trust was TV stations and online sources (Table 3). Interestingly, there was both very little and little trust (ranging between 60% and 80%) in sources of information from celebrities, social media (such as Twitter, Facebook, and WhatsApp), and conversations with family and friends (Table 3).\n\nSmall percent of students indicated that they update themselves about COVID-19 multiple times a day (8.4%); whereas, most of students do that either once a day (34.4%) or once a week (40.3%). In comparison, 25.9% of staff update themselves multiple times a day; whereas 43.8% and 25.3% do that once a day and once a week, respectively.\n\nWe listed number of questions related to formulation of policies and regulations about COVID-19. Most of the participants (62.6% and 83.3% of students and staff, respectively) follow ‘very much so’ the recommendations issued by the authorities to prevent the spread of SARS-CoV-2. On questions related to taking COVID-19 vaccination if becomes available, 50% of students strongly agreed and an additional 25.8% agreed to taking it. Interest in vaccine uptake was lower among the staff (28.4% and 34.6% strongly agreed or agreed, respectively).\n\nParticipants strongly agreed or agreed (72.5% and 19.6% of students as well as 68.5% and 22.2% of staff) that wearing face mask in public should be obligatory. On the role of government, 67.4% of students strongly agreed and 23.1% agreed on enforcement of quarantine of contacts of COVID-19 cases (compared to 45.7% and 38.2%, respectively among staff). Moreover, 81.4% strongly agreed and 14.4% agreed that there should be a legal punishment for those who know they are infected but continue to socialize with others without wearing protective equipment; whereas, lower percentage of staff strongly agreed or agreed (69.1% and 22.8%, respectively). Furthermore, 55.4% strongly agreed or agreed (i.e., 27.4%) that legal punishment should be taken against those who share/spread falsified information about COVID-19 without verifying the source (compared to 46.3% and 33.3% among staff).\n\nOn COVID-19 testing policies, 45.1% and 29.6% strongly agreed or agreed that more COVID-19 testing should be carried out, which is similar to the responses by the staff (40.1% and 30.9%, respectively). Among students, 32.5% and 41.0% strongly agreed or agreed that people should have more information about COVID-19 cases in Kuwait since what is being currently reported has minimal information (i.e., number of new cases, number active and recovered cases, number of PCR tests, number of cases in ICU, and number of deaths). These figures were very similar to the responses by the staff (i.e., 32.8% and 37.7% strongly agreed or agreed). Figure 2 shows the responses about what type of information the participants would like to know about SARS-CoV-2 infectives.\n\nA: students and B: staff.\n\nWe asked the participants about their opinion about resuming the university classes on-campus (in person) at the start of the semester in August 2020. Approximately 43% of students and 46% of staff indicated that they are very worried. In addition, 35% of students and 20% of staff indicated that they are slightly worried. Regarding the option of resuming teaching online/distance learning, 75.4% of students and 84% of staff agreed with this option.\n\nWe asked questions to assess social stigma related to SARS-CoV-2 infection. Interestingly, in response to the question “If anyone you know personally got infected with SARS-Cov-2, what would be your reaction?”, approximately 68% of student and staff participants responded that they would advise the infected to seek medical care immediately; whereas about 29% of both participant groups indicated they would advise to wait for the symptoms to appear before seeking medical care. Interestingly, on the question “Do you think COVID-19 patients did something wrong and consequently got infected”, 12.8% and 18.9% of students strongly disagreed or disagreed; however, 51.8% of them were neutral about it. For the same question, higher proportion of staff 35.2% and 27.8% strongly disagreed or disagreed, compared to 27.2% were neutral about it.\n\nOn the question “If you knew a person (e.g., a relative or a friend) has COVID-19, what would you do” with multiple answers permitted, most of the responses by students and staff indicated that they will avoid contact with infected person (83.3% and 80.9%) but understand his/her condition (81.1% and 77.2%). Only 1% or less indicated they would blame the person for getting infected with the virus, but 29% would tell others about the infected person. Moreover, around 20% of students and staff thought that infection with COVID-19 would lead to social rejection; however nearly 75% of students and staff thought COVID-19 would not affect the person’s medical care coverage, job security, finding a job, or getting married. Finally, participants (82.8% of students and 84.6% of staff) indicated that raising awareness about COVID-19 disease will reduce the unjustified stigma toward COVID-19 patients.\n\n\nDiscussion\n\nOur study focused on COVID-19 awareness and attitude among Kuwait University Health Science students and staff. The university was impacted by the country control measures that included complete closure of its facilities between March and August 2020, followed by partial reopening for staff but continues to offer distance learning for students. Many countries worldwide have gone through school closure and some continue to have it in effect.7 Our findings indicated that students and staff of HSC in Kuwait University followed precautionary measures to control spread of COVID-19, understood the risk of the virus transmission, sought reliable source of information about COVID-19, and were willing to help those infected with the virus.\n\nCOVID-19 epidemic in Kuwait started on February 28, 2020 with imported cases from Iran followed by widespread community transmission that started around March 30, 2020 and still in effect. We revealed that a small percent (1-3%) of the participants reported that have been/currently infected with SARS-CoV-2; however, a larger percent of the participants (>50%) reported knowing someone infected within their immediate social environment indicating a large community transmission in Kuwait. During the study period (May to June 2020), COVID-19 incidence per 100,000 people ranged between 20–25 daily new cases with positivity rate between 15–20%.6 Interestingly, self-reported chronic condition among student was high especially asthma (an underlying condition for COVID-19 severe complications). One study reported that the prevalence of asthma among students (age 18 – 24 years) at Kuwait University was 11.9% (n = 1135).13\n\nMost of students and staff indicated that they believe that probability of getting infected was low and if they got infected, they likely will not develop severe disease. While this finding was anticipated from young adults (i.e., students), it is less expected from staff especially among those older than 55 years of age.14 Most of students and staff disagreed that when someone is infected with the virus will likely get admitted to hospital, and if admitted will likely need ICU bed, and then likely to die. This indicates our participants awareness of COVID-19 complication and disease progression among those infected that is similar to what have been reported in the literature.15,16 More than 50% of the participants expressed that they will immediately seek medical care if they tested positive for COVID-19 and follow the governmental instructions. This finding is different than what the international health organizations such as Centers for Disease Control and Prevention (CDC) recommend.17,18 For instance, CDC recommend to stay home even if you test positive, isolate yourself from others, and seek medical care immediately when you have emergency warning signs such as trouble breathing and/or persistent pain or pressure in the chest.17 Therefore, health education is needed to promote the recommended measures for those who test positive or negative for SARS-CoV-2 or come in contact with a positive case.\n\nParticipants followed most of the recommended precautionary guidelines by the World Health Organization (WHO).18 Mask wearing, handwashing, social distancing was common among the participants. Since they represent a particular segment of the community, this population is more likely to adhere to guidelines compare to the general public. Similar findings have been observed among health science and medical students in other countries.12,19,20 Some of the participants indicated their own infection probability is high while others indicated as low. The risk of infection and risk of transmission is multi-factorial depending on host factors, factors related to the virus, and the environment that connects the virus and the host together.21,22\n\nTrust in sources of information among participants was mostly in official government sources and information obtained from healthcare workers. Less trust was in information obtained from social media or family and friends. This may reflect the education level of participants and their initiative in seeking information regularly as well as due to the regular dissemination of information by the government communication channel. Similar findings were reported in a study from United States where most of general public participants trusted governmental source (CDC and Food and Drug Administration (FDA)) compared to private TV networks and social media.23 Other studies showed similar trend among health science students in Turkey and Iran24,25; whereas, another study from Jordan reported more trust in social media sources among medical students compared to Ministry of Health and international organization such as WHO.26\n\nThe majority of participants indicated that they will take COVID-19 vaccine if becomes available. This is important findings in preparation of vaccination planning and distribution to reach the local herd immunity threshold. To prevent spread of the virus, vast majority of participants indicated that mask wearing should be mandatory with legal punishment for those who do not wear it in public. At the time of this study, there was no state law that fine people for not wearing mask in public. On the other hand, participants indicated that spreading false information or people who know that they are infected and still socialize with others should receive legal punishment. This suggest that participants are aware of the danger of misinformation and irresponsible behavior in spreading the disease.\n\nSocial stigma has been reported to have impact on people response to disease control and prevention measures.27–30 Many participants were not sure if the reason for people getting infected with COVID-19 was due to something they did wrong. Furthermore, participants indicated that they will not blame the person for getting infected. However, participants would avoid contact with infected person, but try to help them which is an approach needed to control disease transmission in the community. Social rejection of patients was a concern shared by the participants. Participants agreed that raising awareness about COVID-19 will lead to reduce unjustified social stigma toward patients as indicated in other studies.28,29\n\nThis study has several limitations. First, it was based on a convenient sample of students and staff who filled the survey online. The online platform was used due to the pandemic mitigation strategies implemented in Kuwait as well as worldwide. Second, male student participation was low. This limits the generatability of the results to all students. However, the female student population represents about three-fourth of the total student body at HSC. Third, the study assessed the self-reported perception, attitude and awareness. However, this has been the trend among several similar studies in the literature.\n\n\nConclusions\n\nThis study presented a baseline estimate of COVID-19 perception and awareness among health science students and staff in Kuwait. It is among the first developed in this special population and provides evidence that more work is needed to raise knowledge and awareness among students in the health science fields. While the span of this epidemic is not clear, health sciences students and staff may find themselves at the frontlines dealing with patients, especially in the winter season. Universities with similar settings can focus on training their health science students investing in raising knowledge and develop training models for students and staff is a critical and should focus on how to deal with COVID-19 infected people, risk and benefit of treatment and vaccine, and follow precautionary measures recommended by reliable sources of information. Without properly equipped human capital and population commitment, the globe will be at higher risk to consequences of COVID-19 epidemic.\n\n\nData availability\n\nOSF: COVID19 students. https://doi/org/10.17605/OSF.IO/SQXEB.31\n\nThis project contains the following underlying data.\n\n- Dataset of staff questionnaire\n\n- Dataset of student questionnaire\n\nOSF: COVID19 students. https://doi/org/10.17605/OSF.IO/SQXEB.31\n\nThis project contains the following extended data.\n\n- A copy of staff questionnaire\n\n- A copy of student questionnaire\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank all of the participants who consented willingly and enrolled in the study voluntarily. The authors would like to state that this manuscript was published as a pre-print and available at https://www.medrxiv.org/content/10.1101/2020.12.26.20248891v1.\n\n\nReferences\n\nSohrabi C, Alsafi Z, O’Neill N, et al.: World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19). Int J Surg. 2020 2020/04/01/; 76: 71–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoy D, Tripathy S, Kar SK, et al.: Study of knowledge, attitude, anxiety & perceived mental healthcare need in Indian population during COVID-19 pandemic. Asian J Psychiatr. 2020; 51: 102083-. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhagavathula AS, Aldhaleei WA, Rahmani J, et al.: Novel Coronavirus (COVID-19) Knowledge and Perceptions: A Survey of Healthcare Workers. medRxiv. 2020: 2020.03.09.20033381. Publisher Full Text\n\nGeldsetzer P: Knowledge and Perceptions of COVID-19 Among the General Public in the United States and the United Kingdom: A Cross-sectional Online Survey. Ann Intern Med. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhader Y, Al Nsour M, Al-Batayneh OB, et al.: Dentists' Awareness, Perception, and Attitude Regarding COVID-19 and Infection Control: Cross-Sectional Study Among Jordanian Dentists. JMIR Public Health Surveill. 2020; 6(2): e18798-e. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Health-Kuwait: COVID-19 Update.2020. Accessed 10 July 2020. Reference Source\n\nUNESCO: United Nations Educational, Scientific and Cultural Organization. Global monitoring of school closures. 2020. Accessed 3 October 2020. Reference Source\n\nKelly MP, Barker M: Why is changing health-related behaviour so difficult? Public Health. 2016 2016/07/01/; 136: 109–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeldsetzer P: Use of Rapid Online Surveys to Assess People's Perceptions During Infectious Disease Outbreaks: A Cross-sectional Survey on COVID-19. J Med Internet Res. 2020 Apr 2; 22(4): e18790. Epub 2020/04/03. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Elm E, Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: Guidelines for reporting observational studies. Int J Surg. 2014 2014/12/01/; 12(12): 1495–9. PubMed Abstract \| Publisher Full Text\n\nWHO. World Health Organization: Regional Office for Europe. Survey tool and guidance: behavioural insights on COVID-19, 17 April 2020.2020. Accessed 6 June 2020. Reference Source\n\nKhasawneh AI, Humeidan AA, Alsulaiman JW, et al.: Medical Students and COVID-19: Knowledge, Attitudes, and Precautionary Measures. A Descriptive Study From Jordan. Front Public Health. 2020 2020-May-29; 8(253). English. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZiyab AH: Prevalence and Risk Factors of Asthma, Rhinitis, and Eczema and Their Multimorbidity among Young Adults in Kuwait: A Cross-Sectional Study. Biomed Res Int. 2017 2017/08/30; 2017: 2184193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMikami T, Miyashita H, Yamada T, et al.: Risk Factors for Mortality in Patients with COVID-19 in New York City. J Gen Intern Med. 2020 2020/06/30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbate SM, Ahmed Ali S, Mantfardo B, et al.: Rate of Intensive Care Unit admission and outcomes among patients with coronavirus: A systematic review and Meta-analysis. PLOS ONE. 2020;15(7): e0235653. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim L, Garg S, O’Halloran A, et al.: Risk Factors for Intensive Care Unit Admission and In-hospital Mortality Among Hospitalized Adults Identified through the US Coronavirus Disease 2019 (COVID-19)-Associated Hospitalization Surveillance Network (COVID-NET). Clin Infect Dis. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCDC: What to Do If You Are Sick.2020. Reference SourceAccessed 11 October 2020.\n\nWHO: Coronavirus disease (COVID-19) advice for the public.2020. Reference SourceAccessed on 11 October 2020.\n\nAtaş O, Talo Yildirim T: Evaluation of knowledge, attitudes, and clinical education of dental students about COVID-19 pandemic. PeerJ. 2020; 8: e9575-e. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGallè F, Sabella EA, Da Molin G, et al.: Understanding Knowledge and Behaviors Related to CoViD–19 Epidemic in Italian Undergraduate Students: The EPICO Study. Int J Environ Res Public Health. 2020; 17(10): 3481. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhatia R, Klausner J: Estimation of Individual Probabilities of COVID-19 Infection, Hospitalization, and Death From A County-level Contact of Unknown infection Status. medRxiv. 2020: 2020.06.06.20124446. Publisher Full Text\n\nTsui BCH, Deng A, Pan S: COVID-19: Epidemiological Factors During Aerosol-Generating Medical Procedures. Anesth Analg. 2020; 131(3): e175-e8. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFridman I, LuFridmancas N, Henke D, et al.: Association Between Public Knowledge About COVID-19, Trust in Information Sources, and Adherence to Social Distancing: Cross-Sectional Survey. JMIR Public Health Surveill. 2020 2020/9/15; 6(3): e22060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSögüt S, Dolu İ, Cangöl E: The relationship between COVID-19 knowledge levels and anxiety states of midwifery students during the outbreak: A cross-sectional web-based survey. Perspect Psychiatr Care. 2020: 10.1111/ppc.12555. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaghrir MH, Borazjani R, Shiraly R: COVID-19 and Iranian Medical Students; A Survey on Their Related-Knowledge, Preventive Behaviors and Risk Perception. Arch Iran Med. March. 2020 2020/4/1; 23(4): 249–54. PubMed Abstract \| Publisher Full Text\n\nAlzoubi H, Alnawaiseh N, Al-Mnayyis A, et al.: COVID-19 - Knowledge, Attitude and Practice among Medical and Non-Medical University Students in Jordan. J Pure Appl Microbiol. 2020 03/31; 14: 17–24. Publisher Full Text\n\nMahmud A, Islam MR: Social Stigma as a Barrier to Covid-19 Responses to Community Well-Being in Bangladesh. Int Journal of Com WB. 2020: 1–7. eng. Publisher Full Text \| Free Full Text\n\nSotgiu G, Dobler CC: Social stigma in the time of Coronavirus. Euro Res J. 2020: 2002461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdelhafiz AS, Alorabi M: Social Stigma: The Hidden Threat of COVID-19. Front Public Health. 2020 2020-August-28; 8(429). English. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhattacharya P, Banerjee D, Rao TS: The “Untold” Side of COVID-19: Social Stigma and Its Consequences in India. Indian J Psychol Med. 2020; 42(4): 382–386. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlali W, AlFouzan W, Alajmi D, et al.: COVID19 students.2021, June 12. Publisher Full Text" }	[ { "id": "99758", "date": "29 Nov 2021", "name": "Moawiah M. Hussein Khatatbeh", "expertise": [ "Reviewer Expertise Public Health research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article about awareness of COVID-19 among health science students and staff of Kuwait University. The comparisons between students and staff are unique in relation to COVID-19 literature.\nFigure 1 presents \"Participants awareness of consequences of COVID-19 patients\". I assume these are attitudes not awareness. Moreover, I would prefer a 3-point Likert Scale. Participants can express their attitudes towards a particular item more easily.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "281362", "date": "11 Jun 2024", "name": "Mahmoud Abdel Hameed Shahin", "expertise": [ "Reviewer Expertise Critical care", "COVID-19", "Medical Education", "Nursing", "Enteral Nutrition." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview of \"Perception and awareness of COVID-19 among health science students and staff of Kuwait University: An online cross-sectional study \" Strengths of the Research Methodology and Process: The study used a cross-sectional online survey design appropriate for assessing the perception and awareness of COVID-19 among the target population at a specific time. The questionnaire was divided into six relevant sections covering socio-demographics, risk and awareness, preparedness and prevention, sources of information, policies, and social stigma. This comprehensive approach allowed for a thorough assessment of various aspects related to COVID-19. The study included both students and staff of the health science departments at Kuwait University, providing a broader perspective on the topic. The researchers took steps to ensure the study's tool validity, including questionnaire validation and expert reviewers' involvement. Generally, the study provides valuable insights into the perception and awareness of COVID-19 among Kuwait University health science students and staff. However, the research methodology and process could be further strengthened to enhance the generalizability of the findings. Areas for Improvement: You mentioned “HCS” in the methods of the abstract, which needs to be expressed in full for the first time. In the data collection tool, you discussed the questionnaire's validity and piloting, but nothing was mentioned about the tool's reliability testing. It is recommended that the test-retest reliability testing or Cronbach’s alpha coefficient testing be mentioned. After the conclusions, it is advised to add the recommendations of the study, which comprise the essence of the study findings for the readers to implement and utilize. Adding the limitations of the study gives more transparency and guides other researchers in terms of research chances of improvement.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/10-566
https://f1000research.com/articles/10-564/v1	14 Jul 21	{ "type": "Case Report", "title": "Case Report: Mycotic common carotid artery pseudoaneurysm in a child. A case report.", "authors": [ "Ben Mrad Imtinene", "Rim Miri", "Ben Mrad Melek", "Wafa Aloui", "Sobhi Mleyhi", "Neila Ben Aba", "Zairi Ihsen", "Tawfik Kalfat", "Raouf Denguir", "Ben Mrad Imtinene", "Rim Miri", "Wafa Aloui", "Sobhi Mleyhi", "Neila Ben Aba", "Zairi Ihsen", "Tawfik Kalfat", "Raouf Denguir" ], "abstract": "Extracranial carotid artery aneurysms in children are extremely rare, nevertheless associated with a great potential of thromboembolic episodes and rupture especially those with mycotic origin. The surgical treatment is very challenging, and there is still a controversy concerning revascularisation after the resection of the aneurysm. In this manuscript, we report the observation of an 8-year-old boy with the medical history of Leukemia who is admitted urgently for a mycotic right common carotid artery aneurysm, occurring after a chemoport infection who was operated on in our cardiovascular surgery department with surgical resection and ligation. It is the second report in the pediatric literature of a mycotic pseudoaneurysm situated in the common carotid artery, but the first documented by medical imagery. Through this case, we highlight that ligation of the infected carotid artery can be a safe and efficient alternative especially in Children.", "keywords": [ "Mycotic", "aneurysm", "children", "carotid" ], "content": "Introduction\n\nMycotic extracranial carotid artery aneurysm (ECAA) in children is extremely rare but associated with a high potential for rupture and thromboembolic episodes.1 The surgical management is challenging and subject to controversy concerning the adequate technique.\n\nIn this article, we present an 8-year-old boy with a case of mycotic ECAA who was operated on in our cardiovascular surgery department. It is the second report in the literature of a mycotic pseudoaneurysm located in the common carotid artery.2,3\n\n\nCase report\n\nAn eight-year-old male North African child treated for leukemia was referred to our cardiovascular surgery department for management of a right lateral-cervical mass. The patient did not report any history of tonsillectomy or cervical trauma. The child was being followed for acute lymphoblastic leukemia type B for which he was receiving polychemotherapy. He had central venous ports (chemoports) placed via the right internal jugular vein one month prior to admission. The chemoport was complicated by a venous thrombosis with port chamber infection two weeks after its implantation. The chamber and the central venous catheter were removed, and the child received a double intravenous broad-spectrum antibiotic (Amoxicillin-Clavulanic Acid and Sulfamethoxazole) treatment two weeks before admission. The patient subsequently developed a right latero-cervical mass one day before admission.\n\nUpon physical examination, the child was in good general condition, awake and alert. He was feverish (temperature = 38.9 EC); cardiac and pulmonary examinations were normal. Neurological examination was normal, notably without signs of facial weakness. Otorhinolaryngological examination was normal, ears and nose were clean. The pharynx exam was also normal, notably without erythema, exudates, or pharyngeal oedema. The patient had a right latero-cervical tumefaction (Figure 1) painful to palpation, pulsatile and expansive (3 cm-long large axis), with inflammatory signs all around.\n\nRight latero-cervical mass (3 cm of large axis), with inflammatory signs all around (red arrow) with cutaneous ulceration 5 cm in diameter located under the right clavicular (blue arrow) (former location of the implantable chamber).\n\nHe also presented a cutaneous ulceration with a diameter of 5 cm, located under the right clavicle (former location of the implantable chamber), with inflammatory signs all around (Figure 1). Complete blood count revealed anaemia with a haemoglobin level of 9.6 g/dL (Normal: 11.9-15 g/dl), thrombocytopenia with platelet levels of 126,000 per mcL (Normal: 150000-400000 mcL), and hyperleukocytosis with a leukocyte count of 22,3 × 109/L (Normal: 4.5-14.5 × 109/L). The C-reactive protein reached 95 mg/dl. (Normal: < 5 mg/dl).\n\nA cervical ultrasound found a right common carotid artery (CCA) false aneurysm measuring 29-28 mm of axis, with inflammation of the right cervical subcutaneous tissue associated with huge cervical lymphadenopathies. A cervical Angio-Computed Tomography (CT) scan (Figure 2) showed a 3 cm pseudoaneurysm of the right CCA beginning 2 cm from its origin and at a distance from the carotid bifurcation, with the presence of multiple cervical lymphadenopathies and an unimpaired cerebral circulation through the polygon of Willis.\n\nCervical computed tomography angiogram showing a 3 cm right common carotid artery pseudoaneurysm beginning at 2 cm of its origin and at a distance from the carotid bifurcation (red arrow), with the presence of multiple cervical lymphadenopathies.\n\nHaemocultures were carried out but did not find any isolated pathogens.\n\nThe patient was operated on the evening of his admission. The procedure was carried out under general anaesthesia. The surgical incision was a lateral right cervicotomy extended to the sternal fork. First, the CCA was dissected and controlled at its origin and terminated just before the carotid bifurcation (Figure 3). An arterial clamping was made on both sides of the mycotic aneurysm, and resection of the latter, and all-around infected tissues, was performed (Figure 3). Our first strategy was to use the greater saphenous vein for reparation. However, given the extent of the infection with brittle arterial tissue, the existence of cutaneous fistula (Figure 4), and the excellent pulsatile arterial reflux from the CCA, the decision was made to make a simple ligation of the artery to avoid complications, especially graft rupture (Figure 5). The immediate surgical procedure was simple, with an alarm clock on the operating table and without neurological deficit.\n\nPreoperative view, the CCA was dissected and controlled and clamped at its origin and terminated just before the carotid bifurcation (blue arrow). Mycotic aneurysm, and all-around infected tissues were resected (yellow arrow).\n\nPreoperative view showing ligation of the two extremities of the CCA (yellow arrow) after total debridement of the infected area (green arrow).\n\nDuring the postoperative period, the child was hospitalized in the intensive care unit. Excised local lymph nodes and aneurysm tissues were sent for bacteriological and pathologic examination. The culture of bacteriological samples was negative, a predictable result given the probabilistic antibiotic therapy introduced from the first presentation of symptoms. Repeated haematological analysis showed that his leukocyte levels had dropped to normal values.\n\nThe patient made an ordinary recovery and was discharged after two weeks. Low-dose aspirin (75 mg per day) with 6 weeks of oral antibiotics (Amoxicillin/Clavulanate 15 mg/kg every 12 hours) were prescribed. Upon one-year follow-up, the neurological examination was normal, and in particular showed no neurological sequelae.\n\n\nDiscussion\n\nExtracranial carotid aneurysms are uncommon in the paediatric population. Therefore, their natural history remains unclear. A literature review that was published in 2021 revealed that only 26 cases of infectious extracranial carotid artery pseudoaneurysms had been reported since 1990.3\n\nMost of the infectious extracranial carotid pseudoaneurysms occur in the internal carotid artery.1 The first case of an extracranial pseudoaneurysm of the common carotid artery was reported by Willemsen in 1997;2 our report is the second one in the medical literature.\n\nIn childhood, ECCA formation is mainly secondary to infection or trauma.4 Historically, ear/nose/throat infections are associated with some vascular complications, such as the Lemierre syndrome (thrombosis of the internal jugular vein) and, uncommonly, carotid artery false aneurysm.5,6 However, these complications have become exceptional nowadays due to the widespread use of antibiotics.7\n\nThe physiopathogenesis of the carotid false aneurysm in this case is not completely clear. We thought that the contiguous dissemination of infection into the parapharyngeal area appeared to be the main cause. In the study herein, the child had an infection of the chemoport implanted via the right internal jugular vein, which we presumed to be responsible for this deep neck infection. Pseudoaneurysm formation is secondary to fragilization and dilatation of the arterial wall by infectious arteritis.7 This infection was promoted by the state of immunodepression caused by his leukemia. The interval separating the initial infection and the diagnosis of the pseudoaneurysm was 15 days in our case. This is in line with the data reported in the literature.7\n\nPulsatile cervical mass is the most frequent clinical presentation of ECCA in the surgical literature.8 Other clinical signs are dyspnea, dysphagia, and voice hoarseness by compression of adjacent anatomical and nervous structures.10-12 Neurological symptoms have been noted in relation with either a cerebral infraction or Horner’s syndrome.1\n\nIn addition, a patient may present with a haemorrhage secondary to the rupture of the pseudoaneurysm.9 This complication is more frequent with mycotic aneurysm.9 Pourhassan et al., reported a rupture rate of around 42% in their review of literature concerning carotid aneurysms in children.1\n\nThe diagnosis in this case was based on cervical Doppler ultrasound and CT angiogram.5–7 It is highly necessary to assess the Willis polygon and the structures surrounding the aneurysm, before any possible surgical procedure in order to reduce complications. Cervical angiography is performed only if an endovascular treatment is considered.5,7\n\nIt is important to note the lack of evidence-based treatment guidelines for this complication in paediatric patients. However, given the high risk of rupture, urgent intervention is highly recommended.13\n\nSeveral treatment strategies with different levels of efficacy and limitations are available in cases of children with infectious extracranial carotid pseudoaneurysm, including surgical treatment, endovascular treatment, or a combination of the two.3\n\nPseudoaneurysm resection with restoration of the arterial continuity using a saphenous venous graft is the most habitual surgical treatment.8 However, in children, arterial reparation may not be realizable if the greater saphenous vein is small in diameter. In these cases, ligation can be proposed. In addition, reconstructive techniques are challenging because of inflammation and proximity of cranial nerves.13\n\nA few cases of ligation of CCA or ICA among the paediatric population, as in our case, have been reported in the literature.1 The risk of stroke is relatively low in children in contrast with adults.6,14 However, we can only perform ligation if the collateral circulation is intact, in order to minimize neurologic consequences.6,11,15,16\n\nEndovascular techniques such as stenting, and coil embolization have provided a less invasive approach to the treatment of infectious pseudoaneurysms in children.12 However, there is still concern that coil embolization or stenting for infectious area may expose patients to an increased risk of persistent infection.12 In all cases, we must combine a 4–6 weeks broad-spectrum antibiotic therapy with the chosen surgical or endovascular treatment.7\n\nLike our patient, in the majority of paediatric cases, an uneventful early outcome is reported.1 However, there is a lack of data on long-term consequences.\n\n\nConclusion\n\nMycotic carotid pseudoaneurysm is a rare complication in children, but associated with a high risk of fatal rupture. When diagnosed, it should be treated urgently. Surgical ligation seems to be a reasonable and viable choice of treatment, especially in children.\n\n\nConsent\n\nWritten informed consent for publication of the patient’s clinical details and/or clinical images was obtained from the father of the patient.", "appendix": "Acknowledgments\n\nThis paper would not have been possible without the exceptional support of our friend Jack Hukill, especially for his help in verifying English this article.\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\nReferences\n\nPourhassan S, Grotemeyer D, Fokou M, et al.: Extracranial carotid arteries aneurysms in children: single-center experiences in 4 patients and review of the literature. J Pediatr Surg. 2007 Nov; 42(11): 1961–8. PubMed Abstract \| Publisher Full Text\n\nWillemsen P, De Roover D, Kockx M, et al.: Mycotic common carotid artery aneurysm in an immunosuppressed pediatric patient: case report. J Vasc Surg. 1997 Apr; 25(4): 784–5. PubMed Abstract \| Publisher Full Text\n\nSundarrajan C, Isa SA, Caruso JP, et al.: Treatment of large infectious extracranial carotid artery pseudoaneurysms in children: a systematic review of the literature. Childs Nerv Syst. 2021 May; 37(5): 1461–1470. Epub 2021 Feb 15. PubMed Abstract \| Publisher Full Text\n\nDeFatta RJ, Verret DJ, Bauer P: Extracranial internal carotid artery pseudoaneurysm. Int J Pediatr Otorhinolaryngol. 2005 Aug; 69(8): 1135–9. PubMed Abstract \| Publisher Full Text\n\nStevens HE: Vascular complication of neck space infection: case report and literature review. J Otolaryngol. 1990 Jun; 19(3): 206–10. PubMed Abstract\n\nLueg EA, Awerbuck D, Forte V: Ligation of the common carotid artery for the management of a mycotic pseudoaneurysm of an extracranial internal carotid artery. A case report and review of the literature. Int J Pediatr Otorhinolaryngol. 1995 Aug; 33(1): 67–74. PubMed Abstract \| Publisher Full Text\n\nReisner A, Marshall GS, Bryant K, et al.: Endovascular occlusion of a carotid pseudoaneurysm complicating deep neck space infection in a child. Case report. J Neurosurg. 1999 Sep; 91(3): 510–4. PubMed Abstract \| Publisher Full Text\n\nEl-Sabrout R, Cooley DA: Extracranial carotid artery aneurysms: Texas Heart Institute experience. J Vasc Surg. 2000 Apr; 31(4): 702–12. PubMed Abstract \| Publisher Full Text\n\nSiablis D, Karnabatidis D, Katsanos K, et al.: Extracranial internal carotid artery aneurysms: report of a ruptured case and review of the literature. Cardiovasc Intervent Radiol. 2004 Jul-Aug; 27(4): 397–401. Epub 2004 Jun 16. PubMed Abstract \| Publisher Full Text\n\nChambers N, Hampson-Evans D, Patwardhan K, et al.: Traumatic aneurysm of the internal carotid artery in an infant: a surprise diagnosis. Paediatr Anaesth. 2002 May; 12(4): 356–61. PubMed Abstract \| Publisher Full Text\n\nHazarika P, Sahota JS, Nayak DR, et al.: Congenital internal carotid artery aneurysm. Int J Pediatr Otorhinolaryngol. 1993 Dec; 28(1): 63–8. PubMed Abstract \| Publisher Full Text\n\nBrochu B, Dubois J, Garel L, et al.: Complications of ENT infections: pseudoaneurysm of the internal carotid artery. Pediatr Radiol. 2004 May; 34(5): 417–20. Epub 2004 Jan 14. PubMed Abstract \| Publisher Full Text\n\nLopez D, Sarac T, Lorenz R: Primary internal carotid artery aneurysm in a 15-year-old male: case report and review of the literature. Ann Vasc Surg 2015 Jan; 29(1): 126. e1–4. Epub 2014 Oct 7. PubMed Abstract \| Publisher Full Text\n\nPearson SE, Choi SS: Pseudoaneurysm of the internal carotid artery: a case report and review of the literature. Arch Otolaryngol Head Neck Surg. 2005 May; 131(5): 454–6. PubMed Abstract \| Publisher Full Text\n\nAntar KA, Keiser HD, Peeva E: Relapsing arterial aneurysms in juvenile Behçet's disease. Clin Rheumatol. 2005 Feb; 24(1): 72–5. Epub 2004 Jul 17. PubMed Abstract \| Publisher Full Text\n\nTovi F, Leiberman A, Hertzanu Y, et al.: Pseudoaneurysm of the internal carotid artery secondary to tonsillectomy. Int J Pediatr Otorhinolaryngol. 1987 Jun; 13(1): 69–75. PubMed Abstract \| Publisher Full Text" }	[ { "id": "96201", "date": "27 Oct 2021", "name": "Fabien Koskas", "expertise": [ "Reviewer Expertise Vascular Surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, an original pediatric case of mycotic aneurysm of the common carotid is presented and well documented. Given the seldomness of such cases, the case deserves publication. Strategy and tactics in emergency are well explained, justified and post-hoc-approved by the uneventful outcome. The paper will be particularly useful for a young vascular surgeon confronted with such an unusual challenge, especially in emergency. Moreover, the discussion on whether or not revascularize is particularly relevant from a medicolegal point of view. Young surgeons may be confronted with septic disasters affecting arteries of potentially vital arteries. There is then a choice between ligating to minimize the hemorrhagic risk but with the sacrifice of an artery of vital or functional importance or revascularizing, a much more complex procedure with an eventual risk of hemorrhagic, therefore vital complications. Strategic decisions must then be taken often in emergency and such decisions medicolegally expose the taker if eventually the vital of functional prognosis of the patient is affected. In our societies where the medicolegal issue is more and more a concern, it is important that published papers like this one show that the decision of ligating a potentially vital artery like the common carotid does not always result in a cerebrovascular disaster. I strongly recommend indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "96569", "date": "03 Nov 2021", "name": "Maurizio Domanin", "expertise": [ "Reviewer Expertise Vascular surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case presented here is well described by the authors, such as all the possible options for its repair have been deeply analyzed.\nIn my personal experience, in particular in children or younger people, I always suggest exploring the possibility of arterial reconstruction. My personal experience on a similar case, but without any spread of local infection that could be eventually inserted among the references, was to reconstruct the whole carotid bifurcation using the greater saphenous vein and one of its major tributaries (Giacomini’s vein) (please see Domanin et al., 20181). If feasible, biological grafts are resistant to infections and guarantee high rates of patency in the long term.\nAnyway, also the choice of the ligature of the carotid artery can be successful, if a valid collateral circulation, sustained by the contralateral carotid or by the vertebral circle, is present and well documented both by Doppler Ultrasound and Computed Tomography Angiography (or Magnetic Resonance Angiography). Moreover, a valid backflow, together with the measurements of the stump pressure from the ipsilateral internal carotid, can give further valuable information regarding the status of collateral supply to the brain.\nThe background of the case’s history and progression is well detailed even if, in all these cases, the real event which leads to pseudoaneurysm development can only be supposed.\nAnyway, the work of Imtinene et al. is clearly presented with updates on the most recent literature regarding this niche topic and conclusions are correct and well balanced. The major problem is related to the extreme rarities of such cases and, as reported by the authors, the complete lack of evidence and the absence of guidelines.\nIn this way, I think that every report on bailout treatments for arterial disease in newborns or children can be helpful for all the surgical community in order to provide and share therapeutic ideas for these really challenging cases.\nI congratulate the authors for their very interesting study which turns the lights on in this disputed topic.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-564
https://f1000research.com/articles/10-562/v1	14 Jul 21	{ "type": "Research Article", "title": "Annotating clause boundary labels to the written composition corpus of Japanese elementary and junior high school students", "authors": [ "Mizuho Imada", "Takumi Tagawa", "Chang-Yun Moon", "Akio Nasu", "Takumi Tagawa", "Chang-Yun Moon", "Akio Nasu" ], "abstract": "To evaluate the development of children’s writing ability, it is necessary not only to examine quantitative indices such as the dependency distance, but also to inquiry the types of structures they use. We conducted clause boundary labeling using Support Vector Machine (SVM) on a corpus of Japanese students' compositions to investigate the change in the tendency of clause use with the progression of school age. The analysis of clause label frequency per sentence exhibited an increase in attributive clauses, nominal clauses, quotation clauses, and continuous clauses, and a decrease in parallel clauses, conditional clauses, reason clauses, time clauses, indirect interrogative clauses, and main clauses. The analysis of dependency distance demonstrated that most of the clauses that increased had short dependency distances, while most of the clauses that decreased had long dependency distances, and that the frequency of clauses with small dependency distances increased relatively with increasing school age. In addition, there was a shift in clause selection among functionally similar clauses, such as from “-te” to continuous forms, from “-tara” to “-ba”, and from “-kedo” and “-keredo” to “-ga”. These results suggest a change in the children’s lexical and grammatical choices, from coordinate to subordinate structures, and from spoken to written vocabulary.\n児童の作文能力の発達を評価するためには、単に係り受け距離などの計量的指標を評価するだけでなく、どのような種類の構造を使うかを考慮する必要がある。そこで「児童・生徒作文コーパス」に対してSVMによる節境界ラベル付与を行い、学齢の進行に伴う節の使用傾向の変化を調査した。文あたりの節ラベル頻度の分析では、連体節、補足節、引用節の増加と、並列節、条件節、理由節、時間節、間接疑問節の減少が見られた。係り受け距離の分析では、増加した節の多くは係り受け距離が小さく、減少した節の多くは係り受け距離が大きいこと、および学齢が上がるほど係り受け距離が小さい節の頻度が相対的に増大することを確認した。また、機能的に類似する節の間で、「て」から連用形へ、「たら」から「ば」へ、「けど」「けれど」から「が」へといった節選択の推移が見られた。これらの結果は、等位構造から従位構造への推移、話し言葉的な語彙から書き言葉的な語彙への推移という、児童の語彙・文法選択の変化を示唆する。", "keywords": [ "作文コーパス", "節境界ラベル", "書き言葉", "話し言葉", "言語発達", "corpus of composition", "clause boundary labels", "written language", "spoken language", "language development" ], "content": "1. 緒言\n\n児童は学齢が上がり言語能力が発達するにつれて、より複雑な文を処理することができるようになると予測される。そこで児童の作文データを分析することによって文の複雑さを計量的に評価することを考えるが、そのためには文の複雑さを数値化する必要がある。比較的簡便に計量可能な指標としては文の長さ、係り受け距離、係り受けの深さなどがあり、基本的にはこれらの数値が大きいほど文の構造は複雑だと判断することができる。\n\nしかし予測に反して、児童が学齢の上昇に従ってより複雑な文を書くようになると単純には言えないことを示すデータが存在する。低学年の児童は「〜して、〜して、〜して」のように並列節を多用して長い文を作ることがあり、このような文は「だらだら文」と呼ばれる。作文教育においては、長すぎる文の使用は好ましくないものとみなされており、短い文に分割するように指導されることが多い（松崎, 2016）。書き言葉の計量的な研究においては、小学生の作文における1文あたりの文字数、形態素数が学年によって大きくは増減しないこと（国立国語研究所, 1964: 386; 宮城・今田, 2015）1が報告されている。これらの事実は、児童が成長につれてより複雑な文を書くようになるとは限らず、また複雑な文を書くことが直ちに作文能力の熟達を示すわけでもないことを示唆する。\n\n児童の作文能力の統語的な発達を評価するためには、単に構造の複雑さを数値化するだけでなく、どのような構造が使われているかを考慮する必要がある。英語では、子供は作文の熟達に従って、口語的な接続詞を用いた等位構造の連鎖から、接続詞を多用せず従属節の使用、文や節の名詞化などにより情報が凝縮された節構造へと移行する（Schleppegrell, 2004）。日本語の児童作文でも、逆接の「しかし」などを除けば接続詞は単調な増加傾向を示さず、一方で連体修飾成分が顕著な増加傾向を示す（宮城・今田, 2015）。また係り受け距離は、文節の種類によって頻度分布が異なる（張・尾関, 1996）。従って児童の統語能力の発達を評価するためには、構造の種類を無視して一律に複雑さを数値化するのではなく、発達に従ってどのような構造を用いるようになるかを重視しなければならない。\n\nそこで本研究では、児童作文において使用される節の頻度分布を調査する。節は単文に相当する基本的な情報伝達単位であり、節を等位的に接続するか、埋め込みを多用するかといった構造の選択において、発達段階に応じた文法的方略の変化が観察されることが予測される。分析対象としては「児童・生徒作文コーパス」（宮城・今田, 2015; 以下「作文コーパス」）を使用するが、節の位置と分類に関する情報（節境界ラベル）は付与されていない。節境界ラベルを付与するシステムとしてはCBAP （丸山他, 2004）、Rainbow （加納他, 2014）があるが、一般に公開されていない。そのため本研究では「現代日本語書き言葉均衡コーパス」（BCCWJ, Maekawa et. al., 2014）の形態論情報と節境界ラベル（BCCWJ-CBL, 丸山, 2013）を訓練データとして、汎用アノテーションツールYamCha （Kudo and Matsumoto, 2003）で作文コーパスに節境界ラベルを付与した。\n\nこのデータを用いて児童作文における節の使用頻度を調査し、使用される節の種類が学年によってどのように変化するかを調べた。調査項目は、1文あたりの節ラベル頻度、節ラベル間の相対頻度、節の係り受け距離である。1文あたりの節ラベル頻度は、節の使用頻度を分析するための基本的な統計量である。節ラベル間の相対頻度は、理由節におけるカラ節とノデ節など、機能的に類似する節の相対的な選択嗜好の推移を分析するための統計量である。理由節全体の頻度が減少する場合、カラ節やノデ節の頻度も基本的に減少傾向が観察されるため、両者の選択嗜好を直感的に把握しやすくするために相対頻度を調べた。節の係り受け距離は、統語的特性の観点から節の使用傾向を分析するために調べた。平均的な係り受け距離は、並列節が多いほど大きく、連体節が多いほど小さくなると予想される。以下、データの作成と調査の方法を説明し、調査結果に対して検討を行う。なお、本研究で使用した作文データは、人名、組織名、地名など個人を識別できる情報があらかじめ被覆され、匿名化されている。また、公開したデータは節境界ラベルとテキスト中における位置情報のみを含み、個人を識別できる情報は含まれていない。従って出版の同意は必要なく、これにより科学的意味あいが歪められていない点を確認した。\n\n\n2. 方法\n\nBCCWJ-CBLを教師データとして、YamChaを用いて節境界ラベルのSVMモデルを作成した。BCCWJ-CBLはBCCWJ本体とは独立したデータであり、節境界ラベルとラベル位置のみを含む。BCCWJには数値表現を解析しやすい漢数字表記に変換したNumTrans版（NT）と原文のままの非NumTrans版（OT）があるが、BCCWJ-CBLのラベル位置はNT版に基づく。一方、作文コーパスの形態論情報はNumTrans処理を行っていないOT形式のデータである。節境界付近に数値表現が現れることはあまり多くないと思われるが、OT形式でモデルを作成した方が作文コーパスにアノテーションをする際に精度がよくなると考えられるため、まずBCCWJ-CBLのラベル位置をNTからOTに変換する処理を行った。併せて、ラベル位置の斉一性のため、句読点や括弧などの記号の直後にラベルがあるときは、その直前の語彙的形態素の直後にラベルを移動した。\n\n次に変換したBCCWJ-CBLとBCCWJの形態論情報（OT版）を重ね合わせ、8割を訓練データ、2割をテストデータとしてYamCha形式のデータを作成した。YamChaで訓練データを学習させてSVMモデルを作成し、テストデータにアノテーションを行って精度を評価した（図 1）。使用した形態論情報は、出現形、語彙素読み、品詞大分類、品詞中分類、活用形である。学習はYamChaのデフォルト設定で行った。すなわち、前後2形態素を含む範囲の形態論情報と直前2形態素の推定済みラベルを素性として使用した。精度の評価は元データのラベルと推定したラベルの種類と位置が一致した場合を正解とし、元データのラベルのうち正解した率を再現率（recall）、推定したラベルのうち正解した率を適合率（precision）、両者の調和平均をF値として以下の式で計算した。\n\n前節と同様の方法で本番用のSVMモデルを作成し、作文コーパスに節境界ラベルを付与した。本研究で使用する作文コーパスは2014年から2016年までの3年間にかけて小学校全学年各2クラス、中学校全学年各4クラスの作文を悉皆的に収集・電子化した165万形態素規模のテキストコーパスである（表 1）。CaboCha/UniDicで形態論情報と係り受け情報を付与し、一部は人手により修正を施してある2。まずBCCWJ-CBLの全データを使用して本番用のSVMモデルを作成した。次に作文コーパスのデータをYamCha形式に変換し、作成したSVMモデルでアノテーションを行った（図 2）。\n\nBCCWJ-CBLのラベルは198種に及ぶが、この中には頻度が低く計量的な分析に適さないものも含まれる。そのため、付与したラベルをグループ化するためにcbl1 （大分類）、cbl2 （小分類）、cbl3 （接頭辞）、cbl4 （接尾辞）の4つの属性に分割したラベルを追加した。接頭辞は「文末」、接尾辞は「助詞」「補足語」を含む（表 2）。以下、分かりやすさのためにBCCWJ-CBLのラベルを鉤括弧、本研究で追加したラベルを二重鉤括弧で示す。\n\n節境界ラベルを付与した作文コーパスを用いて、学年ごとの節の頻度を調べた。ラベルの頻度は作文の長さに比例して増加するため、頻度の調整が必要である。調整方法として、形態素数あたりの頻度と文数あたりの頻度が考えられるが、ここでは文数あたりの頻度で調整した。どちらの方法も文が長くなるほど「文末」以外のラベルの頻度が相対的に大きくなるが、後者の方法では「文末」ラベルがほぼ一定の値（1文あたり1つ）を取るので、直感的に理解しやすいためである。1文あたりの節ラベル数をcps （clauses per sentence）とし、文書dにおける節境界ラベルcのcpsを以下のように定義する。 sdは文書dにおける文数、 nd,cは文書dにおける節境界ラベルcの頻度である。\n\nこの値を全ての文書d、節境界ラベルcについて計算し、学年によるcpsの変化を調べた。cpsは片側に裾の長い分布を取るため、実際の分析にはlog （cps）を使用した。まずcbl1レベルにおけるlog （cps）の分布を箱髭図で可視化し、次に以下のモデルで単回帰分析して各ラベルのlog （cps）の学年による傾きを求めた。\n\n学年による傾きが正の値を取れば学年に従って頻度が増加し、負の値を取れば減少するものと解釈することができる。\n\n単回帰分析の結果cpsの傾きが小さかったいくつかのcbl1について、その下位のcbl2から比較的頻度が高く、かつ機能的に類似するものをいくつか選択し、相対頻度を調べた。節境界ラベルcが複数の節境界ラベルC={c1, c2, …}に占める相対頻度rcは以下のように計算した。fcはcの頻度である。\n\nrの値が大きいほど、節境界ラベルの集合Cの中で相対的に使用頻度が高いと解釈することができる。比較のために、まず各cbl2におけるlog （cps）をcbl1と同様のモデルで単回帰分析し、次に学年別のrを積み上げ棒グラフで可視化した。\n\n文節係り受け構造において、係り元の文節から係り先の文節までの文節数を係り受け距離（dependency distance）とする。作文コーパスに付与されている係り受け情報は全て左から右への係り受けなので、文節sから文節tまでの係り受け距離ddsは両者の文節番号の差idt−idsで計算することができる。文末文節は係り先を持たないのでdd=0とする。\n\nまず、節の種類によって係り先までの距離が異なることを確認するために、節境界ラベルが付与されている文節の係り受け距離を調べ、cbl1ごとに平均を求めた。次に、これらの文節の係り受け距離の分布が学年によってどう変化するかを調べるために単回帰分析を行った。係り受け距離の頻度分布はZipfの法則に従う（Maruyama and Ogino, 1993; 金, 1996; 張・尾関, 1996）とされているので、距離ddと頻度nddはlogndd=a+blogddに従うと考えられる。そのため、以下のモデルで学年ごとに単回帰分析を行った。dd=0の文節は対数が取れないので除外した。\n\nbは負の値を取るが、この値が小さい（絶対値が大きい）ほどddが小さい文節と大きい文節の頻度差が大きい、すなわち係り受け距離の短い文節が多いと解釈することができる。\n\n\n3. 結果\n\nBCCWJ-CBLの8割を教師データ、2割をテストデータとしてアノテーション精度を評価した結果、適合率97.34%、再現率96.21%、F値96.77%だった。CBAP （丸山他, 2004）の解析精度はF値で97.32%〜98.87%とされており、既存の節境界ラベル付与プログラムと比べても大きく遜色ない精度が得られた。日本語の節境界は局所的な形態素列によってかなりよい精度で検出可能（丸山他, 2004）であるため、SVMでも高い精度で検出できたものと考えられる。\n\nBCCWJ-CBLの全データを教師データとしてSVMモデルを作成し、作文コーパスに節境界ラベルを付与した。付与したラベルの総数は203,152ラベル、使用したラベルはBCCWJ-CBLの198種中155種だった（表 3）。\n\n大分類ごとのlog （cps）の分布を図 3に、その単回帰分析の結果を表 4に示す。譲歩節は1例しかないため、単回帰分析から除外した。単回帰分析に基づくlog （cps）の傾きを見ると『連用節』『補足節』『引用節』『連体節』は0.01〜0.07の傾きで増加しており、『条件節』『理由節』『並列節』『時間節』『未定義』『間接疑問節』『主節』は−0.09〜−0.01の傾きで減少している。『連用節』は5%水準で有意、それ以外は0.1%水準で有意だった。\n\ncbl1の単回帰分析で傾きが小さかった4種類のcbl1に属する合計10種類のcbl2を選んで相対頻度を調べた。対象としたcbl1は『連用節』『理由節』『条件節』『並列節』4種類で、単回帰分析の傾きは−0.09〜0.01と比較的小さい。これらのcbl1に属するcbl2のうち、連用節の『動詞型』『テ』、理由節の『カラ』『ノデ』、条件節の『タラ』『ト』『バ』、並列節の『ガ』『ケド』『ケレド』を分析対象とした。これらは各cbl1で比較的頻度の高いcbl2であり、連用節の67%、理由節の100%、条件節の96%、並列節の53%を占める。並列節は『タリ』『シ』も頻度が高いが、逆接の『ガ』『ケド』『ケレド』とは機能が大きく異なるので除外した。\n\nこれらのcbl2をcbl1と同様に単回帰分析したところ、『動詞型』が有意な正の傾きを示し、『ガ』が有意な傾きを示さないことを除いて、他の全てのcbl2の傾きが有意に負の値を示した（表 5）。一方、これらのcbl2について、同じcbl1に属するものに分けて学年ごとに相対頻度rを調べたところ、『動詞型』『ガ』『バ』は学年が上がるほどrが増加しており、『ノデ』『ケレド』も小学校高学年頃まで上昇が見られた（図 4）。\n\ncbl1ごとに係り受け距離の平均を計算したところ、『並列節』が4.38で最も大きく、『主節』が0.11で最も小さかった（表 6）。またlognddを学年ごとに単回帰分析した結果、logddによる傾きは全学年を通じて負の値を取り、かつ学年が上がるほど小さい値をとる傾向があることがわかった（表 7）。ただし、小学2年、小学6年、中学3年では直前の学年より傾きが大きい値を取った。\n\n\n4. 考察\n\ncbl1におけるlog （cps）の単回帰分析（表 4）から、学年の上昇に伴って『連体節』『補足節』『引用節』『連用節』が増加すること、『条件節』『理由節』『並列節』『時間節』『未定義』『間接疑問節』『主節』が減少することが分かる。『連用節』は5%水準で有意、それ以外は0.1%水準で有意だが、箱髭図を見ると必ずしも一様に増加・減少しているとは言えず、ラベルによっては増加する時期、減少する時期、ほとんど変化しない時期があるようである。\n\n『連体節』『補足節』『間接疑問節』は名詞句を形成する節であり、『引用節』も名詞句を作るわけではないが補足節の一種とされる（現代日本語文法研究会, 2008）。これらの節は述語の項として機能し、典型的な従位構造である埋め込み文を形成する。このうち『連体節』『補足節』『引用節』は学年の上昇に従ってcpsが増大しており、学年が上がるほど従位構造を多用する傾向があることを示している。連体節は学齢に従って増加することが報告されており（小熊他, 1998）、本調査でも同様の傾向が確認できる。ただし箱髭図を見ると『連体節』『補足節』とも小 1 から小 3 にかけて若干減少し、その後増加している。また『引用節』も小 1 から小 2 にかけて減少した後増加し、さらに中 1 以降は再び減少している。このような複雑な変化は、単に学齢が上がるに従ってこれらの名詞的な節を多用するようになるという以外の何らかの文法的方略の変化が特定の時期に生じていることを示唆する。『間接疑問節』のみcpsが減少する傾向が見られるが、この原因についてはさらに研究する必要がある。\n\n『並列節』は等位構造を作る節である。『条件節』『理由節』『時間節』は副詞節の一種とされ（現代日本語文法研究会, 2008）、意味機能的には従位的な性格を持つ節だが、名詞節や補足節が述語の項として機能するのと異なり、節と節が統語的には並列に接続するという点で等位構造に近い性質を持つ。これらの節は学年の上昇に従ってcpsが減少しており、学年が上がるほど等位構造の使用が減少することを示している。特に条件節と理由節は2 つの事柄の因果関係を表現する節であり、こうした関係を表現する能力は学齢に従って発達すると予測されるが、本調査の結果からは減少する傾向が見られる。この理由として、文を 2 つに分割して接続表現や指示表現で結束性を表現したり、名詞句や副詞句など別の手段で因果関係を表現する方略の発達により、節による条件、理由の表現が相対的に減少することが考えられる。\n\n『主節』『連用節』は単回帰分析の傾きが−0.01〜0.01でcpsの変化が小さい。『主節』は「文末」「文末_その他」のみを含み、前者は動詞などの通常の述語、後者の多くは名詞で文が終わる体言止めである。これらのラベルは通常文末にのみ付与されるため、大半の文書では『主節』のcpsは 1 である。log （cps）の傾きが0に近いにも関わらず傾きが有意なのはこのためと考えられる。ただし、稀に文中に『主節』が付与されることもあるためcpsが1を超える場合がある。また、文末に「文末_理由節カラ」などが付与される場合は『理由節』などに分類されるため、cpsが1未満になる場合がある。『連用節』は『主節』と同様に log （cps）の傾きは0に近いが、『主節』よりも値のばらつきが大きく、傾きは有意ではない。『連用節』は条件節、理由節、時間節など他の連用的な節に含まれない全ての連用的な節を含むが、最も多いのは『テ』と『動詞型』（動詞連用形節）であり、この 2 つで 67%を占める。この2つの節はいずれも等位構造を作るためによく用いられるが、cbl2におけるlog （cps）の単回帰分析（表 5）では、前者が減少傾向、後者が増加傾向を示している。『動詞型』は、等位構造を作る節の中で増加傾向を示す数少ない例外である。\n\nlog （cps）の単回帰分析の結果は、全体としては学年の上昇によって等位構造が減少し、従位構造が増加することを示している。この結果は、児童はまず等位構造を使用するが次に従属節を使うようになること、述語の名詞化や修飾語化によって文を句や単語に縮約する方略を用いることになること、原因や時間を表す接続詞が減少することなどを指摘する英語での研究（Schleppegrell, 2004）と一致しており、日本語でも同様の傾向があると考えることができる。係り受け距離の分析結果も、この結論を支持する。等位構造的な『時間節』『条件節』『理由節』『並列節』は平均ddが3.35〜4.38と大きく、従位構造的な『引用節』『連体節』『間接疑問節』『補足節』は1.13〜2.12と小さい（表 6）。ddの頻度分布を単回帰分析した結果は、学年が上がるほど傾きが小さな負の値を取る（絶対値が大きくなる）ことを示している（表 7）。これは学年が上がるほど係り受け距離の小さい節が増えることを意味しており、従位構造が増えるというcpsの分析結果と一致する。それと同時に、学年が上がるほど係り受け距離が大きい“複雑な”文が増えるとは一概に言えないことを示唆する。\n\n等位構造から従位構造への文構成方略の変化は『時間節』『条件節』『理由節』『並列節』などの頻度を減少させる。この変化はcbl1レベルに留まらず、それに属するcbl2レベルの節境界ラベルにも波及する。cbl2におけるlog （cps）の単回帰分析の結果（表 5）は、『条件節』『理由節』『並列節』に属する主要なcbl2の多くが、cbl1と同様に減少することを示している。加えて『連用節』に属する『テ』も学年の上昇に従って減少し、唯一『動詞型』だけが増加傾向を示す。\n\n一方で、cbl2レベルの節選択では別の変化が起こっている。cbl2の相対頻度の分析（図 4）は、機能的に類似する節の間で相対的に頻度を増大させるものと減少させるものがあることを示している。最も大きな変化は、話し言葉的な節形態の減少と、それに代わる書き言葉的な節形態の増加である。特に『連用節』『条件節』『並列節』では、『テ』『タラ』『ケド』が減少し、『動詞型』『バ』『ガ』が増加している。これらの形式の文体的特性については書き言葉や話し言葉のコーパスに基づくいくつかの先行研究がある。中俣（2015, 146）は「日本語話し言葉コーパス」と「毎日新聞」のデータで接続助詞の頻度を調査し、前者では「て」が多く、後者では「連用形」が多いことを報告している。また宮内（2012）はBCCWJのジャンル別の接続助詞の頻度を調査し、「たら」「けれど」（「けど」を含む）はフォーマルでない話し言葉的な形式、「ば」「が」はどのジャンルでも出現比率が高いニュートラルな形式としている。これらの研究と照らしても、作文コーパスに見られる変化はおおむね話し言葉的な節形態から書き言葉的な接形態への推移と解釈することができる。\n\n『理由節』はもう少し複雑で、小学 4 年まで『カラ』から『ノデ』への推移が観察されたあと、中学校になると『カラ』がややシェアを取り戻す。宮内（2012）は「から」と「ので」をいずれもフォーマルでない話し言葉的な形式としているが、一方で永野（1952）を引用して「から」は主観的、「ので」は客観的かつ丁寧な形式としている。中学年までの『カラ』から『ノデ』への推移は、主観的な形式から客観的な形式への推移を示している可能性がある。中学校で『カラ』がシェアを取り戻す理由はまだ十分に分析できていない。「~だからだ」のような『カラ』の文末用法の増加が多少は関係しているようだが、『カラ』の再増加の全てを説明するほどには頻度が高くはない（cbl3が『文末』のものを除外しても結果は大きくは変わらない）。別の要因としては『タメ』など他の書き言葉的な理由形式が『ノデ』のシェアを奪っていることが考えられる。『タメ』はおそらく理由以外に目的を表す用法もあるため、BCCWJ-CBLでは『理由節』に分類されていないが、『カラ』『ノデ』に『タメ』を加えて学年別の相対頻度を調べると、『タメ』が小学校高学年以降シェアを増大させることが確認できる。『並列節』の『ケレド』も小学 5 年までは増加するが、その後は減少に転じる。これは話し言葉的な『ケド』からやや書き言葉的な『ケレド』への推移と、ケド・ケレド類から『ガ』への推移という 2 段階の変化が生じていることを示唆する。\n\ncbl2の相対頻度の変化からは、少なくとも 2 つのことを読み取ることができる。第 1 に、等位構造から従位構造へという文法的方略の推移と並行して、類似する機能を持つ節のうちどの形態のものを選択するかという語彙的方略の推移が生じている。どちらの変化も、基本的には話し言葉的な文体から書き言葉的な文体への移行という大きな変化の一部であるように見える。第 2 に、節の使用傾向の変化は単に頻度のみで評価すべきではなく、機能的に類似する節の中での比率で評価する必要がある。例えば『動詞型』はcpsの分析でも増加傾向が見られるが、『テ』と比べるとシェアの増加はより顕著である。逆接の並列節では『ガ』対ケド類、『ケド』対『ケレド』という 2 つの対立があり、『ケレド』は図 1 では増加してから減少しているが、『ケド』と『ケレド』の比率だけで見ればほぼ単調に増加している。こうした節の選択嗜好の変化は、単に個々の節の頻度（あるいはcps）で評価するのではなく、機能的に類似する節の中での相対頻度で評価する必要がある。\n\n加えて、これらの節選択は単に形態の選択の問題ではなく、機能の選択の問題を含んでいる可能性がある。例えば、条件節の諸形式は単に交替可能な形態的バリエーションというわけではなく、それぞれ機能の違いがある。「わからなかったら」は「わからなければ」と交替可能であり、「手をふったら」は「手をふると」と交替可能だが、「六年生になったらもっと頑張ろう」は「なれば」や「なると」と交替することはできない。従って「たら」から「ば」への移行は、単に話し言葉から書き言葉への語彙選択の変化を示しているのではなく、機能の選択の変化を包摂している可能性がある。これらの節の選択傾向をより詳細に分析するためには、どのような機能の節をよく使うかという側面と、類似する機能を持つ節のうちどの形態をよく使うかという側面を考慮しなければならない。\n\n\nデータ可用性\n\nOpen Science Framework: 「児童・生徒作文コーパス」に対する節境界ラベル付与\n\nDOI: https://doi.org/10.17605/OSF.IO/5KCRB （今田, 2020）.\n\nこのプロジェクトは以下の基本データとスクリプトを含む。\n\n• SVMモデル作成 / 節境界ラベル付与\n\n- autoexec.rb （一連の処理を実行するバッチスクリプト）\n\n- cbl2ot.py （BCCWJ-CBLのラベル位置を修正）\n\n- bccwj2yamcha.py （BCCWJ形態論情報とBCCWJ-CBLを重ね合わせてYamCha形式のファイルにする）\n\n- yamcha_fix.py （YamCha出力結果の文字化けを修正）\n\n- cabocha2yamcha.rb （作文コーパスをYamCha形式のファイルにする）\n\n- bunsetsu.rb （統計用基本データbunsetsu.txtを作成）\n\n• 統計処理\n\n- bunsetsu.txt （「児童・生徒作文コーパス」に対して節境界ラベルを付与したタブ区切りテキストファイル、本文および形態論情報を含まない）\n\n- metadata_1.6.txt （「児童・生徒作文コーパスVer.1.6」の統計情報を含むタブ区切りテキストファイル）\n\n- model_test.txt （BCCWJ-CBLによるSVMモデル評価データ）\n\n- statistics.Rmd / html （統計処理と図表の作成に使用したR markdownスクリプト）\n\n以上のデータとスクリプトはクリエイティブ・コモンズ・ゼロの“No rights reserved”著作権放棄条項（CC0 1.0 Public domain dedication）に則って入手することができる。\n\nSVMモデル作成と節境界ラベル付与の一連の処理を実行するには、以下の基本データが必要である。いずれも第三者のライセンスを受けたものであり、入手に制限がある。\n\n• SVMモデル作成\n\n- CORE_NT/core_SUW.txt および CORE_OT/core_SUW.txt （国立国語研究所が有償で提供する「現代日本語書き言葉均衡コーパスVer.1.1 DVD版」に収録されている）\n\n- BCCWJ-CBL_1.0.tsv （BCCWJの利用契約者に限り、https://chunagon.ninjal.ac.jp/ から入手できる）\n\n• 節境界ラベル付与\n\n- 「児童・生徒作文コーパスVer.1.6」CaboCha 形式データ（JSPS科研費JP26285196で構築されたテキストコーパスに形態論情報を付与したもの。本文データの利用がプロジェクト関係者に限り認められているため、形態論情報の利用もそれに準ずる。）\n\n一連のスクリプトの実行は以下の環境で行った。\n\n• YamCha 0.33\n\n• Python 3.8.2\n\n• Ruby 2.7.0p0\n\n• R 4.0.3", "appendix": "参考文献\n\n加納隼人, 佐藤理史: 日本語節境界検出プログラムRainbowの作成と評価. 情報科学技術フォーラム講演論文集. 2014; 13(2): 215–216.\n\n金明哲: 文節の係り受け距離の統計分析. 社会情報. 1996; 5(2): 1–11. Publisher Full Text\n\nKudo T, Matsumoto Y: Fast Methods for Kernel-Based Text Analysis. Proceedings of the 41st Annual Meeting on Association for Computational Linguistics. 2003; 24–31. Publisher Full Text\n\n国立国語研究所: 小学生の言語能力の発達（国立国語研究所報告26）.明治図書.1964. Publisher Full Text\n\nMaekawa K, Yamazaki M, Ogiso T, et al.: Balanced Corpus of Contemporary Written Japanese. Lang Res Eval. 2014; 48: 345–371. Publisher Full Text\n\n松崎史周: 国語教育における「だらだら文」の捉え方と扱い. 日本女子体育大学紀要. 2016; 46, 111–121. Publisher Full Text\n\nMaruyama H, Ogino S: A Statistical Property of Japanese Phrase-to-Phrase Modifications. Mathematical Linguistics. 1993; 18(7): 348–352.\n\n丸山岳彦, 柏岡秀紀, 熊野正, et al.: 日本語節境界検出プログラムCBAPの開発と評価. 自然言語処理. 2004; 11(3): 39–68. Publisher Full Text\n\n丸山岳彦: BCCWJに対する節境界ラベルのアノテーション. 言語処理学会第19回年次大会発表論文集. 2013; 154–157.\n\n丸山岳彦: 現代日本語の多重的な節連鎖構造について: CSJとBCCWJを用いた分析. In: 石黒圭, 橋本行洋[編]: 話し言葉と書き言葉の接点. ひつじ書房. 2014; 93–114.\n\n丸山岳彦, 佐藤理史, 夏目和子: 現代日本語における節の分類体系について. 言語処理学会第 22 回年次大会発表論文集. 2016; 1113–1116.\n\n宮城信, 今田水穂: 『児童・生徒作文コーパス』の設計. 第7回コーパス日本語学ワークショップ予稿集. 2015; 223–232.\n\n宮内佐夜香: 接続助詞とジャンル別文体特徴の関連について: 『現代日本語書き言葉均衡コーパスを資料として』. 国立国語研究所論集. 2012; (3), 39–52. Publisher Full Text\n\n永野賢: 「から」と「ので」とはどう違うか. 国語と国文学. 1952; 29(2): 31–41.\n\n中俣尚己: 日本語並列表現の体系.ひつじ書房. 2015.\n\n日本語記述文法研究会[編]: 現代日本語文法6 複文.くろしお出版. 2008.\n\n小熊利江, 品川直美, 山下直子, et al.: 連体修飾の使用状況に関する一考察. 言語文化と日本語教育. 1998; 16, 70–79. Publisher Full Text\n\nSchleppegrell M: The Language of Schooling: A Functional Linguistics Perspective. 2004; Mahwah, NJ: Lawrence Erlbaum Associates. （石川彰他[訳] : 学校教育の言語: 機能言語学の視点. ひつじ書房. 2017.）\n\n張玉潔, 尾関和彦: 文節間係り受け距離の統計的性質を用いた日本語文の係り受け解析. 自然言語処理. 1996; 4(2), 3–19. Publisher Full Text\n\n\nFootnotes\n\n1 宮城・今田（2015）は学年が上がるにつれて1文あたりの形態素数が増加するとしているが、数値の増減は大きくなく、中学校段階では減少も見られる。\n\n2 形態論情報は現在も整備を継続している。本研究で使用した版（Ver.1.6）では、形態論情報の3分の2程度、係り受け情報の24分の1程度が手作業で修正されている。" }	[ { "id": "89670", "date": "02 Aug 2021", "name": "Naoki Nakamata", "expertise": [ "Reviewer Expertise 日本語学、計量言語学" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nこの論文は、著者が独自に作成した作文コーパスに節境界を自動付与し、児童・生徒の発達に伴って節の使用がどのように変化するかを探ったものです。自動付与の手法は丁寧に説明されており、今後同様の処理を行う際の参考になると思われます。分析結果については、マクロ的な節数の変化ももちろん、機能を同じくする節どうしの間の選好も発達によって変化することを明らかにしたことも、国語教育や言語学において重要な知見であり、価値が認められます。\n以下、ややわかりにくい点、疑問に感じた点を記しますので、適宜参考にしてください。\n①「１．緒言」において、調査項目として、「節ラベル間の相対頻度」が挙げられていますが、わかりにくいです。文に対して、複数の節ラベルを付与するという文脈なので、節Aと節Bの間かと誤解してしまいました。また、２節以降では「節境界ラベルの総体頻度」というように用語が変わっています。用語をもう少し工夫し、統一なさってください。\n②「１．緒言」に「平均的な係り受け距離は、並列節が多いほど大きく、連体節が多いほど小さくなると予想される。」とありますが、連体節が多いと小さくなるというのは機械での係り受け解析になれていない方にはなじみが薄く、少し補足説明があってもよいかと思いました。また、この問題は④の指摘とも関係があります。\n③表 2 節境界ラベルのグループ化において、cbl１、cbl２については下位分類としてよくわかったのですが、cbl3 （接頭辞）、cbl4 （接尾辞）とは何を意味しているのかがわかりませんでした。接頭辞が「文末」というのは理解に苦しみます。作業上、このラベルが必要だったのだろうと思いますが、論文の議論としては、cbl１とcbl2のみで十分に事足りるように感じました。であれば、これらの分類を前面に出す必要はないかもしれません。\n④「４．１等位構造から従位構造へ」の最後の段落。「これは学年が上がるほど係り受け距離の小さい節が増えることを意味しており、従位構造が増えるというcpsの分析結果と一致する。それと同時に、学年が上がるほど係り受け距離が大きい“複雑な”文が増えるとは一概に言えないことを示唆する。」\nこの部分ですが、係り受け距離の定義にもよるが、「係り受け距離が長い」ことのみが複雑度が高いとは言えないのではないでしょうか。今回の分析結果は、学年が上がるほど、距離が小さくなっていますが、それは『引用節』『連体節』『間接疑問節』『補足節』が増えたからだと考えられます。確かにこれらの説は「係り受け距離」は小さいかもしれません。しかし、それは日本語が構造的に埋め込まれた節の主要部が、埋め込み先の名詞の直前に来るということで、表面的な現象であると思います。実際に『引用節』『連体節』『間接疑問節』『補足節』を多用した文は明らかに複雑です。埋め込みの深さなど、認知的負荷は増しています。「係り受け距離が増えるとは言えない」とは言えると思いますが、それをもって「複雑な文が増えるとは一概に言えない」とは言えないのではないでしょうか。\n⑤「ノデ」節の変化は非常に興味深いです。「主観から客観」という議論が行われていますが、これは文体とも関係があるのではないでしょうか。「ので」はより丁寧な形式とされており、丁寧体の文章と相性が良いと思われます。よって、一旦は「ので」が増加するが、作文の文体が発達につれて「です・ます体」ではなくなっていくのにしたがって、「ので」の使用も減少するのでは、と考えました。\n\n本研究は明確かつ正確に提示されたものであり、最新の文献を引用していますか。はい\n\n研究設計は適切で学術的価値がありますか。はい\n\n方法と分析について第三者による再現が可能となるよう十分な詳細が提示されていますか。はい\n\n（該当する場合は要回答）統計分析および解釈は適切ですか。はい\n\n結果の基礎となるソースデータはすべて入手可能で再現性を十全に保証していますか。はい\n\n結論は結果により妥当な裏付けを得ていますか。はい", "responses": [] }, { "id": "90465", "date": "19 Aug 2021", "name": "Yasuhiro Kondo", "expertise": [ "Reviewer Expertise 日本語文法・コーパス言語学" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n本論文は、作文コーパスに機械学習によって節ラベルを付与することを試みている。また、その結果を用いて、児童の学年進行によって、どのように節の種類に変化がみられるかを調査している。以上のように２つの課題からなっているので、査読も２つの部分に分けて示す。\nまず、機械学習（今回はSVM）によって節ラベルを付与する方法については、特に大きな問題はない。このような方法については、言語学的日本語研究においては例が少ないものであるので貴重な成果である。ただし、SVMによる節ラベルの付与の精度のヴァリデーションは、もとのBCCWJについてのテストデータで行っただけである。作文コーパスにおいて、どの程度の精度でそれが実現したかについて、何らかの言及があるとよいと思う（判定の必須条件ではなく、今後の課題あるいは小修正の可能性を示唆する）。\n次に、節の種類と学年進行の関係であるが、おおよそ事前の予想通りの結果が得られており、4節にあるような「等位構造から従位構造」への移行などかなり顕著な事象も観測され、有益である。「話し言葉から書き言葉」への推移とみる考え方にも妥当性がある。今後もこの手法によって、児童作文の分析を行うことのメリットが示されたと考える。\n以上の2点の完成度から、本論文は、適切なものとして公認されるのにふさわしい論文であるとして、承認できる。\nなお、本論文の、SVMモデル作成と節境界ラベル付与の一連の処理を実行するためには、入手に制限のあるデータが必要である。しかし、それらのデータは、第三者のライセンスにより入手できる。\n\n本研究は明確かつ正確に提示されたものであり、最新の文献を引用していますか。はい\n\n研究設計は適切で学術的価値がありますか。はい\n\n方法と分析について第三者による再現が可能となるよう十分な詳細が提示されていますか。はい\n\n（該当する場合は要回答）統計分析および解釈は適切ですか。はい\n\n結果の基礎となるソースデータはすべて入手可能で再現性を十全に保証していますか。一部該当\n\n結論は結果により妥当な裏付けを得ていますか。はい", "responses": [] } ]	1	https://f1000research.com/articles/10-562
https://f1000research.com/articles/10-355/v1	07 May 21	{ "type": "Clinical Practice Article", "title": "Abdominal tuberculosis misdiagnosed as acute surgical abdomen and carcinomatosis", "authors": [ "Edinson Dante Meregildo-Rodriguez", "Rosita Claudia Tafur-Ramirez", "Walter Giovanny Espino-Saavedra", "Sonia Fiorella Angulo-Prentice", "Rosita Claudia Tafur-Ramirez", "Walter Giovanny Espino-Saavedra", "Sonia Fiorella Angulo-Prentice" ], "abstract": "Tuberculosis is a major public health problem worldwide. Tuberculosis can be confused with other diseases and its diagnosis is frequently delayed, especially in areas of low prevalence. We report two cases of young patients who presented preoperatively as acute abdomen due to acute appendicitis. During the intra-operatory these cases were misdiagnosed as “carcinomatosis”, and in the postoperative period these cases were complicated with septic shock. In both cases, histopathology showed caseating granulomas which suggested tuberculous peritonitis and enteritis. Subsequently, RT-PCR in peritoneal fluid confirmed Mycobacterium tuberculosis. In one case the clinical response to treatment was excellent, and the other case was fatal. The aim of this report is to bring attention to the spectrum of tuberculosis, and to serve as a reminder of tuberculosis as the great imitator that can masquerade as cancer. Most tuberculous patients erroneously diagnosed as cancer have extensive “neoplastic” lesions that would suggest an advanced-stage malignancy. Assuming a case as an advanced cancer would reduce the chance of performing more exhaustive studies to get a definitive diagnosis and clinicians would be tempted to offer only palliative treatments.", "keywords": [ "acute abdomen", "gastrointestinal tuberculosis", "tuberculosis", "tuberculous peritonitis", "neoplasm", "cancer" ], "content": "Introduction\n\nTuberculosis (TB) is the most widespread infectious disease and one of the leading causes of death worldwide.1-3 The proportion of patients with extra-pulmonary tuberculosis has risen during the last decades all over the world.1,2 Tuberculosis in Peru ranks fifteenth among the causes of death, and predominantly affects the poorest social strata.2 Abdominal tuberculosis is defined as infection of the peritoneum, omentum, mesentery, lymphatics nodes, and hollow or solid abdominal organs such as the liver, spleen, and pancreas with Mycobacterium tuberculosis.4-7 Abdominal TB accounts for about 5% of all TB cases and 15% of extrapulmonary TB forms and is an increasingly common disease that can have bizarre presentations.8-11\n\n\nCase reports\n\nHerein, we present two cases of abdominal TB mimicking at the presentation an acute abdomen related to surgical aspect of appendicitis, “carcinomatosis”, and septic shock. This report aims to suggest to clinicians the need of including TB in the differential diagnosis of pulmonary and extra-pulmonary malignancies.\n\n\nCase 1\n\nA 32-year-old man presented to the Emergency Department (ED) of Hospital Regional Lambayeque in Peru on July 21, 2019. He had been diagnosed with diabetes mellitus (type 2) two years before, but he did not follow treatment because of financial reasons. He denied risky sexual behavior, smoking, alcoholism, or any previous infectious disease. Furthermore, he denied weight loss, cough, hemoptysis, sweating, diarrhea or constipation. Thirty days before admission, he reported mild colicky hypogastric pain, without irradiation. One week before admission, abdominal pain turned to severe, diffuse, and persistent; and anorexia, general malaise, and premature abdominal fullness after meals were added. Three days before admission, fever and vomiting started.\n\nAt admission, remarkable basic hematology and biochemistry were leukocytosis (15 200/μL), lymphopenia (608/μL), hypoalbuminemia (2.9 g/dL), and a glycated hemoglobin of 6.8 mg/dL. Serologies for human immunodeficiency virus (HIV), human T-lymphotropic virus type 1 (HTLV-1), hepatitis B and C, and rapid plasma reagin/Venereal Disease Research Laboratory (RPR/VDRL), were negative. A chest x-ray (CxR) was normal. An abdominal ultrasound showed free fluid (200 ml) in the pelvic cavity associated with inflammatory signs of mesenteric fat in the right iliac fossa. Acute appendicitis was diagnosed and laparotomy was scheduled. The surgical report described: “multiple and widespread adhesions, linitis plastica, carcinomatosis, hard tumors (0.3-0.5 cm) in the peritoneum, short bowels, and cecum, and multiple abscess in both iliac fossae with purulent and foul-smelling secretion”.\n\nEvolution. One hour after surgery, because of clinical signs of hemodynamic shock, the patient was transferred to the intensive care unit (ICU). At ICU admission the patient was febrile (38.5°C), with hypotension (arterial blood pressure 70/30 mmHg), tachycardia (116 beats per minute), tachypnea (26 breathes per minute) without hypoxia (peripheric saturation 96% in ambient-air), severe dehydration with cold and mottled skin with prolonged capillary refill time, with sleepiness but respondent to verbal stimulus; the abdomen was distended and painful, with reduced intestinal peristalsis, and the wound (8 cm in length) of the recent surgery was macroscopically in order and percutaneous drainages in both iliac fossa were in place showing serohematic secretion. A broad-spectrum antibiotic therapy (meropenem and metronidazole) was started, together with supportive treatment including vasopressors (noradrenaline) and fluid replacement. After a week of the above-mentioned treatment, the clinical condition of the patient was improved and at the seventh day after the surgery, the patient was transferred from the ICU to another hospital division. Then, the result of the histopathological exams of samples collected during surgery showed: “chronic granulomatous inflammation, granulomas, extensive caseous necrosis and Langhans giant multinucleated cells; Ziehl-Neelsen (ZN) stain for Acid Fast Bacilli (AFB) negative”, and a diagnosis of abdominal TB was performed (Figures 1-3). Based on this result quadruple anti-tuberculous treatment (isoniazid 5 mg/kg, rifampicin 10 mg/kg, ethambutol 20 mg/kg, pyrazinamide 25 mg/kg) was initiated in concordance with Peruvian and international guidelines.11,12 Subsequently, the diagnosis was confirmed by a polymerase-chain reaction (PCR) for Mycobacterium tuberculosis in peritoneal fluid. The tuberculin skin test (Mantoux or PPD test) was negative. Serial ZN smears for AFB in urine, sputum, secretion of both pelvic drains, were negative; adenosine deaminase test in peritoneal fluid 292.7 U/L (reference value < 40 U/L). Blood cultures, and cultures for common pathogens, fungi, and M. tuberculosis in urine and peritoneal fluid were negative. Before discharge, the patient was evaluated by an oncologist who suggested some other exams: serum tumor markers (CEA, CA 125, AFP, CA19.9, β-HCG), esophagogastroduodenoscopy, and colonoscopy with ileoscopy, all of which were within normal ranges. In addition, contrast-enhanced chest-abdomen-pelvis computed tomography (CT) and magnetic resonance imaging (MRI) showed “multiple nodular implants in spleen, gut, and peritoneal cavity; but no hepatosplenomegaly or abnormal lymph nodes enlargement” (Figure 4). One week after the start of the antituberculosis treatment, the patient was discharged from hospital because of clinical improvement.\n\n\nCase 2\n\nA 20-year-old man presented to the ED of Hospital Regional Lambayeque on July 18, 2020. He denied any previous disease, but had been incarcerated for almost two years before admission. He presented for six months before admission with fever, productive cough, abdominal pain, nausea and weight loss. One week before admission, he initiated with colicky abdominal pain, nausea, vomiting, and anorexia for three days. One day before admission, abdominal pain turned into intense and diffuse, and fever, persistent vomiting, and absence of passage of flatus and feces were added.\n\nPhysical examination at admission. The patient was emaciated and mildly dehydrated; blood pressure: 100/60 mmHg, respiratory rate: 20 breathes per minute, heart rate 136 beats per minute, temperature: 37.9 °C, and oxygen saturation: 95% at room air. Absent breathe sounds and dullness in both lung bases. Tachycardia. Distended and painful abdomen, bowel sounds diminished in frequency, McBurney’s and Blumberg's signs were positive. Awake and oriented patient, no focal deficit, no meningeal signs.\n\nEvolution. After admission, acute abdomen due to appendicitis was diagnosed. Symptomatic treatment was initiated and preoperative exams and plain chest and abdominal x-ray were ordered, which showed significant bilateral pleural effusion and ascites. Three hours after admission, the patient was transferred to the operating room. During exploratory laparotomy “multiple cerebroid nodules scattered through parietal and visceral peritoneum, small bowel, colon and epiploon; and plenty of ascitic fluid were observed”. The postoperative diagnosis was “carcinomatosis”. After surgery, he was transferred to the ICU, and continued treatment with saline, noradrenaline, imipenem, metronidazole, sedation with midazolam, analgesia with fentanyl, and ventilatory support. Based on clinical data, risk factors, intraoperative findings, and chest x ray imaging, TB was suspected. Then, stains and cultures of multiple samples for Mycobacterium tuberculosis (MTB) and a contrast-enhanced tomographic scan of thorax, abdomen and pelvis were ordered (Figure 5). Three days after surgery thoracocentesis was performed. Five days after surgery RT-PCR resulted positive for MTB in pleural fluid and quadruple anti-tuberculous treatment for MTB was initiated (the same scheme as Case 1). Ten days after surgery histopathology of epiploon was obtained, showing extensive caseous necrosis with granulomas and Langhans multinucleated giant cells. The patient died seven days after initiation of anti-tuberculous treatment due to refractory septic shock (Figure 6). A serial ZN smear for AFB in bronchial secretion, blood cultures, and urine culture, were negative. Serologies for HIV, HTLV-1, hepatitis B and C, and RPR/VDRL, were also negative.\n\n\nDiscussion\n\nAbdominal TB generally presents in four main clinical forms according to the abdominal involvement: enteritis (gastrointestinal), peritonitis, lymphadenitis, and visceral (solid organs) tuberculosis. Most of abdominal TB cases have a combination of these basic forms.5,12,13 Tuberculous enteritis most frequently involves the ileocecal region (80-90% of cases).5,13,14 In our patients we demonstrated enteric and peritoneal involvement.\n\nAbdominal TB occurs by: 1) hematogenous or lymphatic dissemination from a primary lung focus or miliary disease; 2) reactivation from a latent infectious focus; 3) contiguous spread from mesenteric or celiac lymphatic nodes or adjacent organs (i.e. retrograde dissemination from fallopian tubes); 4) direct mucosal invasion by ingestion of tuberculous mycobacteria (ingestion of unpasteurized milk or undercooked meat). Secondary dissemination from a miliary or lung focus occurs in 10-50% of patients, and generally presents with massive involvement of the gastrointestinal tract.5,13-15\n\nApproximately 15-25% of patients with abdominal TB have concomitant active pulmonary TB,1,13-15 more than 50% of patients with abdominal TB patients have some alteration in CxR suggestive of TB,6,13-15 and one third of abdominal TB have evidence of extraperitoneal TB.4,6 This high prevalence of extrapulmonary TB suggests that abdominal TB is a part of disseminated TB. Hematogenous dissemination from a primary lung focus is probably the most important mechanism of peritoneal involvement.6,15 In our patients, we did not find active pulmonary tuberculosis.\n\nAbdominal TB represents a diagnostic challenge and requires a high index of suspicion. Clinical manifestations of abdominal TB are variable and unspecific and can include fever, weight loss, abdominal pain and/or distension, ascites, hepatomegaly, diarrhea, intestinal obstruction, and abdominal mass.5,15 Tuberculosis can be confused with other diseases. The differential diagnosis of abdominal tuberculosis includes abdominal cancers, sarcoidosis and Crohn´s disease. These entities can imitate clinical, radiologic, endoscopic, and even laparoscopic features of entero-peritoneal tuberculosis. Thus, it is of paramount importance to take biopsies.5,15-18 Direct biopsy of suspicious lesions by laparoscopy or laparotomy is the most certain method to obtain diagnostic samples.4-6\n\nHistopathology (necrotizing or caseous granulomas and/or AFB) is highly suggestive and virtually diagnostic of tuberculosis, but it is not specific or pathognomonic. However, in the right clinical and epidemiological context, typical findings can support a presumptive diagnosis of TB1,6,14-16 as occurred in our patients. Hepatic biopsy has a sensitivity of 68% for caseating granulomas in hepatic biopsies.15,18 But, among patients with colonic tuberculosis, only 32.6% have caseous granulomas, and 5% have AFB in colonic biopsies.18\n\nA definitive diagnosis of abdominal TB is made by demonstration of Mycobacterium tuberculosis (in peritoneal fluid or involved organ) by culture and/or PCR. The sensitivity of AFB smear and mycobacterial culture for ascitic fluid is low (<2% and <20%, respectively); whereas, the sensitivity of AFB smear and mycobacterial culture for biopsy specimens is in general <50%.1,16 PCR is more sensitive and specific for diagnosis of TB than AFB smear or mycobacterial culture, but the diagnostic yield of PCR varies depending on the tissue type. The sensitivity and specificity are high for peritoneal fluid and pancreatic and hepatic tissue, but intestinal tissue may be associated with false-positive PCR results.1,15\n\nAbdominal tuberculosis is the great mimicker of abdominopelvic pathology.4,8-10 There exist a few reports of cases initially diagnosed as cancer that subsequently were proved to be tuberculosis.8-10 In a study with 170 patients referred to the National Institute of Neoplastic Diseases of Peru because of a presumed diagnosis of cancer, the final diagnosis was extrapulmonary TB in 77.7% of cases, and pulmonary TB in the 22.3% remaining. Among these 170 patients, three patients were admitted with diagnosis of gastrointestinal cancer and 17 with ovarian cancer; and entero-peritoneal TB was finally diagnosed in nine, and genitourinary TB in seven patients. Despite extrapulmonary TB accountings for up to 20-25% of TB cases in non-HIV infected patients, it is the most common localization that could mimic cancer, probably due to the fact that solid lesions such as tuberculomas —more common in not severely immunocompromised patients— tend to be more frequently confused with malignancies. Besides, in 63.5% of patients the extension of the “neoplastic” lesions would suggest an advanced-stage disease.8 This interpretation could have profound implications in the management of these patients, because assuming a case as an advanced-stage cancer would reduce the chance of performing more exhaustive studies to get a definitive diagnosis. Consequently, most of these patients who are considered erroneously to have advanced cancer are offered only palliative care. Conversely, there exist reports of cancers mistakenly diagnosed and treated as tuberculosis.15\n\nPrompt diagnosis and treatment can reduce morbidity and mortality, and additionally can limit tuberculosis dissemination especially if there exist concomitant pulmonary disease. Almost all abdominal TB cases offered proper treatment result in complete remission and prognosis is excellent. Urgent surgery is only necessary in a minority of cases, if there is obstruction, ischemia, perforation, massive bleeding, or secondary peritonitis.4,15,16\n\nThese cases are enlightening and worthy of reflection because of several reasons: 1) the rare presentation of the clinical picture as an acute surgical abdomen, septic shock, and “carcinomatosis”; 2) the absence of other causes of immunosuppression, except for diabetes in one case; 3) although, histopathology did not establish a definitive diagnosis, it did provide important diagnostic clues to support a presumptive diagnosis and initiation of empiric antituberculosis treatment, until TB was confirmed subsequently by RT-PCR.\n\nIn conclusion, these cases exemplify that we must employ our clinical judgement in conjunction with all diagnostic tools available in order to make an accurate diagnosis within the shortest possible time in patients with a clinical picture suggestive of malignancy. We must be aware that TB can mimic cancer, but the opposite is also true. In consequence, clinicians should always consider TB among the differential diagnosis of malignancy, especially in high endemicity regions or countries with high rates of immigrants.\n\n\nData Availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nInformed Consent: The patients provided written consent to the publication of this clinical practice article and pictures. This study was approved by the Ethics and Research Committee of Hospital Regional Lambayeque (approval number: 0321-095-20 CEI).\n\nDeclaration of Helsinky: The Authors have respected the Declaration of Helsinki as a statement of ethical principles for medical research involving human subjects, including research on identifiable human material and data.", "appendix": "References\n\nFitzgerald DW, Sterling TR, Haas DW: Mycobacterium tuberculosis. In: Bennett JE, Dolin R, Blaser MJ, editor. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 8th Ed. Philadelphia: Elsevier Saunders; 2014. p. 2787–2817.\n\nAlarcón V, Alarcón E, Figueroa C, et al.: Tuberculosis en el Perú: situación epidemiológica, avances y desafíos para su control. Rev. Perú. Med. Exp. Salud Publica. 2017; 34(2): 299–310. Publisher Full Text\n\nReid MJA, Arinaminpathy N, Bloom A, et al.: Building a tuberculosis-free world: The Lancet Commission on tuberculosis. Lancet. 2019; 393(10178): 1331–1384. PubMed Abstract \| Publisher Full Text\n\nDebi U, Ravisankar V, Prasad KK, et al.: Abdominal tuberculosis of the gastrointestinal tract: revisited. World J Gastroenterol. 2014; 20(40): 14831–14840. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanai FM, Bzeizi KI: Systematic review: tuberculous peritonitis —presenting features, diagnostic strategies and treatment. Aliment Pharmacol Ther. 2005; 22(8): 685–700. PubMed Abstract \| Publisher Full Text\n\nBorhanmanesh F, Hekmat K, Vaezzadeh K, et al.: Tuberculous peritonitis. Prospective study of 32 cases in Iran. Ann Intern Med. 1972; 76(4): 567–72. PubMed Abstract \| Publisher Full Text\n\nSabooni K, Khosravi MH, Pirmohammad H, et al.: Tuberculosis peritonitis with features of acute abdomen in HIV infection. Int J Mycobacteriol. 2015; 4(2): 151–3. PubMed Abstract \| Publisher Full Text\n\nVillena-Suarez JR, Vicente W, Taxa L, et al.: Tuberculosis que imita cáncer: casos derivados al Instituto Nacional de Enfermedades Neoplásicas, Lima-Perú. Rev Peru Med Exp Salud Publica. 2018; 35(1): 77–83. Publisher Full Text\n\nPitlik SD, Fainstein V, Bodey GP: Tuberculosis mimicking cancer —a reminder. Am J Med. 1984; 76(5): 822–825Publisher Full Text .\n\nIbarra GF, Ibarra IGA: Tuberculosis Intestinal Simulando Cáncer de Colon: Reporte de Caso y Revisión. Rev Cient Cienc Méd. 2016; 19(1): 52–59.\n\nMeregildo-Rodríguez ED, Méndez-Florián KJ, Espino-Saavedra WG: Duodenal obstruction and upper gastrointestinal bleeding as the initial presentation of an isolated duodenal tuberculosis. Rev Chilena Infectol. 2019; 36(3): 387–391. PubMed Abstract \| Publisher Full Text\n\nNahid P, Dorman SE, Alipanah N, et al.: Official American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America Clinical Practice Guidelines: Treatment of Drug-Susceptible Tuberculosis. Clin Infect Dis. 2016; 63(7): e147–e195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKgomo MK, Mwantembe O: Abdominal Tuberculosis in Sub-Saharan Africa (SSA). In: Segal I. editor. Digestive Diseases in Sub-Saharan Africa: Changes and Challenges. 1st Ed. India: Academic Press; 2014; p. 115–118.\n\nAbu-Zidan FM, Sheek-Hussein M: Diagnosis of abdominal tuberculosis: lessons learned over 30 years: pectoral assay. World J Emerg Surg. 2019; 14: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBae KM, Lim SC, Kim HH, et al.: Possible misdiagnosis of HIV associated lymphoma as tuberculosis among patients attending Uganda Cancer Institute. AIDS Res Ther. 2017; 14(1): 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBae KM, Lim SC, Kim HH, et al.: The relevance of biopsy in tuberculosis patients without human immunodeficiency virus infection. Am J Trop Med Hyg. 2015; 92(3): 636–640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlvares JF, Devarbhavi H, Makhija P, et al.: Clinical, colonoscopic, and histological profile of colonic tuberculosis in a tertiary hospital. Endoscopy. 2005; 37(4): 351–6. PubMed Abstract \| Publisher Full Text\n\nHickey AJ, Gounder L, Moosa MY, et al.: A systematic review of hepatic tuberculosis with considerations in human immunodeficiency virus co-infection. BMC Infect Dis. 2015; 15: 209. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "85112", "date": "01 Jun 2021", "name": "Uzair Yaqoob", "expertise": [ "Reviewer Expertise Surgical and public health 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\nGeneral comments:\nThe authors describe two cases of abdominal tuberculosis, initially presenting as acute abdomen due to appendicitis, resembling carcinomatosis intraoperatively, and later diagnosed as tuberculosis on investigations. I have a few suggestions for revision:\n\nSpecific comments:\nAbstract:\nI do not think \"intra-operatory\" is the correct word.\n\nAbstract is starting with tuberculosis in general and there is no transition to abdominal tuberculosis.\n\nIntroduction:\nThe following sentence should be split into 2 or 3 sentences \"At ICU admission the patient was febrile (38.5°C), with hypotension (arterial blood pressure 70/30 mmHg), tachycardia (116 beats per minute), tachypnea (26 breathes per minute) without hypoxia (peripheric saturation 96% in ambient-air), severe dehydration with cold and mottled skin with prolonged capillary refill time, with sleepiness but respondent to verbal stimulus; the abdomen was distended and painful, with reduced intestinal peristalsis, and the wound (8 cm in length) of the recent surgery was macroscopically in order and percutaneous drainages in both iliac fossa were in place showing serohematic secretion.\"\n\n\"diagnosis of abdominal TB was performed\" - I do not think 'performed' is the correct verb here.\n\nDiscussion:\nNeeds more of the text relating literature with the two presented cases.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [] }, { "id": "85111", "date": "29 Jun 2021", "name": "Virgilio Efrain Failoc Rojas", "expertise": [ "Reviewer Expertise Medicine clinical and 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\nIt is an interesting case from an academic point of view. Two cases start as acute abdominal pain due to appendicitis, resembling intraoperative carcinomatosis, and later diagnosed as tuberculosis.\nI have some suggestions:\n\nI suggest that the sentences of the evolution of the case, both the first and second, should be structured in two or three paragraphs.\n\nIn the discussion I suggest highlighting a little more the fact that tuberculosis is the great imitator and in such an endemic country it should be sought in patients that in the face of a bad evolution this pathology should be sought, as it could be economically beneficial (Foster et al., 20211).\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-355
https://f1000research.com/articles/10-557/v1	12 Jul 21	{ "type": "Study Protocol", "title": "Musculoskeletal disorders in basketball players and associated factors: a systematic review protocol with meta-analysis", "authors": [ "Silvia Cristina de Carvalho Borges", "Michele da Silva Valadão Fernandes", "Priscilla Rayanne e Silva Noll", "Vinicius Diniz Azevedo", "Regina Márcia Ferreira Silva", "Carolina Rodrigues Mendonça", "Matias Noll", "Michele da Silva Valadão Fernandes", "Priscilla Rayanne e Silva Noll", "Vinicius Diniz Azevedo", "Regina Márcia Ferreira Silva", "Carolina Rodrigues Mendonça" ], "abstract": "Introduction: Musculoskeletal disorders play an important role in athletes’ health and success in sports; they are the main contributors to disability, and back pain is a common problem in athletes. Back pain and spinal injuries occur in players because of the repetitive and unilateral body overload during sports practice. Reviews focused on musculoskeletal disorders in basketball players are scarce in the literature. We describe a systematic review protocol for assessing the prevalence of musculoskeletal disorders and the associated factors in basketball players. Methods and analysis: The protocol will be based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) items and articles will be searches in the databases EMBASE, PubMed, and Scopus. Studies will be included if they are written in English and without restriction regarding the search period. All research stages will be performed by two independent reviewers and will be conducted using Rayyan. Potential disagreements will be analyzed by a third reviewer. The inter-rater reliability will be determined through the Cohen's kappa coefficient. Evidence quality will be ascertained by using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) system, while the Downs and Black checklist will be used for assessing the risk of bias. A descriptive summary and presentation of the characteristics and findings of all included studies will be provided in a table. Regarding these data, if there are sufficient high-quality studies, we plan to perform a meta-analysis. Discussion: The obtained results might possibly relate back pain to postural changes in this population. In addition, the data will be compared based on gender and different age groups. It is expected that the results can support the implementation of programs and actions for prevention, ultimately aiming at improving health, quality of life, and sports performance. Registration: PROSPERO CRD42020201653 on August, 31, 2020.", "keywords": [ "back pain", "postural deviations", "athletes", "basketball" ], "content": "Introduction\n\nMusculoskeletal disorders and injuries are prevalent throughout life and account for more than 150 diagnoses concerning the locomotor system.1,2 These conditions are typically characterized by pain and limitations in mobility, dexterity, and functional capacity. Musculoskeletal disorders are the main contributors to disability worldwide,3 with low back pain being the primary global cause.1,4 Furthermore, one in five people suffers from musculoskeletal pain.1\n\nPain is an important warning signal for the body that something is wrong,5 and back pain is a common problem in athletes.6-9 According to a study of 590 basketball athletes, 12.9% reported low back pain.10 In many cases, pain can even prevent subjects from performing daily activities.1 In elite basketball players aged 13-19 years, the prevalence of back pain (49% vs. 28%) and neck pain (42% vs. 20%) is higher in women than men.11 The high prevalence of neck pain and low back pain in basketball players can lead to an increased risk of spinal cord injuries.12\n\nOn the other hand, lumbar spine injuries are also common in basketball players.13 Players of the National Collegiate Athletic Association (NCAA) often have lumbar spine injuries which might cause chronic pain.14 These injuries account for 11.4% and 13.5% of all injuries in competition and practice, respectively, in college basketball athletes,13,15 and for 10.2% of all injuries in the National Basketball Association (NBA) over 17 years.8 Postural deviations play an important role in the occurrence of back pain16 and are common in sportsmen due to the repetitive and unilateral body overload during sports practice.17,18\n\nConsidering the above, it is of great importance to know the prevalence of back pain and postural changes in basketball players, since they can affect athletic capacity of athletes by limiting mobility. The hypothesis is that basketball, as an asymmetric sport requiring a repetitive and unilateral overload, such as throwing and repeating each training day, may cause injuries and/or postural changes. Despite the importance of this topic, studies focused on back pain and postural changes in basketball players are scarce in the literature. In this context, the objective of this study was to address the following questions: What is the prevalence of back pain and postural deviations in basketball players? What are the associated factors? Therefore, this article describes a systematic review protocol aimed at assessing the prevalence of musculoskeletal disorders in basketball players and associated factors. We expect that the results of this review can support the implementation of programs and actions for injury prevention or pain treatment, ultimately aiming at improving health, quality of life, and sports performance. This study is both valuable for sports’ and players’ health.\n\n\nMethods\n\nThis study is a systematic review protocol on postural changes and back pain in basketball players. The review will follow the structure of the PECO (Population, Exposure, Comparator, and Outcome) as explained in the recommended systematic review notification items.19 Therefore, “P”, “E”, “C”, and “O” represent athletes, basketball, gender and age, and prevalence of back pain and postural changes in basketball players, respectively. In this context, back pain will be defined as pain in the cervical, thoracic and/or lumbar areas.20–22 Postural changes were considered to have been caused by scoliosis, kyphosis, lordosis and more.\n\nThis systematic review protocol will follow the PRISMA (preferred reporting items for systematic reviews and meta-analyses) methodology23 and the MOOSE (meta-analysis of observational studies in epidemiology) Guidelines.24 The protocol has been registered on PROSPERO (CRD42020201653) on August, 31, 2020. Any changes made to this protocol throughout the study will be reported in PROSPERO and in the final article.\n\nSystematic searches will be conducted in the Scopus, PubMed, and EMBASE databases in November 2020 by two independent reviewers (S.C.C.B. and M.S.V.F.). Other manual article searches will be conducted by checking the references of each study eligible for inclusion, as well as searches in Google Scholar.\n\nStudies with the following characteristics will be eligible: a) involving players of both genders aged up to 50 years; b) observational studies (longitudinal and cross-sectional, cohort and case-control), c) musculoskeletal disorder assessments in players (back pain and postural deviations); d) publications in English; and e) studies with players in international communities.\n\nStudies will be excluded under the following criteria: a) studies with basketball players with injuries on other regions of the body (knee, shoulder, hip, ankle); b) Paralympic athletes and/or players with physical or mental disabilities; c) samples including mixed sports, except when data on basketball players are presented separately or can be calculated; and d) experimental studies.\n\nThis protocol will include full published articles without restrictions on the search period, and will exclude books, book chapters, case reports, comments, letters, editorials, and duplicate articles. For those articles where the full text cannot be retrieved in online databases, the articles will be requested to libraries or to the study authors via email.\n\nThe searches will be adapted according to database. The following search term blocks will be used: ‘musculoskeletal disorders’, ‘athletes and basketball’. The following standard search strategy will be used:\n\nIn an independent and consensual manner, two reviewers (S.C.C.B. and M.S.V.F) will conduct the search, selection, and evaluation of study quality. A third reviewer (M.N.) will be consulted in cases of disagreement. The retrieved articles from the databases will be grouped using Mendeley.25 After removing duplicate articles, the initial screening will be performed by reading titles and abstracts using Rayyan,26 excluding those not meeting the eligibility criteria. The remaining articles will be read in full and evaluated for determining their eligibility based on inclusion and exclusion criteria. Lastly, eligible articles will be included in the systematic review. The inter-rater reliability for classifications of individual components will be determined by calculating the percentage of agreement and the Cohen's kappa coefficient.27 The flowchart of this systematic review is depicted in Figure 1.\n\nThe authors assessing the eligibility criteria are being trained on the study's inclusion/exclusion criteria and will complete a practice eligibility assessment of 50 test abstracts before starting the review. The authors will also complete a training on risk of bias assessment tools in five non-included articles, in addition to standardized analyses using Mendeley and Rayyan.\n\nOnly included studies will be submitted to data extraction by two reviewers (S.C.C.B. and M.S.V.F.), in an independent and consensual manner. Using a standard data extraction spreadsheet, the following information will be extracted: study characteristics (author, year of publication, nationality, study design, and sport); participant characteristics (gender, age group) and results (prevalence, incidence, and assessment tools for back pain and postural changes; and relationship between back pain and postural changes). If data are insufficient, the study authors will be contacted to enquire on availability of complete data.\n\nThe included articles will be evaluated considering the evidence quality and the methodological quality following the recommendations of GRADE (grading of recommendation, assessment, development and evaluation) and the Downs and Black checklist. GRADE is a methodology implemented for providing transparency and simplicity, since it classifies evidence quality in four categories: very low quality, low quality, moderate quality, and high quality. It is a system widely adopted by organizations worldwide for its rigorous methodological classification and ease of use.28\n\nAn adapted version of the Downs and Black29 checklist, proposed by Noll,30 will also be employed. The Downs and Black checklist has been one of the two most employed tools by systematic reviewers in PROSPERO registered reviews from 2011 to 2018.31 The studies will be scored, allowing the identification of those with methodological quality, by considering the following five aspects: presentation, external validity, internal validity - bias, internal validity – confusion, and statistical power for inferences.\n\nThe risk of bias will be assessed independently by two reviewers. For the data synthesis strategy, the data prevalence found in the studies will be used. In addition to the proposed primary analyses, if sufficient barriers are identified, a comparison between genders and different age groups can be made either in groups or subgroups.\n\nA descriptive summary and presentation of the characteristics and findings of all included studies will be provided in a table. For general group analysis, the results on prevalence and associated factors will be presented. When possible, differences between genders and age groups will be compared.\n\nRegarding these data, if there are sufficient high-quality studies, we plan to perform a meta-analysis.32 Firstly, we will analyze differences in the prevalence of back pain and postural changes between genders and age groups. Secondly, if possible, we will carry out a meta-analysis with data on the associated factors. The percentage of variability attributable to heterogeneity will be estimated with the I2 test. I2 values <40, 40-60, 60-90 and> 90% correspond, respectively, to levels of “unimportant”, “moderate”, “substantial” and “considerable” heterogeneity.32\n\nThirdly, publication bias will be assessed using the Egger test33 and funnel plot. The meta-analysis will be performed using STATA 16.0 (StataCorporation, College Station, TX, USA) with a 95% confidence interval. Publication bias will be assessed by visual inspection of a funnel chart. We plan to present a meta-analysis for back pain and another for postural changes. If meta-analyses are not appropriate, the data will be summarized in a descriptive manner using the synthesis of best evidence.32\n\nThis systematic review is based on the analysis of published articles (secondary data) and does not require approval by an ethics committee. Upon completion, the systematic review will be sent for publication in a peer-reviewed journal.\n\nThis study is currently at the stage of study selection and data extraction.\n\n\nDiscussion\n\nThis systematic review protocol will search for publications on postural changes and back pain in basketball players. It will also analyze the results to relate back pain and postural changes in this population. Moreover, the data will be compared according to gender and age. It is expected that the results can support the implementation of programs and actions for preventing the injuries caused by postural changes and eventually lead to back pain, ultimately aiming at improving health, quality of life, and sports performance.\n\nSine limitations may be expected. First, various measurements and tools might lead to varying levels of musculoskeletal disorders and outcomes. Second, a lack of clarity in the definition of musculoskeletal disorders might complicate the comparisons. Third, if no study meets our eligibility criteria, the review will be reported as an “empty review”. However, even an “empty review” is relevant and might help stimulate appropriate future investigations.34\n\nThe strengths of this study include assessment of a wide range of musculoskeletal disorders, absence of year restrictions, evaluation of quality and the risk of bias analysis. This systematic review will provide critical insights into the prevalence and associations between musculoskeletal disorders in basketball players that may be important for supporting development of clinical practice guidelines and health programs.", "appendix": "Acknowledgments\n\nWe thank the Federal University of Goiás and Institute Federal Goiano for their support.\n\n\nAuthors’ contributions\n\nS.C.C.B., M.S.V.F., P.R.S.N., R.M.F.S., V.D.A., C.R.M. and M.N. conceived the idea of the study and contributed to the design of the research. All authors contributed to the writing, editing, and approval of the final protocol.\n\n\nReferences\n\nWHO: Musculoskeletal Condition [Internet].World Health Organization; 2019 [cited 2021 Dec 15]. Reference Source\n\nVos T, Abajobir AA, Abbafati C, et al.: Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet. 2017; 390(10100). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShahrezaee M, Keshtkari S, Moradi-Lakeh M, et al.: Burden of musculoskeletal disorders in Iran during 1990–2017: estimates from the Global Burden of Disease Study 2017. Arch Osteoporos. 2020; 15(1). PubMed Abstract \| Publisher Full Text\n\nBlyth FM, Briggs AM, Schneider CH, et al.: The global burden of musculoskeletal pain—where to from here? Am J Public Health. 2019; 109(1). 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeynon AM, Hebert JJ, Lebouef-Yde C, et al.: Potential risk factors and triggers for back pain in children and young adults. A scoping review, part II: Unclear or mixed types of back pain. Chiropr Man Therap. 2019; 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeynon AM, Hebert JJ, Hodgetts CJ, et al.: Chronic physical illnesses, mental health disorders, and psychological features as potential risk factors for back pain from childhood to young adulthood: a systematic review with meta-analysis. Eur Spine J. 2020; 29. PubMed Abstract \| Publisher Full Text\n\nNoll M, Wedderkopp N, Mendonça CR, et al.: Motor performance and back pain in children and adolescents: A systematic review and meta-analysis protocol. Syst Rev. 2020; 9(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: The PRISMA statement. PLoS Med. 2009; 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStroup DF, Berlin JA, Morton SC, et al.: Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group. JAMA. 2000 Apr; 283(15): 2008–2012. PubMed Abstract \| Publisher Full Text\n\nRuiz D: Manual de Mendeley 2020. Biblioteca Universitaria Universidad de Murcia. 2020: 1–23.\n\nOuzzani M, Hammady H, Fedorowicz Z, et al.: Rayyan-a web and mobile app for systematic reviews. Syst Rev. 2016; 5(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStefani L, Galanti G, Padulo J, et al.: Sexual activity before sports competition: A systematic review. Front Physiol. 2016; 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuyatt GH, Oxman AD, Vist GE, et al.: GRADE: An emerging consensus on rating quality of evidence and strength of recommendations. BMJ. 2008; 336(7650). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDowns SH, Black N: The feasibility of creating a checklist for the assessment of the methodological quality both of randomised and non-randomised studies of health care interventions. J Epidemiol Community Health. 1998; 52(6). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoll M, De Mendonça CR, De Souza Rosa LP, et al.: Determinants of eating patterns and nutrient intake among adolescent athletes: A systematic review. Nutr J. 2017; 16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarrah K, Young K, Tunis MC, et al.: Risk of bias tools in systematic reviews of health interventions: an analysis of PROSPERO-registered protocols. Syst Rev. 2019; 8(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins J, Chandler J, Cumpston M, et al.: Cochrane Handbook for Systematic Reviews of Interventions. Cochrane Handbook for Systematic Reviews of Interventions. 2019.\n\nLin L, Chu H: Quantifying publication bias in meta-analysis. Biometrics. 2018 Sep; 74(3): 785–794. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYaffe J, Montgomery P, Hopewell S, et al.: Empty reviews: a description and consideration of Cochrane systematic reviews with no included studies. PLoS One. 2012; 7(5): e36626. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCristina de Carvalho Borges S: PRISMA-P-checklist.doc. figshare. Figure. 2021. Publisher Full Text" }	[ { "id": "153377", "date": "24 Oct 2022", "name": "Gopal Nambi", "expertise": [ "Reviewer Expertise Musculoskeletal and Sports physiotherapy." ], "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 giving me this opportunity to review this article. The article is well written, though I have some concerns regarding the article. Use the mesh keywords.\nAbstract:\nThe objective or hypothesis of the study is not clear.\n\nInclude the character of the study articles.\n\nMention the outcome measures measured for the review.\nManuscript\nThe research question or the gap was not formulated properly with suitable recent references.\n\nThere are many recent similar articles in MEDLINE and PubMed databases, please justify the need for this review.\n\nThe database searched for the review is much limited.\n\nMention the duration of the data search.\n\nThe selection criteria are not specific – include exclusion criteria.\n\nInclude the outcome measures analyzed for the review.\n\nMention the data extraction procedure in detail.\n\nMention the software used for the statistical analysis.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "153379", "date": "07 Nov 2022", "name": "Antimo Moretti", "expertise": [ "Reviewer Expertise rehabilitation and sport medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, authors performed a systematic review protocol with meta-analysis to investigate musculoskeletal disorders in basketball players and associated factors.\nGeneral comments: There are several limitations which make the paper unsuitable for indexing in the present form. Better identification of the suitable population must be made (sportsmen, non-agonists, or amateurs.) as well as the age range to be considered. In the introduction, the authors could add epidemiological data about musculoskeletal injuries among the athletic population citing doi: 10.3390/medicina571011181. Most of the references cited referred to adolescents so maybe the age characteristics of the population included must be reviewed.\n\"Introduction\"\nThe first sentence is not clear what the authors mean for \"150 diagnoses\"? Among basketball players?\n\nThe hypothesis that the authors supported is not justified or supported by proper scientific literature, particularly about the basket as an “asymmetric sport requiring a repetitive and unilateral overload”\n\nIn the second question \"What are the associated factors ?\", what do the authors mean for these? Please describe\n\n\"This study is both valuable for sports' and players' health\": There are two populations exposed to different risk factors (training intensity and frequency of competitions) and therefore the evidence and results are not overlapping.\n\"Methods\"\n\nSeveral methodological limitations, such as inclusion and exclusion criteria, detailed data extraction, and range of data search.\n\nThe methodological criteria are not reproducible and verifiable by third parties.\n\n\"Postural changes were considered to have been caused by scoliosis, kyphosis, lordosis and more\": do the authors mean that scoliosis is a consequence of basketball?\n\n\"Studies with the following characteristics will be eligible: involving players of both genders aged up to 50 years; \": The age cut-off is too high, so specify if you want to study sub-populations of professional competitive sportsmen, non-agonists, or amateurs.\n\"References\"\nReference no. 1 seems not specific, replace it with a precise one. References 22 and 27 maybe are not appropriate with text contents.\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-557
https://f1000research.com/articles/10-79/v1	08 Feb 21	{ "type": "Research Article", "title": "A pilot study of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, evaluated by MinION platform", "authors": [ "Boy M. Bachtiar", "Citra F. Theodorea", "Dicky L. Tahapary", "Cindy Astrella", "Natalina -", "Endang W. Bachtiar", "Boy M. Bachtiar", "Citra F. Theodorea", "Dicky L. Tahapary", "Cindy Astrella", "Natalina -" ], "abstract": "Background: Subgingival niche is one biofilm habitat containing rich microbiota, which plays an active role in maintaining the health of periodontal tissue and determining host response. As such, a study of changing subgingival biofilms is important for understanding the effect of a systemic condition. In this study, we compared the occurrence of six bacteria cohabiting in the subgingival area of periodontitis subjects, with (DP, n = 8) and without (NDP, n = 4) diabetes. Methods: The six genus and species of targeted bacteria were confirmed by 16S rRNA amplicon sequencing on MinION nanopore platform. Descriptive statistic was used to describe the obtained data. Results: We found that the six genus and species of targeted bacteria were detected but in different quantities in either group's periodontal pocket. Our data showed that Tannerella forsythia was the most abundant species in subgingival biofilms of the DP group of the red complex bacteria. In contrast, Aggregatibacter sp., which belongs to the phylum of proteobacteria, was present at a relatively lower level. In contrast, Fusobacterium sp., which belongs to orange complex bacteria, showed relative similarities in subgingival biofilms of both groups tested, while Veillonella sp., were abundant in the DP groups. Conclusions: Our data show that the diversity of classic periodontopathogens increased in the subgingival niche of periodontitis subjects with diabetes. It is the first study in Indonesia to apply MinION-based, full-length 16S rRNA amplicon sequencing in periodontitis patients with and without diabetes.", "keywords": [ "MinION", "16S rRNA", "Red Complex bacteria", "Diabetes", "Periodontitis", "Subgingival Microbiome" ], "content": "Introduction\n\nTaking DNA straight from oral samples, without culture samples, is a fundamental principle of oral microbiome study. Currently used molecular methods generally rely on PCR, which can be used to target specific bacterial species. However, detection is only enabled for those that have primers. PCR can also detect all bacterial species using a broad range of 16S primers followed by sequencing, but problems can arise from contamination1.\n\nThe advancement of sequencing technology has led to the mainstream in oral microbiology due to the increasing affordability and improvement in the speed of the sequencing process and quality of data obtained. In this context, MinION is the first sequencer available commercially that uses nanopore technology. Unlike other sequencing technology, the sequencing method provided by the MinION device does not rely on the synthesis of nucleotides. For this reason, we decide to use this long-read nanopore sequencing device to identify the dominant bacteria in diabetic condition-related periodontitis.\n\nOne of the systemic conditions that have been asserted to affect the host immune response to dental plaque is diabetes2,3. However, the effect of diabetes on the composition of the subgingival microbiome is still inconclusive4. A previous study showed that an increased number of periodontal pathogens had been isolated from the subgingival plaque of diabetic patients5. However, another study showed a decrease of bacterial diversity associated with periodontitis6, indicating a putative role of specific oral bacteria species in the oral niche that might be correlated to the condition due to diabetes7.\n\nAmong the subgingival microbial community of periodontitis, the red complex bacteria (Phorphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) have been considered as major periodontal pathogens8,9. Their presence in the subgingival environment indicates that a selected bacteria species occurs due to the suitable anaerobic microenvironment that has been formed10. However, these red complex bacteria are usually preceded by members of other complex oral bacteria species, including those associated with a healthy periodontal pocket11. This indicates that ecological stress has occurred, leading to the presence of periodontitis-related microorganisms leading to an imbalance of bacteria species in the dental plaque10. Consequently, in subgingival samples, the red complex bacteria could be measured as multiple oral bacteria species due to their role in progressive periodontitis.\n\nThe main goal of the current study was to use MinION of the full-length 16S rRNA gene to compare the profile of red complex bacteria and three other genera (Aggregatibacter, Fusobacterium, and Veillonella) in two different subgingival niches of periodontitis, subjects with (DP) and without (NDP) diabetes, in order to assess their microbiome.\n\n\nMethods\n\nThis study was conducted between November 2018 and early June 2019. Twelve patients, 20 to 50 years of age, from consecutive participants were recruited from the Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital (FKUI-RSCM), Jakarta, Indonesia. The Ethics Committee of FKUI-RSCM has approved this study's protocol (No.1062/UN@.F1/ETIK/2018). The investigation procedure has been conducted according to the Declaration of Helsinki. Written informed consent was obtained from each subject to participate in this study, and all participants had not received periodontal treatment or taken antibiotics during the last three months.\n\nSubjects excluded from this study were those who (i) had systemic disease other than diabetes mellitus; (ii) had received periodontal treatment or had taken antibiotic within the previous three months; (iii) and were smokers or pregnant. Participants in this study were those with periodontitis complicated by diabetes (DP, n = 8) and non-diabetes (NDP, n = 4), and diabetes criteria were determined from their medical record.\n\nAll patients consented to provide a subgingival dental plaque for this study. All subjects were diagnosed for periodontitis or healthy periodontal tissue according to criteria described by the American Academy of Periodontology12. Subgingival dental plaque was collected by a registered dentist using sterile periodontal scalers and placed in individual Eppendorf tubes containing phosphate-buffered saline (PBS) buffer (pH 7.4). Samples were stored at -20°C until further processing. Extraction of DNA was performed using a DNA extraction kit Qiagen Q1Amp1 DNA Mini Kit, as per the manufacturer’s instruction. The obtained DNA’s concentration and quality were determined using Qubit assay reagents (Invitrogen; Carlsbad, CA, USA). After dissolving in Tris-EDTA buffer, the DNA was cooled to -20°C for further analysis.\n\nThe effects and efficiency of pooling samples have been investigated in many studies13,14. Therefore, in this study, we analysed pooled samples of subgingival biofilm of the two group patients with periodontitis for shifts in the subgingival community in response to a diabetic condition.\n\nSubgingival biofilm samples were taken from three sites in DP subjects and three periodontal pockets in NDP subjects. The presence at genus and species level of the red complex bacteria (Phorphyromonas gingivalis, Treponema denticola, and Tannerella forsythia), and three other selected genera (Aggregatibacter, Fusobacterium, and Veillonella) were determined in subgingival biofilm samples.\n\nBacterial subgingival biofilm was collected from one diseased site (when present) with probing depth ≥5 mm with bleeding on probing. The collection area was isolated with cotton rolls, and a supragingival plaque was carefully removed with curettes. The collection was done with a sterile endodontic paper point by inserting the point to the depth of the sulcus and moving it laterally along the tooth's axis. Immediately following sampling, the paper point was placed in an Eppendorf tube and stored at -70°C until processed.\n\nWe tested DNA barcoding using the MinIONTM on two different sets of samples. The samples A and sample B comprised of PCR products from 8 (periodontitis patients with diabetic/DP) and 4 (periodontitis patients without diabetic/NDP) patients, respectively.\n\nThe purified DNA were amplified by Polymerase Chain Reaction (PCR) using specific primers (27F and 1492R) in a commercially available kit (16S Barcoding kit;SQK-RAB204; Oxford Nanopore Technologies/ONT, Oxford, UK). The procedure was conducted according to the protocol provided by 16S Barcoding Kit (SQK-RAB204; Oxford Nanopore Technologies/ONT, Oxford, UK). All PCR products for each subgingival biofilm sample obtained from their respective groups (DP and NDP) were pooled and purified. The concentration of each purified pool was measured using the Qubit dsDNA HS Kits by the Qubit Fluorometer (Invitrogen). For each purified pool, library preparation was prepared using the 16S Barcoding Kit mentioned above.\n\nTwo sequencing libraries were further prepared, one for a sample from DP and one for a sample from NDP. The amount of initial DNA used for both barcoding kits was 100 ng. Finally, each sequencing library was loaded into MinION flow cell of the MinION sequencing device (ONT) to be sequenced for 48 hours. Subsequently, the base calling of the generated data (fastq format) was analysed by using EPI2ME Desktop Agent (ONT). For microbiota profiling analysis, we followed the EPI2ME platform by selecting a workflow of 16S alignment for real time analysis. Alternatively, the obtained data can be analysed by a freely available software, NanoPipe (RRID:SCR_016852) (www.bioinformatics.uni-muenster.de/tools/nanopipe2). Since the base calling process is the central to improving the accuracy of nanopore sequencing technology15, in this study, only reads designated as ‘pass’ were included for further analyses. The analysis results were further generated in the form of a report in the EPI2ME platform.\n\nDescriptive analyses were performed with GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA). If the six bacteria were detected in the two sample groups, the group was regarded as positive for these bacteria.\n\n\nResults and discussion\n\nThe ONT has been reported for its potential benefits to analyse microbial communities' composition and dynamics, including oral pathogen16. In this pilot study, we provide a new information on the state of ONTs MinION device for whole genome sequencing of some periodontal bacterial organisms. To describe the main finding of the results, we used the online EPI2ME platform, which contains a 16S workflow for analysing MinION reads.\n\nWe endeavoured to determine if the oral microbial community would reveal the different profiles of the six selected periopathogens in pooled samples collected from periodontitis subjects with (DP) and without (NPD) diabetes. We used plaque subgingival biofilm samples for practical and economic reasons, which have often been employed17.\n\nThe results of 16S rRNA amplicons on MiniION sequencing revealed a total of 113,654 sequence reads after base-calling, with more reads classified than unclassified in either group. However, the subgingival biofilms obtained from the DP group were found better classified and a greater number of species compared to those found in the subgingival microbiome of the NDP group. By comparing the read count, we found that the classified sequence reads in the pooled sample of DP were 112.173 and 1988, respectively, while in the pooled sample of NDP were 1478 and 172, respectively (Figure 1A). We found that the accuracy in pooled DP was 87% compared to those in pooled NDP that showed 85%. These results indicate that the long-read amplicons for sequencing on ONT covered nearly the full length of V1-V9 hypervariable regions of the 16S rRNA gene. In this study, all the reads were 1-directional base-calling, representing a sequence in the forward or reverse direction. Thus, the application of 16S rRNA-based using MinION platform has allowed tracking of bacterial cells' identity in subgingival niches, as shown in this study.\n\n(A) Taxa-level; (B) phylum; (C and D) root taxa in DP (C) and NDP (D) groups.\n\nIn the current study, the long length sequences were taxonomically assigned using a workflow from the cloud-based EPI2ME, which supported the NCBI 16S database, allowing classification down to the phylum, genus and species level, respectively. Therefore, we searched which taxa were responsible for overall community differences between DP and NDP groups. Our data showed that in general, subgingival microbiome patterns were similar between DP and NDP groups, a phenomenon that have been reported by18,19. Other studies20 showed differences between glycaemic status and the proportion of several phyla. This study did not separate the tested sample according to the glycaemic level in diabetic subjects. We aimed to examine whether diabetes mellitus might affect composition of six targeted bacterial communities in the subgingival niche. Thus, we observed these subgingival bacteria by Phylum to species level.\n\nThe obtained sequences were first analysed on taxonomic basis at the phylum level. Eleven phyla (Firmicutes, Proteobacteria, Fusobacteria, Bacteroides, Actinobacteria, Spirochaetes, Synergistetes, Tenericutes, Acidobacteria, Planctomycetes, and Verrucomicrobia) were detected in DP group, while the last five phyla were not detected in DNP group. This finding indicates that the ONT technology allowed sequencing of the entire 16S rRNA gene region of the bacteria belonging to these phyla associated with the disease processes in our diabetic patients. This result agrees with previous findings in the subgingival bacterial microbiome diversity study in diabetic patients21. Interestingly, the long sequence of Synergistetes, which were only found in the DP group, has been identified in the area of periodontitis22. The colonization are located in the outermost region of subgingival biofilm, indicating they are opposed to inflammatory cells23. However, no genus belonging to Synergistic phylum was detected in this study. We assumed this is because of the low-read accuracy by MinION platform, which complicates our complex samples' analysis.\n\nIn this study, the subgingival microorganisms' samples were collected from the periodontal pocket at the same depth (≥ 5mm). Firstly, we found that the seven major phyla detected in subgingival microbiota of DP group were Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes, Actinobacteria, Spirochaetes, and Synergistetes, contain 99% of the taxa. The remaining phyla, Tenericutes, Acidobacteria, Planctomycetes and Verrucomicrobia containing the remaining 1% of the Taxa. Compared to the subgingival microbiota in the NDP group, containing four major phyla (99%): Firmicutes, Proteobacteria, Fusobacteria, and Bacteroides, while the Actinobacteria was the remaining bacteria containing 1% in NDP group. At the phyla level, the phyla occurrences belong to the six targeted bacteria in both groups are the same, but the proportions differed. The Firmicutes Phylum dominated the microbial community with relative abundance higher than 80% in both pooled samples (Figure 1B). This result demonstrated that using MinION, it is possible to associate the single cell level for nearly all subgingival plaque bacteria, from each group tested, to one of the major taxonomic units.\n\nFurthermore, as shown in Figure 1C and D, the subgingival microbiota profiles observed at the phyla level were relatively similar between DP and NDP groups, indicating that the predominant cooperative network microbiome is still conserved . However, when the six targeted bacteria were analysed at family level, the DP and DP groups' bacterial profiles were predominantly by Pasteurellaceae, followed by Veillonellaceae, Porphyromonadaceae, Tannerellaceae, and Spirochaetaceae. All bacteria belonging to these phyla were increased in the DP group compared with the NDP group (Figure 2A). Therefore, the obtained length-sequences were further analysed to determine if diabetes, which alters the nature of inflammatory response24, also influenced the relative abundance of six genera of periodontal pathogens selected in this study.\n\nRelative abundance of (A) six bacterial families; (B) genera of the red complex bacteria; (C) genera of selected non-red complex bacteria.\n\nIn this study, we focused to on identifying the red complex (P. gingivalis, T. denticola, and T. forsythia), which a lot of studies have described as the most important pathogens in adult periodontal disease25,26. We analysed the red complex at genus and species levels of the subgingival niche, as species identification is important because it provides information regarding periodontal disease's pathogenicity and a detailed description of the subgingival microbiome in a diabetic subject. We found that among the three genera belong to these species, the topmost prevalent genera were Porphyromonas, followed by Tannerella, and Treponema (Figure 2B). As shown in Figure 2B, the cumulative reads belong to these genera were significantly increased in DP subjects compared with the NDP group, and more specifically, Treponema was only found in the DP group. This result might indicate that the different quantities of red complex bacteria are more likely due to host diabetic-related responses. However, as with other systemic factors, there are very diverse clinical and medical parameters that might affect the composition of the oral microbiome in systemic disorder patients27. Hence, it is more likely that the red complex bacteria in subgingival plaque microbiome observed in periodontitis subject was affected by several concurrent factors, which we did not include in this study.\n\nIn addition to the red complex bacteria differences observed between DP and NDP subjects, we studied the differences in the individual microbial genera belong to Fusobacteria, Veillonella, and Aggregatibacter. In general, we found that the most abundant bacteria in both subject groups (DP and NDP) belonged to Fusobacterium sp. (Figure 2C). This species has a role in the progression of periodontal disease due to its ability to build a physical relationship (co-aggregation) with other oral bacterial species, notably with P. gingivalis and T. denticola, in the formation of biofilm28.\n\nWhen species analysis was focused to Phorphyromonas, our data indicated that these genera, where its sequences were abundant in the DP group, comprise mainly species P. catoniae, followed by P. pasteri, P. gingivalis, and P. endodontalis, while only P. catoniae and P. endodontalis sequences was detected in NDP group (Figure 3A–C). For Treponems, which are typically restricted to the subgingival crevice29, the full length of 16S RNA gene belonging to this genus were found in six species and one subspecies in DP group, i.e. T. medium, T. denticola, T. lecithinolyticum, T. maltophilum, T. amylovorum, and T. socranskii as well as it subspecies (Figure 4A and B). Lastly, sequences identified as Tannerella was Tannerella forsythia, which was more abundant in the DP than in the NDP group (Figure 4C). However, by comparing to P. gingivalis and T. denticola, the cumulative reads of T. forsythia were found to be higher (not shown), suggesting that individuals with diabetes may have an increase in the subgingival abundance of the T. forsythia,\n\n(A) Abundance of Porphyromonas sp; (B and C) dendrograms showing the variability of Porphyromonas sp. in DP (B) and NDP (C) groups.\n\nRelative abundance of (A) six Treponema sp; and (B) Dendogram showing the variability of Treponema sp., across pooled samples. (C) Tannerella forsythia.\n\nWhen species analysis was focused on P. gingivalis, literature shows that this species has been proposed as an important keystone pathogen-induced dysbiosis in periodontitis conditions30. It has the ability to modify the oral microbiota composition31. In this study, the bacterium was only found in a sample collected from DP patients, while we obtained all the samples from chronic periodontitis patients. Additionally, in the EPI2ME 16S workflow, nanopore sequence reads are blasted against the NCBI database for 16S DNA. Although it is possible that certain species are not represented in the database, this was not the case for P. gingivalis, as its 16S rRNA gene sequence can be retrieved from NCBI refSeq database. Thus, our study contradicts the previous report showing that P. gingivalis was associated with periodontitis in patients without diabetes32.\n\nAs also shown in literature, the presence of red complex bacteria in subgingival niche are usually found with consortia, which include various species belong to the “orange, green, and purple complex”33,34 as well as non-pathogenic microorganisms35. Since this polymicrobial consortium comprising the mix species induced significant increased alveolar bone resorption than the mono species36, our result may suggest the difference in host response between the DP and NDP groups, and we did not explore this in this study. Furthermore, our results are in line with a previous study that recovered several periodontal pathogens, including A. actinomycetemcomitans, Campylobacter rectus, F. nucleatum, and P. intermedia, which was similar in both diabetic and non-diabetic subjects, but P. gingivalis was more frequently detected in individuals with diabetes37. Our finding is also consistent with previous reports, in which P. gingivalis is a quantitatively minor constituent of biofilms associated human periodontitis38–40, in addition to its association with progressive bone loss in periodontitis patients41, particularly those with diabetes40.\n\nOther studies showed that the red complex species can be detected in higher numbers when the disease reaches the advanced state8. However, this study showed that only the read counts of T. forsythia were found higher in patients with diabetes than the other red complex bacteria species. Our result supports the idea of polymicrobial synergy and dysbiosis for periodontitis, which highlights the importance of other bacterial species in keystone pathogenesis42. Thus, species other than the red complex species may have similar keystone role in periodontitis30, as shown in this study. Another interesting finding was that we observed T. forsythia to be associated with periodontitis, and it did not relate to diabetes as its DNA was detected in all samples obtained from DP and NDP, and to lesser extent was the DNA of P. endodontalis. Similar findings have been reported previously43,44. Similarly, P. catoniae, which has been found in the mouth of infants before eruption of their teeth45, was detected in both DP and NDP groups in our study.\n\nIn this study, we observed that although the presence of the red complex species in DP group had similar trends as was seen in NDP group, two of them (T. denticola and T. forsythia) showed differences in abundance. This result might indicate that the different quantity is more likely due to host diabetic-related response. However, there are very diverse clinical and medical parameters that might affect the composition of the oral microbiome in systemic disorder patients27. Hence, it is more likely that the subgingival plaque microbiome observed in this study was affected by several concurrent factors.\n\nIn addition to the red complex bacteria differences observed between DP and NDP subjects, we studied the differences in the individual microbial species belong to Aggregatibacter, Fusobacteria, and Veillonella.\n\nRegarding Aggregatibacter sp., A. actinomycetemcomitans have been officially designated as aetiology agents of periodontitis, together with P. gingivalis, and T. forsythia46,47. Hence, our aim was to find out the presence of A. actinomycetemcomitans in a sample obtained from DP and NDP groups. While the DNA sequence of A. acitnomycetemcomitans was not present at any samples tested, we did find A. aphrophilus and A. segnis. These two species have been known to belong to the genus of Aggregatibacter, in addition to A. actinomycetemcomitans48. Therefore, this finding is the first step towards understanding the potential contribution and a partnership between A. aphrophilus and A. segnis with P. gingivalis, and T. forsythia in periodontitis patients with and without diabetes. Comparison of the cumulative reads of the two species (A. aphrophilus and A. segnis), between DP and NDP groups is shown in Figure 5A and B. Additionally, despite the presence of A. aphrophilus and A. segnis, our data are consistent with a previous report that species belonging to genus Aggregatibacter were present at a relatively low level compared to other periodontal pathogenic species49. Another study also showed that both A. actinomycetemcomitans and P. intermedia are of only minor importance in periodontal disease progression50.\n\n(A) Abundance of Aggregatibacter sp.; (B) Dendogram showing the variability of Treponema sp., across pooled samples. (C) Tannerella forsythia.\n\nIn terms of Fusobacteria, within oral cavity F. nucleatum is the most abundant species, in both diseased and healthy individuals51,52. This species has a role in the progression of periodontal disease due to its ability to build a physical relationship (co-aggregation) with other oral bacterial species, notably with P. gingivalis and T. denticola formation of biofilm28. Also, in the subgingival model, the count of P. gingivalis and some tested bacteria significantly decreased in the presence of Fusobacterium sp./spp.53. Our data showed that, although the species variability of Fusobacterium sp. was relatively similar between the two groups tested, the cumulative reads of F. nucleatum was found more abundant in the DP group (Figure 6A–C). In contrast, the reverse was found for P. gingivalis (Figure 3A). Hence, it is important to carry out studies that evaluate the possibility of host response-associated diabetes regulating the interaction between F. nucleatum and P. gingivalis.\n\n(A) Abundance of Fusobacterium sp.; (B and C) dendrograms showing the variability of Fusobacterium sp. in DP (B) and NDP (C) groups.\n\nIn this study, Firmicutes bacteria at genus level to be analysed was Veillonella sp. We found that the annotation accuracy for Veillonella at the genus level was 88%. The cumulative reads of sequences belonging to Veillonella sp. consisted of eleven and five species in DP and NDP, respectively (Figure 7A–C). We found that V. parvula was the predominant Firmicutes bacteria in subgingival microbiota of both groups, with more abundance in the DP group. Additionally, the results of this study was similar with a previous report elsewhere, in which V. rogosae was detected at a low number in DP patients54, and was not detected in NDP individuals. Although it had been proposed to be used as an index for the state of chronic periodontitis54, there is no clear explanation at present regarding the increased number of V. parvula in subgingival biofilms of diabetic patients. Our result, however, may indicate different environment conditions due to diabetes that my lead to increased number of V. parvula in subgingival niche. Interestingly, Veillonella sp. have been reported to have the ability to inhibit the host-cell effect of P. gingivalis55, the red complex species that we found in lower abundance in the subgingival niche of DP patients in the current study. Thus, the difference in the amount and other bacterial species is not sufficient to explain the difference in periodontitis severity in a patient with diabetes. Although the host's immunological response may be influenced by diabetes24, in the case of our subjects, other risk factors, including genetic background56 may also affect inflammation and periodontal disease expression57, which we did not include in this study. Considering these facts, we suggest that in periodontitis patient with diabetic, the subgingival microbiota formed by a low level of red complex and other representative bacteria may indicate that the red complex bacteria are necessary but insufficient to be linked to diabetes.\n\n(A) Abundance of Veillonella sp.; (B and C) dendrograms showing the variability of Veillonella sp. in DP (B) and NDP (C) groups.\n\nThere are some limitations of this study. First, we compared the subgingival microbiome profile based on pooled PCR amplicons separated by the DP and NDP groups, respectively, not with health and disease sites as controls. It is also well known that detailed site-specific information might be lost when using pooled samples for microbial analysis58. Although being inferior to the non-pooling sample, this study suggests the pooling approach for sequencing studies, particularly if there are budgetary constraints that do not permit individual sample runs' analytical execution. Lastly, the descriptive analysis prevented us from testing the directional relationship between diabetes and periodontitis.\n\n\nConclusion\n\nThis is the first study in Indonesia to show that using the Nanopore MinION sequencing technology, we can investigate the presence of a consortium of red complex bacteria (P. gingivalis, T. forsythia, and T. denticola) that includes three genera (Aggregatibacter, Fusobacterium, and Veillonella) in periodontitis subjects with and without diabetes. The present study revealed that the abundance of the sequence reads of six selected bacteria in subgingival microbiome were strongly affected by diabetic condition. All sequences observed in a large number were derived from the DP group. However, the six selected periodontal pathogens profile was relatively similar between DP and NDP pooled DNA samples. Therefore, we reject the hypothesis that the composition of subgingival biofilm in DP patients is more variable than in periodontitis subjects without diabetes. Additionally, one species belonging to the red complex bacteria (P. gingivalis) was only found in the subgingival microbiome of DP. Lastly, the capability of differentiating bacterial species, and even subspecies, as shown in this study, makes the MinION sequencer useful for pathogen detection in periodontitis subjects since it enables full-length 16S rRNA amplicon sequencing, while the reads can be analysed in real-time. However, we suggest, when investigating the subgingival microbiome of periodontitis patient with diabetes, there should be evidence in the presence of the targeted bacteria before the detection of attachment loss or bone loss\n\n\nData availability\n\nOpen Science Framework: A pilot study of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, evaluated by MinION, https://doi.org/10.17605/OSF.IO/DQE6F59.\n\nThis project contains the following underlying data\n\n- Subject data\n\n- Fastq files\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to acknowledge to Astri Deviana for MinIon Nanopore work at Department of Oral Biology, Faculty of Dentistry Universitas Indonesia.\n\n\nReferences\n\nBöttger EC: Frequent contamination of Taq polymerase with DNA. Clin Chem. 1990; 36(6): 1258–9. 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PubMed Abstract \| Publisher Full Text\n\nBachtiar EW: A pilot study of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, evaluated by MinION. 2021. http://www.doi.org/10.17605/OSF.IO/DQE6F" }	[ { "id": "79850", "date": "02 Mar 2021", "name": "Wan Izlina Binti Wan Ibrahim", "expertise": [ "Reviewer Expertise Oral Biology (Oral Biochemistry", "Oral Microbiology", "Oral Histology", "Oral Physiology", "Oral Environment)", "Traditional Medicine and Natural Products (Free radical biochemistry & DNA damage)", "Biotechnology", "Medical Biotechnology (Gel- & non-gel-based proteomics", "liquid chromatography & mass spectrometry)", "Proteins (Proteomics & glycoproteomics - cancer", "disease biomarkers", "human and mammalian milk)" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments:\nThe manuscript presents interesting findings regarding the presence of red complex and three genera subgingival microbiome in periodontitis subjects with and without diabetes, using ONT MinION platform, which is one of the most recent sequencing technologies.\n\nWhile detailing the advantages of the platform, it would be great to also describe about the limitations or drawbacks of this technology, because the authors made quite a number of postulations, some can be confounding, based on literatures that used other technologies or techniques. This platform is known to be error prone, particularly in studying small or rare sequence differences.\n\nIt would be also be really great if the authors consider to include a method for bacterial identification and quantification that is able to validate these results, perhaps a preliminary study, if there is any?\n\nSuggest proofread again to recheck incorrect sentence structure, missing preposition, as well as minor spelling and grammar mistakes. E.g. phylum are sometimes capitalized when it is not actually a proper noun.\nSpecific comments:\nMethod\nMicrobial assessment:\nEppendorf is a brand. Should be written as 'a microcentrifuge tube' instead of \"an Eppendorf tube\".\n\nI suggest changing the subheading \"Microbial assessment\", to 'Microbial sample collection'.\n\nIt is unclear what the authors meant by \"The presence at genus and species level of the red complex bacteria (Phorphyromonas gingivalis, Treponema denticola, and Tannerella forsythia), and three other selected genera (Aggregatibacter, Fusobacterium, and Veillonella) were determined in subgingival biofilm samples.\" - How did the authors determine \"the presence\" of these bacteria? Or the authors are actually already referring to the MinION sequencing already? If yes, kindly consider revising the sentence.\n\nThird paragraph \"MinIONTM\" - The authors should be consistent, whether to use MinION with TM (superscript) or without, throughout the text.\n\nMinION sequencing and data analysis:\n\"Polymerase Chain Reaction\" is not a proper noun, so there is no need to capitalize each word.\nResults and discussion\nRead analysis:\nSecond paragraph, third sentence \"By comparing the read count, we found that the classified sequence reads in the pooled sample of DP were 112.173 and 1988, respectively, while in the pooled sample of NDP were 1478 and 172, respectively (Figure 1A)\". Consider revising this sentence.\n\nPerhaps the authors intended to write 'we found that the classified and unclassified sequence reads in the pooled sample of DP were 112.173 and 1988, respectively, while the classified and unclassified sequence reads in the pooled sample of NDP were 1478 and 172, respectively.'?\nDistribution of the subgingival microbiota at the phylum and family level\nSecond paragraph, \"All bacteria belonging to these phyla were increased in the DP group compared with the NDP group (Figure 2A).\". I believe 'phyla' should be replaced with 'families'.\nAnalysis of Aggregatibacter, Fusobacterium, and Veillonella\nFigure 5 - In the text, the authors described Figure 5A and 5B as showing the cumulative reads of A. aphrophilus and A. segnis between DP and NDP groups. However, the legend says (A) Abundance of Aggregatibacter sp.; (B) Dendogram showing the variability of Treponema sp., across pooled samples. (C) Tannerella forsythia. Kindly revise the legend.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6413", "date": "02 Jun 2021", "name": "Boy Muchlis Bachtiar", "role": "Author Response", "response": "General comments: We highly appreciate the reviewers’ insightful and helpful comments on our manuscript. According to the reviewer suggestions, we will carefully rewrite the incorrect sentence structure, missing preposition, as well as minor spelling and grammar mistakes. For example, as suggested, the typo of “Phylum” will be been changed to 'phylum', in the new version of this manuscript. We also would like to correct some mistake we made in the original manuscript. In the new version of this manuscript, we will explain the limitations of the MinION platform, as well as adding the needed reference, and will put it in the last sentence of the second paragraph when explain the Synergistic phylum, as follows: \"However, no genus belonging to Synergistic phylum was detected in this study. We assumed this is because of the low-read accuracy by MinION platform (Laver 2015), which complicates our complex samples' analysis.\" Regarding the other identification and quantification method to validate this sequencing platform, our responce is we did not include bacterial identification and quantification method, such as real time-PCR to validate the sequencing result, as our main purposed was to estimate microbial community diversity using MinION platform. Specific comments: Method Microbial assessment: As suggested, the word “Eppendorf tube\" has been changed to \"microcentrifuge tube\". Thank you. The sub heading \"Microbial assessment\" has been changed to \"Microbial sample\". Thank you. As suggested, the sentence, “The presence at genus and species level of the red complex bacteria (Phorphyromonas gingivalis, Treponema denticola, and Tannerella forsythia), and three other selected genera (Aggregatibacter, Fusobacterium, and Veillonella) were determined in subgingival biofilm samples, will be changed in the new version. Thus, the new sentence become,” in order to detect the The presence at genus and species level of the red complex bacteria (Phorphyromonas gingivalis, Treponema denticola, and Tannerella forsythia), and three other selected genera (Aggregatibacter, Fusobacterium, and Veillonella) were determined in subgingival biofilm samples. According to the reviewer suggestion, we have changed the word MinIONTM, and only write it as MinION throughout the text. Thank you. Yes, we agree, we will rewrite the “Polymerase Chain Reaction” without the capital word. Thank you. C. Results and discussion Read analysis: ”By comparing the read count, we found that the classified sequence reads in the pooled sample of DP were 112.173 and 1988, respectively, while in the pooled sample of NDP were 1478 and 172, respectively (Figure 1A)\". Consider revising this sentence. Author response: Yes, thank you for this important correction. As suggested, we will change the wrong sentence, In the new version, the corrected sentence will become “By comparing the read count, we found that the classified and unclassified sequence reads in the pooled sample of DP were 112.173 and 1988, respectively, while the classified and unclassified sequence reads in the pooled sample of NDP were 1478 and 172, respectively”. Under the subtitle; within the second paragraph, “Distribution of the subgingival microbiota at the phylum and family level”. Author response: We agree with the suggestion. Hence the word phyla have been changed to be families. Thank you. Analysis of Aggregatibacter, Fusobacterium, and Veillonella: Figure 5 - In the text, the authors described Figure 5A and 5B as showing the cumulative reads of A. aphrophilus and A. segnis between DP and NDP groups. However, the legend says (A) Abundance of Aggregatibacter sp.; (B) Dendogram showing the variability of Treponema sp., across pooled samples. (C) Tannerella forsythia. Kindly revise the legend. Author response: Thank you very much for this very important correction. Accordingly, in the new version, the legend will been revised." } ] } ]	1	https://f1000research.com/articles/10-79
https://f1000research.com/articles/10-1/v1	04 Jan 21	{ "type": "Method Article", "title": "WIND (Workflow for pIRNAs aNd beyonD): a strategy for in-depth analysis of small RNA-seq data", "authors": [ "Konstantinos Geles", "Domenico Palumbo", "Assunta Sellitto", "Giorgio Giurato", "Eleonora Cianflone", "Fabiola Marino", "Daniele Torella", "Valeria Mirici Cappa", "Giovanni Nassa", "Roberta Tarallo", "Alessandro Weisz", "Francesca Rizzo", "Konstantinos Geles", "Domenico Palumbo", "Assunta Sellitto", "Giorgio Giurato", "Eleonora Cianflone", "Fabiola Marino", "Daniele Torella", "Valeria Mirici Cappa", "Giovanni Nassa", "Roberta Tarallo" ], "abstract": "Current bioinformatics workflows for PIWI-interacting RNA (piRNA) analysis focus primarily on germline-derived piRNAs and piRNA-clusters. Frequently, they suffer from outdated piRNA databases, questionable quantification methods, and lack of reproducibility. Often, pipelines specific to miRNA analysis are used for the piRNA research in silico. Furthermore, the absence of a well-established database for piRNA annotation, as for miRNA, leads to uniformity issues between studies and generates confusion for data analysts and biologists. For these reasons, we have developed WIND (Workflow for pIRNAs aNd beyonD), a bioinformatics workflow that addresses the crucial issue of piRNA annotation, thereby allowing a reliable analysis of small RNA sequencing data for the identification of piRNAs and other small non-coding RNAs (sncRNAs) that in the past have been incorrectly classified as piRNAs. WIND allows the creation of a comprehensive annotation track of sncRNAs combining information available in RNAcentral, with piRNA sequences from piRNABank, the first database dedicated to piRNA annotation. WIND was built with Docker containers for reproducibility and integrates widely used bioinformatics tools for sequence alignment and quantification. In addition, it includes Bioconductor packages for exploratory data and differential expression analysis. Moreover, WIND implements a \"dual\" approach for the evaluation of sncRNAs expression level quantifying the aligned reads to the annotated genome and carrying out an alignment-free transcript quantification using reads mapped to the transcriptome. Therefore, a broader range of piRNAs can be annotated, improving their quantification and easing the subsequent downstream analysis. WIND performance has been tested with several small RNA-seq datasets, demonstrating how our approach can be a useful and comprehensive resource to analyse piRNAs and other classes of sncRNAs.", "keywords": [ "piRNA", "small RNA sequencing", "ncRNA-expression", "workflow" ], "content": "Introduction\n\nAdvances in the field of Next-Generation Sequencing and big data analysis have led to the identification of several small non-coding RNA (sncRNA) classes, some of which are still poorly characterised1,2. Among others, the most investigated include microRNAs (miRNAs), small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), small nuclear (snRNAs) and small nucleolar RNAs (snoRNAs). Increasing evidence demonstrates that the different sncRNAs constitute interconnected networks of molecules with key-regulatory functions in multiple biological processes, including physiological events, organism development or even disease3.\n\npiRNAs represent an heterogeneous group, ubiquitous in most animal's germline cells, with lack of conserved sequences and few common structural features in the various species, due to the highly adaptive nature of the piRNA pathway4. Germline piRNAs typically have a 21–35 nt length, a strong bias for 5'-end uridine signature and a 2'-O-methyl group at their 3'-end5. Most of them are transcribed by either mono-directional or bidirectional genomic clusters, specific regions ranging from <1 kb to >100 kb, giving rise to a long, single-stranded precursor and further processed in multiple mature piRNAs through enzymatic cleavage. A subset of piRNAs present an adenosine bias at position 10, a feature indicating their biogenesis through the ping-pong cycle, a mechanism by which the cleavage of the target RNA is coupled with the production of a second population of target-specific piRNAs. They interact with PIWI proteins of the Argonaute (AGO) family, forming a silencing complex able to suppress transposable elements, regulate target’s gene expression at both epigenetic and post-transcriptional level and defend from viral infections6. These piRNA functions are well studied in the animal germline, however in somatic cells, their role needs to be further elucidated. Additional studies have revealed that piRNA dysregulation can contribute to the onset of several diseases7. Notably in cancer, the abnormal expression of piRNAs has been associated with tumour initiation, progression, and metastasis formation and these molecules have shown the potential to be useful diagnostic tools and therapeutic targets as well as biomarkers for cancer prognosis8.\n\nA limitation in understanding their function and use in clinical practice is the lack of a comprehensive and reliable method for their identification in tissues others than germline. A common strategy for piRNAs identification is based on mapping the reads obtained from high-throughput small RNA libraries to the genome and then annotate to small RNA databases for quantification. Most of the piRNA sequences identified so far have been deposited in databases such as piRNABank9, piRNAdb (https://www.pirnadb.org/), piRNAclusterDB10 and others. However, data collected in these repositories mainly include germline piRNAs, while somatic piRNAs represent a minor fraction. In addition, piRNAs in somatic tissues and human cancers are less abundant than in germline, thereby leading to a more difficult identification and characterisation. Although piRNAs were initially confounded with fragments of longer RNAs, functional piRNAs have been identified to derive from fragments of rRNAs, tRNAs, snoRNAs, and post-transcriptionally processed mRNA11–13. Another level of complexity is represented by their genomic origin(s) and their actual amount, since identical sequences of piRNA can be produced by multiple genomic loci, resulting in very low precision and sensitivity.\n\nFor all the reasons stated above, and since existing workflows and tools for piRNA-analysis, usually, focus on the identification and quantification of piRNA clusters (PILFER14, unitas15) or use outdated piRNA databases, we decided to implement a useful workflow for small RNA sequencing data analysis, able to analyse all classes of sncRNAs but especially designed for piRNA identification. We created a workflow that provides a quick method to integrate different piRNA databases in one annotation track, a two-method approach for small RNA identification, annotation and quantification, and an output with several ready-to-publish plots and statistics. Additionally, we packaged the entire workflow in several Docker16 containers avoiding the annoying problems related to the installation and libraries dependencies. Finally, we applied it to different small RNA datasets, highlighting that piRNAs are dysregulated in breast cancer tissues and may play an important role in maintaining the stemness of MCF7 spheroid-enriched cancer stem cells (CSCs).\n\n\nMethods\n\nIn this study, we implemented a workflow for small RNA sequencing data analysis, defined WIND (Workflow for pIRNAs aNd beyonD)17, designed for a comprehensive identification and quantification of small-RNAs and especially of piRNAs. We deployed it exploiting the Docker containerisation approach, allowing us to integrate multiple bioinformatics tools. In detail, we created two Docker images where we adopted broadly used tools for pre-processing, reads alignment, identification and quantification of sncRNAs, and all downstream analyses. We also integrated the already available container made for Salmon18 for transcriptome analysis. This solution takes into account best practices for reproducibility, versatility and ease of use, as the software deployment is fast and efficient. It can be used in various operating systems with only the requirements of the Docker engine and some minimum adjustments for processing power and RAM for the most memory demanding tools.\n\nThe workflow consists of three significant steps (Figure 1):\n\nThe Annotation forging step, represented in blue, is the creation of a GTF file, where the two input databases (piRNABank and RNAcentral) are merged to produce the new small RNA annotation track, that together with the Fasta files constitute the inputs of the following step. In Pre-processing & Quantification step (light blue area), the user's fastq files undergo through the quality check, and the adapter removal followed by the two quantification approaches (completed by Salmon, and STAR with FeatureCounts software) that perform in parallel alignment and the quantification of reads. In the green box, representing the Exploratory data analysis phase, are displayed all the possible results produced by the workflow. The data analyst could also pursue differential expression analysis if that is the desirable outcome.\n\n1. Annotation forging: the generation of the annotation files for small RNA sequences used in the next quantification step.\n\n2. Pre-processing and quantification: pre-processing, alignment and quantification of the reads assigned to sncRNAs (using a dual approach: genomic and transcriptomic analysis).\n\n3. Exploratory data analysis: result exploration of both quantification methods in parallel and Differential Expression (DE) with two different methodologies (edgeR19,20 and limma-voom21).\n\nAnnotation forging. The first step of WIND, the Annotation forging (blue box in Figure 1) is the creation of the annotation track. In this step, we tried to reduce and potentially correct the issues regarding piRNA annotation, such as the presence of multi-mapped piRNA IDs, the inconsistencies among piRNA databases, and the misleading annotation of small RNA fragments. In particular, 667,944 human and 1,399,813 mouse sequences were acquired from piRNABank (02-May-2007, Version 1, hg19). Duplicates and multi-mapped sequences were collapsed, leading to 23,439 and 39,986 unique sequences for human and mouse, respectively. Afterwards, these unique sequences were realigned to the latest version of the reference genome (Gencode22 primary assembly, GRCh38.p13 for human, and GRCm38.p6 for mouse) using STAR aligner23 with the following parameters: --alignIntronMax 0, --outFilterMultimapNmax 100, --outFilterMatchNmin 16, and --outFilterMismatchNmax 0. Further on, 446,265 human and 180,780 mouse small RNA sequences from RNAcentral24 (v16, 28/09/2020) were utilised to complete the database (for details see Extended data: Supplementary Table 1). Then, the sequences from both databases were filtered with respect to their length, keeping only those with less than 100 bases in length, since our primary interests are piRNAs and sncRNAs, and keeping only those that correspond to standard chromosomes. Moreover, a re-classification of the piRNABank sequences was made. According to Tosar et al.25, a small percentage of annotated piRNAs are probably piRNA-sized fragments of sncRNAs (rRNAs, tRNAs, YRNAs, snRNAs, and snoRNAs) or intermediates of miRNA biogenesis and potentially act as contaminants in the quantification step of the workflow. For this reason, piRNABank sequences matching sequences from RNAcentral with different sncRNA types (biotype) other than piRNA are re-categorised with the biotype from RNAcentral.\n\nSubsequently, as it is well established that mature piRNAs have a length of around 21~35 bases26, before the final assembly of the sequences from both databases, the piRNABank sequences are filtered for the length of shorter than 69 bases (<69bp) and exported to Fasta and GTF (Gene Transfer Format) files format. These tracks, for human and mouse species, have been included in the GitHub repository of the workflow and are available for users (https://github.com/ConYel/wind); therefore, the annotation forging step can be skipped. The workflow can also be used for any other species, but in this case, it would be necessary for the user to run the Annotation forging step with the specific genome and small RNA sequences, respectively.\n\nThe mapping of these piRNA sequences to the genome has revealed that the piRNAs can derive from two types of genomic locations: discrete genomic loci (the piRNA clusters) and protein-coding genes (e.g. UTRs, exons, introns)27–29. Using bumphunter30 package in the workflow, we were able to obtain piRNA origin information and provide it as the first additional file of the annotation. Since piRNAs are involved in the maintenance of genome stability through the silencing of transposable elements31, in this step, we also report a GTF file with the intersection between the genomic positions of small RNAs, the various categories of Transposable Elements (TEs) and information about the TE class, family and gene. Briefly, the GTF file is created with the related Fasta file; then the Genomic_Region_info, Multimapping_piRNA_info and Transposable_Elements_info files are reported which carry information about the genomic topology for the sequences in the GTF file. Likewise, these files are available in the GitHub repository (GRCh38 and GRCm38), for future usage by the data analyst.\n\nPre-processing and quantification. The second step of the workflow, Pre-processing and quantification (light blue box in Figure 1), consists of a quality control check of the small RNA-seq data, carried out using the FastQC tool32, followed by the adapter removal using Cutadapt33 and by another quality control check using FastQC again. After these initial steps, the workflow exploits two different approaches for the quantification of sncRNAs. In particular, one uses alignment to a reference genome with STAR and then quantification of aligned reads with FeatureCounts34; the other one uses Salmon (an aligner-free method) for the estimation of transcript-level abundance. We named the two approaches “genomic” and “transcriptomic” based on how the two methods work. Both approaches have positive and negative features. Undoubtedly, with STAR, the reads are aligned on a reference genome, Salmon instead is an alignment-free quantification method, able to prioritise the association of a feature with a specific site on a transcriptome. On the one hand, STAR could associate a read on multiple sites creating a complete list of identified regions, but this makes it more difficult to determine the genomic locations of origin, thus requiring more computational work. On the other hand, Salmon is a transcriptome quantifier able to correct for fragment GC-content and positional bias, which improves the accuracy of abundance estimates and potentially the sensitivity of subsequent DE analysis.\n\nTo ensure the proper alignment of sncRNA reads to the genome, we used the following options for STAR aligner (as used in SPAR workflow35): --alignIntronMax 1, --outFilterMultimapNmax 100, --outFilterMismatchNmax 1 and --outFilterMatchNmin 14. For Salmon, to be suitable for small RNA reads, the following options were applied: --seqBias, --gcBias, --numBootstraps 100, and --validateMappings as was suggested from the work of Wu et al.36. Resulting files, from the previous step, are imported to R using the Bioconductor packages: tximport (for Salmon) and Rsubread (for FeatureCounts), as DGEList objects (edgeR). After reads count, a FeatureCounts object is reported as an R object (.RDS) for an easy and fast way to import it in R. Moreover, we decided to record the assigned reads from both Salmon and FeatureCounts as BAM files.\n\nExploratory data analysis. The last step of the workflow is the Exploratory data analysis (EDA), which includes the filtering of low expressed small RNAs, the normalisation procedures performed in parallel for both FeatureCounts and Salmon, and then the visualisation of the results according to the suggested EDA workflows37–40 from Bioconductor41. Finally, the workflow provides several useful output files: text and RDS files with filtered or normalised reads, information about the filtering step, Multidimensional Scaling (MDS) plots, biodetection plots, expression per small RNA category plot (countsbio), distance-matrix plot, hierarchical clustering plots with various normalisation methods, Principal Components Analysis (PCA) plots, Relative Log Expression (RLE) plots42,43, voom-derived plots, sequence length histograms, and piRNA sequence logos. Briefly, for each dataset analysed, 9 RDS files, 17 tab-delimited files with all the statistics from alignment, filtering and annotation plus the filtered and normalised reads in counts per million (CPM), and 24 PDF files with several exploratory data analysis plots for each of the two methods used are generated.\n\nEventually, the gene expression data can be further compared using the DE analysis module, which allows calculating logarithmic fold change values using limma-voom or edgeR methods, and finally, both results (Salmon and FeatureCounts) can be merged and visualised together. Then, the data analyst can choose to use the union of the results, and either consider all the molecules identified by at least one of the two methods, or use the intersection of the results and consider only the molecules supported by two methods. The differentially expressed molecules can be further used for piRNA target prediction analysis (included in the code) which was inspired by the similar module of iSMART tool44.\n\nThis workflow is structured to provide maximum flexibility to the user, who can modify several elements. In each step, alterations can be made regarding the tools or the databases used according to the needs of the data analyst, while the workflow strategy remains the same. Specifically, in the first step, the GTF file can be enriched with more sequences of interest or a completely new GTF file could be created for any species. Currently, the first step has been performed on human and mouse sncRNA sequences for the generation of the GTF files (included in the GitHub repository), but the same approach could be utilised for any well-annotated genome that has enough small RNA sequences reported. In the second step, it is possible to use different tools for quality control, adapter trimming, aligning of the reads, e.g. Subread45 or HISAT246 or a different \"alignment-free\" RNA-seq quantification method, as Kallisto47.\n\nThe workflow was run on CentOS Linux release 7.8.2003 (Core) with Docker Engine - Community v19.03.13 and in R v4.0.0, with Bioconductor v3.12.\n\nThe complete workflow has been tested on several datasets to evaluate whether this worked in the identification of known piRNAs, low abundant molecules and in different species. Specifically, we have evaluated the performance of the transcriptomic approach on sncRNA identification, and particularly on piRNAs, for which this method has been tested here for the first time. We created a small dataset where spike-in sequences of piRNA-like molecules were added to the input RNA. For this purpose, RNA of metastatic colon cancer cell line (COLO 205), where piRNA's population has been already characterised48, was used. To mimic the behaviour of true piRNAs, a synthetic set of 4 piRNA-like molecules was used, including two non-methylated (SS-22 and SS-28) and two methylated (mSS-22 and mSS-28) of different lengths (22 nt and 28 nt). Spike-ins were chemically synthesised at Exiqon, adapting the sequences described in Locati et al.49 to our conditions and the pool of 4 molecules was used at three different concentrations, with a final amount of 0.3 × 10^9 (dil_A), 0.3 × 10^10 (dil_B) and 0.3 × 10^11 (dil_C) molecules/ug of RNA. Library preparation and sequencing were performed as previously described in Sellitto et al. 201948 (samples are available on ArrayExpress, Accession number E-MTAB-9772: COLO205_Dil_A, COLO205_Dil_B, COLO205_Dil_C).\n\nTo test the performance of WIND, in both high-piRNA and low-piRNA expression conditions, we used Human Testis RNAs (BioChain Institute Inc, Newark, CA, USA) and COLO 205 cell line RNAs (samples are available on ArrayExpress. Accession number E-MTAB-8115: Non_treated_Testis_1 and Non_treated_COLO205_1, Non_treated_COLO205_2, Non_treated_COLO205_3; Accession number E-MTAB-9782: Non_treated_Testis_2 and Non_treated_Testis_3). To test the workflow on mouse data, we used two samples of mouse adult Cardiac Myocyte (samples are available on ArrayExpress, Accession number E-MTAB-9866: aCM1, aCM2)50,51.\n\nFurthermore, we also exploited two public datasets to test our workflow thoroughly including the differential expression module dataset, consisting of two experimental conditions in triplicates, MCF-7 enriched CSCs spheroids and monolayer cultures (Accession number GSE6824652,53); and a subset of 18 samples from TCGA-BRCA54,55, using 9 Primary Solid Tumour versus 9 Solid Tissue Normal corresponding samples.\n\n\nResults\n\nThe goal of this study was to create a robust workflow for the identification and quantification of piRNA sequences in small RNA sequencing data. It focuses on elucidating and solving one of the most challenging issues of this kind of analysis, the annotation controversies of piRNAs, thus providing relatively accurate detection of the piRNA expression patterns. As a first point, a unique GTF file was generated for human and mouse species, starting from sequences obtained from the two widely used databases (piRNABank and RNAcentral) for piRNAs and sncRNAs, respectively. The GTF file was created as described in the Methods obtaining 155,143 different genomic locations corresponding to 43,973 sequences in human and 932,645 distinct genomic locations corresponding to 99,624 sequences in mouse for all small RNA types (see Extended data: Supplementary Table 1). Furthermore, in humans, from the 43,973 sequences coding for small RNAs, 32,161 were classified as piRNA, and 22,718 of them were found in common between RNAcentral and piRNABank; instead, 9,045 were found only in RNAcentral and 398 only in piRNABank. Additionally, in the mouse genome, from 99,624 sequences of small RNAs, 70,051 were categorised as piRNA, 36,960 were in common between the two databases and on the contrary, 30,834 were unique to RNACentral, and 2,257 were exclusive to piRNABank.\n\nTo test WIND thoroughly, we used several datasets with different characteristics: data produced in house, data available in a public repository, samples which include internal controls (spike-ins), datasets from different species (human and mouse), a dataset including different experimental conditions, and a dataset of tumour tissues (for more details see Methods and Data availability). First, we compared the quantification capability of the two methods implemented in the workflow. In particular, we evaluated the performance of the transcriptomic approach on piRNA quantification, as this method to our knowledge, has not yet been used to analyse this sncRNA class. For this reason, we decided to apply the workflow on an own-made dataset, in which spike-in sequences of four piRNA-like molecules, two non-methylated and two methylated at three different concentrations, were included (see Methods for details). Exploiting this feature, we were able to assess the high efficiency of both (genomic and transcriptomic) approaches in quantifying the spike-ins, as demonstrated by the very similar results obtained by the different methods. Supplementary Table 2 (Extended data) summarises the results obtained for the 4 piRNA-like molecules calculated using three methods: iSMART, FeatureCounts, and Salmon. The results show that all approaches can identify and quantify all the types of piRNA-like sequences (methylated, non-methylated, and of different length) correctly.\n\nFor a long time, piRNAs have been considered exclusively expressed in germline cells, but recently, it has been reported by several studies their presence also in somatic and pathologic tissues5,48,56–58. Germinal cells generally show the most significant number and a higher level of expression of piRNAs. Starting from this knowledge, we tested the workflow on small RNA data obtained from human testis samples and tumour cell line (COLO205) to assess the capability to detect piRNAs in high and low concentration. Using WIND, we analysed the dataset and represented the results as plots of biodetection (Figure 2A and B) and countsBio (Figure 2C and D) per sample from NOISeq59 package. Biodetection plots are made from raw data in order to explore: a) the percentages of each small RNA type (named \"biotypes\") in the genome (referred to the whole set of small RNAs provided); b) the proportion detected in each sample; c) the percentage of each biotype within the sample. The countsBio plots, instead, show the count distribution for each biotype displayed as box plots, and the number of sncRNAs detected per biotype. Here, the two biodetection plots show, as expected, the presence of higher percentage of piRNA in testis sample respect to the COLO 205 cells (~80% in testis and ~25% in COLO 205; Figure 2A and B). Moreover, considering the countsBio plots (Figure 2C and D), it is also possible to assess piRNAs higher median expression in testis if compared to the COLO 205 cells. Finally, we also produced sequence logos for the expressed piRNAs in the two sample types. These plots indicate if the bias for uridine at first position base or the adenine at the 10th position of the sequence exists and if there are other biases in the 15 first bases of the sequences60. As expected, both groups showed a strong bias for uridine at the first position (drawn as thymine in the plots), in accordance with the preferential binding of PIWI proteins to transcripts starting with U. A bias, albeit with a much weaker signal, was also evident toward adenine at position 10 in testis group, a hallmark of piRNAs generated by the ping-pong cycle and typical feature of germinal cells (Figure 2E and F).\n\nA) and B) Biodetection plots (genomic approach) from NOIseq reporting: percentages of each sncRNA type called \"biotype\" on the genome (grey bar) for one of the samples; the proportion detected in each sample (red stripes bar); the percentage of each biotype within the sample (red bar). The biotypes on the right side of the green dashed line are the least abundant. C) and D) CountsBio plots (genomic approach) from NOIseq showing the count distribution for each biotype displayed as boxplots. Numbers on top of the plot show how many sncRNAs are detected per biotype in the entire dataset analysed. Different colours indicate different sncRNA classes. E) and F) Sequence Logo (1-15 bps) extracted from the piRNA sequence of the expressed piRNAs found in each group of samples (transcriptomic approach). A, C, E represents the results obtained for one representative testis samples, while B, D, F represent one representative COLO 205 sample.\n\nFor this analysis, we applied a stringent approach; thus, we considered as expressed only those molecules that were identified by both methods (genomic and transcriptomic). Then, we found that 6982 piRNAs were identified in testis and 240 in COLO 205 cells. Therefore, this workflow was able to efficiently identify a good number of piRNAs in somatic cells, where low levels of expression make the procedure more complicated, even when very stringent analysis parameters are used.\n\nIt is worth mentioning that, as detailed in the Methods, this workflow operates using two methods in parallel, each of which is able to identify sncRNAs with different performance. Applying the two algorithms together (considering the union of the results) allows the identification of an enriched number of molecules. The final user can decide, based on specific interests, which results should take into consideration, the union of the two approaches, only one, or the intersection.\n\nWe also evaluated the performance of the workflow for piRNA identification in the mouse. Specifically, we analysed small RNA-seq samples from mouse adult cardiac myocyte (aCM). In these samples, we were able to identify, considering the union of the genomic and transcriptomic approach, ∼ 470 different piRNAs per samples (see Extended data: Supplementary Table 3 for the details of the two analysis in comparison). We found that the piRNA population identified in aCM represents 11% of all reads assigned to small RNAs, and the top 100 expressed molecules are listed in Supplementary Table 3 (Extended data).\n\nMoreover, to test the accuracy of the workflow across diverse sets of data, we moved to a public dataset. Recent findings have indicated that the role of piRNAs may not be only limited to germ cells, but may be extended to the regulation of cancer, promoting a stem-like state of tumour cells61. Therefore, we selected a dataset (GSE68246) to compare the piRNA profiles of breast spheroid-enriched CSCs against parental MCF7 cells and also generated in this case files, statistics and plots with WIND that are all available on the GitHub repository. On the expression data, filtering for low-expressed features was first carried out, then two of the NOIseq filters (1 count per million, and proportional filtering) or the EdgeR were applied, filtering by group with and without the specific batch. The resulting objects were reported as RDS files and, for all the analysed sequences, a histogram (Figure 3A and B) with the average log2 CPM before and after filtering of the counts was made using the edgeR filtering. Finally, the normalisation of all the counts was carried out with multiple methods: TMM62, TMMwsp (TMM with singleton pairing), RLE63, limma-Voom, with and without quality weights quantile64, Voom, with and without quality weights using the TMM normalisation, and Voom with and without quality weights exploiting the TMMwsp normalisation. To visualise the unforeseen sources of variation and to control whether the normalisation applied was correct, RLE plots (Figure 3C and D) were generated for all the sequenced data, for each normalisation method and for the not normalised, filtered data. An RDS object was also exported with the list of all normalised objects, and hierarchical clustering (Figure 3E and F) was then performed on previous data with various normalised methods. We applied the Euclidean distance and the methods of Ward's, complete and average linkage. Furthermore, a correlation plot (Figure 4A) with sample-to-sample distances was made to show the similarities and dissimilarities between samples on all sncRNA data. In order to check for batch effects and get the summarised effects of the experimental categories, MDS plots (Figure 4B) and the first two Principal Components on a PCA plot were reported (Figure 4C). From the GTF file, sequences' lengths were extracted and combined with information about the expressed molecules to draw the histograms (Figure 5), allowing to underline the differences between the two methods or between the groups of interest. Alongside, in this case, the sequences' logos for only the expressed piRNAs were generated. Moreover, we reported a tab-delimited file with the mean CPM per biological group, as it is useful to know these values for further studying or visualisation. All the files, plots and statistics are available in the GitHub repository.\n\nA–B) Histograms of average log2 Counts Per Million (CPM) among all samples before (A) and after (B) filtering with one of the selected methods (EdgeR filtering in this case) for sncRNA data. C–D) Relative Log Expression (RLE) plots for each normalisation method, made with the use of plotRLE function for all the sncRNA data. As an example, only the first two plots (with TMM (D) and without normalisation (C) for the filtered counts derived from FeatureCounts) are shown. E–F) Hierarchical Clustering plots, exploiting all the sequenced sncRNA data, with multiple clustering methods and different normalisation methods. As an example, only the first two plots (with TMM (D) and without normalisation (C) for the filtered counts derived from FeatureCounts). In black and brown are shown the two different groups (monolayer and spheroid).\n\nA) Correlation plot showing samples' distances in GSE68246 dataset. The darker the colour, the more correlated they are. B) Multidimensional Scaling (MDS) plot using all the sequenced data and one of the normalisation methods applied in the workflow (in this case, TMM) made with plotMDS() function from EdgeR. In black and brown are shown the two different groups (monolayer and spheroid). C) Principal Components Analysis (PCA) plot displaying the first two Principal Components using all the sncRNA molecules data. Each sample is shown with different colours (depending on the group) and different symbols (depending on the batch).\n\nThe colours indicate the two different methods of quantification (genomic and transcriptomic).\n\nUltimately, we performed the differential expression analysis on the results of both methods (genomic and transcriptomic), and the union of the comparisons was reported (Extended data: Supplementary Table 4). Our workflow identified 399 differentially expressed sncRNAs (p-value ≤ 0.05) using both methods and 382, considering the adjusted p-value ≤ 0.05. 68 miRNAs were found DE, in common with Boo et al.52. Most importantly, we were able to identify 81 expressed piRNAs, 20 of which differentially expressed (adj. p-value < 0.05) between spheroids and parental cells, with 17 of them up- and 3 down-regulated (Figure 6A). Their log-fold changes were varying from -3.12 to 6.67, and all of them derive only from the sequences found in RNAcentral, none in piRNABank, thus showing the importance of including both databases in the final GTF file. Of these 20 DE piRNAs, five of them (DQ570339, DQ570326, DQ584904, DQ582231, DQ593270) have also been found DE in the work of Vella et al.65 in cardiosphere-derived cells. This suggests a possible functional role of this group of piRNAs in the stemness of cancer cells, independently from the tissue type.\n\nA) Heatmap of differentially expressed piRNAs in 3 MCF7 Spheroid samples versus 3 MCF7 Monolayer (GSE68246 public dataset) found in common with both approaches (genomic and transcriptomic). B) Heatmap of differentially expressed piRNAs among 9 Primary Solid Tumour versus 9 Solid Tissue Normal from TCGA found in common with both approaches (genomic and transcriptomic).\n\nAs a final test, we exploited sncRNA data of 18 samples from TCGA-BRCA, 9 Primary Solid Tumor versus 9 Solid Tissue Normal (Extended data: Supplementary Table 5). We identified 11 piRNAs DE out of 198 DE sncRNA molecules with both approaches (genomic and transcriptomic). In the heatmap (Figure 6B), it is possible to note that the two approaches obtained equivalent results, and the clustering approach showed a good clustering between tumour and normal samples. Interestingly, some of the identified piRNAs have been previously described as related to cancer progression in breast (DQ57069858) or other tissues like kidney, ovary, and lung (DQ58103366, DQ593398 - DQ59293267, piR-5272968). In addition, from the 198 DE small RNAs, 55 are reported as miRNA and most importantly, we found the cancer-specific MIR-8 (now reported as mir-141 and mir-200) upregulated as previously reported by Hoadley et al.55. In order to acquire possible functional information about the DE piRNAs, we predicted their possible target RNAs (using the code included in the workflow), and we identified 12 genes, 9 long-coding RNAs and 3 protein-coding genes (Extended data: Supplementary Table 5). All of them are predicted to bind their targets at the CDS region, two at the 3' UTR and one at the 5' UTR. The functional enrichment analysis of the 12 predicted piRNA targets, using the EnrichR online tool69, revealed that they might be involved in regulating \"signal transduction that contributes to a DNA damage checkpoint\" (GO:0072422), a biological process that has a vital role in cancer progression.\n\n\nConclusions\n\nIn this paper, we describe a novel bioinformatics workflow, WIND17, for the identification and analysis of piRNA from small RNA sequencing data. The main innovations of WIND are: a Docker containerisation approach for the complete analysis, the integration of two databases for piRNA annotation, a dual-method for detection and quantification of piRNAs (named as \"genomic\" and \"transcriptomic\" in this article), and the creation of ready-to-use plots and statistics for the interpretation of the results. The idea was born in order to cope with the absence of a gold-standard pipeline for piRNA identification and annotation. We tried to solve many issues related to small RNA sequencing data analysis and, in particular, piRNA identification and quantification. For this reason, the first step was to deploy multiple Docker containers set up to run all the steps of the workflow without installing tools, software or libraries. After this, we focused on the creation of an easy method to integrate data from distinct databases (RNACentral and piRNABank). As described in methods, we were able to assess the deep diversity between the databases. Indeed, it was possible to notice not only differences in numbers of piRNAs annotated between the two databases (both in human and mouse genome) but also inconsistencies in the annotation or in the classification (e.g. the same molecule is classified as piRNA in one and as miRNA in the other). Actually, combining databases usually produces discrepancies and working with sncRNA sequences that have multiple annotations is troublesome. However, with this step, it is possible to obtain a unique GTF file that merges this information (all ids and genomic locations associated with that specific molecule) that can be used for piRNA identification and annotation. The main part of our workflow consists of two detection methods for piRNAs described above as \"genomic\" and \"transcriptomic\". For the genomic part, we decided to perform an alignment using STAR. STAR is a well-known genomic aligner that uses a reference genome to compute read alignments. For the transcriptomic part, we used Salmon to produce accurate transcript-level quantification estimates from sncRNA sequencing data. Salmon's main innovation is the use of quasi-mapping (accurate and very fast-to-compute read alignments). However, even if the transcriptomic approach proved to be working well, it has been demonstrated that for the identification of some sncRNAs might not be as efficient as the genomic approach36. For this reason, we set the methods to improve sncRNAs identification, following the suggestions of previous works35,36. Our idea was to combine the two approaches in order to evaluate the similarities between the results obtained and then ameliorate the identification of piRNAs. The last step was to create a Differential Expression module and, most importantly, the automatic creation of plots and statistics useful for the interpretation of data and results.\n\nTo test WIND, we applied it to several sncRNA datasets. Working on the first dataset, were we used the spike-in approach, we found a good consistency between the different methods in the detection of piRNA-like molecules, highlighting the efficiency of both approaches in piRNA quantification. Furthermore, the test on germline and somatic tissues revealed that the two methods, even when a stringent filter is applied, are able to assess the presence of piRNAs also in tissues where they are not abundant. In addition, the workflow is also functional in different species, as shown by the results obtained on the mouse genome. Finally, we also tested WIND on two published datasets, comprising tumour cell lines and tissues. Our workflow, also in this instance, was able to efficiently identify piRNAs and find differentially expressed molecules (not previously investigated) and to recognise, in general, a significant number of sncRNAs.\n\nIn conclusion, WIND is a complete dockerised workflow, usable by bioinformaticians and data analysts who want to explore small RNA sequencing data globally, but specifically designed and optimised for piRNAs. WIND allows going from raw data to plots and statistics ready for publication thanks to fast and efficient software implementation, making it very useful in the field of small RNA research.\n\n\nData availability\n\nArrayExpress: Monitor the efficiency of \"WIND: A Workflow for pIRNAs aNd beyonD\" for the identification of single-stranded (SS) spike-in piRNA-like molecules in smallRNA-seq, Accession number E-MTAB-9772: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9772/\n\nArrayExpress: Monitor the efficiency of \"WIND: A Workflow for pIRNAs aNd beyonD\" for the identification of piRNA molecules in small RNA-seq, Accession number E-MTAB-9782: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9782/\n\nArrayExpress: Monitor the efficiency of \"WIND: A Workflow for pIRNAs aNd beyonD\" for the identification of piRNA in mouse samples, Accession number E-MTAB-9866: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9866/\n\nArrayExpress: Analysis of the 3’-end of piRNAs in the COLO 205 cell line through sodium periodate (NaIO4) /β-Elimination treatment and small RNA-Seq, Accession number E-MTAB-8115: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-8115/\n\nNCBI Gene Expression Omnibus: miRNA transcriptome profiling of spheroid-enriched cells with cancer stem cell properties in human breast MCF-7 cell line, Accession number GSE68246: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68246\n\nSelected samples from the Genomic Data Commons Data Portal70 have been accessed and analysed from the TCGA-BRCA project: https://portal.gdc.cancer.gov/projects/TCGA-BRCA\n\nZenodo: Supplementary tables, http://doi.org/10.5281/zenodo.426838571.\n\nThis project contains the following extended data:\n\nSupplementary Table 1. Statistics of GFT files obtained for human and mouse genome. The file reports the data of the filtering process and the final GTF data.\n\nSupplementary Table 2. Spike-in quantification. For each sample are shown the percentage of each piRNA-like molecules, respect to the raw reads count, using three quantification methods.\n\nSupplementary Table 3. Statistics of sncRNA data analysis for mouse cardiomyocytes. The file reports the results obtained using the two methods applied in the workflow and the list of top 100 expressed piRNAs.\n\nSupplementary Table 4. Differentially Expressed molecules found for GSE68246 dataset. In yellow are highlighted miRNA DE in common with Boo et al.52, in red and green are highlighted the up- and down-expressed molecules respectively, in light blue and cyan the molecules with a p-value and adjusted p-value less than 0.05 respectively.\n\nSupplementary Table 5. Differentially Expressed molecules found for BRCA TCGA dataset. In red and green are highlighted the up- and down-expressed molecules respectively, in light blue and cyan the molecules with a p-value and adjusted p-value less than 0.05 respectively. For DE piRNA molecules, the predicted possible target RNAs are also provided.\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\nWorkflow available from: https://github.com/ConYel/wind\n\nArchived workflow as at time of publication: http://doi.org/10.5281/zenodo.428990917.\n\nLicense: MIT\n\nAll software packages used throughout this workflow are publicly available through the Bioconductor project (http://bioconductor.org), or the Comprehensive R Archive Network (https://cran.r-project.org) and all bioinformatics tools are freely available as Docker containers on https://hub.docker.com/r/congelos/.", "appendix": "Acknowledgements\n\nWe acknowledge ELIXIR-IIB (http://elixir-italy.org/), the Italian Node of the European ELIXIR infrastructure (https://elixir-europe.org/), for the computational power support provided.\n\nWe acknowledge Domenico Giosa, for his valuable input on using awk programming for the generation of specific files.\n\nThe results shown here are in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.\n\n\nReferences\n\nDuarte Junior FF, Bueno PSA, Pedersen SL, et al.: Identification and characterization of stem-bulge RNAs in Drosophila melanogaster. 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Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4268385" }	[ { "id": "76394", "date": "14 Jan 2021", "name": "Juan Pablo Tosar", "expertise": [ "Reviewer Expertise Molecular and cell biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors developed a workflow for small RNA-seq data analysis, especially intended for the study of Piwi-interacting RNAs or piRNAs. The authors called their workflow WIND (for Workflow for pIrnas aNd beyonD) which is a nice name but should not be presented as an acronym because it is not. The manuscript is professionally written and reads very well, and I think the workflow is complete and can be a useful resource. However, I have major concerns in its design that I will try to explain below.\nOne of the motivations of the authors was to develop a reliable package for piRNA analysis that can be also applied for piRNA identification in somatic cells. The authors cite our 2018 study (ref. 25) so they are aware that piRNA databases contain a small percentage of contaminating entries that are probably not piRNAs. They considered this information in the design of their workflow and removed all piRNA reads in piRNABank that also have an alternative annotation in RNA Central. However, it is not clear to me whether they did the same with the piRNA sequences in RNA Central that also have an alternative annotation in RNA Central. Figure 2A shows that roughly 80% of the sequences in testis are piRNAs, and also 80% of the sequences in testis are annotated as tRNAs. So this is the proof that, if the authors really intended to depurate their GTF file from piRNAs also having an annotation in RNA Central, they were not effective in doing so. And this completely alters the author’s conclusions regarding non-germinal piRNAs.\nAnother concern is that RNA Central is explicitly a database of non-coding RNAs. Thus, by removing entries in piRNABank that have an alternative annotation in RNA Central, they are not removing those piRNAs in piRNABank that are mRNA-fragments. How to distinguish secondary piRNAs generated from cleavage of coding sequences from mRNA fragments contaminating piRNA databases? It is not surprising, therefore, that the authors found that the remaing piRNAs in their GTF file are either derived from piRNA clusters or from protein-coding genes. This is a methodological bias and does not seem to be a deliberate decision based on the biology of the piRNA pathway. Again, this can completely alter the authors’ results and conclusions when analyzing RNA-seq data obtained in somatic cells using WIND.\nThe authors affirm that they focused in “solving on of the most challenging issues of (small RNA) analysis, the annotation controversies of piRNAs”. I’m afraid that, in my opinion, this controversy is still not solved. I would suggest the authors to read our last contribution in this topic (Tosar et al. 20201) and reconsider the design of their workflow based on what we discussed in that paper. My suggestion is to take the union of piRNAs from piRNABank and RNA Central, and remove those sequences that have alternative annotations in RNA Central. This can be used to construct GTF file 1 containing “canonical” piRNAs derived from piRNA clusters and also mRNA fragments (whether truly piRNAs or not). Then, remove sequences matching to human or mouse mRNAs from RefSeq to make GTF file 2, containing sequences that can only be classified as piRNAs. Repeat their analysis and compare the results shown in this manuscript with what they see based on my suggested approach.\nMinor comments:\nConsider adding “testis” and “COLO 205 cells” as a headline in Figure 2.\n\nThe authors refer that the problem of detecting piRNAs in COLO 205 cells is their low expression. However, there are some sequences which are highly expressed according to Figure 2, D. Are these sequences really piRNAs?\n\nA brief description of the sequencing library preparation should be supplied. If the authors spiked in methylated RNAs, treating the samples with sodium periodate before NGS could have been an interesting control.\n\nSequence logos are nice and can be informative, but the workflow could be more powerful if it included plots showing ping-pong signals.\n\nWhy 69 nt as a cut-off?\nI hope the authors find this suggestions useful and my comments constructive.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6638", "date": "14 May 2021", "name": "Francesca Rizzo", "role": "Author Response", "response": "Q.1 One of the motivations of the authors was to develop a reliable package for piRNA analysis that can be also applied for piRNA identification in somatic cells. The authors cite our 2018 study (ref. 25) so they are aware that piRNA databases contain a small percentage of contaminating entries that are probably not piRNAs. They considered this information in the design of their workflow and removed all piRNA reads in piRNABank that also have an alternative annotation in RNA Central. However, it is not clear to me whether they did the same with the piRNA sequences in RNA Central that also have an alternative annotation in RNA Central. Figure 2A shows that roughly 80% of the sequences in testis are piRNAs, and also 80% of the sequences in testis are annotated as tRNAs. So this is the proof that, if the authors really intended to depurate their GTF file from piRNAs also having an annotation in RNA Central, they were not effective in doing so. And this completely alters the author’s conclusions regarding non-germinal piRNAs. A.1 We thank the reviewer for the helpful suggestion. We removed all piRNA sequences in piRNABank that also have an alternative annotation in RNA Central. However, checking the sequences in RNA Central that also have an alternative annotation in RNA Central, we found only six molecules and none of them is a piRNA. These sequences have been deduplicated in the final GTF, and corresponded to snoRNAs. We apologize to the reviewer for the misunderstanding, in Figure 2, the categories on the right of the green dashed line should be referred to the axis on the right. In this specific case, the piRNAs in the testis sample are ~75% while tRNA are ~4% as shown on the axis on the right of the plot. To clarify this point, we specified this in the legend as follows: << The biotypes on the right side of the green dashed line are the least abundant, and the reference values are reported on the Y right axis.>> Q.2 Another concern is that RNA Central is explicitly a database of non-coding RNAs. Thus, by removing entries in piRNABank that have an alternative annotation in RNA Central, they are not removing those piRNAs in piRNABank that are mRNA-fragments. How to distinguish secondary piRNAs generated from cleavage of coding sequences from mRNA fragments contaminating piRNA databases? It is not surprising, therefore, that the authors found that the remaining piRNAs in their GTF file are either derived from piRNA clusters or from protein-coding genes. This is a methodological bias and does not seem to be a deliberate decision based on the biology of the piRNA pathway. Again, this can completely alter the authors’ results and conclusions when analyzing RNA-seq data obtained in somatic cells using WIND. A.2 We want to thank the reviewer for this challenging question. As suggested in question 3, we have created a new GTF that takes this into account by removing the sequences matching to human or mouse mRNAs. For more details see “answer 3”. Q.3 The authors affirm that they focused in “solving one of the most challenging issues of (small RNA) analysis, the annotation controversies of piRNAs”. I’m afraid that, in my opinion, this controversy is still not solved. I would suggest the authors to read our last contribution in this topic (Tosar et al. 20201) and reconsider the design of their workflow based on what we discussed in that paper. My suggestion is to take the union of piRNAs from piRNABank and RNACentral, and remove those sequences that have alternative annotations in RNACentral. This can be used to construct GTF file 1 containing “canonical” piRNAs derived from piRNA clusters and also mRNA fragments (whether truly piRNAs or not). Then, remove sequences matching to human or mouse mRNAs from RefSeq to make GTF file 2, containing sequences that can only be classified as piRNAs. Repeat their analysis and compare the results shown in this manuscript with what they see based on my suggested approach. A.3 We want to thank the reviewer for this very illuminating article regarding the piRNA annotation challenges. We agree with the reviewer that the problem has not been completely resolved, but we are trying to move in that direction and above all, we are trying to highlight that this problem must be considered and addressed in order to study the piRNAs correctly. Using the suggestions proposed, we created a new GTF file removing the sequences matching to human or mouse mRNAs. Using this approach, it is possible to note that some differences exist between the previous and the new results. For this reason, we modified all the tables, figures and data in the text accordingly. We are confident that now our workflow is stronger and more robust. However, it is possible to obtain the previous GTF from GitHub or change the code in order to produce the preferred GTF. Finally, we have also added on the method section the details about the creation of this new GTF file: <> Minor comments: Q.4 Consider adding “testis” and “COLO 205 cells” as a headline in Figure 2. A.4 We modified the figure as suggested. Q.5 The authors refer that the problem of detecting piRNAs in COLO 205 cells is their low expression. However, there are some sequences which are highly expressed according to Figure 2, D. Are these sequences really piRNAs? A.5 When we talk about the low expression of piRNAs in COLO205 we are referring to an average expression of all identified molecules, however, among these, there are also molecules with a higher expression. In any case, after the creation of the new GTF file, as described before, we reanalysed all the datasets and we were still able to identify some molecules highly expressed and classified as piRNAs. Obviously, WIND exploits the previous knowledge about sncRNAs, this means that the obtained results, even if more accurate thanks to these new improvements, are still limited due to the primary databases used. However, as always suggested, a wet-lab validation should be necessary to confirm the in silico results and to really establish the correct identification and function of the discovered molecules, but this is beyond the scope of this article. Q.6 A brief description of the sequencing library preparation should be supplied. If the authors spiked in methylated RNAs, treating the samples with sodium periodate before NGS could have been an interesting control. A.6 As suggested by the reviewer, we added three samples treated with sodium periodate before library preparation and now, in Supplementary Table 2, is available the “Treated” table with the percentages of all the spike-ins used. Furthermore, we modified the method section accordingly: Q.7 Sequence logos are nice and can be informative, but the workflow could be more powerful if it included plots showing ping-pong signals. A.7 We added in the GitHub repository a code called “pinp_pong.Rmd” that creates a ping-pong plot or a coverage plot for both the strands from a BAM file selected for piRNAs. Finally, we modified the Methods section as following: Q.8 Why 69 nt as a cut-off? A.8 We have selected 100 nts as a filter for sncRNAs sequences on both databases to filter out all other sequences that were too long to be sncRNAs. Following, we added a new filter to the sequences deriving from the piRNABank alignments to the genome (hg38). Although the piRNABank sequences that we used are shorter than 34 nts, when these sequences were aligned to the genome, the genomic ranges were in some cases ≥ 69 nts due to gap-opening, specifically, we found few sequences aligning on genomic regions longer than 33nts (69, 75, 81, 87 and 99 nts). This could be due to the fact that the original piRNABank database was built on genome version hg18. In order to exclude these genomic ranges, which correspond to molecules that do not align correctly on the new version of the genome, we applied the filter at 69 nts as piRNAs are considered to be about 28-34 nts. However, the users can easily change this number or remove this filter as they prefer." } ] }, { "id": "80430", "date": "25 Mar 2021", "name": "Wen Yao", "expertise": [ "Reviewer Expertise Genomics", "Bioinformatics", "Genetics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 developed a new computational pipeline for analysis of piRNAs in small RNA sequencing data. The methods were elaborated clearly in the manuscript. The pipeline was tested with real small RNA sequencing data of human and mouse. The manuscript is well written and the WIND pipeline will be a good tool facilitating the analysis of piRNAs. I have several comments and suggestions for the authors.\nMajor:\nThe title should be revised as “a strategy for in-depth analysis of small RNA-seq data” is confusing. The WIND pipeline mainly focuses on the analysis of piRNA, which is only a category of all small RNA species. For example, miRNA is ignored by WIND.\n\nI agree with the author that the key to piRNA analysis is the comprehensive and accurate identification of piRNAs and piRNA precursor. However, only piRNABank and RNAcentral were used in the “Annotation forging” step of WIND while updated databases of piRNA and piRNA cluster had been published. I suggest the authors integrating piRBase1 and piRNA cluster database2 in the WIND pipeline.\n\nFigure 1. “small RNA Fastq files” were not used in the “Annotation forging” step, and should be placed in the “Pre-processing and quantification” step.\n\nIn the “Annotation forging” step, I noticed two length thresholds (100 bp and 69 bp) were used when building the “final Fasta file”. So why 100 and 69?\nMinor:\nFigure 5 is bar plot rather than histogram.\n\nFigure 6B - part of the legend at the bottom is obscured.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6639", "date": "14 May 2021", "name": "Francesca Rizzo", "role": "Author Response", "response": "Q.1 The title should be revised as “a strategy for in-depth analysis of small RNA-seq data” is confusing. The WIND pipeline mainly focuses on the analysis of piRNA, which is only a category of all small RNA species. For example, miRNA is ignored by WIND. A.1 The most problematic part of this workflow was, without any doubt, creating a good GTF file and removing all the possible noise from piRNA sequences. We agree that, in this paper, we focused our attention mostly on piRNAs but our workflow is made for analysing all species of sncRNAs as shown in the figures. A user can easily focus his attention on another small RNA species and perform all the analyses exploiting the genomic and the transcriptomic approach. For this reason, we choose to write in the title “piRNAs and beyond”, to indicate that our workflow is suggested not only for the study of piRNAs but can also be used to analyse other sncRNAs molecules. Q.2 I agree with the author that the key to piRNA analysis is the comprehensive and accurate identification of piRNAs and piRNA precursor. However, only piRNABank and RNAcentral were used in the “Annotation forging” step of WIND while updated databases of piRNA and piRNA cluster had been published. I suggest the authors integrating piRBase and piRNA cluster database in the WIND pipeline. A.2 Thank you for your valuable comment. Initially, we have incorporated piRNAbank in our WIND pipeline as the first building block for the piRNAs annotation track. After that, we incorporated RNACentral to include also sncRNAs sequences in the annotation track in order to have a complete view of the sncRNAs species (known until now). However, regarding the additional piRBase database, we are facing a major bottleneck in including it. In detail, the database provides the largest and more comprehensive resource of piRNA sequences annotation, including more than 8 million sequences for human and about 60 million sequences for mouse. A major problem is that many of these sequences are located in the same genomic locus, and they differ only in one or a few nucleotides opening the possibility, not yet demonstrated, that they could be piRNA isoforms produced by variations on 5’ or 3’ end, including nucleotides extension, addition or trimming. This situation raises the question of how these molecules should be quantified and including all of them as independent molecules (“In piRBase, if a piRNA sequence is a subsequence of another piRNA, both of them were considered as different sequences and were assigned distinct piRBase names.” cited from Wang et al. (2019)) would significantly falsify the abundance ratio in the quantification step when measuring gene expression with Featurecounts or Salmon. Indeed, this could be an additional methodological constraint that would generate biased counts for any further downstream analysis (e.g. differential expression analysis). On this premise, we are considering including piRBase in a future version of WIND, but some issues about the quantification need to be solved. Nevertheless, about piRNAClusterDB (piRNAcldb), as suggested, we integrated the information included in piRNAcldb as metadata in the final GTF file produced by the annotation forging step. We updated the workflow shown in Figure 1 and we added the following sentence in the Method section: Q.3 Figure 1. “small RNA Fastq files” were not used in the “Annotation forging” step, and should be placed in the “Pre-processing and quantification” step. A.3 We modified the figure as suggested. Q.4 In the “Annotation forging” step, I noticed two-length thresholds (100 bp and 69 bp) were used when building the “final Fasta file”. So why 100 and 69? A.4 We have selected 100 nts as a filter for sncRNAs sequences on both databases to filter out all other sequences that were too long to be sncRNAs. Following, we added a new filter to the sequences deriving from the piRNABank alignments to the genome (hg38). Although the piRNABank sequences that we used are shorter than 34 nts, when these sequences were aligned to the genome, the genomic ranges were in some cases ≥ 69 nts due to gap-opening, specifically, we found few sequences aligning on genomic regions longer than 33nts (69, 75, 81, 87 and 99 nts). This could be due to the fact that the original piRNABank database was built on genome version hg18. In order to exclude these genomic ranges, which correspond to molecules that do not align correctly on the new version of the genome, we applied the filter at 69 nts as piRNAs are considered to be about 28-34 nts. However, the users can easily change this number or remove this filter as they prefer. Minor comments: Q.5 Figure 5 is bar plot rather than histogram. A.5 We corrected the error in the figure legend and in the text. Q.6 Figure 6B - part of the legend at the bottom is obscured. A.6 We corrected the issue." } ] } ]	1	https://f1000research.com/articles/10-1
https://f1000research.com/articles/10-554/v1	09 Jul 21	{ "type": "Research Article", "title": "Cultural heritage buildings for urban tourism destinations: portraits of Siantar, Indonesia, in the past", "authors": [ "Erond Litno Damanik", "Daniel H.P. Simanjuntak", "Daud Daud", "Daniel H.P. Simanjuntak", "Daud Daud" ], "abstract": "Background: This study was motivated by the failure to use historic buildings, plantations heritage, and modernization of Siantar. The problem is focused on the optimization of historic buildings, icons for urban tourism destinations. The study contribution is useful for the protection, utilization, and development of cultural heritage buildings into a tourist destination in urban areas. More specifically, the study aims to explore and discuss the optimization of urban tourism to support economic and territorial growth. Methods: The study was carried out qualitatively with a pragmatic methodological approach according to the tourism paradigm. The study departs from the colonial archives: photographs, maps, notes, and field research focused on the identification, significance, and contribution to urban history. The data were transcribed verbatim and analyzed thematically. Raw information was reduced and coded according to the relevance of the study. Data are combined into categories and themes reflecting descriptive analysis, classification, and interpretation. Data validation was done through triangulation strategies, member checking, rich descriptions, and saturation. Results:The Historic Tours of Siantar and Its Surroundings, the findings of this study were carried out in three stages; development based on national consensus in law, utilization into public space, appreciation for managers, and management incentives, and determining urban tourism designs. Conclusions: Utilization of cultural heritage buildings for urban tourism destinations reflects the urban with plantation characteristics, portraits of cities in the past, packed into urban tourism experiences.", "keywords": [ "Heritage", "buildings", "urban", "tourism" ], "content": "Introduction\n\nIn Pematangsiantar, Indonesia, based on an infrastructure map dating back to 1926, at least 57 monumental buildings were recorded during the plantation period1,2. All of these buildings were offices, schools, worship places, hospitals, hotels, cinemas, train stations, cafes, swimming pools, and banks. Initially, the 57 buildings belonged to the colonial government, plantation companies, and missionaries. The exception is that the map does not include buildings belonging to the native Siantar’s autonomous elites or shops scattered around the city. The construction of buildings was motivated by three main factors: (1) plantation expansions, (2) transformation of villages into cities, and (3) evangelism.\n\nPematangsiantar is a city of plantations, its growth having been determined relatively by plantations, notably since 1907. Except for Medan, no other region in the Eastern part of Sumatra claims to be more developed than Siantar. As a plantation area, the number of historic building units undoubtedly increased with the construction of shops, houses, offices, bridges, warehouses, and factories around the city. The entire heritage is estimated to consist of more than 300 buildings1.\n\nIn this study, the heritage of the plantation, according to Law number 11 of 2010 concerning Cultural Heritage, is Cultural Heritage Buildings. After the independence in 1945 and the Nationalization policy, especially against Dutch assets in 1958, most of the buildings were controlled by the government, private sector, and individuals. Some of them were returned to their original function and some of them switched functions2.\n\nDuring 1970–2015, 22 buildings were abandoned, 11 were damaged and 17 were destroyed. Today, at the time of this research, 36 iconic units have been found concentrated around the heart of the city, at ground zero, to the north and south sides of the city. In general, the remaining heritages are government-owned offices, churches, hotels, and private schools.\n\nThe reality of heritage is summarized in six pejorative assumptions; (1) relatively high resistance to urban development, (2) irrelevance to modernity and social change in urban areas, (3) obsolete, worthless, and out of date, (4) symbols of colonialism, slavery and exploitation, (5) no longer relevant nor under the spatial planning and building layout of an urban setting, and (6) an eyesore to the city's aesthetics.\n\nIn this study, heritage is seen as having at least six urgencies and significances; (1) an icon of the city, containing memory and identity, (2) urban monuments, history of development and modernization, (3) source of knowledge, architecture, land use, and spatial planning, (4) relevance to territorial development, (5) historic zonation in urban areas, and (6) urban nostalgia. All of the above six urgencies and significances contributed to economic and territorial development through their use as a heritage tourist destination.\n\nOptimization of heritage tourism in Siantar correlates with its geographical position and absolute advantages; (1) being adjacent to Lake Toba which offers panoramic views of nature3, (2) being known as a student-friendly, tolerant, multicultural city, and urbanization destination, (3) surrounded by tea, rubber, palm oil, and cocoa, packaged into a single plantations-tourism, (4) as a bridge connecting the west and south coasts of North Sumatra.\n\nThis study emphasizes the use of heritage as anurban tourist destination. Urban tourism is correlated with economic benefits based on position and strategic advantage4. Urban tours require planning and design, the latter being intended to protect, preserve, and take advantage of heritage, especially buildings that exist. Heritage becomes an urban icon, containing memory and identity4, sustainability and development of the city5, contains architecture6, aesthetics, land use, climate adjustment and becomes the cradle for designing a historic area7, requires conservation8,9 developed into a tourist destination in urban areas10.\n\nThe focus of the study is formulated on the following first question: why is heritage tourism important? The answer is summarized in the following four points; (1) preserving the remaining heritage as historic evidence, memory and city identity, (2) helping the development of the city through the tourism sector, (3) increasing the attractiveness and tourist destinations of the city, and (4) encouraging economic and territorial growth. The second question is how to design heritage-based urban tourism, which is at the core of this study.\n\nBased on these two questions, the two main assumptions of the study are formulated as follows; (1) the implementation of urban tourism contributes significantly to economic and territorial growth, (2) urban tourism is correlated with the conservation of icons, monuments, and nostalgia. Our goal is to design urban tourism based on existing buildings according to a strategic position.\n\nAlthough the political will of the government or stakeholders is relatively low, a logical and rational tourism design and model is very helpful for heritage conservation in urban areas. At a minimum, the government and stakeholders must change their perspectives on historic buildings. Heritage-based urban tourism is nostalgia, a portrait of Siantar in the past, in addition to providing economic incentives, as well as contributing greatly to territorial growth.\n\n\nLiterature review\n\nHistoric buildings or cultural heritage buildings become the basis for tourism development in urban areas and their surroundings. Heritage-based urban tourism is the type of tourism that is most developed today11. Heritage tourism is the fastest-growing travel segment and is changing the nature and trends of tourism in the world. Recent studies have shown a relatively high and more competitive demand to visit places that have a heritage in the tourism domain12. Heritage, thus, contributes to local income and development and at the same time preserves the history of an area’s past13.\n\nHeritage is the cultural wealth of an area. Cultural property includes both movable and non-transferable cultural property14. The conception of cultural wealth refers to all aspects, including works of art, customs, museums, monuments, construction, folk art, natural history, and even wildlife, all of which are considered to be heritage15. In the World Heritage Convention in 1972, heritage includes architecture, groups of buildings, and sites16. More specifically, historic monuments are not just any single architecture but also include urban settings in which evidence of certain civilizations or developments of historic importance is found16. Based on this statement, the entire urban is a monument, an accumulation of cultural creations and expressions over the years.\n\nThe historic nuance of the city is recorded in its heritage, a mainstay of tourism in the city, and its surroundings. Urban tours are different from other types of tourism. Urban tourism reflects high population density and diverse cultural offerings in a relatively small area but attracts many types of tourists. Urban tourist attractions and facilities lead to the interaction between tourists and residents. Tourism is directed at all things about the past that anyone can visit17. Urban tourism includes museums, districts, reconstructions, sculptures, monuments, and shrines, using a historic perspective in various manners18. Both, historic heritage and urban tourism are collaborative industries, turning the location into a destination and making it economically viable19–21.\n\nHeritage-based tourism is rooted in nostalgia, an economic determinant for urban development22. Tourism is an agent and tool for social and cultural change in urban areas. Heritage tourism reflects the growth and success of historic heritage management17. Heritage tourism is seen as optimizing the use of the cultural heritage of a community, region, country, or even continent in the global tourism market23. Global interest in downsizing public spaces increases the privatization of goods and services. Urban tourism is becoming a trend that is oriented towards entrepreneurship and entertainment24, two determinants of tourism success in urban areas. Tourism, in many cases, becomes a force of rejuvenation and a loss of heritage integrity.\n\nUrban tourism has consistently been one of the fastest-growing segments of the travel phenomenon in developed economies. This fact is relevant and in line with the rise of mass tourism in the 1960s25–26. The trips of tourists to cities with high population density are short-duration, usually 1–3 days27. Cities are a destination for migration, not only for business, trade, education, or those looking for work but also as places where people gather intending to seek pleasure27. The growth of urban tourism is driven by spatial, social, economic, and technological forces28. The urbanization process has led to an increase in the number of visits and to feel more connected to the urban lifestyle29. Urban tourism is a significant driver of the economy. Facilitated with access to cellular information, the boundary between tourists and residents becomes increasingly blurred and encourages tourists to increasingly seek, find, and consume local experiences30.\n\nBeing cultural centres, the largest tourist destinations in the world are cities. More than half of the world's population lives in cities, and it is calculated that by 2030, about 5 billion people will live in cities31. Every year, hundreds of thousands of villagers visit cities. Based on Euromonitor, international tourist arrivals in 2013 had increased by 4.8%31. In the same year, the top 100 cities in the world experienced a growth of 5.4%31. Urban vacations include business, education, and family visits. Sun and beach holiday activity grew by 31% in 2013-2018, representing 29% of all vacation trips31. The holiday tourism segment grew by 28% and reached a market share of 23%. Since 2013-2018, urban trips have increased by 72%. In fact, in some cities, travel goes beyond sun and beach holidays as the most popular holiday31.\n\nIn the UK for example, urban vacations reached 42% in 2014 compared to 38% for sun and beach holidays in the same year32. The reality in developed countries is different from Indonesia. 1,530,268 international tourist visits in August 2019, decreased by 89.22% or 164,970 in August 202032. The largest numbers of international visitors were from Timor Leste, Malaysia, China, the United States, and the Netherlands33. This decline is closely related to the coronavirus disease 2019 (COVID-19) pandemic, national political conditions, and the gradual decline in the quality of tourist attractions.\n\nIn this study, urban tourism is intended to encourage urban growth through the utilization of heritage. Heritage is important to remind the city of nostalgia and all aspects that influence it. Siantar, for the record, the territory of the Damanik clan in Simalungunese, was assigned as a municipality in 19171. The transformation into a city started in 1907, when Sang Naualuh Damanik, a local ruler, was exiled to Bengkalis, Riau34. After the arrest, six other local rulers in Simalungun recognized Dutch authority35,36. The motivation for the conquest was the expansion of plantations on the east coast of North Sumatra, starting from Medan, extending to Binjai, Langkat, Serdang, Bedagei, Tebingtinggi, Simalungun, Batubara, Asahan, Kisaran to Labuhanbatu37–39.\n\nBefore 1907, Siantar, as Anderson noted in 1823, had a low population of fewer than 5,00039. The area was completely untouched by modernization until 190540,41. Siantar, especially since 1907, transformed into a city42, as well as a colonial plantation economic centre43. On the one hand, plantations are an indicator of capitalism44, economic globalization and legal changes45, and urban modernization46,47, but on the other hand, they marked the beginning of colonialism48, ethnic polarization49, agrarian disputes48, and slavery50.\n\nPlantations are indicators of modernization51–53. In Siantar and its surroundings, initially, historic buildings belonged to the companies Amsterdam Trade Union (HVA), the Amsterdam Rubber Culture Company (RCMA), the Dutch-American Plantation Company (HAPM), Rubber Plantation Investment Trust, Sumatera Rubber Plantation Ltd, and Harrison and Crossfield.\n\nOn the one hand, this study correlates with the improvement of the tourism-based urban economy, and on the other hand, it contributes to the preservation of cultural heritage buildings. Both of them are relevant, apart from being adjacent to the Lake Toba Caldera Geopark and tourism trends of the 21st century, potentially supporting growth and economic incentives offered by the tourism industry.\n\n\nMethods\n\nThe study was conducted between April-September 2020 and carried out qualitatively with a pragmatic approach54,55. The qualitative nature of the study was intended to identify and inventory all heritage units from the colonial period that still exist to this day. Identification and inventory were carried out by tracing references back to the colonial period, internet, and field studies. Field studies are required to obtain data on existing and demolished buildings. 36 buildings that still exist today were identified and registered, the data includes its state of the art, location, function, architectural style, owner, height, and area of the building. This strategy aims to obtain an accurate number of historical buildings and projected use as a tourist destination.\n\nThe pragmatic approach was intended to obtain a causal-functional explanation56, regarding the use of heritage to find locality-based tourist designs. Historic buildings are seen as having pragmatic, economic, and social values. Tourism design requires an interdisciplinary, anthropological, history, tourism, and economic development approach. This study is significant, especially in unravelling the potentials of historic buildings as well as for planning urban tourism according to geographical characteristics.\n\nArchival research. We searched the Dutch-language colonial archives for records of buildings, notes, photos, maps and sketches, in June 2020 at the office of the Dutch Language, Land and Ethnology Institute (KITLV), Jakarta. The archives are magazines (Tijdschrift), reports (Verlag), historical records (geschiedenis), notifications (mededeelingen), handovers (overgave), encyclopedia, and newspapers. Besides, the KITLV and Tropenmuseum Institute websites were accessed to enrich resources. Maps, especially photos of building units are matched through observation to obtain details, architecture, area, height, recent conditions, functions, and sustainability.\n\nField study. Field studies were carried out between April-September 2020 by observing the existing buildings to obtain the latest conditions. Researchers and assistants were divided into three groups to record buildings in 4 segments. Field studies were summarized in a logbook, recorded by date, recent situation and building details57.\n\nInterview. The interviews were conducted in two steps by the lead researcher (ED). Firstly, interviews with three heads of agencies for 2–3 hours, from the City Development Planning Agency (Bappeda), the Education and Culture Office, and the Tourism Office in their respective offices to obtain an explanation of the historical buildings in Siantar. The three of them were interviewed separately, in their respective offices. The interview was conducted after sending a formal letter and finding the right time. The interview was guided by a structured list and was not repetitive58. The focus of the interview was government policies on historical buildings, revitalization programs and long-term building utilization. A non-governmental organization, Sumatra Heritage Home, was also interviewed to obtain information and views on historical buildings in Siantar.\n\nThe four institutions were interviewed separately in a structured manner in May 2020 based on their authority, main tasks and functions as well as capacity. The interviews were recorded using a tape recorder and field notes were taken. The interview was terminated when the information was repeated, i.e. data saturation was achieved. Logbooks were compiled during the research process59.\n\nSecondly, a focus group discussion (FGD) was held for 5 hours, inviting 20 participants outside of government officials and journalists, consisting of academics, urban planners, architects, historians, travel agents, building owners, and non-governmental organizations. All participants were officially invited by email and reminded via the chat application. FGD is focused on the utilization of historical buildings as city tourist destinations. Each participant was asked to provide views and opinions on the urgency, significance, mechanisms and strategies of building utilization. The FGD committee is the researcher in charge of preparing a list of questions, managing discussions, taking notes and recording. In addition to developing questions, 16 main questions were asked to obtain information from each participant60. FGD was held in August 2020 at Siantar Hotel.\n\nAll data were compiled and tabulated for chronological analysis, to measure the economic contribution of an existing building. Data were identified and organized into tree coding, and arrange in excel. Bias or subjectivity is reduced over comparisons between informants. The data were transcribed verbatim and analyzed thematically. Data are combined into categories and themes reflecting descriptive analysis, classification, and interpretation. Data validation was done through triangulation, member checking, rich descriptions, and saturation. Results were presented to participants to obtain feedback.\n\nThree researchers are male and lecturer, one doctorate, and two masters. The three of them have research experience during 2015–2020.\n\nSiantar and its surroundings have more than 300 units of historic buildings as heritages from the plantation period. This number was obtained after calculating the building units in the city centre and its surrounding areas, including the Sidamanik tea plantation, Marjandi, Nagahuta, palm oil and rubber plantations in Sarbelawan, Bandar, and Tanohdjawa. The building units consist of offices, hotels, cinemas, banks, shops, schools, museums, houses of worship, hospitals, factories, warehouses, bridges, railway stations and yards, military and police barracks, employee official homes, residences, markets, and wildlife parks.\n\nIn the city centre, buildings are spread across three main settlements: (1) European buildings around Simbolon area at the South, (2) Chinese buildings around Cipto area at the city centre, and (3) Simalungunese buildings on Pamatang at the city centre. The city became the base for colonial government and administration which was adjacent to the Siantar self-government centre. The population of the city is European, Chinese, and indigenous. Around the city, buildings include factories, warehouses, coolie barracks, and homes of entrepreneurs.\n\nIn the colonial era, Siantar was divided based on ethnicity; the Muslims resided in Timbanggalung, Christians lived in East Siantar, Toba in North Siantar, Karonese in Tomuan, Javanese on Jalan Bali and Simalungunese on Jalan Asahan. Plantation workers, especially Javanese, occupied the barracks provided by the company at the plantation. Historic buildings around the city are still being discovered, even though most of them have been destroyed.\n\nAll buildings were the supporting infrastructures of the city, such as offices, hotels, banks, cinemas, ice factories, schools, hospitals, and houses of worship. Based on the Siantar City infrastructure map of 1926, there were 57 such buildings were recorded. However, the map does not include shops with Chinese architecture and the Simalungunese noble houses in Pamatang. Until today, of the 57 building units, 22 have been abandoned, 11 damaged and 17 destroyed.\n\nThis study found 36 iconic buildings that still exist. The 36 buildings are grouped into four segments: (1) Flower Gardens, (2) Pamatang, (3) Simbolon, and (4) Asahan. Table 1 shows historic buildings from the plantation era in 4 segments of Siantar.\n\nAll 36 buildings are currently controlled or owned either by the government or social institutions. These remaining buildings are concrete evidence that marks the history of the city’s growth and development. All of them have the potential to become objects and tourist destinations for the city if managed properly. It should be underlined that Siantaris located in the Flower Garden segment, the heart of Siantar which showcases European civilization. As the heart of the city, it interconnects government infrastructures, businesses, services, entertainment, and transportation. Precisely in the centre of the Flower Garden lies Ground Zero, the geographical point used for calculating the distance to other areas,connects the mayor's office, banks, Simalungun International Club, hotels, train stations, telecommunication offices, and cinemas. Figure 1 shows the heart of Siantar City in 1930.\n\nSource https://www.kitlv.nl.\n\nThe figure to the right shows a Siantar Hotel, owned by a Swiss national, built-in 1925. Parallel to the hotel is a railway station, the first means of public transportation in North Sumatra, built-in 1883. The railway network to Siantar was built-in 1917. In its day, trains played an important role in the mobility of goods from Siantar to the Port of Belawan. Simalungun International Club was a White Collar lounge frequented during the plantation period. The two-storied building is the mayor's office. It was built in 1917 when Siantar was declared a city. At the time of its establishment, Siantar City covered an area of 1,024 hectares, donated by Riah Kadim Waldemar Damanik, the King of Siantar, to Nagoriraad, the forerunner of the City Council. The mayor's office serves as historical evidence, marking the transformation of a village into a city.\n\nThe current path that separates the yard from the mayor's office is Jalan Sutomo. The tamarind tree on the left side of the road was imported from Medan to add to the city's aesthetics. In the distance, there is an unmanaged forest area. The Flower Garden segment is the embryo of the city adjoining the autonomous dominion of the local ruler, the Damanik clan in Siantar. Siantar, starting from the Flower Garden segment, has expanded to the south (Simbolon segment) and north (Asahan segment).\n\nMeanwhile, the Pamatang segment is a self-governing area, separated by the Bah (River) Bolon, upstream at Bah Sijengesan and emptying into the Malacca Strait. In 1938, Siantar began to develop rapidly. The orientation of the city’s development to the north and south had an impact on the unique layout of Siantar, extending to north and south and narrowing to the west and east. This uniqueness is due to the two main roads in the city centre, Jalan Sutomo, and Merdeka, which serve as economic centres since the plantation period.\n\nBased on city characteristics, by taking into account the surrounding environment and geographical position, the most relevant type of tourism in Siantar is urban tourism. As a medium-scale city, this heritage-based tourism is nostalgia-oriented, intended to preserve the city's collective identity and memory. Usually, in medium-scale cities, the urban’s cradle and history, icon, and relative identity are preserved.\n\nCompared to urban tourism where tourism tends to be directed at shopping, consumerism, and a modern lifestyle without much local experience, city tourism tends to reflect a complex recreational experience. In a medium-scalecity, visitors can still find the cradle of the city, buildings from a past period that are hundreds of years old. The cuisine tends to reflect different local peculiarities and is not as homogeneous as it is in metropolitan cities. Urban tourism introduces the grandeur of the city through its historical heritage, culinary delights that reflect the uniqueness of the local community, cultural attractions that describe local characteristics that tend to be different and attractive to tourists.\n\nUrban tourism tends to prioritize historical objects, packaged into tourist destinations. Thus, the essence of tourism is to sell nostalgia or memories of the past as a city identity. Cities in Turkey (such as Istanbul), Israel (such as Bethlehem and Jerusalem), Saudi Arabia (such as Mecca and Medina), Egypt (such as Cairo), Vietnam (such as in Ho Chi Min and Hanoi), Philippines (such as Intramuros), or Malaysia (such as on the island of Penang), and others, all sell their heritage as tourist destinations. The same fact is also found in the medium-scale city, Siantar in North Sumatra.\n\nTourism in Siantar does not offer the beauty of geographic or topographical landscapes. However, Siantar has historic nuances, namely as the seat of the autonomous Damanik clan, a plantation city, surrounded by plantations and adjacent to the Toba Caldera Geopark.\n\nUrban tourism in a medium-scale city is an activity that monetizes nostalgia, converting collective identity, and memories into economic value. Nostalgia intends to package destinations that reflect the level of civilization, knowledge, and human intelligence from the past. Urban tourism, thus, involves human activities that are designed to shift the ordinary tastes of visitors to the extraordinary, fostering enthusiasm for the city, to spend more time, and to shop. Tourism is directed towards visiting buildings that still exist to this day, especially at the heart of the city. The stories behind the buildings are developed so they make an interesting attraction. Each building has its own story which is recorded in its architecture, construction, function, layout, building layout, and current conditions.\n\nVisitors are directed to enjoy Siantar delicacies at the 105-year-old Kok Tong Cafe, stay at the 95-year-old Siantar Hotel, or enjoy a drink from the 95-year-old Siantar ice factory. Christian visitors can visit the 116-year-old Protestant Church on Jalan Gereja or the 86-year-old Catholic Church on Jalan Sibolga. Furthermore, visitors are directed to visit the two main hospitals in Siantar, namely the 87-year-old Djasamen Saragih, or the 82-year-old Army Hospital. Visitors can see the 91-year-old boarding school in Siantar or visit the 67-year-old Siantar Zoo. Besides, tourists are directed to the 82-year-old Simalungun Museum. The architectural style of plantation period buildings in Siantar ranges from art deco, modern to the renaissance.\n\nAfter visiting the historic buildings, visitors are directed to the tea, rubber, and oil palm, and cocoa plantations located around Siantar. At the plantation location, some factories process raw materials into production goods, for example, the Bah Butong Tea Factory, or rubber in Sarbelawan and palm oil processing in Tanohdjawa. Plantation offers knowledge of raw materials, processing, and produce goods for consumption. This experience is especially indescribable for foreign tourists.\n\nFurthermore, tourists are guided to visit the Toba Caldera Geopark61. This legendary destination was formed by a volcanic movement62. The lake tourism experience is coupled with mystical stories; the legend of the hanging stone, Samosir Island, ancient mountain peak which erupted 75,000 years ago, Pusukbuhit, the holy mountain where the ancestors of the Tobanese descended from the sky63 and the observation tower of Tele.\n\nMore specifically, the concept of urban tourism offered in Siantar according to the city's character, local experience, and the geographical position, the findings of this study, is The Historic Tours of Siantar and Its Surroundings. This concept offers a combination of heritage tourism, plantation-tourism, and the Toba Caldera Geopark which covers four main points; (1) to safeguard, protect and utilize heritage as tourist objects and destinations; (2) to encourage the creative economy and providing incentives for managers, owners, and stakeholders with an interest in heritage, (3) to preserve the collective memory and identity of the city, namely plantation heritage as the embryo of modernization, urban growth and development, and (4) to provide city tourist destinations supported by plantation-tourism and Toba Caldera Geopark.\n\n\nDiscussion\n\nThe implementation of The Historic Tours of Siantar and Its Surroundings can only be implemented through a synergy between the city government, the private sector, and the cultural community. Companies such as 4th State Plantation Companies which controls tea, rubber, and oil palm should work together with London Sumatra (Lonsum) for plantations-tourism purposes. The city government designates 36 sites as heritage, and cultural communities engaged in entrepreneurship and entertainment. Figure 2 shows the steps for utilizing heritage in the concept of The Historic Tours of Siantar and Its Surroundings.\n\nThe Historic Tours of Siantar and Its Surroundings could potentially be developed based on historic buildings. The use of buildings not only preserves the heritage of the plantation but also provides destinations for urbanites. The opportunities for The Historic Tours of Siantar and Its Surroundings will be wide if there is the synergy between the city government, private sector, and stakeholders. The government needs the political will to designate 36 buildings as protected and preserved sites in local regulations. After this stipulation, the synergies with stakeholders to design tourism programs, incentives, and stimuli. Urban tourism is necessary to open informal sector employment opportunities for 211.017 peoples, two-third of the city's population2.\n\nApart from presenting economic values as well as cultural experiences, The Historic Tours of Siantar and Its Surroundings also serve as the basis for leisure activity to acquire the values of modern society. In this context, heritage plays an important role as a meaning factory for visitors, namely plantations. Cultural motivation in urban tourism parallels increasing interest in culture in general. Short visits to the city, for example, play a role in increasing the number of visitors to all kinds of attractions64. The creation of destinations is not limited to cities or regions that offer a rich heritage background. The revival of tourism associated with nostalgia has an impact on increasing conservation and interest in visiting64. Nostalgia, it seems, remains a lucrative business.\n\nThe need for The Historic Tours of Siantar and Its Surroundings lies in the following five points: (1) the existence of heritage sites, (2) the opportunity for heritage preservation, (3) supports collective memory and identity, (4) a transit area, student city and urbanization destination from the north of Lake Toba and the South of the Malacca Strait and the industrial area of the special Sei Mangkei area, and (5) the availability of plantation commodities; tea, rubber, palm oil, and cocoa as well as the Toba Caldera Geopark which was set in May 2020 around Siantar.\n\nFigure 3 shows a model of The Historic Tours of Siantar and Its Surroundings, a typical tourism concept combining with historical building objects, plantations-tourism as well as nature tourism with the Toba Caldera Geopark.\n\nThe Historic Tours of Siantar and its Surroundings are focused on experiencing the cultural environment of a plantation in the city centre covering landscaping, visual arts, special lifestyles, values, traditions, and events. Furthermore, from the city centre visitors can visit tea, oil palm, or rubber plantations, the legacy of colonial plantations around Siantar, ending with the highlight of their tour at the Toba Caldera Geopark. Through this concept, Siantar as a buffer for the East coast of North Sumatra and Lake Toba should be able to seize tourism opportunities that take advantage of historic buildings in the city centre.\n\nIn this way, heritage tourists will spend longer and spend more, making tourism an important economic development tool65. This strategy was chosen primarily to optimize cultural resources that are used and promoted to increase the number of jobs and gross national product created by the tourism industry66,67. The visitors are those who are busy at school, business, or want to spend their spare time. Visitors are differentiated based on their special interests. This tourism is distinguished from other special interests such as nature and adventure.\n\nThe Historic Tours of Siantar and Its Surroundings focuses on 12 items at once; (1) language, (2) people, (3) handicrafts, (4) food and eating habits, (5) music and art, (6) the history of a place, (7) ways of working and technology, (8) religion that is stated in the story, (9) special architectural forms and characters, (10) the dress code of the local population, (11) the education system, and (12) activities during free.\n\nHowever, the difficulties faced today regarding the use of historic buildings as tourist destinations are summarized in the following four points; (1) there is no historical building that is legally protected as cultural heritage. This situation results in the vulnerability of buildings to abandonment, vandalism, and destruction. (2) No city government regulation establishes historic sites. This has a certain impact on the vulnerability of eliminating traces of urban embryo. (3) Not all historical buildings are used as public spaces accessible to visitors. (4) Historic buildings have not been included as tourist destinations. The reality is that tourism in Siantaris still focused on government-funded annual exhibitions, carnivals, and cultural attractions.\n\nThe four problems above lead to two main obstacles in designing The Historic Tours of Siantar and Its Surroundings; (1) low public and private involvement in the preservation, protection, and use of heritage sites as tourist destinations, and (2) lack of political will of the municipal government to issue regulations related to the preservation and protection of heritage sites. Midian Sianturi, Head of Bappeda, personal communication on August 23, 2020, stated67:\n\n\"We are reviewing 36 historical buildings in downtown Siantar. To protect, we are currently drafting a regional regulation. Next year, 2021, we hope it will be final. Protection is intended for various things, for example, the interests of urban tourism.\"\n\nUrban tourism contributes to the development and implementation of inward integrative strategies for future development68. Through urban tourism, local developments are designed based on historical zoning, industry, and development. The development of advanced tourism has an impact on the future growth of Siantar. The growth of urban tourism is reflected in four interrelated processes: (1) tourists, (2) local authorities, (3) attitudes to tourism, and (4) travel agency69.\n\nThe increase in tourism is influenced by four factors: (1) urbanization, (2) the development of low-cost airlines, (3) the tendency of the proportion to travel per year, and (4) the availability of internet-based services70–72. These four items reflect the synergy between government, stakeholders, and tourism actors. Synergy is needed to protect historic buildings, mobile-based optimization, and travel guides.\n\nBased on the above, the government and city stakeholders should have the political will to protect and conserve the urban’s tourism resources. Historic buildings have economic potential if managed properly and properly. Once the historic building is lost, it is difficult to rebuild it. Even if it can be built, some nuances are lost, and the storyline and scenario become irrelevant. Heritage sites require great attention, conservation policies, and appropriate use.\n\nOptimization of heritage sites is packaged into heritage tourism, as a development tool to encourage economic growth through the participation of visitors from other regions, who are motivated partly or wholly by history, artistry, science, lifestyle, community, regional, group, or institutional worship73. The stories behind the buildings invoke awe, respect, and pride in the city’s past. In it, they find value and meaning, namely nostalgia. Activities that package historic buildings as destinations are nostalgic tourism, activity summaries, and historic expressions embedded in monuments throughout the city.\n\nThe Historic Tours of Siantar and Its Surroundings has a high potential for urban-development due to several reasons: (1) adjacent to the Toba Caldera Geopark and the Strait of Malacca; (2) a student city, a migration destination for junior high school, high school, and university students; (3) a plantation city marked by historic buildings that still exist today, (4) surrounded by tea, rubber and oil palm plantations, (5) a pluralistic and multicultural city, and (6) a trading city which has developed drastically in the last 2 decades, marked by the Sei Mangkei industrial area and the Teluk Nibung port.\n\nThe Historic Tours of Siantar and Its Surroundings, thus, offer three main tourism component:(1) historic building tours, supported by souvenir shops, cultural attractions, culinary delights, (2) plantations-tourism, visits to tea, rubber, and oil palm around Siantar, and (3) natural tourism in the Toba Caldera Geopark.\n\nThe research finding, based on the description above, the historic buildings in the centre of Siantar have an impact on economic development impact if they are managedas tourist destinations. In this case, the tourism concept offered is The Historic Tours of Siantar and Its Surroundings, which is a tourism concept that offers, among others, the following destinations; urban tours centred on historic buildings, plantations-tourism centred on tea, oil palm, and rubber plantations, and natural tourism centred on the Lake Toba Caldera Geopark. To discover the impact of The Historic Tours of Siantar and Its Surroundings, the steps that need to be considered are the protection and revitalization of historic buildings from damage and destruction. In this step, the city government should issue local regulations protecting historic buildings, so that they can be managed into public spaces or at least made accessible to the public.\n\nThe political will of the government and stakeholders is expected to make The Historic Tours of Siantar and Its Surroundings a reality. Then, synergies with stakeholders such as entrepreneurs and agencies must builts to guide tourists while in Siantar. Without political will and synergy, historic buildings in the city centre will disappear and be replaced with new ones. This will undoubtedly erase traces of urban history, identity, and collective memory. The Historic Tours of Siantar and Its Surroundings offers a tourism concept that is intended as an economic development tool. Based on this study, through the concept of The Historic Tours of Siantar and Its Surroundings, nostalgia becomes an economically-empowered memory and identity. Urban tours, thus, offer the Siantar-based tourist content of the past.\n\n\nConclusions\n\nThe existence of historic buildings only is maintained or preserved through their use as an urban tourist destination. Without the management and utilization of it to become an urban tourist destination, the historic buildings will undoubtedly be destroyed. The historic building is a plantation heritage that the city characterizes contains nostalgia, and reflects modernization, and transformation to a plantation city. Utilization as a tourist attraction correlates with the preservation of historic, economic, and regional development values. This study concludes that The Historic Tours of Siantar and Its Surroundings are an optimization tool that links urban, plantations, and nature tourism. It's planning as urban tourism destinations reflect the history of development and modernization according to the characteristics of plantations, the portrait of Siantar in the past. This study recommends the protection of historic buildings through local regulations, revitalization, and utilization as public spaces or at least accessible to the public. Without the protection and revitalization, historic buildings will be lost.\n\n\nData availability\n\nFigshare: Interview data. https://doi.org/10.6084/m9.figshare.14236202.v558.\n\nThis project contains the following underlying data:\n\n- Anonymised interview transcripts from interviews\n\nFigshare: Interview-Focus Group Discussion (FGD) Data. https://doi.org/10.6084/m9.figshare.14315768.v160.\n\nThis project contains the following underlying data:\n\n- Anonymised interview transcripts from focus group\n\nFigshare: Research logbook April-September 2020 and 36 existing historic building in Pamatangsiantar until 2020. https://doi.org/10.6084/m9.figshare.14316164.v157.\n\nThis project contains the following underlying data:\n\n- Logbook for field study\n\nFigshare: Research logbooks. https://doi.org/10.6084/m9.figshare.14236250.v159.\n\nThis project contains the following extended data:\n\n- Researcher logbook/study timeline\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe researcher expressed his gratitude and appreciation to the Rectors, Chair of the Institute for Research and Community Service, and Dean of the Faculty of Social Sciences of the Universitas Negeri Medan. 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Publisher Full Text\n\nTripadvisor: TripBarometer 2015: Five Key Traveler Trends. www.tripadvisor.com/TripAdvisorInsights/n2582/tripbarometer-2015-five-key-traveler-trends. Accessed August 25, 2020. Reference Source\n\nSilberberg T: Cultural Tourism and Business Opportunities for Museums and Heritage Sites. Tour Manag. 1995; 16(5): 361–365. Publisher Full Text" }	[ { "id": "89406", "date": "02 Aug 2021", "name": "Rita Margaretha Setianingsih", "expertise": [ "Reviewer Expertise Archaeologists and Cultural Heritage Environment" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 no other resources or activities located in urban areas and providing entertainment. For example, what is in the Simbolon area or in the China area around Jalan Dr. Cipto, or in the Simalungun area? More discussion on matters related to The Historic Tours of Siantar and its Surroundings. So the discussion talks about Geopark and others - we recommend discussing the potential that exists in Pematang Siantar urban area.\n\nCultural heritage is indeed a primary element in urban tourism in Siantar City, but it must also be supported by secondary elements related to the combination of attractiveness that is felt to be unique and becomes a motivation for tourists. Secondary elements describe urban facilities that support and complement the tourist experience. For example, Pematang Siantar has old transportation facilities (such as: BSA = Birmingham Small Arms Company, Java, and others) which may be accessible by tourists for short distances.\n\nThere is less description of what tourists should do in Siantar City – something to see, something to do, and something to buy. Something to see – cultural heritage. Something to buy – culinary at Cipto Street, Ganda Bakery, Horas Market. Something to do – walk in the garden, city park walks, Goddess Kwam Im Statue (Vihara Avalokitesvara), Maha Vihara Vidya Maitreya.\n\nIn the abstract section, it is mentioned about the research results, one of which is the determination of urban tourism design. But there is nothing in the conclusion and discussion section. It is better to discuss this, especially based on locality and it is better to focus on Siantar City (limited research).\n\nFor urban tourism, an itinerary should be made, so that tourists know the list of activities and budget estimates (this is for management incentives). It has been listed, but it extends outside the city of Siantar, for example to the areas of Sarbelawan and Tanohdjawa. This is not in accordance with the title of urban tourism. Maybe the title should be The Historic Tours of Siantar and its Surroundings.\n\nAs stated in the abstract on the results of research on the use of public space, the appreciation of managers and incentives for managers has not been discussed and is not included in the conclusions.\n\nIt is better to use a library about the city of Siantar, not a library about the Simalungun area or plantations. .\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": "6991", "date": "03 Aug 2021", "name": "Erond Litno Damanik", "role": "Author Response", "response": "Thank you very much for the reviewer's suggestions. Here is our response to reviewers' suggestions: (1) More specifically, this study discusses historical buildings in Pematangsiantar City and its surroundings. In the manuscript, we mention that urban development marginalizes historical buildings, icons, collective memory, and identity as plantation cities. We describe that historical buildings must be preserved and can be optimized as tourist destinations, whether office buildings, cafes, gift shops, and others. In developing this city destination, we propose for a tourist attraction with its surroundings where there are plantations and also Geoparks. All three can be combined as a destination in North Sumatra. (2) We agree that city tourism must be supported by secondary elements, whether BSA, culinary, or other cultural attractions. However, our focus, the most important and first is how to save historic buildings, especially through local regulations. If this potential is maintained, both in the city core and its surroundings, plantations and Geoparks, it will move to the next stage, namely development, both the use of historical buildings, BSA, culinary and other attractions. (3) Appropriate. We are actually aware of this reviewer's suggestion. However, as we mentioned earlier that the initial motivation for writing this article was to save historical buildings that were managed to become city tourist destinations. If all the remaining buildings, these 33 units are designated as cultural heritage, we move to the next stage, namely development. These 33 buildings are to behold. This is the initial potential. All stakeholders and especially the city government must understand this first, furthermore move on to the next stage. (4) The result of this research is historical tourism in Pematangsiantar City and its surroundings which combines historic buildings in the city core, plantations in the main buffer, and Geopark in the secondary buffer. The three are complementary and interrelated. In the plantation area are found gardens and historical buildings such as tea and oil palm factories, hospitals and research centers. In Geopark, especially Parapat, we can find hotels since the colonial era as well as the Pinus Research Centre. (5) Sarbelawan, Marihat, and Balimbingan are an inseparable part of the building in the heart of the city. In addition to being a unit in the colonial era, at this time, the area is also a part of Pematangsiantar City today. In these areas historical buildings were found such as a Oilpalm Research Centre (Marihat), a hospital (Balimbingan), as well as emplacements and train stations in Sarbelawan. In our thinking, when talking about historical buildings in Siantar City, all the areas covered should be included as a single unit. Then, plantations and Geoparks are added values, where Siantar is traffic that is impossible to pass. These three potentials become one unit. Then, in Parapat, for example, the Pine Research Center and hotels built since the colonial era were found. All this must be explained in advance to develop a real tourism. (6) Appropriate. Historical buildings, 33 units were regulated and designated as cultural heritage through local regulations, then continued as public spaces so that the public can see them as evidence of history and culture. If all these remaining buildings can be saved and used as public spaces that can be enjoyed by the community, tourism managers will receive incentives from this utilization. For the time being, only a few buildings have survived and most of them are buildings managed as government offices and hotels. (7) Appropriate. Two things we want to say; (1) Siantar is the Simalungun area which was liberated as a city in 1917. All colonial archives always write \"Siantar-Simalungun” as a single unit, (2) references, more specifically to historical buildings in Siantar City, are minimal. The only reference is Tideman (1922). In addition, there were no other writings that specifically discussed historical buildings. I (Erond L. Damanik, one of the authors) have several articles about Siantar that I use as a reference. If we search on the internet, we can hardly find any journal articles or reliable sources about historical buildings in the city. The main sources of photographs are KITLV.NL, or the archives of both colonial and newspaper records at the National Archives of the Republic of Indonesia, Jakarta. However, the number is very limited. The limitations of this reference, on the one hand, become the motivation for this study, while on the other hand it is a challenge. We thank the reviewers for their reading and suggestions for this article. We can consider a number of suggestions for further follow-up studies." } ] }, { "id": "91291", "date": "18 Aug 2021", "name": "Michael Hitchcock", "expertise": [ "Reviewer Expertise Tourism", "heritage", "social science research methodology" ], "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\nGenerally, the paper is descriptive, and it is not clear what the contribution is to debates about urban heritage and tourism. This is partly a consequence of a lack of an adequate literature review. For example, the paper mentions the World Heritage Site issue, but overlooks some critical texts such as (some I have been involved in): Harrison and Hitchcock (2005)1; Hitchcock, King and Parnwell (2010)2, and King (2016)3.\nThe methodology is a bit disorganised and there is no explanation as to why this one and not another one was selected. It also does not engage sufficiently with other papers on research methods. The name of the approach needs to be stated clearly and close to the beginning of the section. It takes a while to work out what is being done. The results are written in a largely descriptive manner and there is a curious lack of critical engagement. It was not quite clear what the aim of the paper is. It is also inconclusive even though there is an attempt at a Conclusion.\nIn its current form the paper is not indexable, and the authors would need to thoroughly re-write it for it to be accepted. It needs to be thoroughly rewritten with much more development of its analytical purpose and more critical engagement with the existing literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "91863", "date": "31 Aug 2021", "name": "Milena Ivanovic", "expertise": [ "Reviewer Expertise cultural tourism development", "development and management of cultural heritage", "urban tourism", "historical cultural heritage", "tourism destination development" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article presents an overview of the heritage buildings in Siantar from the plantation period, the need for their conservation and possible utilisation as attractions in urban tourism, and the development of the heritage tours through the Historic Tours of Siantar and its Surroundings. It could have been an interesting article, but the argument put forward by the researcher falls short of expectations.\nThe article lacks focus, organisation, and constructive argument. Sections presenting the study results and discussion of the findings are intertwined with the literature. Since the aim of the article is not clearly stated, the literature review is misguided and too generalised. In addition, the site description should not be discussed in the literature. Statistical data meant to highlight the growth of urban tourism is confusing, and none is referring to Sumatra and Siantar. The authors cannot use a percentage of urban tourism growth in Europe to argue the potential growth of urban tourism in Sumatra. Europe is the most visited continent globally, the continuous cultural heritage destination with the most famous cities in the world. What exactly are the points for comparison with Sumatra or Siantar?\nThe study is well designed, especially because historical records of historical buildings were checked, compared, and verified on the ground. This approach gives credibility to a study. In addition, the study design follows a strict procedure of the inventory phase of the cultural attractions selection process. The clustering of attractions into four districts is the result of this process. Still, the primary historical significance of each cluster, the linkage corridors and the relationship between the clusters are not explained. The geographical map should be presented showing each cluster and how they are linked. The unique selling point is an offering of European, Chinese, and local heritage clusters surviving in a medium-sized city.\nThe study is not designed to be replicated because it implements the well-known selection process of determining cultural attractions.\nThe sources of data are submitted.\nGeneral comments:\nThe aim of the article is not clear. Also, the reason for the conservation of cultural heritage is misinterpreted and cannot be for tourism.\nThe main reason should be for education and in building national identity and pride. Tourism is just one of the uses of cultural heritage, but when heritage is negatively impacted by tourism numbers, it should be conserved and protected. Another way of conserving cultural heritage through tourism use is by creating clusters and possibly by theming the areas and, in turn, creating functional tourism precincts. This should be better explained, given the richness of the data obtained on the ground. The inventory is just a starting point - the first phase of the selection process in turning historic buildings into a tourist attractions. See chapter 7 of [ref 1].\nThe conceptual framework is too broad. Urban tourism destination is not synonymous with historical heritage destination. It is unclear how nostalgia fits in; it was not well integrated. It is not clear the link between Siantar as a student-friendly city and the further development and inclusion of tourism clusters into urban tours.\nThe size and population of the city and its main urban functions are not explained.\nIf the article is completely rewritten and restructured, it can present a valuable contribution to applying the selection process in creating viable tourism attractions. In its current form, the article is not suitable for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-554
https://f1000research.com/articles/10-553/v1	09 Jul 21	{ "type": "Research Article", "title": "Health profession readiness for interprofessional education in the Central Appalachia: a cross-sectional study", "authors": [ "Amanda Blankenship", "George Asimellis", "Bhargavi Joshi", "Briana Rodriguez", "Margaret Wright Sidle", "Amanda Blankenship", "Bhargavi Joshi", "Briana Rodriguez", "Margaret Wright Sidle" ], "abstract": "Background: This study on interprofessional relationships took place in Eastern Kentucky analyzing optometry, medical and nursing students at the University of Pikeville. The Readiness for Interprofessional Learning Scale (RIPLS), regarding all three healthcare professional schools, was used to measure and determine students' views on working with one another. The purpose of the study was to examine similarities and differences in student attitudes across the three health professional programs within the same university. Methods: Second year University of Pikeville (UPIKE) nursing, optometry, and medical students were given survey questions that followed the validated 19-item Readiness for Interprofessional Learning Scale (RIPLS). Results: While the optometry and medical students demonstrated statistically similar attitudes, key statistical findings included that nursing students were more likely than medical students to believe that clinical problem solving can only be learned effectively with students/professionals from their own school/organization (p = 0.015); nursing students were more likely than medical students to welcome the opportunity to work on small group projects with other health and social care students/professionals (p = 0.018); and nursing students were more likely than both optometry and medical students to not be sure what their professional role will be/is (p=.005). Conclusions: At the UPIKE, there is an observable difference between the attitudes toward IPE. Nursing students appeared to have a more positive attitude toward IPE than medical and optometry students, with the medical and optometry students having similar attitudes.", "keywords": [ "Online questionnaire", "interprofessional education", "survey", "IPEC" ], "content": "Introduction\n\nHealth care has become an integrated environment, consisting of a kaleidoscope of professions, working together for the goal of quality care and improved health care outcomes for patients. The importance of establishing a common structure early in a medical professional student’s career is crucial in maintaining the integrity of this type of work environment. Interprofessional education (IPE) prepares health professional students with the skills for collaborating with various health professionals. Curriculum intervention through IPE training has had positive effects on the education of health professionals1 Optometry students, at least in most of the US Schools, have IPE built into their curriculum during their third year. Medical school and nursing students have the option of joining a seminar to educate themselves further.\n\nDuring the present time, when the world is facing a shortage of healthcare workers, global leaders in healthcare and policymakers are looking for innovative ideas and ways that can help solve the problem and develop programs to bolster the global health workforce. A key aspect of any solution in sight for solving such personnel and workforce issue is by developing collaboration across the various disciplines in healthcare. At present, patient care is a multidisciplinary approach with various teams focused on providing patient needs and improving quality of life during serious medical conditions. In any healthcare setup and at any given time, teams of doctors, social workers, nurses, and paramedics work in collaboration to provide medical and logistical support to everyone involved in patient care.\n\nThe delivery of effective and high-quality patient care tends to be demanding, requiring a good understanding between the team members and is a complex activity. But as seen in various situations, the understanding and collaboration between several healthcare givers can be difficult and problematic. IPE is a way to improve understanding and teamwork. It is not a new concept but has been known for over 50 years and has been endorsed by the World Health Organization for more than 20 years.2 IPE is defined as educational experiences in which members or students of two or more professions learn with, from, and about each other to improve collaboration and the quality of care and services. In academics or universities, IPE is realized when students from two or multiple professional fields come together and learn from, about, and with each other collaboratively.3\n\nIPE has the potential to improve health outcomes, could be a powerful tool to help students in professional courses to understand their importance on the team, and to provide them with their professional identity. At the same time, it could help them understand the importance of the roles others play within the healthcare provider’s team.4\n\nAt present, IPE education is extremely single-tracked with emphasis on professional courses and making sure that students learn and understand the importance of one’s course in isolation, while completely neglecting the importance and requirement of other professionals. Today, patients have complex health issues and typically require more than one healthcare provider to understand and address the full spectrum of their disease condition and treatment modalities. The delivery of such patient care occurs in compartmentalized silos and with many misunderstandings and significant miscommunication between different team members. With so much history, IPE is still a style of education that has remained obsolete, underutilized, and often misunderstood.\n\nIPE can provide a practical framework for learning and clinical practice. This can help bridge the gap between several individuals by creating an environment where the primary focus is on patient care. Early implementation of IPE is important and may prevent the development of negative interprofessional attitudes.\n\nOur school, the University of Pikeville, is located in central Appalachia, USA. It has the distinction of being the only school across a very large geographical area that has the combination of the three health professional schools that emphasize primary care, namely Osteopathic Medicine, Optometry, and Nursing. No such study has taken place either in the geographical area of our school or in the combination of medical, optometry, and nursing schools. Particularly considering the shift of optometry into primary care ophthalmology, the underlying trends about an interprofessional collaboration among the future health professionals are unknown. The purpose of this study was to assess and analyze the student’s perception of interprofessional importance and respect for learners studying various professional courses. Thus, we evaluated how different medical professional school students view the importance and requirement of interprofessional care.\n\n\nMethods\n\nThe study was designed and implemented at the University of Pikeville (UPIKE), due to the presence of three health professional colleges, the Kentucky College of Optometry (KYCO), the Kentucky College of Osteopathic Medicine (KYCOM), and the Elliot School of Nursing (ESN). UPIKE is unique in the greater Appalachian geographical area and the Commonwealth of Kentucky for having the combination of these three different professional schools of optometry, medicine and nursing.\n\nThis is a cross-sectional descriptive study that was conducted in the spring of 2019 by using a survey questionnaire which was provided to students over a semester at the three different professional schools.\n\nThe students recruited were all second year students enrolled in optometry, osteopathic, and the nursing programs. There were no other inclusion or exclusion criteria used for the selection of the sample students for this research.\n\nThe study was extended to a total of 240 students across the three schools and was administered online via surveyhero.com; a link to the survey was sent to the student’s school email address. Due diligence was exercised to prevent any duplication of responses and any responder can fill the form only once by clicking the link provided. The surveys were sent to students with adequate time to respond at baseline with two subsequent weekly reminders.\n\nThe survey questions were developed based on the KYCO objectives for IPE and included a few open-ended questions and a few questions involving the Likert scale for answering. Such questionnaires are used to explore the use of IPE as a strategy to reform health professional education to become more collaborative.5,6\n\nThe survey questions followed the validated 19-item Readiness for Interprofessional Learning Scale (RIPLS).7,8 The questions were designed to study the perceptions of students regarding the knowledge and respect about students from other professional courses across various topics. The questions investigated the student’s perception about how they feel about interprofessional learning, is teamwork essential, how will clinical problem solving get better with cross-professional collaboration and help them overcome their course limitations. The survey required a maximum of 5 to 10 minutes to complete. A copy of the questionnaire can be found in the Extended data.13\n\nThe statistical software SPSS v 26 was used to analyze the data from the study. The results were expressed as mean and standard deviation and any differences were considered as significant if the p-value for that determinant was greater than 0.05. Analysis of variance was used as the statistical test.\n\nThe study was approved by the institutional review board (IRB# IRB00009652IRB) of the University of Pikeville. Participation (i.e., responding to the request) was voluntary; completing the questionnaire was taken as consent to participate. The data were collected and processed anonymously, and consent was taken from the students to use their data for analysis.\n\n\nResults\n\nThe survey for this study was administered to 240 students over a semester, of which 103 returned their response. In total, 61% of the total responders were female students with 38% being male students; 1% did not identify gender (Table 1).\n\nBased on the data analyzed following the survey (Table 2 and Figure 1), the nursing students were more likely than medical students to believe that clinical problem solving can only be learned effectively with students/professionals from their own school/organization (p = 0.015). The nursing students were also more likely than medical students to welcome the opportunity to work on small group projects with other health and social care students/professionals (p = 0.018) compared to optometry or osteopathic medical students. Also, the nursing students were more likely than both optometry and medical students to not be sure what their professional role will be/is (p = 0.005).\n\nThe maximum score was 10.\n\n\nDiscussion\n\nThe strength of this work is that it is the first study performed in our area attempting to measure the perceptions of IPE among health professional students. The survey measured and compared the attitudes of divergent healthcare professional students. Because IPE is influential to provide patients with the best care, the end goal is to provide knowledge about the attitudes of our professional medical students and to foster more research in this area.\n\nIn this study, the optometry and medical students did not demonstrate statistical differences in their attitudes toward IPE, while the nursing students had more positive attitudes. The statistical data supporting that fact could be influenced by the circumstance that nurses coordinate with physicians and other healthcare professionals more than the other professions surveyed within this study. It is noteworthy that optometry students and medical students shared similar results, although only the optometry students have IPE classes built into their curriculum. Nurses have diverse, important duties that allow them to work in different healthcare fields, and this, perhaps, explains similar findings in the literature that supports the nursing school program's higher IPE positive attitudes.6\n\nSimilar studies involving Physician Assistant professional school students9 indicated very positive attitudes toward IPE. Interestingly enough, these attitudes appear to decline between matriculation and graduation; such findings have also been reported among undergraduate students as well.6 It is important; therefore, that the strengthening of IPE in the health professional school’s curricula will serve to make full use of the students’ initial positive attitudes and focus on skill development for IPE competencies. A well-placed IPE curriculum, particularly during clinical training, can be associated with greater receptivity toward collaborative care.10,11\n\nWe hope that the findings of this study will foster the determination of academic institutions to strengthen IPE in their curricula by instituting formal interprofessional educational activities that involve students across all programs for health professionals. The incorporation of IPE into the core curricula can be implemented at the singular level of each student and within the clinical setting as a whole, relying on faculty activities and coursework and collective activities at the school level exert influence in the health education and practice system.3,12 The valuable collaborative learning opportunities will enhance respect for other professionals and insight into the value of IPE in healthcare delivery.\n\n\nConclusion\n\nThis study revealed that, at UPIKE, nursing students had the best attitudes toward IPE compared to optometry and medical students. Nursing students also differed in their attitudes about clinical education involving interprofessional education techniques compared to optometry and medical school professional students.\n\nThis study showed that there is room for improvement for all UPIKE healthcare professional students. Because the data showed a discrepancy between professional students, another study discerning if the IPE course taught within the schools could have influenced this phenomenon will be conducted over the next couple of years at UPIKE.\n\n\nData availability\n\nDANS: Interprofessional study questionnaire data, https://doi.org/10.17026/dans-xe2-tufn.13\n\nDANS: Interprofessional study questionnaire data, https://doi.org/10.17026/dans-xe2-tufn.13\n\nThis project contains the following extended data:\n\n- IPE study questions\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\nWang Z, Feng F, Gao S, et al.: A Systematic Meta-Analysis of the Effect of Interprofessional Education on Health Professions Students’ Attitudes. J Dent Educ. 2019 Dec; 83(12): 1361–1369. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Framework for action on interprofessional education and collaborative practice.World Health Organization; 2010. Reference Source\n\nGonzález-Pascual JL, Icaran E, Saiz-Navarro EM, et al.: Impact of the first interprofessional education undergraduate program in Spain. J Interprof Care. 2018 May 4; 32(3): 374–377. PubMed Abstract \| Publisher Full Text\n\nMéndez MJ, Armayor NC, Navarlaz MT, et al.: The potential advantages and disadvantages of introducing interprofessional education into the healthcare curricula in Spain. Nurse Educ Today. 2008 Apr 1; 28(3): 327–336. Epub 2007 Sep 18. PubMed Abstract \| Publisher Full Text\n\nDow AW, DiazGranados D, Mazmanian PE, et al.: An exploratory study of an assessment tool derived from the competencies of the interprofessional education collaborative. J Interprof Care. 2014 Jul 1; 28(4): 299–304. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoster S, Norman I, Murrells T, et al.: Interprofessional attitudes amongst undergraduate students in the health professions: a longitudinal questionnaire survey. Int J Nurs Stud. 2008 Nov; 45(11): 1667–1681. PubMed Abstract \| Publisher Full Text\n\nMcFadyen AK, Webster VS, Maclaren WM: The test-retest reliability of a revised version of the Readiness for Interprofessional Learning Scale (RIPLS). J Interprof Care. 2006 Dec; 20(6): 633–639. PubMed Abstract \| Publisher Full Text\n\nda Costa MV, Vilar MJ, de Azevedo GD, et al.: Interprofessional education as an approach for reforming health professions education in Brazil: emerging findings. J Interprof Care. 2014 Jul; 28(4): 379–80. PubMed Abstract \| Publisher Full Text\n\nLohenry K, Lie D, Fung CC, et al.: Interprofessional Education: What Measurable Learning Outcomes Are Realistic for the Physician Assistant Profession? J Physician Assist Educ. 2016 Jun; 27(2): 63–67. PubMed Abstract \| Publisher Full Text\n\nPinto A, Lee S, Lombardo S, et al.: The impact of structured inter-professional education on health care professional students' perceptions of collaboration in a clinical setting. Physiother Can. Spring 2012; 64(2): 145–156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWellmon R, Gilin B, Knauss L, et al.: Changes in student attitudes toward interprofessional learning and collaboration arising from a case-based educational experience. J Allied Health. Spring 2012; 41(1): 26–34. PubMed Abstract\n\nHo K, Jarvis-Selinger S, Borduas F, et al.: Making interprofessional education work: the strategic roles of the academy. Acad Med. 2008 Oct 1; 83(10): 934–940. PubMed Abstract \| Publisher Full Text\n\nAsimellis G (University of Pikeville, Kentucky College of Optometry): Interprofessional Study Questionnaire Data. DANS. 2021. Publisher Full Text" }	[ { "id": "136322", "date": "25 May 2022", "name": "Takatoshi Makino", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\nThe purpose of this study was to assess and analyze the student’s perception of interprofessional importance and respect for learners studying various professional courses. As a result, nursing students were more likely than medical students in items 12, 14, and 18.\nGeneral comment\nIt is interesting the results found using the Readiness for Interprofessional Learning Scale (RIPLS) for undergraduate students. However, before accepting for indexing the authors should address the following points which are fundamental.\nSpecific comments\nIt should be integrated by either typing in “0” for all amount of statistics or typing in “0” for the amount of statistics that exceed “1” according to APA style. I recommend typing in “0” for all amount of statistics including Abstract. Because previous studies in this journal typed in “0” for all amount of statistics.\n\nI'm confused about the purpose of your article. Because you wrote different purposes in Abstract and Introduction. Please write the same purpose of your article in both the Abstract and Introduction.\n\nYou set up the purpose to assess and analyze the student’s perception of interprofessional importance, and to use RIPLS according to McFadyen (2006)1. Please describe the effect of IPE used by RIPLS for undergraduate students in the Introduction to explain the association between the purpose of your article and the scale.\n\nYou didn’t write the significance of the study in the Introduction. Please describe the significance of the study in the Introduction to explicitly understand the discussion points in the Discussion.\n\nYou state it was conducted in the spring of 2019. Please explicitly state the month in the Study design.\n\nYou didn’t write the number of recruited students. Please explicitly describe the number of recruited students for each department in the Participants\n\nYou didn’t write your IPE program as an intervention. Please describe the detail of the IPE program at your university. For example, the purpose, behavioral goal, students who take the course, contents, term, and credit (mandatory, select, transfer system) in Methods to understand the influencing factors in your article.\n\nI am confused about the results in your article. Because you wrote a few open-ended questions, few questions, and RIPLS as the survey question in Data collection. However, I couldn’t confirm all results for a few open-ended questions, a few questions, and RIPLS in Results. Please describe all survey questions in Data collection to understand the results related to the purpose of your survey.\n\nPlease describe in more detail the RIPLS in Data collection to understand evaluation content, likert, and score of analysis.\n\nPlease describe more specifically what data you used and how you analyzed it. In particular, please describe how you analyzed data to make Table 2.\n\nYou made Table 2 and Figure 1 in this article. However, I think it is the same data in Table 2 and Figure 1. Please select either one.\n\nYou made Table 2 for only 5 items. However, you should describe all 19 items in Table 2 to understand the influencing factor.\n\nYou wrote the response rate and the result of data analysis for only items 12, 14, and 18 in Results. However, you should describe all data analysis for all results for a few open-ended questions, a few questions, and RIPLS in Results to understand the influencing factor related to the purpose of your survey.\n\nPlease describe the discussion for the results which relate to the purpose of your survey in the Discussion to understand the significance of the study in the Introduction.\n\nIs the work 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "153147", "date": "07 Nov 2022", "name": "Joan Groessl", "expertise": [ "Reviewer Expertise Interprofessional Education", "Problem based learning", "Scholarship of Teaching and Learning", "Ethics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn reviewing the article, it was unclear if this was meant to be a research brief or a thorough analysis of the results. The only data I was able to access was a bar chart of several of the questions. There was no evidence of the stated ANOVA or results with significance level. Having done research myself with this tool, the statistical strategies would seem appropriate, at least for the RIPLS portion. [This is why I put \"not applicable\" for the statement below related to the statistical analysis being appropriate.]\nThe conclusions outlined in the article were inconsistent with what appeared on the included Table. In both the abstract summary of the results and the results section, the authors note that \"nursing students were more likely than medical students to believe that clinical problem solving can only be learned effectively with students/professionals from their own school/organization\". The remainder of the sentence in the abstract (after the semi-colon) appears contrary to that statement and to findings of other research on the topic. In the table enclosed in the article, nursing students scored more positively on each dimension of the few prompts reported.\nWhen discussing data collection, it would be helpful to know what questions were created to \"explore the use of IPE as a strategy to reform health professional education to become more collaborative\". [What kinds of questions would lead to determine this?] The statement would seem to be a conclusion rather than a focus of questions for student respondents. The article fails to address the results of these in the narrative.\nIn two areas of the introduction, this reviewer wonders if a different word might make the meaning of the declaration clearer. I underlined the word in question.\n\"At present, IPE education is extremely single-tracked with emphasis on professional courses and making sure that students learn and understand the importance of one’s course in isolation\"\n\nEmphasis is on professional skills/development, not the course per se.\n\n\"the underlying trends about an interprofessional collaboration among the future health professionals are unknown\"\n\nThe authors are discussing a shift in one focus but linkage to the selected portion is unclear.\n\nAre the authors discussing attitudes? Response to IPE?\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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-553
https://f1000research.com/articles/8-498/v1	16 Apr 19	{ "type": "Research Article", "title": "Patient treatment pathways of multidrug-resistant tuberculosis cases in coastal South India: Road to a drug resistant tuberculosis center", "authors": [ "Priya Rathi", "Kalpita Shringarpure", "Bhaskaran Unnikrishnan", "Abhinav Pandey", "Abhirami Nair", "Kalpita Shringarpure", "Bhaskaran Unnikrishnan", "Abhinav Pandey", "Abhirami Nair" ], "abstract": "Background: Delays in initiating multidrug-resistant tuberculosis (MDR TB) treatment adds risk to individual patients and the community due to disease progression, and on-going transmission. The Government of India offers free TB diagnosis and treatment, however many presumptive MDR TB patients wander within the Indian healthcare system and delay accessing the programme. To better improve access to care, it is imperative to understand the treatment pathways taken by MDR TB patients. We aimed to describe the diagnostic and treatment pathway taken by presumptive MDR TB patients registered in the Programmatic Management Drug-resistant TB Program (PMDT). Methods: We conducted a cross-sectional study of all patients registered during August 2016 – April 2017 at one PMDT center of a district in Karnataka, India. A semi-standardized questionnaire collected the number, type (private vs public sector), and dates of healthcare facilities (HCFs) visits prior to the initiation of MDR TB treatment. Delays were the number of days, expressed in medians and interquartile range (IQR), from the date of onset of illness until the date of initiation of MDR TB treatment. Results: We found that patients preferred private HCFs; however, due to lack of treatment and unaffordability they shifted to public HCFs. Median delay to register under the program was more in private HCFs (180days) in comparison with public HCFs (120 days). We also found that the diagnosis of MDR among presumptive MDR cases (detection rates) were much higher (80%) in public HCFs. Conclusion: Awareness regarding the availability of free standard diagnosis and appropriate treatment under the TB program should be increased in the general population and private practitioners. The government should involve private HCFs to provide standard diagnostics and treatment to the patients seeking a private facility.", "keywords": [ "Diagnostic delay", "Treatment delay", "Patient delay", "Health care system delay Programmatic management of Drug resistant TB", "detection rate", "Private health care facility", "Public health care facility." ], "content": "Introduction\n\nMultidrug-resistant tuberculosis (MDR TB), is defined as tuberculosis (TB) bacilli resistant to at least two first-line drugs — rifampicin and isoniazid. According to the Global TB Report 2017 published by the World Health Organization (WHO), India ranks first globally in the burden of TB and MDR TB with an incidence rate of 27% and 24% of global burden1,2. Nearly 3% of new TB cases and 12% of previously treated patients in India are MDR TB1,2.\n\nTo reduce transmission of MDR TB, the Government of India developed and implemented a national policy for the programmatic management of drug-resistant TB (PMDT) in 20073. The strategies and objectives of PMDT complement the National Strategic Plan for TB Elimination (2017–2025), and include treatment of MDR TB, enhancing laboratory systems for faster diagnosis, and offering social protection and supportive systems to ensure uninterrupted treatment with shorter, less toxic regimens3–5.\n\nDelays in MDR diagnosis and appropriate treatment initiation impacts individual patients, through advance disease progression, additional costs, and poor quality of life; but also in the community through increased risk of ongoing transmission to other individuals6 The time taken to seek care, type of health care facility being sought and reasons for shifting from one health care facility to other are important factors for understanding delays in diagnosis, PMDT treatment initiation, poor outcomes. Describing health seeking behaviors and pathways taken by persons presumed tobe MDR TB case is a vital step in developing and implementing interventions that bridge the gap between timely diagnosis and treatment, and policies that improve the overall health system\n\nIn India, approximately 20% of the persons needing MDR TB treatment actually receive it, and among those who do receive treatment, less than half (48%) who start treatment finish successfully1,2. These poor outcomes are largely due to persons who are lost to follow-up and premature death. In 2017, the proportion of deaths during MDR TB treatment in India was higher than the global average (21% vs. 14%)1\n\nTherefore, in this study we aimed to determine the health-seeking pathway of presumptive MDR TB patients prior to treatment at the PMDT center. We also assessed the median time taken, and reasons for shifting from one heath care facility (HCF) to another.\n\n\nMethods\n\nWe conducted a cross-sectional study of all patients registered during August 2016 – April 2017 at one PMDT center of a district in Karnataka, India. The PMDT center is on the campus of a 150-year-old district hospital, which has recently been awarded as one of the best government run hospitals in India, by Kayakalp6. This PMDT center caters to three nearby districts. It has ten inpatient beds, facilities for diagnosis, pre-treatment evaluation and treatment of MDR TB, monitoring complications associated with second-line antituberculosis treatment. After inpatient care to initiate treatment (approximately 2 weeks), the patient is released to outpatient care for continuation of care at Directly Observed Treatment (DOT-Plus) centers throughout the community.\n\nAfter obtaining approval from the Institutional Ethics Committee (IEC) of Medical College Mangalore (IEC KMC MLR 11-16/328) and permission from DR-TB center, all patients registered under PMDT program were asked to enroll in the study. A line list was taken from DR-TB center, every day, and patients present in DR-TB center were approached by the Principal investigator (PI) at the DR-TB center. The purpose of the study was explained to the patients in their vernacular language and written informed consent was obtained from patients. PI collected data using a questionnaire by interviewing the patients.\n\nFace to face interviews were guided by a semi-structured questionnaire, which had been developed based on a literature review and the content was validated by experts in epidemiology (CDC Atlanta), a PMDT medical officer, and a layperson (local ground staff member working in the tertiary care hospital). The interview enquired about the various healthcare facilities (HCFs) visited by the patient from the time they experienced TB symptoms, designated as a presumptive MDR patient, until they registered for treatment at the PMDT center. We noted the time interval and reason for shifting from one facility to another. All information was penned down on the questionnaire at the time of the interview without any audio or video recording.\n\nData collected was entered and analyzed using Statistical Package for Social Science (SPSS) version 11.5. Kolmogorov-Smirnov test was done to find the normality of data. Results were expressed in median and inter-quartile range (IQR). Chi-square test was performed to find out the association between type of HCF visited by patients and reasons for shifting from one HCF to the other. The patient treatment pathway has been used to express health seeking behavior of the patients and was created using Adobe Illustrator trial version. The pathway was created using vector images with the visits being represented with different colors and different kinds of lines used for the diagnostic status of the patient. The meaning of both the lines as well the colors have been explained in the legends accompanying the pathways. Moreover, the number of patients shifting between HCF has been represented using the numbers accompanying the respective lines.\n\nOperational definitions. Multidrug resistant TB (MDR-TB): Patients with sputum-smear positive pulmonary TB, and at least one M. tuberculosis isolate with demonstrated resistance to at least isoniazid and rifampicin\n\nPathway: The various type of HCF visited by a presumptive MDR patient before registering for PMDT treatment in a chronological sequence. The various HCF were merged into two broad types: private and public health care sectors. Public HCFs include Primary Health Centers (PHCs) and Public Referral Hospitals (PRHs), including secondary and tertiary referral centers. Private HCF include secondary and tertiary referral center participating in Revised National Tuberculosis Control Program, non-participating allopathic clinics and practitioners (registered and unregistered) and Ayurveda Yoga Unani Sidda Homeopathy (AYUSH) practitioners7\n\nTime delays in the health care pathways8: The total delay is the time interval from the onset of illness until the initiation of anti MDR-TB drugs. It is the sum of two-time intervals: 1) diagnostic delay (time interval between the onset of symptoms and labelling of the patient as a MDR-TB patient); 2) treatment delay (time interval between MDR-TB diagnosis and initiation of anti MDR-TB drugs).\n\nThe total delay is also the sum of patient delay (time interval between onset of symptoms and presentation to first health care provider) and healthcare system delay (time interval between the date of health-seeking behavior at a health care provider and the initiation of anti MDR-TB treatment), since it can be attributed to these types of delay,\n\nReasons for shifting from one HCF to another: treatment not available, the appropriate treatment for the symptoms was unavailable in that HCF; treatment not affordable, cost of treatment was beyond the paying capacity of patient and its family; referred, patient was asked to visit another HCF for review/consultation or appropriate treatment; not satisfied, symptoms did not alleviate or the patient perceived that the services being provided were inadequate.\n\n\nResults\n\nDuring the study period, a total of 55 patients were initiated on treatment at the PMDT center; however only 40 patients consented to participate in the study. The mean age in our study was 40 years (SD: 13.9). There were 28 (70%) male patients and 12 (30%) female patients. In total, 35 patients were educated at least until primary level, while only 12.5% of patients were illiterate. A total of 65% of the study participants belonged to rural areas while only 14 patients lived in an urban area.\n\nOut of 40 patients interviewed, 15 went to a public HCF and 25 went to a private HCF as their first clinic encounter (Figure 1 and Figure 2). In total, 23 (57.5%) were diagnosed with MDR TB during this encounter.\n\nAmongst the 15 patients who went to the public HCF at the first encounter, 12 (80%) were diagnosed with MDR and were transferred directly to the PMDT center., whilst the remaining three patients required additional visits before MDR diagnosis (two went to public HCF and one went to private HCF).\n\nAmong the 25 patients who went to the private sector at the first clinical encounter, 11 (44%) were diagnosed with MDR, and were transferred directly to the PMDT center. The remaining 14 required addition visits before MDR diagnosis (9 went to public HCF and 5 went to private HCF).\n\nAll 40 patients underwent a second visit: 18 went to DRTB center, 15 opted for public HCF, while 7 went to private HCF. Out of the 17 undiagnosed patients, 14 (82.3%) were diagnosed.\n\nOut of the 15 who went to public HCF for second visit, four were already diagnosed (two from public HCF and two from private HCF) and 11 were undiagnosed. Of the 11 undiagnosed, 10 were diagnosed on this visit and were referred to the DR TB center. One undiagnosed patient went to another public sector. Out of four previously diagnosed patients, two were referred to DR TB center and two again shifted to another public sector HCF. Hence a total of 12 patients out of 15 shifted to DRTB center and three visited other public HCF.\n\nSeven patients went to private HCF for a second visit. Out of these seven, one was previously diagnosed in other private institution and six were undiagnosed (five came from private HCF and one from public HCF). In this visit, of the six patients who were undiagnosed, four were diagnosed. Five patients (one previously diagnosed and four newly diagnosed) visited DR TB center from a private HCF. Two still remained undiagnosed. One went to the DR TB center. The other undiagnosed patient visited another private sector HCF.\n\nOverall, 18 patients (12 from public HCF and six from private HCF) visited DRTB center in their third visit.\n\nOf the 40 patients, 22 patients underwent a third visit to HCF. Among them, 18 came to DR TB Center, three went to public HCF while one went to the private sector. Out of the three who went to public HCF, one was undiagnosed and two were previously diagnosed (1st visit). The undiagnosed case received a diagnosis and went to the DR TB center. The two previously diagnosed patients also went to DR TB center in their fourth visit.\n\nOne undiagnosed patient from the private HCF was diagnosed and referred to DR TB center in the fourth visit.\n\nThe median (IQR) patient delay was found to be 25 (10, 60) days. For patients contacting public HCF as their first point of health care contact, the median patient delay was 30 days, while for those contacting private HCF, it was found to be 20 days.\n\nIn the first visit among the 23 patients who were diagnosed to have MDR TB, the median delay in reaching the DR TB center after contacting the first HCF was 120 (30, 240) days. Median (IQR) delay in a public health care facility was 105 (60, 382) days with the highest being 1825 days, while in a private health care facility the median delay was 180 (10, 240) days with the highest being 300 days.\n\nAmong the patients diagnosed in second visit, the median delay in reaching DRTB center was 210 (82,270) days from the day of first HCF contact. In public HCF, this delay was found to be 195 (82,247) days with the highest being 365 days while it was 255 (105, 341) days in private HCF with the highest being 365 days.\n\nOne patient who remained in the public sector was diagnosed at his third visit with a delay of 210 days. Similarly, a patient who remained in the private sector was diagnosed after a delay 120 days on his third visit.\n\nFurthermore, median delay among female patients (30 days) was more than male patients (20 days). Also patients aged more than 45 years had more median delay of 30 days as compared to those below 45 years.\n\nThe most common reason for shifting from first to second HCF was referral (both public and private) followed by non-affordability (only seen in private HCFs) and non-satisfaction (seen more in private HCFs). Similarly, the reasons for shifting from 2nd to 3rd HCF were referral (mostly in public), non-satisfaction and non-affordability in private. In the third shift, only reason given was referral to DR TB center (Table 1).\n\n\nDiscussion\n\nMDR TB is an emerging disease in India. The disease is difficult to treat and treatment outcomes are poor, making it a potential public health threat in the future. Our study sheds light on patients’ treatment pathway and reasons for shifting between health care providers for diagnosis and treatment for MDR TB.\n\nIn our study, 63% of patients went to private health care facilities (HCF) as first point of health care contact, which was slightly more than the average 48% as seen in a systematic review from India9,10. This pattern is also seen in other studies done in India and in other developing countries10–16. The health seeking behavior of a patient depends on the knowledge about the disease, severity of symptoms and social support available, especially in a communicable and stigmatized disease like TB17. Studies conducted in India showed that most people had poor awareness about TB-related symptoms, transmission, and the services offered by the national TB control programs18,19. Moreover, patients in India have reported treatment barriers, such as, long distance between the TB centers and their homes, lack of confidence in the efficacy of government supplied medication, and the lack of privacy during directly observed treatment sessions12,18,20. All these reasons suggest a preference for private HCF as their preferred health provider for TB diagnosis and treatment.\n\nA study done by Arinaminpathy et al.21 shows that the quality of care meted out to TB patients is sub-optimal22. This negligence in treatment of TB cases later gives rise to DR TB as the bacterial strains achieve antimicrobial resistance. Further, the private sector poses many hurdles in TB control. Studies show that the sectors’ disorganization produces gaps in TB surveillance due to irregular notification of cases and absence of guidelines22.\n\nHowever, there was a delay on the part of patients to report to their first point of health care contact after appearance of symptoms. The median delay was found to be 20 days which is slightly more than two weeks of the cough criterion issued by RNTCP for TB screening. This was in line with studies done by Dhanvij et al. (20 days), Nimbarte et al. (19 days) and Ananthkrishnan et al. (18.3 days), while at the same time was quite less than shown in studies done by Goel et al. (30 days), Kulkarni et al. (8 weeks), Selvam et al. (28 days) and Shamim et al. (>30 days)12,22,–27. This shows the diversity in patient delay across India. Also, the median delay was found to be more among female patients as compared to male patients. This has been shown in various studies done in India as well as in some developing countries8,14,28. However study done by Nimbarte et al. in Central India shows that the delay is less among females than in males23. Further, we also found that patients above the age of 45 years postponed seeking health care as compared to younger patients. This is in line with the study done by Charles et al.28 The median delay was found to be greater among those who had public HCF as their first point of contact which has also been seen in the study done by Nimbarte et al.23.\n\nIn our study, 57.5% of the patients were diagnosed at their first point of contact, while 82.3% were diagnosed at their second point of contact. The results are similar to those seen in the study done by Ananthakrishnan et al.26. Among the 55% of patients who made a second visit to a different HCF other than DR TB center, 68% of the patients went to a public HCF. This is also seen in the study done by Charles et al.28. Also we find that in all the visits, the yield of diagnosis at public HCF was always more than that at private HCF. This could be due to improper tests done for detection or lack of technologies for doing the same, as seen in a review by Satyanarayana et al.29.\n\nThe most common reason for shifting between HCF other than referrals was unaffordability followed by dissatisfaction. This is in contrast to the study done by Charles et al. where the major reason was found to be dissatisfaction with the available HCF followed by unaffordability28. In public HCF, the most common reason was referral which was in accordance to the PMDT guidelines. This was followed by patient reasons such as dissatisfaction.\n\n\nConclusion\n\nThe present study found that there was substantial patient delay and total delay in diagnosis and treatment of DR TB patients. Since the patient delay was more in females and elderly people, they can be prioritized for screening and diagnosis by active and enhanced case finding methods.\n\nThe study also projects the importance of a public-private collaboration in treating DR TB cases. As stated by many patients that their reason for shifting from one private HCF to other was non-affordability of the treatment, hence provisions of standard diagnostic services and treatment at low to no costs can reduce the delay in treatment. This can be achieved by incentivizing treatment and providing standard diagnostic modalities to private sector under RNTCP.\n\nActions should be taken from the government side to improve the quality of services provided in a public HCF, better infrastructure and human resources. Hence stigma and dissatisfaction related to public health services can be tackled and better awareness about government disease control programs can be created.\n\n\nData availability\n\nAccording to the IEC of Medical College Mangalore, we are not permitted to share data with any external agency for protection of data as it contains information which is personal and can be identified. However, if required by anyone, the data can be requested from the corresponding author after full justification of usage of the information. Conditions of access: researchers must use the data for similar research or sufficiently anonymize and give due credit to the authors of this study.\n\nOpen Science Framework: Patient treatment pathways of multidrug-resistant tuberculosis cases in coastal South India: Road to a drug resistant tuberculosis center: Structured questionnaire, https://doi.org/10.17605/OSF.IO/TQNUE30\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe acknowledge Dr Patrick K Moonan, U.S. Centers of Disease Control and Prevention, Division of Global HIV and Tuberculosis, Atlanta USA for his valuable inputs and contributions in the study.\n\n\nReferences\n\nWorld Health Organization: Global tuberculosis report 2018. 2018; [cited 2018 Dec 28]. Reference Source\n\nMinistry of Health and Family welfare: National Anti-TB Drug Resistance Survey. [cited 2018 Dec 28]. Reference Source\n\nMinistry of Health and Family Welfare: Guidelines on Programmatic Management of Drug-Resistant Tuberculosis in India. 2017; [cited 2018 Dec 29]. Reference Source\n\nBhavan N: National Strategic Plan for Tuberculosis Elimination 2017-2025. Ministry of Health and Family welfare; 2017; 109. Reference Source\n\nFalzon D, Jaramillo E, Schünemann HJ, et al.: WHO guidelines for the programmatic management of drug-resistant tuberculosis: 2011 update. Eur Respir J. 2011; 38(3): 516–528. PubMed Abstract \| Publisher Full Text\n\nZerbini E, Chirico MC, Salvadores B, et al.: Delay in tuberculosis diagnosis and treatment in four provinces of Argentina. Int J Tuberc Lung Dis. 2008; 12(1): 63–68. 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Int J Tuberc Lung Dis. 2007; 11(2): 161–167. PubMed Abstract\n\nBonadonna LV, Saunders MJ, Zegarra R, et al.: Why wait? The social determinants underlying tuberculosis diagnostic delay. PLoS One. 2017; 12(9): e0185018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinto LM, Udwadia ZF: Private patient perceptions about a public programme; what do private Indian tuberculosis patients really feel about directly observed treatment? BMC Public Health. 2010; 10: 357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSreeramareddy CT, Qin ZZ, Satyanarayana S, et al.: Delays in diagnosis and treatment of pulmonary tuberculosis in India: a systematic review. Int J Tuberc Lung Dis. 2014; 18(3): 255–266. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJayachandran V: A case study on tuberculosis treatment defaulters in Delhi: Weak health links of the community with the public sector, unsupported migrants and some misconceptions. Annals of Tropical Medicine and Public Health. 2014; 7(2): 124–129. Reference Source\n\nArinaminpathy N, Batra D, Khaparde S, et al.: The number of privately treated tuberculosis cases in India: an estimation from drug sales data. Lancet Infect Dis. 2016; 16(11): 1255–1260. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhanvij P, Joshi R, Kalantri S: Delay in diagnosis of tuberculosis in patients presenting to a tertiary care hospital in rural central India. J MGIMS. 2009; 14(ii): 56–63. Reference Source\n\nNimbarte SB, Wagh V, Selokar D: Health seeking behaviour among pulmonary tuberculosis patients in rural part of central India. Int J Biol Med Res. 2011; 2(1): 394–397. Reference Source\n\nShamim H, Vidyasagar, Shalini S, et al.: Health Care seeking behavior of cough symptomatics (pulmonary tuberculosis suspects) attending medicine outpatient department of a tertiary care hospital of Jharkhand. IJIMS. 2015; 2(8): 42–49. Reference Source\n\nKulkarni PY, Kulkarni AD, Akarte SV, et al.: Treatment seeking behavior and related delays by pulmonary tuberculosis patients in E-ward of Mumbai Municipal Corporation, India. Int J Med Public Health. 2013; 3(4): 286. Publisher Full Text\n\nAnanthakrishnan R, Jeyaraju AR, Palani G, et al.: Care Seeking Behavior of the TB Patients who were Registered in an Urban Government Tuberculosis Control in Chennai, Tamilnadu, India. J Clin Diagn Res. 2012; 6(6): 990–993. Reference Source\n\nKapoor SK, Raman AV, Sachdeva KS, et al.: How did the TB patients reach DOTS services in Delhi? A study of patient treatment seeking behavior. PLoS One. 2012; 7(8): e42458. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCharles N, Thomas B, Watson B, et al.: Care seeking behavior of chest symptomatics: a community based study done in South India after the implementation of the RNTCP. PLoS One. 2010; 5(9): pii: e12379. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSatyanarayana S, Subbaraman R, Shete P, et al.: Quality of tuberculosis care in India: a systematic review. Int J Tuberc Lung Dis. 2015; 19(7): 751–763. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRathi P: Patient Treatment Pathways of Multidrug-Resistant Tuberculosis Cases in Coastal South India: Road to a Drug Resistant Tuberculosis Center. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/TQNUE" }	[ { "id": "52457", "date": "16 Sep 2019", "name": "Sharath Burugina Nagaraja", "expertise": [ "Reviewer Expertise Tuberculosis", "HIV", "Diabetes - Operational 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\nI congratulate your efforts on working on treatment pathways of drug resistant TB patients. Overall, the manuscript reads well. However, I have few comments for your consideration which will help in betterment of the manuscript. The comments are mentioned separately below.\n\nComments:\nThe authors in the introduction state that “we aimed to determine the health- seeking pathway of presumptive MDR TB patients prior to treatment at the PMDT center”. The manuscript lacks clarity on identifying the presumptive MDR-TB patient under the programmatic settings. Probably, the authors should specifically say that the presumptive MDR-TB is based on laboratory diagnosis rather than a clinical presumption.\n\nStudy settings: The authors have to specify the name of the hospital, location of the PMDT centre with the names and population of the districts it is catering to. They should also briefly describe about the types/numbers of public and private health facilities in the districts.\n\nThe statement “the patient is released to outpatient care for continuation of care at Directly Observed Treatment (DOT-Plus) centers throughout the community” needs to be modified because under programmatic conditions the patients are referred for domiciliary care in the community.\n\nThe name of the institution which has accrued ethics approval needs to be mentioned.\n\nData collection: It is found that the details were collected from the time the patients experience TB symptoms. These details are not analysed and was beyond the scope of the study. For a patient, there is a thin line of difference from being a presumptive TB case and a presumptive DR-TB case. There could be a lot of subjective variability in remembering the things which have occurred in the past. It is better if the authors explain the measures taken to negate such variability during the process of interview and increased their accuracy of findings.\n\nData analysis: Please mention the reference for adobe illustrator trail version.\n\nTime delays in health care pathways: I feel the definitions needs further clarity. The starting point for delay calculations is the point of onset of illness. We presume that the authors are taking into account that it is from the point when the patient has a laboratory diagnosis of DR-TB. The authors should explicitly mention in their definitions.\n\nReasons for shifting from one HCF to others: These are the options or answers expected from the patients. They have to be presented in the results as the findings of patients’ interviews. Hence, should be removed from the method section.\n\nResults: For better understanding the authors have to describe their findings as numbers (percentages). The ‘p’ values mentioned in table 1 should be removed, it has no relevance.\n\nAmong the 25 patients who went to private sector, 11 were diagnosed as MDR TB. What type of diagnosis did the private sector facilities made? Clinical or laboratory diagnosis? It is also mentioned that the patients are transferred to PMDT centres. Under programmatic settings, the transfer happens from one RNTCP administrative unit to the other.\n\nThere is a scope for authors to present the findings of proportions of patients who underwent 1,2,3 and 4 HCF visits as a bar diagram/table stratified by public and private facilities.\n\nDiscussions: The authors should remain focused on the study context. There has been much comparison with other studies. This tends to lose the focus of the readers. The recommendations have to be relevant to the study findings.\n\nLimitations: The authors can add a paragraph on limitations of the study (response rate, sample size, generalizability, definitions-subjective variability).\n\nConclusion: The conclusion should be specific and should be described in a paragraph.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5476", "date": "13 May 2020", "name": "Priya Rathi", "role": "Author Response", "response": "I congratulate your efforts on working on treatment pathways of drug resistant TB patients. Overall, the manuscript reads well. However, I have few comments for your consideration which will help in the betterment of the manuscript. The comments are mentioned separately below.We thank the reviewer for the kind words of appreciation and recognising the work behind the manuscript. We have answered all query/comments point to point and have indicated its current location in the modified manuscript.The authors in the introduction state that “we aimed to determine the health- seeking pathway of presumptive MDR TB patients prior to treatment at the PMDT centre”. The manuscript lacks clarity on identifying the presumptive MDR-TB patient under the programmatic settings. Probably, the authors should specifically say that the presumptive MDR-TB is based on laboratory diagnosis rather than a clinical presumption.We have now included the exact definition of a presumptive diagnosis of DRTB based on PMDT guidelinesMethods data collection paragraph 2 and operational definitionStudy settings: The authors have to specify the name of the hospital, location of the PMDT centre with the names and population of the districts it is catering to. They should also briefly describe about the types/numbers of public and private health facilities in the districts. We have now specified the facility name, location and other details as suggested by the reviewerMethods. Study setting paragraph 1The statement “the patient is released to outpatient care for continuation of care at Directly Observed Treatment (DOT-Plus) centers throughout the community” needs to be modified because under programmatic conditions the patients are referred for domiciliary care in the community. We have now changed this based on the PMDT guidelinesMethods. Study setting paragraph 2The name of the institution which has accrued ethics approval needs to be mentioned.Name of the IEC has been addedMethods. Study setting paragraph 3Data collection: It is found that the details were collected from the time the patients experience TB symptoms. These details are not analysed and was beyond the scope of the study. For a patient, there is a thin line of difference from being a presumptive TB case and a presumptive DR-TB case. There could be a lot of subjective variability in remembering the things which have occurred in the past. It is better if the authors explain the measures taken to negate such variability during the process of interview and increased their accuracy of findings.We have mentioned the measures taken for alleviating subjectivity as followsWe validated the starting point with the reports and patient file from the DDR-TBC to look for HIV co infection, previous lab reports, history of contact, previous treatment history, if available. Data collectionData analysis: Please mention the reference for adobe illustrator trail version.We have incorporated the reference and linkData analysisTime delays in health care pathways: I feel the definitions needs further clarity. The starting point for delay calculations is the point of onset of illness. We presume that the authors are taking into account that it is from the point when the patient has a laboratory diagnosis of DR-TB. The authors should explicitly mention in their definitions.We have changed the definition to make it clearer to understand.Operational DefinitionsReasons for shifting from one HCF to others: These are the options or answers expected from the patients. They have to be presented in the results as the findings of patients’ interviews. Hence, should be removed from the method section.These were the options given to the patients hence we have mentioned in both the sections Results:For better understanding, the authors have to describe their findings as numbers (percentages). The ‘p’ values mentioned in table 1 should be removed, it has no relevance.We have included the numbers and also removed p-value from table 1Among the 25 patients who went to private sector, 11 were diagnosed as MDR TB. What type of diagnosis did the private sector facilities made? Clinical or laboratory diagnosis? It is also mentioned that the patients are transferred to PMDT centres. Under programmatic settings, the transfer happens from one RNTCP administrative unit to the other.There are private sectors with diagnostic facilities, hence the diagnosis refers to laboratory diagnosis. We meant, that the patient was referred to the public sector from the private sectorDetails of first visit and shift from one HCF to other HCF Paragraph 3There is a scope for authors to present the findings of proportions of patients who underwent 1,2,3 and 4 HCF visits as a bar diagram/table stratified by public and private facilities.Since our objective was to map the pathway, we have incorporated the different color lines for different number of visit (1,2,3,4)This has been explained in the data analysis section as followsThe meanings of both the lines as well the colours have been explained in the legends accompanying the pathways. Moreover, the number of patients shifting between HCF has been represented using the numbers accompanying the respective lines. Data-analysisFigure 2 legendsDiscussions: The authors should remain focused on the study context. There has been much comparison with other studies. This tends to lose the focus of the readers. The recommendations have to be relevant to the study findings.We have now removed the comparing figures and have kept only relevant textDiscussionLimitations: The authors can add a paragraph on limitations of the study (response rate, sample size, generalisability, definitions-subjective variability).Thank you for your insight. We have incorporated the paragraph for limitation.Limitation reads asThis study was conducted among the 40 patients from one DDR-TBC of Karnataka therefore the findings can be generalized to the population seeking health care from the same DDR-TBC. Second limitation would be the subject variability of the definition, however we have made full effort to validate the findings with medical reports of the patients from DDR-TBC to ensure the starting and end point of the pathway. Since the study was based on recall of the patients, there are chances of inherent recall bias.LimitationConclusion: The conclusion should be specific and should be described in a paragraph.We have made it specific and conveyed it in a single paragraph.Conclusion read as-The present study found that there was substantial patient delay and total delay in diagnosis and treatment of DR TB patients. The study projects the need of a public-private collaboration in treating DR TB cases; in terms of linkages between public and private sector for diagnosis and treatment of drug resistance TB, provisions of standard diagnostic services and treatment at low to no costs.This may be achieved by incentivising treatment and providing standard diagnostic modalities to private sector under NTEP." } ] }, { "id": "59044", "date": "06 Feb 2020", "name": "Douglas Fraser Wares", "expertise": [ "Reviewer Expertise Programmatic care and control of tuberculosis", "drug-resistant tuberculosis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview of “Patient treatment pathways of multidrug-resistant tuberculosis cases in coastal South India: Road to a drug resistant tuberculosis center” paper for F1000Research. The authors have written about an important topic related to the treatment pathways of drug-resistant TB patients in India. The manuscript reads well, however I have a number of major comments which need to be addressed to ensure the scientific level of the manuscript is raised. My major and minor comments follow, namely:\nMajor comments:\nIn the flowchart and text, the authors talk about “Presumptive MDR-TB patients”. However the patients enrolled in the study are diagnosed and registered MDR-TB cases. These are not the same thing. How is “Presumptive MDR-TB patients” defined and is the same definition being used in both the private and public sector facilities in the study? This needs to be stated by the authors and how the “Presumptive MDR-TB patients” are being identified in the facilities of the 2 sectors and whether similar levels of identification of “Presumptive MDR-TB patients” was being done in the different sector facilities?\n\nTime to diagnosis will be determined to a degree by what diagnostic capacity is available in the respective health facility. Hence if a facility has the capacity for rapid molecular diagnosis of TB and rifampicin resistance (RR-TB) e.g. if it has a GeneXpert machine on site, then diagnosis of RR-TB can be done within the day. If not and the patient or sample has to be sent to another facility which then does only culture and phenotypic drug susceptibility testing (DST) and not rapid molecular DST, there will be an inherent time difference between the patients seen at the initial health facility. So did all the health facilities involved in the study have the same access to diagnostic capacity?\n\nEven if the diagnostic capacity available to all health facilities involved in the study, where any RR-/MDR-TB cases diagnosed but not registered? If yes, were the proportions the same in the different sectors analysed?\n\nWere the 15 DR-TB patients who did not consent to be participate in the study similar to the 40 patients who did participate in the study? Was there any difference in these 15 patients between those who initially visited a public sector facility versus those who visited a private facility initially?\n\nMinor comments:\nReference 1 and related text could be updated as the WHO 2019 Global TB Report is available.\n\nIntroduction, 2nd paragraph. It should be stated that PMDT is part of the Revised National TB Control Programme (now renamed as the National TB Elimination Programme) and is not a separate programme as implied by the current text.\n\nIntroduction, 2nd paragraph. Reference 5 should be updated as it refers currently to a 2011 WHO document which has been updated since then.\n\nIntroduction, 3rd paragraph. Reference 6 is from a paper dealing with Argentina. Is there no relevant reference paper from India that could be used instead? Also does the current reference paper talk about DS-TB and/or DR-TB?\n\nMethods, Study settings, 1st paragraph. The district name and hospital name where the PMDT centre is located should be given. As should the names of the 3 districts that the centre caters for.\n\nMethods, Study settings, 1st paragraph. For non-Indian readers, the meaning of “… by Kayakalp.” needs to be explained. Also the reference given is no. 6 which refers to a paper dealing with Argentina and hence seems incorrect.\n\nMethods, Study settings, 1st paragraph. The authors mention “.. DOTS-Plus centres” and only mention about initial in-patient care. DOTS-Plus is outdated terminology and initial in-patient care practice alone is outdated practice under RNTCP. The text should be amended so the reader understand what is current RNTCP terminology and practice. Especially as in Figure 1, the term of DR-TB Centre is used.\n\nMethods, Study settings, 2nd paragraph. 12.5% of patients in the study were illiterate, hence how was “written consent” given?\n\nMethods, Data collection, 1st paragraph. Is it correct then that there was no actual field testing of the questionnaire prior to the study being conducted? What training did the PI have on the questionnaire prior to conducting the patient interviews?\n\nMethods, Operational definitions, reasons for shifting from one HCF to another, 1st paragraph. As pointed out by the previous reviewer, what is listed here are options or answers expected to be provided by the patients. Hence this part of the methods section needs revising.\n\nResults, 1st paragraph. Need to be consistent in the use of both numbers and % in the last sentence.\n\nResults, Details of first visit and shift from one HCF to other HCF, 2nd paragraph. Although 12 of the 15 who initially visited a public HCF were diagnosed with MDR, only 10 were immediately referred to the DR-TB centre. Why did the 2 other patients have to attend a HCF for a 2nd time before referral to the DR-TB Centre?\n\nDiscussion. As stated by the previous reviewer, the authors spend a lot of the discussion section comparing with other studies. Many of the times this is just in relation to confirming previous observations. Such text could be reduced (e.g. 2nd and 4th paragraphs).\n\nDiscussion, 2nd paragraph. Should ease of access to service be more prominent amongst important issues surrounding health seeing behavior of patients? Is this not a major reason why patients initially seek care in the private sector rather than the public sector?\n\nDiscussion, 3rd paragraph. DR-TB is now more of a “transmitted” disease rather than the “acquired resistance” as discussed in the first few sentences of the paragraph. This needs to be noted. Also the meaning of the sentence “Further, the private sector poses many hurdles in TB control.” is unclear?\n\nDiscussion, 5th paragraph. For clarity of reading, the authors should to include the actual numbers involved when they state “57.5%” (=23/40) and “82.3” (=14/17).\n\nDiscussion, 6th paragraph. Did the study by Charles et al. (ref 28) include DR-TB cases as well as DS-TB? If not, how relevant is the reference to the current study?\n\nLimitations. The authors need to include a paragraph or two on “Limitations” of the study.\n\nConclusions. Some of the statements made are pretty sweeping with little data or evidence from the study to support them. For example the statements in the 1st paragraph made about female and the elderly – these are based on very small patients numbers and hence how valid and generalizable are they? In the 2nd paragraph, an alternative view is that there may be better awareness in the community of the disease and availability of services. Hence a higher proportion of those who actually have the disease present to the public sector facilities for their 1st visit. And surely better linkages between the HCFs of the two sectors needs to be established, in addition to the option of providing everything in all private HCFs?\n\nConsistency of language. DOTS-Plus centers / DR-TB Centre / PMDT center are all used in the manuscript. Firstly I would suggest the authors use “centre” and “programme”, and also are consistent in the term they use for the centre where the DR-TB patients were registered.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5477", "date": "13 May 2020", "name": "Priya Rathi", "role": "Author Response", "response": "We have answered all query/comments point to point and have indicated its current location in modified manuscript.In the flowchart and text, the authors talk about “Presumptive MDR-TB patients”. However, the patients enrolled in the study are diagnosed and registered MDR-TB cases. These are not the same thing. How is “Presumptive MDR-TB patients” defined and is the same definition being used in both the private and public sector facilities in the study? This needs to be stated by the authors and how the “Presumptive MDR-TB patients” are being identified in the facilities of the 2 sectors and whether similar levels of identification of “Presumptive MDR-TB patients” was being done in the different sector facilities?This study was a retrospective inquiry on pathway taken to reach Drug Resistance TB Center. The starting point of this inquiry was taken from the time these patients were presumptive DR case which by definition as per Programmatic Management of Drug Resistant Tuberculosis Guidelines is: Presumptive MDR case are Any of the following:It refers to the following patients in order of their risk: TB patients found positive on any follow-up sputum smear examination during treatment with first-line drugs including treatment failures; - TB patients who are contacts of DR-TB; - previously treated TB patients; - new TB patients with HIV co-infection.These definitions were used in the study.Method –Data collection, Operational definition.Time to diagnosis will be determined to a degree by what diagnostic capacity is available in the respective health facility. Hence if a facility has the capacity for rapid molecular diagnosis of TB and rifampicin resistance (RR-TB) e.g. if it has a GeneXpert machine on site, then diagnosis of RR-TB can be done within the day. If not and the patient or sample has to be sent to another facility which then does only culture and phenotypic drug susceptibility testing (DST) and not rapid molecular DST, there will be an inherent time difference between the patients seen at the initial health facility. So did all the health facilities involved in the study have the same access to diagnostic capacity?Not all the health facilities involved in the study have the same diagnostic capacity. This is what we are trying to bring out from our study, that those patients who visited such health care facility which lacked Standard Diagnostic facilities were diagnosed late as compare to those who visited Government Health facility which has better diagnostic facility with Standard Operating Procedures (SOPs) and standard of care with respect to DR-TB. Under PMDT programme, DDR- TB centers are developed which complies with Standard Diagnostic and Treatment guidelines.Even if the diagnostic capacity available to all health facilities involved in the study, where any RR-/MDR-TB cases diagnosed but not registered? If yes, were the proportions the same in the different sectors analyzed?We have only considered those patients who were registered at DDR- TB Center and enquired which health care facilities they visited prior to DRTB Center and their sequence to what happened there with respect to diagnosis and treatment and referral.For the patients in the Government Sector, registration is done under the National TB Program. For patients who are initiated with treatment in the private sector, the private sector notifies to the government. In this study, our objective was to find out the pathway and the source of information was the patient themselves and not the health care providers. Hence, we did not enquire on the notification of the cases if diagnosed at private sector.Were the 15 DR-TB patients who did not consent to participate in the study similar to the 40 patients who did participate in the study? Was there any difference in these 15 patients between those who initially visited a public sector facility versus those who visited a private facility initially?Since the direction of inquiry was retrospective and the unit of inquiry was the patient or close relative, in the 15 patients who did not consent for the study, we were unable to find their first point of contact to Health care facility. However, with respect to the current diagnosis of TB and treatment regimen, they were the same as both were done at DRTB center with standard protocol and SOP. Reference 1 and related text could be updated as the WHO 2019 Global TB Report is available.We have changed the reference to the latest report.Reference 1Introduction, 2 paragraph. It should be stated that PMDT is part of the Revised National TBControl Programme (now renamed as the National TB Elimination Programme) and is not aseparate programme as implied by the current text.We have mentioned it now and have highlighted it as well.Introduction, 2nd paragraph.Introduction, 2 paragraph. Reference 5 should be updated as it refers currently to a 2011 WHO document which has been updated since then.We have updated the same.Introduction, 2nd paragraphReference 5.Introduction, 3 paragraph. Reference 6 is from a paper dealing with Argentina. Is there norelevant reference paper from India that could be used instead? Also, does the current reference paper talk about DS-TB and/or DR-TB?We have not taken any data from the Argentina study, we have just cited the conceptual knowledge of delay in diagnosis, hence the same reference can be used, the study included DS and Previously treated TB patients. We have also added one Indian study as well, however it covers only a few points as compared to Argentina studyIntroduction, 3rd paragraph. Reference 6 and 7Methods, Study settings, 1 paragraph. The district name and hospital name where the PMDTCentre is located should be given. As should the names of the 3 districts that the Centre caters for.The DRTB center is situated in district hospital of Dakshina Kannada District, Karnataka State , India which also caters to two neighboring districts of Chikmagalur and Shimoga. This has been included now in the first paragraph.Methods, Study settings, paragraph 2Methods, Study settings, 1 paragraph. For non-Indian readers, the meaning of “… by Kayakalp.”needs to be explained. Also, the reference given is no. 6 which refers to a paper dealing with Argentina and hence seems incorrect.This has been removed and the above statement has been mentioned instead.Methods, Study settings, 2nd paragraphMethods, Study settings, 1 paragraph. The authors mention “.. DOTS-Plus centers” and only mention about initial in-patient care. DOTS-Plus is outdated terminology and initial in-patient care practice alone is outdated practice under RNTCP. The text should be amended so the reader understands what is current RNTCP terminology and practice. Especially as in Figure 1, the term of DR-TB Centre is used.We have changed the terms to District Drug resistance TB center (DDR-TBC) and Peripheral Health Institution (PHI) as per PMDT guidelines.Methods, Study settings, paragraph 3Study settings, 2nd paragraph. 12.5% of patients in the study were illiterate, hence how was “written consent” givenThose who were illiterate, the informed consent process was conducted in front of literate impartial witness as per ICMR ethics guidelines.( https://www.icmr.nic.in/sites/default/files/guidelines/ICMR_Ethical_Guidelines_2017.pdf) Reference 8Methods, Data collection, 1st paragraph. Is it correct then that there was no actual field testing of the questionnaire prior to the study being conducted? What training did the PI have on the questionnaire prior to conducting the patient interviews?The tool was pilot tested in two patients. PI holds an MD degree in Community Medicine, where there is training in epidemiology and has conducted multiple projects in the past. The questionnaire was content to validate independently by two experts and discussed for feasibility along with the pilot testing.Not applicableMethods, Operational definitions, reasons for shifting from one HCF to another, 1st paragraph. As pointed out by the previous reviewer, what is listed here are options or answers expected to be provided by the patients. Hence this part of the methods section needs revising.We have modified the section as Follows-These were the options provided to the patients-1. treatment not available, the appropriate treatment for the symptoms was unavailable in that HCF; 2. treatment not affordable, cost of treatment was beyond the paying capacity of the patient and its family; 3. referred, the patient was asked to visit another HCF for review/consultation or appropriate treatment; 4.not satisfied, symptoms did not alleviate or the patient perceived that the services being provided were inadequate. Also, along with this, they were also asked if any other reasons not included in the list.Methods- Operational definitionResult-1st paragraph. Need to be consistent in the use of both numbers and % in the last sentence.We have improvised the result with consistency.Result-1st paragraphResults, Details of first visit and shift from one HCF to other HCF, 2nd paragraph. Although 12 of the 15 who initially visited a public HCF were diagnosed with MDR, only 10 were immediately referred to the DR-TB center. Why did the 2 other patients have to attend a HCF for a 2nd time before referral to the DR-TB Centre?These two patients were from the other districts, they were referred from PHC to District hospital of the concerned district and from there to the DDR-TBC.Discussion. As stated by the previous reviewer, the authors spend a lot of the discussion section comparing with other studies. Many of the times this is just in relation to confirming previous observations. Such text could be reduced (e.g. 2nd and 4th paragraphs).We have modified the discussion as suggested, we have removed the comparing figures and have only mentioned relevant texts, we have reduced 2nd and 4th paragraphDiscussionDiscussion, 2nd paragraph. Should ease of access to service be more prominent amongst important issues surrounding health seeing behavior of patients? Is this not a major reason why patients initially seek care in the private sector rather than the public sector?Yes, we have incorporated the point in the discussionDiscussion Paragraph 2Discussion, 3rd paragraph. DR-TB is now more of a “transmitted” disease rather than the “acquired resistance” as discussed in the first few sentences of the paragraph. This needs to be noted. Also the meaning of the sentence “Further, the private sector poses many hurdles in TB control.” is unclear?There are both kind of cases, acquired as well as transmitted, we have now included your point in our discussion, also we have explained how the private sector can hinder the outcome of DRTBDiscussion paragraph 2Discussion, 5th paragraph. For clarity of reading, the authors should to include the actual numbers involved when they state “57.5%” (=23/40) and “82.3” (=14/17).Changes have been incorporatedDiscussion paragraph 4Discussion, 6th paragraph. Did the study by Charles et al. (ref 28) include DR-TB cases as well as DS-TB? If not, how relevant is the reference to the current study?We have removed this part of the discussion.Not applicableLimitations. The authors need to include a paragraph or two on “Limitations” of the study.Thank you for the insight. We have incorporated the paragraph for limitation.Limitation reads as:This study was conducted among the 40 patients from one DDR-TBC of Karnataka therefore the findings can be generalized to the population seeking health care from the same DDR-TBC. Second limitation would be the subject variability of the definition, however we have made full effort to validate the findings with medical reports of the patients from DDR-TBC to ensure the starting and end point of the pathway. Since the study was based on recall of the patients, there are chances of inherent recall bias.LimitationConclusions. Some of the statements made are pretty sweeping with little data or evidence from the study to support them. For example, the statements in the 1st paragraph made about female and the elderly – these are based on very small patients numbers and hence how valid and generalizable are they? In the 2nd paragraph, an alternative view is that there may be better awareness in the community of the disease and availability of services. Hence a higher proportion of those who actually have the disease present to the public sector facilities for their 1st visit. And surely better linkages between the HCFs of the two sectors needs to be established, in addition to the option of providing everything in all private HCFs? We have modified the conclusion based on your suggestion.Conclusion read as-The present study found that there was substantial patient delay and total delay in diagnosis and treatment of DR TB patients. The study projects the need of a public-private collaboration in treating DR TB cases; in terms of linkages between public and private sector for diagnosis and treatment of drug resistance TB, provisions of standard diagnostic services and treatment at low to no costs. This may be achieved by incentivizing treatment and providing standard diagnostic modalities to private sector under NTEP. ConclusionConsistency of language. DOTS-Plus centers / DR-TB Centre / PMDT center are all used in the manuscript. Firstly I would suggest the authors use “centre” and “programme”, and also are consistent in the term they use for the centre where the DR-TB patients were registered. We have changed it to District drug resistance TB center (DDR-TBC), We have also clarified and changed center and programme.All throughout the manuscript" } ] } ]	1	https://f1000research.com/articles/8-498
https://f1000research.com/articles/10-207/v1	12 Mar 21	{ "type": "Research Article", "title": "The diagnostic yield of whole exome sequencing as a first approach in consanguineous Omani renal ciliopathy syndrome patients", "authors": [ "Intisar Al Alawi", "Mohammed Al Riyami", "Miguel Barroso-Gil", "Laura Powell", "Eric Olinger", "Issa Al Salmi", "John A. Sayer", "Intisar Al Alawi", "Mohammed Al Riyami", "Miguel Barroso-Gil", "Laura Powell", "Eric Olinger", "Issa Al Salmi" ], "abstract": "Background: Whole exome sequencing (WES) is becoming part of routine clinical and diagnostic practice. In the investigation of inherited cystic kidney disease and renal ciliopathy syndromes, WES has been extensively applied in research studies as well as for diagnostic utility to detect various novel genes and variants. The yield of WES critically depends on the characteristics of the patient population. Methods: In this study, we selected 8 unrelated Omani children, presenting with renal ciliopathy syndromes with a positive family history and originating from consanguineous families. We performed WES in affected children to determine the genetic cause of disease and to test the yield of this approach, coupled with homozygosity mapping, in this highly selected population. DNA library construction and WES was carried out using SureSelect Human All Exon V6 Enrichment Kit and Illumina HiSeq platform. For variants filtering and annotation Qiagen Variant Ingenuity tool was used. Nexus copy number software from BioDiscovery was used for evaluation of copy number variants and whole gene deletions. Patient and parental DNA was used to confirm mutations and the segregation of alleles using Sanger sequencing. Results: Genetic analysis identified 4 potential causative homozygous variants each confirmed by Sanger sequencing in 4 clinically relevant ciliopathy syndrome genes, (TMEM231, TMEM138, WDR19 and BBS9), leading to an overall diagnostic yield of 50%. Conclusions: WES coupled with homozygosity mapping provided a diagnostic yield of 50% in this selected population. This genetic approach needs to be embedded into clinical practise to allow confirmation of clinical diagnosis, to inform genetic screening as well as family planning decisions. Half of the patients remain without diagnosis highlighting the technical and interpretational hurdles that need to be overcome in the future.", "keywords": [ "renal ciliopathy", "cystic kidney disease", "Oman", "whole exomes sequencing" ], "content": "Introduction\n\nThere are over 750 million people worldwide affected with chronic kidney disease (CKD), a disease burden that is much higher than those living with diabetes, cancer or even AIDS/HIV1. Inherited kidney diseases and renal ciliopathy syndromes are one of the major contributors to CKD burden, where up to 10% of adults and over 70% of children reaching end stage kidney disease (ESKD) are expected to harbour genetic causes2. However, studying such rare diseases has considerable challenges mainly due to the small size of patient cohorts negatively affecting progress of treatments and commercial feasibility. Collaborative research and progress of new technologies and methodologies are strategic to overcoming these challenges.\n\nWES is becoming part of routine clinical and diagnostic practice2. Focusing only on protein-coding regions through WES decreases the sequencing costs and produces manageable genetic data for interpretation, which enhances its extensive usage in diagnosis leading to the discovery of previously unrecognized renal disease genes and disorders2,3. In the case of heterogeneous renal ciliopathies, WES has been extensively applied in research studies as well as for diagnostic utility to detect various novel genes and variants4,5. In this study, WES was used to determine the genetic causes of cystic kidney disease and renal ciliopathy syndromes in a group of 8 unrelated Omani children from consanguineous families. As this study shows, the focus of nephrogenetics in Oman is primarily to establish an accurate genetic diagnosis to explain clinical phenotypes using the significantly improved diagnostic power of genomic technologies.\n\n\nMethods\n\nThis study was approved by the North East-Newcastle & North Tyneside 1 Research Ethics Committee (18/NE/350).\n\nPatients were identified and recruited from paediatric referrals for investigation of inherited kidney disease to the nephrology services within the Ministry of Health Hospital, Muscat, Oman between 2015 and 2018. Whole blood (1.5-2.5 ml in EDTA) samples were collected specifically for this study and used for extraction of genomic DNA. DNA samples from affected and other family members were given an anonymised sample number. All patients had clinical features strongly suggestive of an inherited renal ciliopathy. Written and informed consent was obtained from the parents / guardians of each patient, and any family members (including parents and siblings) involved in this study.\n\nClinical information relating to patient presentation, phenotype and family pedigree structure, with an emphasis on familial kidney disease was obtained, following informed consent for access to the medical records. Family pedigrees were drawn using Invitae© online tool (https://familyhistory.invitae.com).\n\ngDNA was isolated from whole blood of patients and the available family members using Hamilton’s Microlab® STAR™, according to the manufacturer protocol. DNA extraction was performed in the National Genetic Centre in Oman. DNA library construction and WES were outsourced to EuroFins GATC Biotech (Germany) or Novogene Co., Ltd (China). SureSelect Human All Exon V6 Enrichment Kit (Agilent Technologies, CA, USA) and Illumina HiSeq platform (Illumina, San Diego, CA, USA) were used. Analyses of raw data (FASTQ format) were performed including sequence reads mapping to the human reference genome hg19 using BWA (Li and Durbin, 2009), removal of PCR duplicates using Picard (http://broadinstitute.github.io/picard/), alignment refinement using GATK, coverage analysis and SNP and indel calling using GATK’s Haplotype Caller (McKenna et al., 2010).\n\nSNP and indel VCF files were investigated using Qiagen Variant Ingenuity tool for variants filtration and annotation. Nexus copy number software from BioDiscovery (9.0) was used for CNVs analysis and visualization. To detect regions of homozygosity, WES genotype data were used to create homozygosity mapping using the online homozygosity mapper tool (http://www.homozygositymapper.org/).\n\nSanger sequencing was utilized to confirm suspected disease-causing variants and their segregation if DNA samples from parents and other family members were available. Primer3 was utilized to design primer sequences (http://primer3.ut.ee/) (Extended Data Table 16). PCR amplification was performed using Taq PCR master mix (Qiagen) kit, as per the manufacturer instructions. Sanger sequencing was outsourced to EuroFins GATC Biotech (Germany). The obtained sequences were assembled and aligned compared to a reference sequence using the SequencePilot 4.2.2 software (JSI Medical Systems GmbH).\n\n\nResults\n\nWES was carried out for 8 unrelated paediatric patients with an age range of 3 months to 6 years of age (5 female, 3 male) with a clinical suspicion of a renal ciliopathy syndrome and known consanguinity as demonstrated by pedigree diagrams (Extended data Figure 16). This was a diagnostic-naïve population without prior genetic analysis. Patients had a variety of clinical features, renal and extra-renal, with 5 probands reaching ESKD within 5 years of life (Table 1). Seven out of 8 had a positive family history of kidney disease and 6 had extra-renal manifestations typical of ciliopathy syndromes which included Senior-Løken syndrome, Joubert syndrome, Meckel syndrome and Bardet-Biedl syndrome (Table 1).\n\nCKD, chronic kidney disease; DD, developmental delay; ESKD, end stage kidney disease; F, female; M, male; m, month; y, year.\n\nQuality control of WES revealed that >99% of the reads were properly mapped to the reference genome. The details of the depth, coverage and target sequences covered are summarized in Extended data Table 26. The average coverage depth was 145.9. Comparable coverage of target coding regions was achieved among the 8 cases with an average of 96.4% of the exome being covered at least 20-fold (Extended data Table 26). Homozygosity mapping of all patients confirmed large regions of homozygosity, typical of known parental consanguinity (Extended data Figure 26).\n\nA molecular genetic diagnosis was obtained in 4 out of the 8 patients (Figure 1), leading to an overall diagnostic yield of 50% (Table 2). Four different homozygous single nucleotide variants (SNVs) were detected in 4 known ciliopathy genes (TMEM231, TMEM138, WDR19 and BBS9) and were confirmed by Sanger sequencing (Figure 1). Three of the mutations were missense mutations affecting highly conserved amino acids (Extended Data Figure 36) whilst the fourth was a splice-site mutation (Figure 2). All tested samples were examined for mutations in ACMG actionable genes but none were identified.\n\nPedigrees of solved families with Sanger sequecing chromatograms confirming the disease causative variants that were identified by WES in four families. A. M46 with homozygous missense variant in TMEM231. B. M48 with homozygous missense variant in TMEM138. C. P18 with homozygous missense variant in WDR19 D. N36 with homozygous splice site variant in BBS9.\n\nReference sequence IDs: TMEM231: NM_001077416; TMEM138: NM_016464; WDR19: NM_025132; BBS9: NM_198428\n\nAbbreviations: CADD score, combined annotation dependant depletion; conser, conservation; gnomAD, Genome Aggregation Database; Hom, homozygous; N/A, not available\n\nPositions and the predicted protein alterations are shown for A. TMEM231 B. TMEM138 C. WDR19 D. BBS9. Exon structure is marked by a dashed line. Protein domains are shown in colored bars. Known mutations are shown above the gene/protein structure with the number showing frequency (if >1) of probands reported for each mutation. Mutations identified in this study are shown below the gene/protein structure.\n\nThe identified causative variant in M46 was novel (c.710A>G; p.Y237C in TMEM231) and has not been previously reported in any databases. This homozygous missense change is found in a large region of homozygosity on Chromosome 16 (Extended data Figure 26) and is predicted by Sorting Intolerant from Tolerant (SIFT) to be damaging, PolyPhen-2 to be possibly damaging and MutationTaster to be disease causing. The Y237 amino acid in TMEM231 is conserved to Caenorhabditis elegans (Extended data Figure 36). Mutations in TMEM231 are known to cause both Joubert syndrome and Meckel syndrome (Extended Data Table 36), and the clinical phenotype of patient M48, which included encephalocele, polydactyly and polycystic kidney disease and early onset ESKD, is consistent with a Meckel-like ciliopathy syndrome.\n\nThe identified causative variant in M48 was a known allele (c.389A>G; p.Y130C in TMEM138) and has been previously reported in a child with Joubert syndrome and a cerebello-retinal-renal phenotype7. This homozygous missense change is found in a narrow region of homozygosity on Chromosome 11 (Extended data Figure 26) and is predicted by SIFT to be deleterious, PolyPhen-2 to be probably damaging and MutationTaster to be disease causing. The Y130 amino acid in TMEM138 is conserved to Danio rerio (Extended data Figure 36). Mutations in TMEM138 are known to cause Joubert syndrome (Extended data Table 46), and the clinical phenotype of patient M48, which included molar tooth sign, visual loss and cystic kidney disease, is consistent with a Joubert syndrome ciliopathy.\n\nThe identified causative variant in P18 was a known allele (c.3553G>A; p.R1178Q) in WDR19 and has been previously reported in cases of nephronophthisis (NPHP)-related ciliopathies with retinal and liver involvement8–10, Senior-Løken syndrome11 and more complex ciliopathies12. This homozygous missense change is found in a large region of homozygosity on Chromosome 4 (Extended data Figure 26) and segregation of the pathogenic causative allele in WDR19 with P18’s family members was confirmed. The missense allele is predicted by SIFT to be tolerated, PolyPhen-2 to be probably damaging and MutationTaster to be disease causing. The R1178 amino acid in WDR19 is conserved to C.elegans (Extended data Figure 3 6). Mutations in WDR19 are associated with a wide spectrum of ciliopathies (Extended data Table 56), and the clinical phenotype of patient P18, which included NPHP and early onset ESKD and retinal dystrophy is consistent with a Senior-Løken syndrome.\n\nThe identified causative variant in N36 was a known splice-site allele (c.1789+1G>A in BBS9) and has been previously reported in patients with Bardet-Biedl syndrome (BBS)13,14. This homozygous missense change is found in a region of homozygosity on Chromosome 7 (Extended data Figure 26) and is predicted to cause loss of splice donor site. Mutations in BBS9 are known to cause BBS (Extended data Table 66), and the clinical phenotype of patient N36, which included features of BBS including post-axial polydactyly affecting all limbs and cystic kidney disease is consistent with a BBS ciliopathy.\n\n\nDiscussion\n\nIn paediatric populations, CKD is a major contributor to health-care burden leading to severe morbidity and mortality. At least 17% of those with ESKD are considered as CKD with unknown aetiology, where the primary kidney disease is not clear15. In addition, the primary clinical diagnosis of CKD patients is often inaccurate15. Thus, in the developing era of precision medicine, WES is used as an essential tool that provides novel diagnostic perspectives for the detection of the causes of CKD. Knowledge of genetic causes has valuable clinical implications in therapeutic intervention, improving prognosis, guide family counselling or managing settings of kidney transplantation16. Despite being rare, inherited kidney diseases represent one of the most common causes of CKD and ESKD, accounting for up to 10% of adults and almost all children commencing renal replacement therapy17. The possibility of monogenic causes in those with unknown aetiology of CKD or with atypical clinical presentation is assumed to be high15. At least 500 different genetic causes have been associated with childhood CKD18.\n\nIn this pilot study, we examined the utility of WES in the diagnosis of 8 different Omani children with childhood onset CKD related to cystic kidney disease and a suspected inherited renal ciliopathy. A conclusive genetic diagnosis was achieved in half of the cases. Positive WES findings allow a precise molecular diagnosis and targeted clinical management as well as informing family planning and facilitating proper genetic counselling. In four of the children (M46, M48, P18 and N36) the molecular genetic findings confirmed the suspected clinical diagnosis. The identification of a molecular genetic diagnosis in all these families can provide accurate genetic advice about the parent’s reproductive choices and the possibility of preimplantation genetic diagnosis (PGD) or early genetic testing of a foetus in future pregnancies.\n\nA wide range of genetic studies have been performed in childhood CKD populations and different diagnostic yields were achieved due to differences in the inclusion criteria or patients and the study design. In a study of families with inherited kidney disease, Mallett, et al.19 reported a diagnostic yield of 46%, reflecting the significant ability of WES in underlying the potential genetic causes of most renal phenotypes. In another recent study2, Groopman et al. reported higher diagnostic yield in patients with congenital and cystic kidney disease (23.9%). Furthermore, regardless of the primary kidney diagnosis, higher diagnostic yield was associated with a positive family history of CKD, history of parental consanguinity and presentations of extra-renal features2,5. Thus using a combination of homozygosity mapping along with WES genotype data is always recommended as a powerful approach for consanguineous families to identify rare genetic causes20.\n\nAlthough WES provides massive amounts of genetic data, 4 patients remained unsolved in this study. Interpretation of many novel and extremely rare variants is still limited by the incomplete knowledge of the total human protein-coding genes as well as the incorrect annotation of variants pathogenicity and incorrect association of genes with the disease in the literature. At present, up to 70% of protein-coding genes have no recognized human disease phenotype21. False gene-disease associations are present in the literature22,23 and clinically valuable databases of variants pathogenicity, such as Human Gene Mutation Database (HGMD®), comprise various errors causing benign variants being falsely selected out of the data and allocated as plausible diagnosis24. This situation is predicted to improve as further genomes are sequenced, including large data collections containing populations of both healthy individuals and patients with rare diseases. In addition, studying more families with similar clinical phenotypes from the same population may facilitate linking novel undiscovered genes to the disease phenotype in those unsolved patients.\n\nIn this study, WES confirmed the clinical diagnosis in 4 children. In a similar study of large consanguineous or familial cohort (n = 79) of children clinically diagnosed with NPHP, genetic diagnostic yield of 63% was reported, of which the clinical diagnosis was confirmed in 64% and changed to different molecular diagnosis in the remaining 36%10.\n\nThis study has some limitations, including small sample size that does not give a generalized image of broader childhood renal ciliopathy in the population from Oman. However, an enhanced assessment of the utility of WES in the clinical diagnostic practice of these disorders may be given through systematic WES analysis of a larger, unselected cohort. Moreover, the diagnostic gap in this study may be caused by the common technical limitation of WES, including the missed detection of structural variant breakpoints, sequencing difficulties for regions with repetitive elements or guanine-cytosine (GC)-rich regions, and limited discrimination between highly homologous genomic regions with pseudogenes. These limitations are attributed to the short-read lengths that are utilized to generate high genomic coverage and depth25. These limitations are assumed to be resolved through using long-read sequencing platforms that compromise these technical challenges and improve the detection of genetic variants25. Thus, the emerging future of long-read sequencing based whole genome sequencing (WGS) could enhance the diagnostic yield of patients with inherited renal ciliopathies and provide more conclusive primary kidney disease diagnosis. This can be supported by recent reports of WGS obtaining higher molecular diagnostic yield compared with WES, where 20–40% of those unsolved by WES were genetically conclusive by WGS26.\n\nRecent advancements in medical genetics through the use of massively parallel sequencing have not only advanced the discovery of novel causative variants, genes and phenotypes, but also contributed to the re-classification of diseases and phenotypes into novel gene-based ontologies27. However, all types of next generation sequencing (NGS)-based testing (Target panel, WES and WGS) have some shared limitations, including the inability to obtain enough coverage of genomic regions with highly repetitive GC-content sequence, such as that in MUC1 gene. In his study of six unrelated families with medullary cystic kidney disease type 1 (MCKD1)28, Kirby et al. highlighted the challenges of these technologies in detecting the causative monogenic causes of some Mendelian disorders, such as MCKD1, where only long-range polymerase chain reaction and molecular cloning successfully performed the task. Moreover, in many patients with acquired diseases, NGS testing is of limited importance and transformation of genetic results into clinical setup may be challenging27. In the field of kidney disease, the majority of genetic testing studies are narrowed to a research setting, thus until now the knowledge of its diagnostic efficacy in clinical practice is still limited15. In addition, managing the medical ethics raised by these technologies, including uncertain variants and incidental findings, and balancing the social concerns is still challenging29.\n\n\nConclusion\n\nWES of patients with different inherited cystic kidney diseases and renal ciliopathies shows promise as a diagnostic tool, especially in well selected patients with a high coefficient of inbreeding and/or with a syndromic presentation. It has the potential to resolve those cases with clear suspicion of renal ciliopathies, as well as those with uncertain aetiology causing CKD. The fact that ~50% of patients remain without genetic diagnosis after WES highlights the need for improved sequencing techniques and interpretation tools, driven by constantly evolving knowledge regarding the genetic architecture of diseases. The clinical impacts of positive WES results on therapeutic choice, genetic counselling and guidance of kidney transplant are critical. Indeed, professional genetic counselling on the prospective effects of a positive test result is crucial, bearing also in mind the possibility of incidental findings. Although further studies from the Omani population are required, we predict an expanding impact of NGS-based diagnosis, both gene panels and WES in clinical practice in the very near future.\n\n\nData availability\n\nFigshare: The diagnostic yield of whole exome sequencing as a first approach in consanguineous Omani renal ciliopathy syndrome patients, https://doi.org/10.6084/m9.figshare.13696750.v130.\n\nThis project contains the following underlying data:\n\nM46.snps.vcf\n\nM46.indels.vcf\n\nM48.snps.vcf\n\nM48.indels.vcf\n\nJAS_P18.GATK.snp.vcf\n\nJAS_P18.GATK.indel.vcf\n\nJAS_N36.GATK.snp.vcf\n\nJAS_N36.GATK.indel.vcf\n\nFigshare: The diagnostic yield of whole exome sequencing as a first approach in consanguineous Omani renal ciliopathy syndrome patients, https://doi.org/10.6084/m9.figshare.c.5287753.v16.\n\nThis project contains the following extended data:\n\nExtended data Table 1. Forward and reverse primer sequences used for WES variants verification (https://doi.org/10.6084/m9.figshare.13675201)\n\nExtended data Table 2. Whole exome sequence alignment and coverage profile by sample (https://doi.org/10.6084/m9.figshare.13675222)\n\nExtended Data Table 3. TMEM231 alleles (https://doi.org/10.6084/m9.figshare.13675471)\n\nExtended data Table 4. TMEM138 alleles (https://doi.org/10.6084/m9.figshare.13675504.v1)\n\nExtended data Table 5. WDR19 alleles (https://doi.org/10.6084/m9.figshare.13675540)\n\nExtended data Table 6. BBS9 alleles (https://doi.org/10.6084/m9.figshare.13675546)\n\nExtended data Figure 1. Pedigree diagrams (https://doi.org/10.6084/m9.figshare.13675552.v1)\n\nExtended data Figure 2. Homozygosity mapping (https://doi.org/10.6084/m9.figshare.13675558.v1)\n\nExtended data Figure 3. Clustal alignments of amino acids associated with identified missense mutations (https://doi.org/10.6084/m9.figshare.13675561.v1)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors thank the staff at the National Genetic Centre, Muscat, Oman, and the cooperative nephrologists and nurses from different referral hospitals throughout Oman for their kind collaboration.\n\n\nReferences\n\nCrews DC, Bello AK, Saadi G: 2019 world kidney day editorial - burden, access, and disparities in kidney disease. J Bras Nefrol. 2019; 41(1): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGroopman EE, Marasa M, Cameron-Christie S, et al.: Diagnostic utility of exome sequencing for kidney disease. N Engl J Med. 2019; 380(2): 142–151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBraun DA, Hildebrandt F: Ciliopathies. 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Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.13696750.v1" }	[ { "id": "81405", "date": "30 Mar 2021", "name": "Enza Maria Valente", "expertise": [ "Reviewer Expertise neurogenetics", "ciliopathies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article, the authors show the importance of Whole Exome Sequencing (WES) approach as a first genetic screening for patients with renal ciliopathy syndromes. They focus on a small cohort of 8 consanguineous Omani probands, who underwent WES. The diagnostic yield was 50%: four out of 8 probands were found to carry homozygous pathogenic variants in known genes. The study was carried out in a proper way, with sound methodology. The identified variants were adequately named and classified following ACMG guidelines. The discussion correctly addresses limitations of WES.\n\nThere are two main limitations in this article. The first is the very small cohort size, which does not really allow making a correct estimate of the diagnostic yield of WES in renal ciliopathies. Expansion of the study to a larger group of patients would clearly provide more useful information, also regarding the genetic background of genetic renal ciliopathies in Oman. Second, even maintaining this cohort, it would be very interesting to know more about “negative” cases: where there any single heterozygous pathogenic variants in known genes? Any missense variants classified as VUS? Moreover, since families were consanguineous, the authors should report whether they checked for genes included within stretches of homozygosity by descent, and list the potentially interesting homozygous variants in candidate genes lying within these regions. This would definitely improve the study which, in its present form, does not add substantial new data to current knowledge on the topic, and therefore it remains largely confirmatory.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6855", "date": "09 Jul 2021", "name": "John Sayer", "role": "Author Response", "response": "Yes we agree that this is a small study size, but we were careful only to include subjects that had not had any previous genetic investigations and fulfilled the criteria of a suspected renal ciliopathy syndrome. In this way this study was seeing the added value in terms of genetic diagnosis to perform whole exome sequencing as a first line approach in contrast to a targeted renal genetics panel. We have added some comments regarding the size of the cohort. In response to the comments regarding the “negative cases’ we have now expanded the paper to discuss these further as there may, as the reviewer suggests be some useful learning points. As all the families were consanguineous, we focussed on homozygous variants but we have commented now on any significant heterozygous variants in cystogenes. Similarly, we have looked at missense alleles and synonymous changes that have been exclude by pathogenicity filters and comment on these also. Finally, as suggested we report homozygous variants in regions of homozygosity by descent in candidate genes that may shed new light on renal ciliopathies. A new table detailing variants in unsolved cases has been added and the discussion expanded to account for these new data." } ] }, { "id": "81403", "date": "06 Apr 2021", "name": "Paraskevi Goggolidou", "expertise": [ "Reviewer Expertise Renal Ciliopathies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Al Alawi et al. provides an interesting application of Whole Exome Sequencing (WES) in Ciliopathy patients in Jordan. The authors have looked at a small number of Ciliopathy patients and identified mutations of interest in 50% of them. As they are working with rare disease patients, the limitations in sequencing bigger patient populations are understandable. Furthermore, this work provides a very useful proof of concept in the application of WES for genetic diagnosis of rare diseases, such as Ciliopathies.\nHowever, as discussed in the discussion section, WES has got limitations and other approaches such as WGS might have been more insightful for this study. The manuscript would thus benefit from a more thorough discussion of the possibility of identified by WGS cases of non-exonic mutations causing Ciliopathies and a comment on the mechanisms behind this. Furthermore, it is important to comment on whether there were any single heterozygous pathogenic variants identified in known genes and if they checked for genes within stretches of homozygosity in consangeneous families. Finally, for the non-specialist audience a brief description of the key characteristics and genes associated with renal ciliopathies in the introduction section would have been useful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6856", "date": "09 Jul 2021", "name": "John Sayer", "role": "Author Response", "response": "We agree that WGS may be more powerful than WES in order for us to solve the unsolved cases. We have added some discussion regarding this point as suggested. In response to the comments regarding the “negative cases’ we have now expanded the paper to discuss these further as there may, as the reviewer suggests be some useful learning points. As all the families were consanguineous, we focussed on homozygous variants but we have commented now on any significant heterozygous variants in cystogenes. Similarly, we have looked at missense alleles and synonymous changes that have been excluded by pathogenicity filters and comment on these also. Finally, as suggested we report homozygous variants in regions of homozygosity by descent in candidate genes that may shed new light on renal ciliopathies. A new table detailing variants in unsolved cases has been added and the discussion expanded to account for these new data. We have now added a brief introduction to renal ciliopathies and the common genetic causes has now been added as suggested." } ] } ]	1	https://f1000research.com/articles/10-207
https://f1000research.com/articles/10-546/v1	07 Jul 21	{ "type": "Research Article", "title": "Killer immunoglobulin-like receptor (KIR) genes are associated with the risk of episodes of high-level and detectable viremia among HIV controllers", "authors": [ "Nathalia Beatriz Ramos De Sá", "Karina dos S. Silva", "Marcelo Ribeiro-Alves", "Diogo Gama Caetano", "Fernanda Heloise Côrtes", "Suwellen S. D. de Azevedo", "Brenda Hoagland", "Beatriz Grinsztejn", "Valdilea G. Veloso", "Mariza G. Morgado", "Sylvia Lopes Maia Teixeira", "Karina dos S. Silva", "Marcelo Ribeiro-Alves", "Diogo Gama Caetano", "Fernanda Heloise Côrtes", "Suwellen S. D. de Azevedo", "Brenda Hoagland", "Beatriz Grinsztejn", "Valdilea G. Veloso", "Mariza G. Morgado", "Sylvia Lopes Maia Teixeira" ], "abstract": "Background: HIV controllers (HICs) constitute a heterogeneous group of HIV-1 individuals able to suppress plasma viremia to low or undetectable levels in the absence of antiretroviral therapy. Host genetic factors may be involved in the sustained control of viral replication observed. We investigated the distribution and the potential impact of human leukocyte antigens (HLA)-B and -C alleles, killer immunoglobulin-like receptor (KIR) genes, single nucleotide polymorphisms (SNPs) of the NLRP3, CARD8 and IL-1β inflammasome genes, and CCR5Δ32 mutation on the viral control among HICs. Methods: In total, 28 HICs were categorized as persistent elite controllers (PECs, n = 7), ebbing elite controllers (EECs, n = 7), and viremic controllers (VCs, n = 14) according to the level of natural suppression of viremia. HLA alleles were assigned by sequencing-based typing, KIR alleles by polymerase chain reaction (PCR) sequence-specific amplification, SNPs by real-time PCR, and the CCR5Δ32 mutation by PCR. Results: Significant differences were observed in the pairwise comparisons of protective HLA-B alleles, KIR Bx genotype, KIR2DL3 + C1 pair, KIR2DL5, and KIR2DS5 allelic carrier frequencies among the HIC groups. Multivariate models showed that HICs without the KIR2DL3 allele or without KIR2DL3 + C1/C2 pair, with the HLA-C08 allele or with the NLRP3 rs10754558-G SNP had a higher mean hazard of a viral load above 2,000 copies/mL, while a lower mean hazard of this event was observed for HICs with KIR2DL5, KIR2DS1, KIR2DS5, and KIR3DS1 alleles. Moreover, HICs with the KIR2DS5 allele had less risk of undergoing viral load (VL) blips within the same normalized period than those participants without this allele, while HICs without the KIR2DL3 allele had a mean higher risk of experiencing VL blips. Conclusions: These results indicate that innate immune mechanisms may play an essential role in modulating the sustained control of viral replication in HICs.", "keywords": [ "HIV controllers", "viral load", "HLA", "KIR", "CCR5 Δ32", "Inflammasome SNPs." ], "content": "Introduction\n\nDifferent levels of viremia control are observed among human immunodeficiency virus (HIV)-1-infected individuals. Approximately 1% of the HIV-1-seropositive population suppresses viral replication to extremely low or undetectable levels in the absence of antiretroviral therapy and are termed HIV controllers (HICs) or elite controllers (ECs) (Deeks & Walker, 2007; Lambotte et al., 2005). HICs constitute a heterogeneous group of HIV-1-infected individuals, even when compared to persons with low-level viremia, they exhibit marked genetic and immunologic heterogeneity (Pereyra et al., 2008; Côrtes et al., 2015). Heterogeneity in viral load (VL), classification criteria and follow-up time to define the EC profile is also observed among the studies (Gurdasani et al., 2014; Navarrete-Muñoz et al., 2020). The mechanisms involved in the control of viral replication remain to be fully elucidated. Some studies point to the role of viral features in this phenomenon, while others focus on the role of host genetic in the efficient control of HIV-1 observed among HICs (Côrtes et al., 2015; Balasubramaniam et al., 2019).\n\nHuman leukocyte antigens (HLA) class I molecules have a crucial role in the cytotoxic T-lymphocyte (CTL) response. Genes that encode these molecules have been consistently associated with distinct patterns observed in the dynamics of HIV-1 infection (McLaren & Carrington, 2015), among which we can highlight the protective role attributed to the HLA-B27 and B57 alleles in EC cohorts (Lécuroux et al., 2014; Migueles et al., 2000; Fellay et al., 2007; Pereyra et al., 2010). However, the presence of HLA-B27 and B57 cannot be directly related to viral control, since some HIV-1-infected individuals who harbor these alleles present absence of viremia control (Pereyra et al., 2008; Emu et al., 2008). While HLA-B alleles are well-reported markers of HIV disease progression and control (McLaren & Carrington, 2015), HLA-C alleles have been neglected. HLA-C alleles were generally considered inferior in restricting CTLs responses compared to HLA-A and –B (Kulpa & Collins, 2011). On the other hand, they act as excellent ligands for killer immunoglobulin-like receptor (KIR) receptors on natural killer (NK) cells and protect target cells from lysis mediated by NK cells (Kulpa & Collins, 2011). In this context, the HLA-C locus emerged as an important host genetic determinant of HIV infection outcomes. The HLA-C08 and HLA-C18 alleles have already been associated with better disease control in a study of HIV controllers (Lazaryan et al., 2011), and the HLA-C15 allele was a protective factor in a cohort of exposed uninfected infants (Bardeskar et al., 2018). Moreover, a single-nucleotide polymorphism 35 kb upstream the HLA-C locus (rs9264942) showed the most significant association with viral load (VL) control (Fellay et al., 2007; Thørner et al., 2016; Malnati et al., 2017). This variant has been associated with high HLA-C mRNA (messenger ribonucleic acid) levels and higher surface expression of HLA-C alleles (Fellay et al., 2007; Thørner et al., 2016; Malnati et al., 2017).\n\nInflammasomes are cytosolic multiprotein complexes of the innate immune system responsible for activating inflammatory responses (Rathinam & Fitzgerald, 2016). These complexes regulate the maturation of cytokines of the IL-1 family, including IL-1β and IL-18 (Rathinam & Fitzgerald, 2016). Activation of the inflammasome during HIV-1 infection mainly contributes to immune hyperactivation, which is the main pathogenic mechanism of HIV-1 progression (Marín-Palma et al., 2018). However, the relationship between inflammasome single nucleotide polymorphisms (SNPs) and viral load control in the context of HIV-1 infection is still unclear. The genetic restriction to HIV-1 infection caused by polymorphisms in the chemokine receptor CCR5 is another important factor that can modulate HIV-1 dynamics. Homozygosity for a 32 base-pair deletion in the CCR5 gene (Δ32 mutation) is considered as a resistant phenotype leading to the protection of individuals against infection with HIV-1 R5 tropic lineages (Dean et al., 1996; Liu et al., 1996; Samson et al., 1996; Marmor et al., 2011), while heterozygosity may result in in slower progression to acquired immune deficiency syndrome (AIDS) (Huang et al., 1996; Sullivan et al., 2001).\n\nInnate immunity mechanisms also participate in the sustained control of viral replication observed among ECs, mainly effector responses mediated by NK cells (Tomescu et al., 2012). The activity of these cells contributes to reduce viral replication in acute infection, thereby cooperating to the ability of ECs to control viremia (O'Connell et al., 2009). Consistent associations between KIR genes — expressed on the surface of NK cells — and viral control have already been described, highlighting the protective role conferred by KIR3DL1 associated with HLA-B molecules carrying the Bw4 motif (Martin et al., 2007). The inhibitory KIR2DL1, 2DL2, and 2DL3 recognize HLA-C ligands (Kulkarni et al., 2008), but no consistent association between HLA-C/KIR alleles and viral control has been described so far. Other innate immunological mechanisms have also been studied in ECs cohorts, including the ability of NK cells to induce cell death by antibody-dependent cellular cytotoxicity (ADCC) (Lambotte et al., 2013).\n\nStudies with HICs may provide novel insights regarding host mechanisms of virus control (Hatano et al., 2009). Different from previous studies with HICs from the United States and Europe (Lambotte et al., 2005; Pereyra et al., 2008; Walker & Yu, 2013; Pernas et al., 2018), here, we have the opportunity to examine the role of these genetic markers in a HICs cohort that contains a high degree of miscegenation, which characterizes the Brazilian population. Besides this, other genetic-related particularities can be observed among Brazilians, such as the association of HLA-B52 with the non-progression to AIDS (Teixeira et al., 2014). Therefore, in this study, we analyzed the distribution of HLA-B and -C, KIR, CCR5 genes, as well as selected SNPs to investigate their impact on the viral control observed in a cohort of Brazilian HICs.\n\n\nMethods\n\nThe individual description of the HICs analyzed in this study had already been published (de Azevedo et al., 2017; Côrtes et al., 2018; Caetano et al., 2020). Individuals who fulfilled the criteria of HICs were identified in a cohort of HIV-1 seropositive individuals followed at the Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz (INI/FIOCRUZ), Rio de Janeiro, Brazil. These individuals were defined as subjects over 18 years old with documented HIV-1 infection for >3 years and RNA viral load below the detection limit (40 or 80 copies/mL, depending on the year of inclusion) in the absence of antiretroviral treatment. As this is a longitudinal study, these individuals were/have been followed up and classified under one of the categories of viral control.\n\nAfter 12 years of follow up, a cohort of 28 HICs could be composed including HIV-1-infected individuals classified in three categories according to the plasmatic viral load (VL): (1) persistent elite controllers (PECs) if 100% of VL measures were below the limit of detection (< LD; 50-80 copies/mL) for the respective available commercial assays (n = 7); (2) ebbing elite controllers (EECs) if subjects had occasional (≤ 30%) episodes of transient low-level (> LD to 400 copies/mL) viremia (n = 7); and (3) viremic controllers (VCs) if most (≥ 70%) of VL determinations were between 51 and 2,000 copies/mL (n = 14). Occasional VL measurements above the upper limits were accepted for the EECs and VCs. Participants were followed at least once every 6-12 months, starting in November 2008, to perform HIV-RNA VL quantification and CD4+ T lymphocytes counts. In each visit, whole blood was collected into an ethylenediaminetetraacetic acid (EDTA)-containing tube, and 1 mL was stored at -20 °C until use. At this moment, from the 28 individuals [median follow-up = 9.02 years (interquartile range (IQR) = 6.46)], 11 are currently being followed up and 17 are no longer study participants due to cART entry (n = 09) or loss of follow up (n = 08).\n\nAll participants provided written informed consent, and the ethical committee of Instituto Nacional de Infectologia Evandro Chagas (INI-FIOCRUZ) approved the study (CAAE 1717.0.000.009-07). The corresponding documents can be found as extended data (de Sá & Teixeira, 2021).\n\nDNA was extracted from whole blood using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Nordrhein-Westfalen, Germany) according to the manufacturer’s instructions. The DNA concentration was determined using the Thermo Scientific Nanodrop 2000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and the filtrates containing the isolated DNA were stored at −20 °C until used for genomic analysis.\n\nHigh-resolution HLA-B and -C alleles typing was performed by automated nucleotide sequencing (sequencing-based typing — SBT) according to the manufacturer’s instructions on the ABI platform using commercial kits (SECORE Sequencing kit, Invitrogen by Life Technologies, Brown Deer, Wisconsin, USA). HLA-B and -C alleles were assigned using a four-digit designation utilizing uTYPE® v6.0 SBT software (Invitrogen by Life Technologies, Brown Deer, Wisconsin, USA). The grouping of HLA-B alleles in HLA Bw4 and/or Bw6 epitopes associated specificities followed the Immuno Polymorphism Database (IPD)-International Immunogenetics Project (IMGT)/HLA nomenclature guidelines (Robinson et al., 2013). The grouping of HLA-C genes in C1 (HLA-C01/03/07/08/12/14/16) and C2 (HLA-C02/04/05/06/15/17/18) epitope-associated specificities was based in the classification currently used in the literature (Mandelboim et al., 1996; Faridi & Agrawal, 2011; de Sá et al., 2020).\n\nQualitative analysis of KIR genes was performed using a commercial kit based on sequence-specific primer amplification methods — SSP (SSP KIR Genotyping Kit, Invitrogen, Brown Deer, Wisconsin, USA). A total of 14 KIR genes and 2 KIR pseudogenes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1) were screened using this approach. KIR AA and Bx genotypes designation followed the current working definition, which characterizes these genotypes based on the combinations of haplotype A (absence of all the activating genes, except KIR2DS4) and haplotype B (presence of one or more of the activating genes) (Uhrberg et al., 1997; Fernandes-Cardoso et al., 2016).\n\nWe selected four single nucleotide polymorphisms (SNPs) in 3 inflammasome genes based on previously published data (Pontillo et al., 2013) and considering the relevance of each gene in the inflammasome pathway: CARD8 (Caspase Activation and Recruitment Domains-8) rs2043211 and rs6509365; NLRP3 (Nucleotide oligomerization domain–Like Receptor family, Pyrin domain-containing protein-3) rs10754558; and IL-1β rs1143634. Besides that, we selected HLA-C rs9264942 C>T (TaqMan® genotyping assay C_29901957_10) based on the association of this SNP with a lower HIV-1 viral load (Fellay et al., 2007, 2009; Wei et al., 2015). SNP genotyping was performed using commercially available TaqMan assays (Applied Biosystems/AB and Life Technologies) using the ABI7500 Real-Time platform (AB). Allelic discrimination was carried out by means of the Thermo Fisher Connect Software® version 1.\n\nDNA samples were PCR amplified to determine the presence of the CCR5 Δ32 mutation. Primers (CCR5-F: GCT GTC TTT GCG TCT CTC CCA GGA and CCR5-R: CTC ACA GCC CTG TGC CTC TTC TTC) were used to amplify a 239 base-pair (bp) fragment covering the Δ32 mutation region. The cycling conditions were: 1 cycle 94°C 5 minutes; 30 cycles 94°C 1 minute, 60°C 30 seconds, 72°C 2 minutes; 1 cycle 72°C 10 minutes. The PCR amplified products (5 ul) were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. A single band of 239 bp indicated the CCR5/CCR5 wild-type genotype, while the heterozygous genotype CCR5/Δ32 was detected by 239 and 207 bp bands, and the homozygous genotype Δ32/Δ32 by a single band of 207 bp.\n\nDirect count estimated the frequencies of HLA, KIR, SNPs and CCR5 alleles and genotypes. Kruskal-Wallis ANOVA by Ranks test was used to compare the sociodemographic, clinical, and laboratory characteristics among the different HICs groups for continuous numerical variables. In contrast, for categorical nominal variables, Fisher's exact test was used in the evaluation of frequencies among the different HICs groups. Tests of equal proportions were used to evaluate the allele carriers’ relative frequencies among the HICs groups. For comparative purposes, HLA-B genomic distribution data from the general Brazilian population were extracted from REDOME (National Registry of Bone Marrow Donors, Brazilian Ministry of Health) data (Rede Brasil de Imunogenética, 2018), and for HLA-C we used data available at Allele Frequencies Net Database.\n\nTo estimate the hazard of a transitory loss of virological control, defined as the observation of the first VL determination above 2,000 copies/mL, we calculated person-years (pY) at risk for each patient between HIV diagnosis and the occurrence of an event. Individuals were censored either at the time of final observation, death, cART (combined antiretroviral therapy) initiation, or the last follow-up visit/exam, whichever occurred first. The effect of various risk factors on the outcome was assessed using hazard ratios (HR) and corresponding 95% confidence intervals (CI), which were estimated through the Cox proportional hazard model (Therneau & Grambsch, 2000).\n\nTo evaluate the occurrence of multiple events of VL blips, herein defined as the number of detectable viral loads counted after HIV diagnosis and until cART initiation, we calculated the incidences/rates of the number of viral loads per pY for each patient, and 95% CI were estimated according to asymptotic standard errors calculated from a Gamma distribution (Lehmann & Casella, 1998). The effect of various risk factors on the outcome was assessed by relative risks (RR) and corresponding 95% CI were estimated employing the Negative Binomial (NB) models. Individual exposure time (in years) was used as an offset in the NB models (Hilbe, 2011).\n\nTwo-tailed levels of significance ≤ 0.01, 0.05 and 0.1 were considered “highly significant”, “significant” and “suggestive”, respectively. All statistical analysis was performed using the R statistical software package, version 3.4.1 (R Development Core Team, 2017).\n\n\nResults\n\nTable 1 depicts the main clinical and epidemiological characteristics of the HICs cohort distributed according to the viral control groups (de Sá & Teixeira, 2021). Overall, we found a similar frequency of females and males (16 [57.1%] and 12 [42.9%], respectively), while 21 (75%) of the individuals have identified themselves as heterosexual and 6 (21.4%) as men who have sex with men (MSM). Most heterosexual male (75%) and MSM subjects (83.3%) belonged to the VCs group. The ethnic background composition was diverse, with an unequal distribution of White, Brown, and Black participants among groups of HICs (P = 0.04). Participants had a median age of 32 (IQR 10.4) years old, with a similar median age observed among the PECs (33 years old; IQR 6.9), EECs (37 years old; IQR 13.9), and VCs (30 years old; IQR 9.8) groups.\n\na For categorical nominal variables, P-values were calculated using Fisher's exact test. For continuous numeric variables, P-values were calculated using Kruskal-Wallis ANOVA by Ranks test. Differences were considered significant with a value of P < 0.05.\n\nb Age at HIV diagnosis.\n\nc Classification according to the International Standard Classification of Education (ISCED) maintained by the United Nations Educational, Scientific, and Cultural Organization (UNESCO).\n\nd Middle cumulative CD4+ T Cells Count and HIV-1 Viral Load.\n\nAbbreviations: HICs = HIV controllers; PECs = persistent elite controllers; EECs = ebbing elite controllers; VCs = viremic controllers. IQR = interquartile range.\n\nAll HICs groups displayed a median CD4+ T cells count above 800 cells/μL (IQR 431). Further details about immunological and virological characteristics of most HICs of this study were depicted in previous studies from our group (Bello et al., 2009; Côrtes et al., 2015, 2018; de Azevedo et al., 2017; Caetano et al., 2020).\n\nTable 2 describes the relative frequencies of HLA-B and -C, KIR, and CCR5∆32 alleles carriers found in the different HICs groups. Due to the dependence between the genotypes, we did not perform this comparative analysis for the SNPs included in this study. However, the complete genetic characterization of the analyzed markers is depicted in Table 3. We observed significant differences between PECs and EECs for protective HLA-B alleles, KIR Bx genotype, KIR2DL5, and KIR2DS5 allelic carrier frequencies. Among EECs and VCs, the frequencies of KIR Bx genotype, KIR2DL3 + C1 pair, KIR2DL2, KIR2DL5, and KIR2DS2 alleles carriers were also significantly distinct. The only genetic marker with significantly different frequencies between PECs and VCs was KIR2DS5. It is noticeable that HLA-B protective alleles (HLA-B27, B52, and/or B57) were found in 71% of the PECs, 43% of the VCs, and only 14% of the EECs.\n\na P-values were calculated using Tests of Equal Proportions. Differences were considered significant with a value of P < 0.05.\n\nb Protective HLA-B alleles included HLA-B27, B52, and B57 alleles.\n\nc Protective HLA-C alleles included HLA-C08, C15, and C18 alleles.\n\nAbbreviations: PECs = persistent elite controllers; EECs = ebbing elite controllers; VCs = viremic controllers. NC = not calculated. N = number of individuals in each HIC group. n = observed number of an allele/genetic marker carriers. Freq = relative frequency of carriers for each allele/genetic marker, calculated as the number of carriers/N.\n\nTo check for an enrichment of protective HLA alleles in our cohort, we compared the classical protective HLA-B and -C frequencies here obtained with those from the general Brazilian population, and with HIV-1-infected individuals who progressed to AIDS (Teixeira et al. 2014; Biberg-Salum et al., 2018), considered as HIV non-controllers (HIV-NC) (Table 4). For HLA-B, we noted that the frequency of carriers of these alleles in our HICs cohort (0.464; n = 13) is on average 3.34-fold higher (P < 0.001) than in the overall Brazilian population (0.139; n = 396,740) and 3.22-fold higher (P = 0.014) in the HIV non-controllers group (0.144; n = 29). Similarly, for HLA-C we noted that the frequency of carriers of these alleles in our HICs cohort (0.571; n = 16) is on average 3.60-fold higher (P < 0.001) than in the overall Brazilian population (0.159; n = 42) and 3.50-fold higher (P < 0.001) than in the HIV non-controllers group (0.161; n = 26). Only two PECs carried the CC genotype in the HLA-C rs9264942 polymorphism. The KIR Bx genotype was found in 86% of PECs, 79% of the VCs, and 29% of EECs. The KIR3DL1+Bw4 genotype was detected in only two VCs, and the KIR3DS1+Bw4 genotype in four participants (one PEC, one EEC, and two VCs). All PECs and EECs had the KIR2DL3 allele. The CCR5∆32 mutation was found in four heterozygous (WT/∆32) participants (one PEC, one EEC, and two VCs), resulting in an overall allelic frequency of 7.1%, and no homozygous (∆32/∆32) participants were identified in this cohort. Therefore, we cannot assign any relation between this genetic marker and the distinct patterns of viremia control observed in these HICs.\n\nP-values were calculated using the unconditional logistic regression model. Differences were considered significant with a value of * P < 0.05.\n\na Data from Brazilian Registry of Bone Marrow Donors (REDOME).\n\nb Data from Teixeira et al., 2014.\n\nc Protective HLA-B alleles included HLA-B27, B52, and B57 alleles.\n\nd Data from Allele Frequencies Net Database (http://www.allelefrequencies.net).\n\ne Data from Biberg-Salum et al., 2018.\n\nf Protective HLA-C alleles included HLA-C08, C15, and C18 alleles.\n\nAbbreviations: HICs = HIV controllers; HIV-NC = HIV non-controllers; BGP = Brazilian general population; N = number of individuals; n = observed number of an allele carriers; Freq = relative frequency of carriers for each allele, calculated as the number of carriers/N; NC = not calculated.\n\nTo test the potential relevance of genetic host factors in the risk of a transitory loss of virological control in some HICs, we conducted Cox proportional hazard multivariate analysis to identify factors associated with the occurrence of the high-level viremia (> 2,000 copies/mL) (Table 5). A higher mean hazard of this event was observed for HICs without the KIR2DL3 allele [aHR = 79.098 (3.236-1933.264); P = 0.007], HICs with the HLA-C08 allele [aHR = 8.379 (1.181-59.472); P = 0.034], HICs with the C/G genotype [aHR = 7.462 (2.294-2505.478); P = 0.042] and G/G genotype [aHR = 75.817 (2.294-2505.478); P = 0.015] in the NLRP3 polymorphism (rs10754558), and HICs without the KIR2DL3 + C1/C2 pair [aHR = 11.78 (1.602-86.619); P = 0.015] than for their counterparts. In the other way, a lower mean hazard of this event was observed for HICs with the following alleles: KIR2DL5 [aHR = 0.131 (0.023-0.738); P = 0.021], KIR2DS1 [aHR = 0.109 (0.014-0.867); P = 0.036], KIR2DS5 [aHR = 0.058 (0.007-0.466); P = 0.007], and KIR3DS1 [aHR = 0.108 (0.013-0.876); P = 0.037] when compared with their counterparts.\n\na Levels = 0 (absent); 1 (present).\n\nb First HIV-1 VL determination above 2,000 copies/mL.\n\nc pY: person-years at risk were calculated for each patient between HIV diagnosis and the occurrence of an event.\n\nd Hazard ratios were adjusted by gender and education whenever applicable, which were variables associated with an uncontrolled viremic event in bivariate analysis (P < 0.2).\n\nAbbreviations: HR = hazard ratios; aHR = adjusted hazard ratios.\n\nThe relative risk of occurring any event of detectable viremia (VL blip) was also evaluated by means of the negative binomial (NB) multivariate models (Table 6). Overall, HICs with the KIR2DS5 allele [aRR = 0.294 (0.122-0.705); P = 0.006] had less risk of undergoing VL blips within the same normalized period than those participants without this allele. Conversely, HICs without the KIR2DL3 allele [aHR = 4.987 (1.174-21.196); P = 0.03] had a mean higher risk of experiencing VL blips within the same normalized period than those with this allele. The results of Cox's proportional hazard multivariate analysis and Negative Binomial (NB) models of the HLA-C and inflamassome SNPs are depicted in Tables 7 and 8.\n\na Levels = 0 (absent); 1 (present).\n\nb Number of events: number of viral load blips (defined as the number of detectable HIV-1 viral loads after HIV diagnosis).\n\nc pY: person-years at risk were calculated from HIV diagnosis until a cART treatment initiation.\n\nd Relative risks were adjusted by gender, age at HIV diagnosis, education, and skin color whenever applicable, which were variables associated with multiple events of viral load blips in the bivariate analysis (P < 0.2).\n\nAbbreviations: RR = relative risk. aRR = adjusted relative risk.\n\na First HIV-1 VL determination above 2,000 copies/mL.\n\nb pY: person-years at risk were calculated for each patient between HIV diagnosis and the occurrence of an event.\n\nc Hazard ratios were adjusted by gender and education whenever applicable, which were variables associated with an uncontrolled viremic event in bivariate analysis.\n\nAbbreviations: HR = hazard ratios; aHR = adjusted hazard ratios; NC = not calculated.\n\na Number of events: number of viral load blips (defined as the number of detectable HIV-1 viral loads after HIV diagnosis).\n\nb pY: person-years at risk were calculated from HIV diagnosis until a cART treatment initiation.\n\nc Relative risks were adjusted by gender, age at HIV diagnosis, education and skin color whenever applicable, which were variables associated with multiple events of viral load blips in the bivariate analysis (P < 0.2).\n\nAbbreviations: RR = risk ratios or relative risk; aRR = adjusted relative risk; NC = not calculated.\n\nOur results point to a protective role for KIR2DL3 and KIR2DS5 alleles since the absence of the former was associated both with a higher risk of a transitory loss of virological control and to a higher risk of any event of detectable viremia (VL blip); while the presence of the latter was associated to a lower risk of these events. Epidemiological features were introduced in the multivariate models for controlling bias in the statistical analysis. The limited sample size and the study design do not allow us to explore the associations involving these epidemiological features depicted in Tables 5 and 6.\n\n\nDiscussion\n\nIn the present study, we characterized the distribution of HLA-B, HLA-C, KIR, SNPs of the NLRP3, CARD8, IL-1β, and CCR5Δ32 mutation in a cohort of HICs with different levels of virological control. Our results show that host genes classically associated with protective mechanisms are present in our HICs cohort. Despite the small number of participants analyzed, we observed a higher frequency of HLA-B protective alleles in this cohort than in the general Brazilian population and Brazilian HIV non-controllers (Teixeira et al., 2014), confirming the results of many studies showing enrichment of protective B27 and B57 alleles in HICs (Migueles et al., 2000; Fellay et al., 2007; Pereyra et al., 2010; Lécuroux et al., 2014). We also observed that B27 and B57 allele carriers’ frequencies were not different among HICs groups. However, when considered together with HLA-B52 composing the ‘protective HLA-B alleles’ group, a significant difference of these alleles frequencies between PECs and EECs was observed (P = 0.002, Table 2). In this study, multivariate models showed that some genetic markers were associated with a transitory loss of virological control and/or of an event of detectable viremia (VL blip). A lower mean hazard for the risk of transitory loss of virological control was observed for KIR2DL5, KIR2DS1, KIR3DS1, and KIR2DS5 alleles. In contrast, the HLA-C08 allele and the absence of KIR2DL3 and KIR3DL3+C1/C2 alleles were associated with an increased risk of this event (Table 5). Additionally, KIR2DL3 allele absence was also associated with a higher risk of an event of detectable viremia (VL blip), and KIR2DS5 was also associated with a lower risk of these events (Table 6), pointing to a protective role of these alleles. Interestingly, no association was found for the CC genotype in the HLA C rs9264942 polymorphism with either the hazard of an uncontrolled viremic event or the risk of undergoing VL blips. This variant (CC genotype) has been associated with a higher viral load control (VL) in previous studies (Fellay et al., 2007; Thørner et al., 2016; Malnati et al., 2017).\n\nStudies have been associated KIR2DL3 receptor and its ligand HLA-C1 with a lower VL and a higher CD4+ T cell count (Körner et al., 2014). In agreement with these studies, we observed an increased HR of a high-level viremic event and a higher risk of experiencing VL blips in HICs without KIR2DL3. Inhibitory KIR receptors (KIR2DL2/KIR2DL3 heterozygosity) were associated with resistance to HIV-1 infection among female sex workers in the absence of their respective HLA ligands. These results indicate that absence of these ligands may reduce the threshold for NK cell activation via activating KIR, resulting in NK cytotoxic activity and early elimination of HIV-1 infected cells (Jennes et al., 2006).\n\nKIR2DS5 allele carriers had a lower mean hazard of high-level viremia (> 2,000 copies/mL) events and, in the mean, less VL blip events than those without the allele (Table 5). This activating gene was associated with the protection against HIV-1 infection of exposed uninfected infants (Chavan et al., 2016) and reducing HIV-1 transmission from mother to child (Omosun et al., 2018). It remains unclear what mechanisms at the KIR2DS5 allele might result in this protection. We speculate that the presence of activating mechanisms driven by this gene could impact viral control. This mechanism may be due to the activation signal delivered to NK cells when activating receptors bind their ligand on the target cell surface, leading to the activation of these cells and eliminating the pathogen (Srivastava et al., 2003).\n\nThe relevance of NK cell function regarding the HIC status has been suggested by genetic studies (Martin et al., 2002, 2007). The effect of the combination of KIR3DS1+ NK cells with the Bw4 epitope reduces the susceptibility to HIV-1 infection, improves viremia control, and decreases CD4+ T cells depletion (Martin et al., 2002; Alter et al., 2007, 2009). Moreover, in agreement with our results, the KIR3DS1 gene has been indicated as a protective factor in the resistance to HIV in different routes of exposure (Boulet et al. 2008; Chavan et al. 2014; Habegger de Sorrentino et al. 2013; Tallon et al. 2014). KIR2DL5 has already been associated with a reduced risk of HIV-1 infection and reduced HIV-1 transmission among Africans (Jennes et al., 2006; Omosun et al., 2018), but there is no previous report associating this allele with virological control. The KIR2DS1 gene bind HLA-C molecules of the C2 group (Folley et al., 2008). It has already been significantly associated with a lower viral load in HIV-1 patients from India (Chavan et al., 2014) and South Africa (Wong et al., 2010).\n\nThe HLA-C08 allele and the rs10754558 G allele were associated with an increased risk of transitory loss of virological control in this study. Several researchers have reported the positive influence of these alleles in HIV disease course (Lazaryan et al., 2011; Shepherd et al., 2015; Mhandire et al., 2018; Pontillo et al., 2010, 2012). The presence of the C08 allele has already been associated with a high CD4+ T cell count (Mhandire et al., 2018), and this allele was enriched among HIV controllers (Lazaryan et al., 2011) and long-term non-progressors (LTNPs) (Shepherd et al., 2015). The NLRP3 rs10754558-G SNP has already been described as a protective factor against HIV-1 infection in different ethnic groups (Pontillo et al., 2010, 2012). Moreover, in vitro assays showed that rs10754558 was associated with NLRP3 mRNA stability since the PBMCs isolated the CG and GG patients increase the NLRP3 expression approximately 2-fold when compared to CC patients (Ravimohan et al., 2018). These results indicate that the protection to the disease exerted by the variant possibly depends on the stable expression of the NLRP3. Although our findings do not corroborate previous studies, it is relevant to note that the current study has some limitations, as the lack of a functional NK cell characterization of HICs and the limited number of participants enrolled in our cohort. EC cohorts are difficult to establish and maintain, given that they represent a rare group of individuals. Therefore, to obtain more reliable results, additional studies with larger populations and functional studies are needed to better understand the importance and role of the HLA-C08 and the rs10754558 G allele in the context of HIV infection.\n\nIn conclusion, our results showed an association between the absence and presence of host immunogenetic markers with the frequencies of high-level viremia episodes and the frequency of VL blips in our HICs cohort, highlighting the protective role of KIR2DL3 and KIR2DS5. Besides, we confirmed that protective HLA-B and HLA-C allelic frequencies are higher in HICs than in HIV-1 non-controllers and in the general population. Although the limitations of this study, the results presented here provide an essential contribution to understanding the genetic factors that can modulate viral control in these individuals. Additional studies aiming to identify the role of host genetics in persistent vs. transient control of HIV-1 replication in HICs will undoubtedly help a more efficient clinical management of this particular cohort.\n\n\nData availability\n\nOpen Science Framework: Killer immunoglobulin-like receptor (KIR) genes are associated with the risk of episodes of high-level and detectable viremia among HIV controllers. https://doi.org/10.17605/OSF.IO/ZGQPA (de Sá & Teixeira, 2021).\n\nThis project contains the following underlying data:\n\n- CCR5 Raw Data.pdf (PDF containing CCR5 raw gel images)\n\n- Epidemiological data of HICs.csv (Epidemiological characteristics of HICs)\n\n- Clinical data of HICs.csv (Immunological and virological characteristics of HICs)\n\n- Genetic characterization of HICs.csv (HLA-B, HLA-C, KIR and SNPs of the NLRP3, CARD8, IL-1β genes raw data of HICs)\n\n- KIR Results.pdf (PDF containing KIR gels raw images of HICs)\n\nOpen Science Framework: Killer immunoglobulin-like receptor (KIR) genes are associated with the risk of episodes of high-level and detectable viremia among HIV controllers. https://doi.org/10.17605/OSF.IO/ZGQPA (de Sá & Teixeira, 2021).\n\nThis project contains the following extended data:\n\n- Consent Form.pdf (PDF containing the original and English version)\n\n- Project consent form (PDF containing the ethical committee approval)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors thank the patients who participated in the study, Dr. Gonzalo Bello for the revision of the manuscript, the Multi-user Research Facility of Real Time PCR, Instituto Oswaldo Cruz, FIOCRUZ, and all the staff involved in the clinical follow-up of these patients for giving support during the medical follow-up, blood collection, HIV-RNA VL quantification, and CD4+ T lymphocyte count. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "89207", "date": "09 Aug 2021", "name": "Roberto Biassoni", "expertise": [ "Reviewer Expertise KIR and other NK receptors." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article could be interesting but some additional analysis is necessary. The sample size is limited so a Statistical Power analysis is needed to understand the strength of the analysis. In this regards many of the reported results are contradictory. As an example, it is intriguing that the analysis of the protective HLA-B alleles is statistically significant both at the cumulative and at the single allele level. Conversely, this is not true for HLA-C alleles (Table 4). It is awkward to use the absence of KIR2DL3 (a marker of A-haplotype) to describe an unprotective phenotype, while markers of Bx-haplotypes (KIR2DS5, KIR2DS1, KIR3DS1) are all significantly associated with protection (Table 5).\nSome analysis are missing: in detail the KIR2DL3 alone is not analyzed (NC??), and it is disturbing that the presence of KIR2DL3+C1 show a statistical significance in the pair analysis (VC vs EECs), but the analysis is missing for the PEC vs VCs (Table 2).\nFinally, KIR2DS5 and KIR2DS1 have been already known to be associated with the status of elite controller but not LTNP, but not correctly cited in this article.\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "96851", "date": "26 Oct 2021", "name": "Nicole F. Bernard", "expertise": [ "Reviewer Expertise Immunologist working in the field of NK cell responses in HIV infected individuals and in HIV infected Elite Controllers." ], "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 manuscript, the authors characterize HLA-B and C allotypes, presence of KIR genes, presence of the CCR5Δ32 mutant, and single nucleotide polymorphism (SNPs) at NLRP3, CARD8, and IFN-1β inflammasome genes in 28 HIV controllers (HICs). Seven of these are classified as a persistent elite controller (PEC), 7 as ebbing elite controllers (EEC), and 14 as viral controllers (VC).\nCritique\nA more effective way to describe the three groups of HICs would be to provide for each of the 28 subjects a graph showing longitudinal follow-up for CD4 counts and viral load in which the viral load blips are shown.\n\nThe rationale for including inflammasome SNPs in this study could be better rationalized, particularly as none appear to be linked with viral load control in HICs.\n\nThis study has several limitations:\nAs mentioned by the authors the size of the HIC study population is small, particularly as it has been split into three subgroups, PECs, EECs, and VCs. This compromises the power to detect between HIC group differences that may be apparent were there larger numbers of study subjects.\n\nThe only between-group difference is in the frequency of all protective HLA-B allotypes for comparisons between PEC and EEC. Comparisons between the general Brazilian population and HICs reveal significant differences in the frequency of all protective HLA-B alleles and each one individually. Comparisons between non-controllers and HICs reveal significant differences in the frequency of all protective HLA-B alleles only. Enthusiasm for this result is diminished by the fact that similar results have been reported in several previous publications.\n\nThere are 2 HLA-B*57:02 positive persons among the HIC population, 1 among PEC, and 1 among VC. This allele is associated with rapid HIV progression and is not a protective allele in the context of HIV infection. This should not be considered as a protective HLA-B allele in either the HIC groups or the non-controllers and the general Brazilian population. Tables 2 and 4 should be modified accordingly.\n\nThere are a large number of comparisons made between HIC groups and with non controllers or the general Brazilian population using the p-value, cut-offs of 0.01 or 0.05 to identify significant differences. As these comparisons are not corrected for multiple comparisons, it is likely that some of the significant between-group differences observed arise by chance and would fall below the level of significance were such corrections implemented.\n\nOn page 3, paragraph 4, the statement is made: “no consistent association between HLA-C/KIR alleles and viral control has been described so far.” This is not accurate as the manuscript by Malnati et al., 1 referenced in this submission, showed an association between HLA-C/KIR and viral control. This should be corrected.\n\nThe authors show that the HIC population studied contains a high degree of miscegenation that characterizes the Brazilian population. While the authors appear to view this as a strength this is also a weakness of the study. The frequency distribution of the highly polymorphic HLA allotypes and KIR haplotypes and allotypes differ in different racial/ethnic populations. Differences in admixture from one person to another and between groups may account for between-group differences in some of the parameters being compared rather than being due to the variables being studied contributing to HIV control.\n\nThe authors mention that lack of KIR2DL3 and lack of KIR2DL3 + HLA-C1/C2 was associated with a higher chance of transitory loss of viral load control (a blip), Absence of KIR2DL3, and expression of its allelic counterpart at this locus, KIR2DL2, is a marker for the centromeric KIR haplotype B. A lower chance for loss of HIV control is associated with the genes encoding KIR2DL5, KIR2DS5, KIR2DS1, and KIR3DS1, which are all in the telomeric KIR haplotype B region, though KIR2DL5 can be found in either the telomeric of centromeric KIR haplotype B regions. As the KIR genes in centromeric or telomeric regions are in close linkage disequilibrium, it is possible that only one KIR gene in the telomeric haplotype B region rather than each of these genes mentioned to be associated with viral load control may actually be associated with HIV control. Additional text on this point could be added to the discussion.\n\nAnother weakness of this manuscript is that combined KIR/HLA genotypes, particularly inhibitory KIR/HLA genotypes that encode receptor/ ligand combinations important in NK cell education and acquisition of anti-HIV function are not considered. On page 10 first paragraph, HLA-Bw4 is not a ligand for KIR3DS1. The ligand for KIR3DS1 is now known to be HLA-F. This should be corrected.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-546
https://f1000research.com/articles/10-400/v1	18 May 21	{ "type": "Case Report", "title": "Case Report: SARS-CoV-2 as an unexpected causal agent of isolated febrile hepatitis", "authors": [ "Paraskevas Filippidis", "Francois van Ouwenaller", "Alberto Cerutti", "Anaïs Geiger-Jacquod", "Christine Sempoux", "Giuseppe Pantaleo", "Darius Moradpour", "Frederic Lamoth", "Paraskevas Filippidis", "Francois van Ouwenaller", "Alberto Cerutti", "Anaïs Geiger-Jacquod", "Christine Sempoux", "Giuseppe Pantaleo", "Darius Moradpour" ], "abstract": "Background: Respiratory symptoms and pneumonia are the predominant features of Coronavirus disease 2019 (COVID-19) due to emerging SARS-CoV-2 virus, but extrapulmonary manifestations are also observed. For instance, some degree of liver injury has been described among patients requiring hospital admission for severe COVID-19. However, acute febrile hepatitis as an initial or predominant manifestation of COVID-19 has been rarely reported. Case presentation: A 34-year-old man without underlying medical conditions presented with fever of unknown origin for two weeks in the absence of respiratory symptoms or other complaints. Laboratory testing revealed isolated acute hepatitis, for which an extensive microbiological work-up did not reveal identification of the causal agent. PCR testing for SARS-CoV-2 on a nasopharyngeal swab was negative on two occasions and initial serology for SARS-CoV-2 (at 15 days from symptoms onset) was also negative. However, repeated SARS-CoV-2 serological testing at 30 days demonstrated seroconversion leading to the diagnosis of COVID-19-related hepatitis. The patient's condition progressively improved, while transaminases steadily declined and eventually returned back to normal within 30 days. Conclusions: We describe here a unique case of SARS-CoV-2 isolated febrile hepatitis in a young and previously healthy man, which was diagnosed by demonstration of seroconversion, while PCR screening was negative. This case report highlights the role of repeated serological testing for the diagnosis of extrapulmonary manifestations of COVID-19.", "keywords": [ "COVID-19", "hepatology", "liver", "serology" ], "content": "Background\n\nSevere acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus disease 2019 (COVID-19) can cause a wide spectrum of clinical presentations ranging from mild flu-like illness to severe pneumonia with acute respiratory distress syndrome1. While the pandemic is ongoing, atypical presentations with extrapulmonary organ involvement, including heart, kidneys, skin, nervous system, hepatobiliary and gastrointestinal tract are increasingly recognized2. Liver injury with mild or moderate elevation of transaminases has been observed in about half of patients requiring hospital admission for severe COVID-193,4. However, acute febrile hepatitis as a predominant manifestation of COVID-19 has been rarely reported and has been mainly observed among patients with underlying liver diseases and/or concomitant signs of upper or lower respiratory tract involvement with diagnosis established by polymerase chain reaction (PCR) in nasopharyngeal swabs5–7. We report here a case of a young previously healthy man presenting with febrile hepatitis as a unique clinical manifestation, for which diagnosis of COVID-19 was established by seroconversion.\n\n\nCase presentation\n\nA 34-year-old previously healthy Caucasian man was admitted to our tertiary care hospital for investigation of fever of unknown origin. He had presented night fever up to 39.5°C with rare chills, night sweats and moderate headache for two weeks prior to admission. His symptoms responded to acetaminophen and nonsteroidal anti-inflammatory drugs. During the first week after fever onset, he had also presented with a mild sore throat and dry cough, which had completely resolved at the time of admission. Exposure history was remarkable with travel to a Greek island one month ago and a superficial scratch from his neighbor’s cat two months ago. Upon admission, his vital signs and physical examination were unremarkable. Laboratory tests showed systemic inflammation with leukocytosis (10.5 G/l), thrombocytosis (501 G/l) and elevated inflammatory markers (C-reactive protein 126 mg/l, ferritin 695 µg/l), as well as acute hepatitis with a three-fold increase of transaminases, a two-fold increase of alkaline phosphatase and a four-fold increase of gamma-glutamyltranspeptidase. Total bilirubin was normal. Cervical, thoracic and abdominal computed tomography was unremarkable. PCR for SARS-CoV-2 on a nasopharyngeal swab was negative on two occasions. Serology for SARS-CoV-2 performed at admission (i.e. at 15 days from fever onset) was negative using the Luminex S protein trimer IgG assay, as previously described8. Serologies for hepatitis A, B, C and E viruses, Epstein-Barr virus, cytomegalovirus and human immunodeficiency virus were negative. Further microbiological diagnostic work-up, including serological testing for bartonellosis, Q fever, brucellosis, tularemia, Lyme disease, rickettsial diseases, toxoplasmosis, syphilis and leptospirosis was negative. Autoimmune and metabolic causes of hepatitis were also excluded. We consequently performed a percutaneous liver biopsy, which showed nonspecific acute lobular hepatitis with negative immunohistochemical staining for herpes viruses (Figure 1). Cultures of the liver tissue were sterile and broad-spectrum bacterial (16S rDNA), fungal (18S rDNA) and mycobacterial PCRs, as well as specific PCRs for Brucella spp., Bartonella spp., Coxiella burnetii, were all negative. Transthoracic echocardiography found no endocarditis-related abnormality. Positron emission tomography (PET) revealed moderate hypermetabolism of the posterior naso-oropharynx and numerous hypermetabolic cervical lymph nodes. However, naso-oropharyngeal endoscopy and cervico-facial magnetic resonance imaging found no structural abnormality. Serologies for hepatitis A, B, C and E viruses, cytomegalovirus and Epstein-Barr virus were tested again at a two-week interval and remained negative. However, repeated SARS-CoV-2 serology (i.e. at 30 days from the onset of fever) turned out clearly positive. This result was confirmed on a subsequent serum sample collected one week later and showing increasing titers. Retrospective SARS-CoV-2 PCR testing in serum and in liver tissue was negative. As illustrated in Figure 2, the patient’s condition progressively improved despite persistent low-grade fever at discharge, while transaminases steadily declined and eventually returned back to normal within 30 days. Fever had completely resolved after six weeks of follow-up.\n\nMild lobular hepatitis with few apoptotic bodies (arrow) surrounded by mononuclear inflammatory cells (arrowhead). Hematoxylin-eosin staining, original magnification X400.\n\nEvolution of fever and liver function tests as well as results of SARS-CoV-2 PCR and serology. Serology testing for SARS-CoV-2 was performed by the Luminex S protein trimer IgG assay, as previously described8. Results are expressed in mean fluorescence intensity ratio with a positive cut-off of 6. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; γ-GT, γ-glutamyltranspeptidase.\n\n\nDiscussion and conclusions\n\nInvestigations of acute hepatitis includes a diagnostic work-up for classical hepatotropic viruses and some other well known, albeit rarer, infectious agents causing liver injury. We report here a clinical observation raising attention to a novel pathogen that should be considered and actively searched for in such situations: the emerging SARS-CoV-2 virus.\n\nWhile concomitant liver injury has been commonly observed among patients with severe COVID-19 pneumonia3,4, clinical presentation of COVID-19 with hepatitis as the predominant feature has been rarely reported5–7. All these cases had positive PCR for SARS-CoV-2 in nasopharyngeal swabs at admission, three had concomitant pulmonary involvement and two had predisposing liver conditions (e.g. chronic hepatitis C or previous liver transplantation).\n\nWhile concomitant liver injury has been commonly observed among patients with severe COVID-19 pneumonia3,4, clinical presentation of COVID-19 with hepatitis as the predominant feature has been rarely reported5–7. All these cases had positive PCR for SARS-CoV-2 in nasopharyngeal swabs at admission, three had concomitant pulmonary involvement and two had predisposing liver conditions (e.g. chronic hepatitis C or previous liver transplantation).\n\nUnlike previous reports5,7, our patient had no underlying liver disease and presented isolated fever and hepatitis with no radiological evidence of pulmonary involvement and negative SARS-CoV-2 PCR in nasopharyngeal swabs. Seroconversion occurring in a timeframe that was consistent with the course of the disease, after exclusion of other infectious and non-infectious causes of hepatitis, led to the diagnosis of COVID-19-related hepatitis in this case.\n\nInterestingly, seroconversion occurred relatively late (between two and four weeks from fever onset). In a recent population-based seroprevalence study, the serological method used in this case (i.e. Luminex S protein trimer IgG assay) showed a sensitivity of 97% and a specificity equal or above 97%, when performed by day 15 from symptom onset8. However, modest or delayed antibody responses (up to day 20) have been reported9, which could be more frequently observed in young patients with less severe forms of the disease. While specific data about the antibody response in patients with extrapulmonary manifestations of COVID-19 are lacking, the present case report highlights the role of serology for the diagnosis of these atypical forms presenting later in the course of the disease, when respiratory symptoms are absent or not predominant, and the need for repeated serological testing (up to 3–4 weeks from symptoms onset) in case of high clinical suspicion and/or absence of alternative diagnosis.\n\nIn the present case, attempts to demonstrate the presence of the virus in blood or liver tissue by PCR were unsuccessful. Indeed, the rate of positive SARS-CoV-2 PCR in non-respiratory samples, such as blood or deep-organ tissues, is notoriously low10. Although the nature of liver injury in COVID-19 remains to be elucidated, abnormal inflammatory response, rather than direct viral cytotoxicity, is suggested as the main pathophysiological mechanism of hepatitis11. A case series of patients deceased from COVID-19 showed lobular hepatitis in 50% of liver autopsies, but found no correlation between histologic findings and a positive PCR assay for SARS-CoV-2 on liver tissue12.\n\nIn conclusion, we report a case of SARS-CoV-2 infection with acute febrile hepatitis as a predominant manifestation. Importantly, this case highlights the need to include COVID-19 in the differential diagnosis of primary hepatitis while the pandemic is ongoing, and the crucial role of repeated serological testing (up to three to four weeks from symptoms onset) for the identification of such atypical extrapulmonary manifestations of the disease.\n\n\nList of abbreviations\n\nSARS-CoV-2 Severe acute respiratory syndrome coronavirus 2\n\nCOVID-19 Coronavirus disease 2019\n\nPCR Polymerase chain reaction\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Author contributions\n\n\n\nP. Filippidis: clinical management of the case, data collection, figure design and drafting of manuscript.\n\nF. Van Ouwenaller, A. Cerruti, A. Geiger-Jacquod: clinical management of the case, review and editing of manuscript.\n\nC. Sempoux: interpretation of histopathology, figure design, review and editing of manuscript.\n\nG. Pantaleo: interpretation of serological testing, review and editing of manuscript.\n\nD. Moradpour: clinical management of the case, review and editing of manuscript\n\nF. Lamoth: clinical management of the case, study design and writing of manuscript.\n\n\nAcknowledgements\n\nNot applicable.\n\n\nReferences\n\nRegina J, Papadimitriou-Olivgeris M, Burger R, et al.: Epidemiology, risk factors and clinical course of SARS-CoV-2 infected patients in a Swiss university hospital: An observational retrospective study. PLoS One. 2020; 15(11): e0240781. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu Z, McGoogan JM: Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention. JAMA. 2020; 323(13): 1239–42. PubMed Abstract \| Publisher Full Text\n\nHao SR, Zhang SY, Lian JS, et al.: Liver Enzyme Elevation in Coronavirus Disease 2019: A Multicenter, Retrospective, Cross-Sectional Study. Am J Gastroenterol. 2020; 115(7): 1075–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang C, Shi L, Wang FS: Liver injury in COVID-19: management and challenges. Lancet Gastroenterol Hepatol. 2020; 5(5): 428–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFiel MI, El Jamal SM, Paniz-Mondolfi A, et al.: Findings of Hepatic Severe Acute Respiratory Syndrome Coronavirus-2 Infection. Cell Mol Gastroenterol Hepatol. 2021; 11(3): 763–770. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWander P, Epstein M, Bernstein D: COVID-19 Presenting as Acute Hepatitis. Am J Gastroenterol. 2020; 115(6): 941–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrevenzoli M, Guarnaccia A, Alberici I, et al.: SARS-CoV-2 and hepatitis. J Gastrointestin Liver Dis. 2020; 29(3): 473–5. PubMed Abstract \| Publisher Full Text\n\nFenwick C, Croxatto A, Coste AT, et al.: Changes in SARS-CoV-2 Spike versus Nucleoprotein Antibody Responses Impact the Estimates of Infections in Population-Based Seroprevalence Studies. J Virol. 2021; 95(3): e01828–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLong QX, Liu BZ, Deng HJ, et al.: Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med. 2020; 26(6): 845–8. PubMed Abstract \| Publisher Full Text\n\nWang W, Xu Y, Gao R, et al.: Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA. 2020; 323(18): 1843–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi J, Fan JG: Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease. J Clin Transl Hepatol. 2020; 8(1): 13–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLagana SM, Kudose S, Iuga AC, et al.: Hepatic pathology in patients dying of COVID-19: a series of 40 cases including clinical, histologic, and virologic data. Mod Pathol. 2020; 33(11): 2147–55. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "85690", "date": "28 Jun 2021", "name": "Werner Albrich", "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\nThe authors describe the rare case of a 34 yo male with acute Covid-19 infection which manifested mainly as acute and self-limiting hepatitis aside from mild upper respiratory symptoms, treated with acetaminophen and NSAIDs. Despite nasopharyngeal testing with PCR and liver biopsy for SARS-CoV-2 PCR, only seroconversion for SARS-CoV-2 was positive. Extensive work-up of alternative causes of the acute hepatitis were all negative.\n\nMinor comments:\nShould provide more detail on doses and frequency of acetaminophen and NSAIDs used for his hepatitis.\n\nDid he take any herbal medications for the cold symptoms?\n\nProvide more detail of tests for autoimmune and metabolic causes of the hepatitis.\n\nWas there any pathological evidence of endotheliitis on liver biopsy?\n\nGenerally speaking, I agree with the authors’ conclusions but would rephrase the title and manuscript text in that the hepatitis was not isolated but predominant.\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? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6860", "date": "07 Jul 2021", "name": "Paraskevas Filippidis", "role": "Author Response", "response": "Dear reviewer, Thank you for your precious feedback about our article and for the questions you submitted. Please find below point-to-point answers to your questions. Should provide more detail on doses and frequency of acetaminophen and NSAIDs used for his hepatitis. Response: Acetaminophen was taken at a dose of 500 mg once or twice per day on demand during 14 days, namely a cumulative dose of 13.5 g upon admission, while the nonsteroidal anti-inflammatory drug nimesulid was taken at a dose of 100 mg once per day on demand during 14 days, namely a cumulative dose of 900 mg upon admission. In fact, these medications were rather used for the febrile syndrome than for his hepatitis and doses were relatively low to explain the liver damage observed. In addition, liver enzymes progressively normalized despite the continuous administration of the above mentioned medication to treat fever. Did he take any herbal medications for the cold symptoms? Response: No herbal medications or dietary supplements were taken by the patient. Provide more detail of tests for autoimmune and metabolic causes of the hepatitis. Response: Autoimmune and metabolic diagnostic work-up included antinuclear antibodies, rheumatoid factor, complement, total immunoglobulin G, complete autoantibody panels for autoimmune hepatitis and vasculitis, copper and ceruloplasmin values and normal thyroid tests, which were all normal. Liver imaging was also normal. Was there any pathological evidence of endotheliitis on liver biopsy? Response: No pathological evidence of endotheliitis was found in liver biopsy. Generally speaking, I agree with the authors' conclusions but would rephrase the title and manuscript text in that the hepatitis was not isolated but predominant. Response: We rephrased the title’s manuscript as to indicate a predominant rather than isolated character of patient's febrile hepatitis. On behalf of my co-authors, Paraskevas Filippidis" } ] } ]	1	https://f1000research.com/articles/10-400
https://f1000research.com/articles/10-300/v1	19 Apr 21	{ "type": "Case Report", "title": "Case Report: Polyvinylpyrrolidone deposition disease from repeated injection of opioid substitution drugs: report of a case with a fatal outcome", "authors": [ "Ida Viken Stalund", "Gro Nygard Riise", "Friedemann Leh", "Tormod Karlsen Bjånes", "Lars Riise", "Einar Svarstad", "Sabine Leh", "Gro Nygard Riise", "Friedemann Leh", "Tormod Karlsen Bjånes", "Lars Riise", "Einar Svarstad", "Sabine Leh" ], "abstract": "Background: Intravenous injection of oral opioid substitution drugs (OSD) is widespread among injecting drug users. Several OSDs contain the polymer polyvinylpyrrolidone (PVP) as an excipient. Parenterally administered PVP of high molecular weight may accumulate in tissues and organs. This phenomenon was first described in the 1950s, when PVP was utilised in medication for parenteral use. We report a case of an opioid-addicted patient with extensive PVP–deposition caused by repeated injections of OSDs. Case presentation: A 30-year-old male drug addicted patient in opioid substitution therapy (OST) was repeatedly referred to his local hospital in a poor general condition. Work-up revealed severe normocytic anaemia, renal insufficiency, pancreas insufficiency and pathological fractures. Biopsies from fractured bones, bone marrow and gastric mucosa showed extensive infiltrates of histiocytes with intracytoplasmic vacuoles. Vacuole content stained slightly bluish in hematoxylin and eosin stain, red in Congo red stain and black in periodic acid methenamine silver stain. The morphological appearance and staining properties were in accordance with the diagnosis of PVP deposition. The patient had been injecting both buprenorphine tablets and a specific methadone syrup for several years. The methadone syrup contained large amounts of high molecular weight PVP, making it the most likely cause of the deposition. His health quickly deteriorated and he died, impaired by multi-organ failure and cachexia, five years after the first diagnosis of PVP-deposition. The autopsy revealed extensive PVP-deposition in all sampled organs and tissues. Conclusions: Histological investigation and the correct identification of PVP in the biopsies led to the discovery of a severe adverse effect from long-standing misuse of a drug. The disseminated PVP deposition likely contributed to multi-organ dysfunction and cachexia with a fatal outcome. The deposited PVP likely originated from repeated injections of a certain methadone syrup.", "keywords": [ "Polyvinylpyrrolidone", "PVP", "povidone", "opioid substitution therapy", "opioid substitution drugs", "methadone", "adverse effect", "case report" ], "content": "Background\n\nInjection of oral opioid substitution drugs (OSD) is a concern in the treatment of opioid dependency. An Australian study found that 7–13% of clients in opioid substitution therapy (OST) injected their medication weekly or more often.1 The OSDs may also be sold on the illegal drug market, and 26.6% of out-of-treatment intravenous drug users in a Norwegian study reported having injected methadone during the past 4 weeks.2 Injection of oral or sublingual drug formulations may lead to vascular and soft tissue damage with a range of secondary complications.3\n\nSeveral OSDs contain the excipient polyvinylpyrrolidone (PVP),4 a water-soluble polymer with a wide variety of applications in the pharmaceutical industry.5 When orally ingested, PVP is not absorbed, and causes no harm.6,7 When injected, PVP is not metabolized, and the only way of excretion is via glomerular filtration.6 While low molecular weight (MW) PVP is freely filtered by the glomerulus, PVP with moderate or high MW will be partly or completely retained in the body.6,8 In the middle part of the last century, PVP was utilized as a plasma expander9 and as a retarding agent in hormone preparations for injection.10 Reports from this time described storage in multiple tissues following repeated parenteral administrations of PVP-containing preparations.9–11\n\nWe report a case of extensive PVP-deposition disease with a fatal outcome following long-term injection of PVP-containing OSDs.\n\n\nCase presentation\n\nA male, 30-year-old drug addicted patient in OST was admitted to the local hospital. He was hepatitis C positive and had a history of hospitalisations for skin infection. At admittance, he was in a poor general condition with nausea, vomiting, abdominal pain and muscle aches. Physical examination revealed a diffusely tender abdomen and poor dental status. Laboratory investigations disclosed non-specific inflammatory signs with an increased erythrocyte sedimentation rate (83 mm/h) and C-reactive protein (CRP, 90 mg/L), severe normocytic anaemia with a haemoglobin of 7.8 g/dL and renal insufficiency (serum creatinine 133 μmol/L, estimated glomerular filtration rate 60.1 mL/min/1.73m2) with microalbuminuria. Blood cultures were negative. Radiological examinations of the thorax and abdomen showed splenomegaly and a pancreatic cyst, but otherwise no radiologic signs of infection, malignancy or kidney pathology. His CRP and serum creatinine levels fell spontaneously, and he left the hospital against the doctor’s advice after four days.\n\nTwo months later, the patient fractured his right clavicle. After a two-week delay, he was admitted to hospital with a fever and a swollen and erythematous clavicular region. His general condition and nutritional status had worsened, nausea and vomiting persisted and anaemia and renal insufficiency had relapsed. Blood cultures were positive for Staphylococcus aureus, and antibiotic treatment against suspected osteomyelitis was initiated. Magnetic resonance imaging (MRI) of the clavicular region and the upper arm showed mottled signal changes with a high signal intensity in the lateral clavicle, the humeral bone and the acromion (Figure 1a). The diagnoses considered at this time were osteomyelitis or malignancy. Biopsies from the fractured bone, bone marrow and gastric mucosa were performed in the work-up of this complex symptomatology.\n\n(a) MRI (T2-blade-sag-FS) of the right humerus showing mottled signal changes. (b) Clavicular bone (H&E): The marrow space is infiltrated by histiocytes with bluish transparent bubbles. (c) Bone marrow (H&E): Massive infiltration of histiocytes and scarce remaining hematopoietic tissue. (d) Gastric mucosa (H&E): Infiltration of vacuolated histiocytes in an extended lamina propria. All scale-bars 50 μm.\n\nThe biopsies all showed similar infiltrates of histiocytes with a cytoplasm extended by vacuoles of different sizes (Figure 1b–d) and eccentrically located nuclei.\n\nBiopsies from the fractured bone revealed reactive changes with ongoing fibrosis. The fibrotic tissue contained the multivacuolated histiocytes as singular cells, small groups or sheets of cells (Figure 1b). The bone marrow biopsy showed massive histiocytic infiltrates (Figure 1c). There was reduced fat cell content, and there was almost no visible hematopoietic tissue. The gastric biopsy showed antrum mucosa with elongated gastric pits and aggregates of multivacuolated histiocytes in both the superficial and deep lamina propria (Figure 1d). A gastric biopsy taken two years previously was re-examined. It showed the same histiocytic infiltrates; however, the findings did not lead to the correct diagnosis at the time.\n\nIn the hematoxylin and eosin (H&E) stain, the vacuoles had distinct membranes and a bluish, transparent looking content. Vacuole content did not stain in the periodic acid Schiff (PAS), Prussian blue or Alcian blue stain. The cells were positive for CD68/PGM1 confirming the histiocytic nature (Figure 2a). The vacuoles stained red in Congo red stain and black in the periodic acid methenamine silver (PASM) stain (Figure 2b and c). The vacuoles did not show birefringence.\n\nAll micrographs are from the same bone marrow biopsy. (a) CD 68/PGM1: The vacuolated cells are CD68-positive histiocytes. (b): Congo red stain: Vacuole content stains faintly red. (b) PASM: Vacuole content stains grey or black. All scale bars 50 μm.\n\nThe microscopic appearance of the vacuolated histiocytes and the histochemical staining properties were consistent with PVP deposition.12,13 At that time, several of the OSDs marketed in Norway contained PVP.4 Our patient had injected both buprenorphine tablets and methadone syrup regularly over several years. The buprenorphine tablets contained PVP K30 (MW 44–54 kDa). The specific methadone syrup he had been injecting contained large amounts of PVP K90 (MW 1 000–1 500 kDa).14 While much of the PVP K30 is expected to be excreted within weeks, PVP K90 will not be excreted and consequently accumulates in the body.6 It is therefore plausible that injections of the PVP K90-containing methadone syrup were the cause of the PVP-deposition disease in this patient. Based on the history of this patient and other similar cases,15,16 the European Medicines Agency suspended this methadone syrup in 2014.14\n\nAfter the diagnosis was made, the patient’s health gradually declined, in part due to his poor self-care and underlying chronic drug addiction. There is no specific treatment for PVP deposition disease. His persistent anaemia was treated with regular blood transfusions and erythropoietin-stimulating agents with limited effect. The kidney failure was managed by supportive treatment, and serum creatinine levels fluctuated between 150 and 450 μmol/L.\n\nWithin the first year of the diagnosis, the patient suffered a left sided pathological femoral neck fracture with impaired fracture healing, complicated by chronic osteomyelitis. In the right hip, he developed severe bone destruction of the acetabulum with dislocation of the femoral head and a subsequent femoral neck fracture (Figure 3a). MRI scans of both hips and the pelvis (not shown) revealed the same changes as those detected in the shoulder region. Biopsies from the greater trochanter region showed extensive histiocytic infiltrates (Figure 3b). Surgical treatments were unsuccessful. Henceforth his mobility deteriorated to such a degree that he needed a wheelchair and required daily activity support and care in a palliative care centre. Furthermore, he developed endocrine pancreas insufficiency with insulin-dependent diabetes mellitus. He had a gradual weight loss of 22 kg during four years despite adequate food intake. Malabsorption due to exocrine pancreas insufficiency was suspected, but supplementation of pancreas enzymes had little effect on the weight loss.\n\n(a) Pelvic radiograph. Left hip: The hip prosthesis has been removed due to loosening and replaced by a Girdlestone hip. Right hip: Extensive lytic and sclerotic changes in the proximal femur and acetabulum leading to medial dislocation of the femoral head. (b) Biopsy from the right greater trochanter (H&E): The marrow space is filled with histiocytes with the bluish vacuoles characteristic of PVP-deposition. The bone trabecula has empty lacunar spaces and contains a bluish material. Scale bar 50 μm.\n\nFive years after the diagnosis of PVP deposition disease, the patient died, impaired by multi-organ failure and advanced cachexia with a body mass index below 15 kg/m2.\n\nThe autopsy concluded that the immediate cause of death was multi organ failure due to PVP deposition disease, which in turn was a consequence of the patient’s illicit substance abuse. The following organs and tissues were sampled during the autopsy: the pericardium and myocardium, the pleura and lungs, kidneys, liver, pancreas, gastric mucosa, adrenal glands, peritoneum and abdominal adipose tissue, the spleen, lymph nodes, and femoral bone and bone marrow. Microscopically, we observed PVP-deposition in all sampled organs and tissues (Figure 4). The PVP-containing histiocytes were partly scattered in the interstitium, e.g. in the myocardium (Figure 4a), and partly organised like larger, nodular lesions, e.g. in the pleura (Figure 4b). Peri- and intra-neural distribution was seen in several organs. Autopsy findings corresponded well with the clinically observed pancreas and kidney insufficiency. In the pancreas, dense fibrosis was interspersed by heavy infiltrations of histiocytes (Figure 4c). There was little preserved pancreas parenchyma. The kidney showed moderate to severe interstitial fibrosis and tubular atrophy, and only minor glomerular changes. There were infiltrates of PVP-containing histiocytes in the interstitium, mostly in atrophic areas (Figure 4d). There were no amyloid depositions or signs of other renal diseases.\n\nInfiltrates of histiocytes with the bluish vacuoles characteristic of PVP in all organs. (a) Myocardium: Interstitial fibrosis and histiocytic infiltrates. Scale bar 50 μm. (b) Pleura: Nodular lesion composed of fibrotic tissue and histiocytes. We found similar lesions in the pericardium and peritoneum. These were macroscopically visible. Scale bar 200 μm. (c) Pancreas: Pronounced fibrosis with histiocytic infiltrates. Poorly preserved pancreatic parenchyma. Scale bar 50 μm. (d) Kidney: Cortical tissue showing interstitial fibrosis, tubular atrophy, and an infiltrate of histiocytes in the extended interstitium. Scale bar 50 μm.\n\n\nDiscussion\n\nWe present the case of a drug-addicted patient with widespread PVP-deposition disease. We describe the patient’s clinical course from the first diagnosis of PVP-deposition to his death five years later and present biopsy and autopsy findings. The PVP deposition disease in this patient was likely caused by repeated intravenous injections of a methadone syrup containing high MW PVP.\n\nThe characteristic appearance and staining properties of PVP in tissue samples are well known.17 Positivity for Congo red and PASM and negativity for PAS distinguishes PVP deposits from those seen in hereditary storage diseases. The histiocytic nature of the cells and the PAS negativity rule out metastatic signet ring cell carcinoma as a differential diagnosis.17 Furthermore, PVP is not birefringent, unlike most other foreign materials commonly observed in tissue samples from injecting drug users.18 Hence, making the diagnosis of PVP deposition is straightforward if this option is considered.\n\nWhether PVP deposition causes disease was controversial for a long time. The first reports of PVP- deposition described storage in the tissue that persisted years after the administration of PVP, but disputed whether the storage was harmful to the functioning of the target organs.19 Later reports described clinically relevant adverse effects. Those most frequently reported were cytopenias, bone destruction, polyneuropathy and granulomatous lesions of the skin.17,20–23 PVP deposition in internal organs such as the liver, kidneys, pancreas and the gastrointestinal tract has also been described, but the adverse effect of the deposition in these organs is less well known.24,25\n\nOur patient experienced a complex clinical syndrome and rapidly deteriorating health. The reason for his health decline was multifactorial, and his continued drug addiction likely aggravated the clinical course. Many of the clinical conditions associated with the fatal outcome correspond to findings in biopsies and the autopsy showing extensive PVP deposition. We believe that the extensive PVP deposition contributed to his health decline through several mechanisms. The widespread PVP deposition in bone and bone marrow was likely the main reason for the patient’s anaemia, pathological fractures and impaired fracture healing, fitting well with previous reports.20,22,23 The impaired mobility and chronic osteomyelitis that resulted from these fractures gravely affected his health and quality of life. Furthermore, progressive cachexia was an important part of his health decline. The reason for the weight loss was not established during his lifetime, but his continued drug use likely contributed. Other possible contributing factors were pancreas insufficiency, progressive kidney failure, malabsorption, chronic infections and continued problems with vomiting, all likely related to the extensive PVP deposition. In summary, the PVP deposition probably played a major role in causing the patients’ multiple organ dysfunction ultimately leading to the fatal outcome.\n\nInjection of oral OSDs is common among injecting drug users and has long been a concern in the treatment of opioid addiction.2 As an attempt to prevent injections, the previously mentioned methadone syrup was made highly viscous.14 However, the increased viscosity did not prevent such unintended use. As a consequence, the choice of high MW PVP as thickener caused further severe adverse effects from injection.\n\n\nConclusions\n\nThis case revealed an unanticipated explanation for anaemia and pathological fractures in a drug-addicted patient. The correct identification of the observed foreign material as PVP revealed that injection of a certain methadone syrup containing PVP probably caused the patient’s deposition disease. Based on the clinical history, biopsy and autopsy findings, we conclude that the widespread PVP deposition likely contributed to the patient’s severe morbidity and death.\n\n\nList of abbreviations\n\nPVP: Polyvinylpyrrolidone\n\nOSD: Opioid substitution drug\n\nOST: Opioid substitution therapy\n\nH&E: Hematoxylin and eosin stain\n\nPASM: Periodic acid methenamine silver stain\n\nPAS: Periodic acid Schiff stain\n\nCRP: C-reactive protein\n\nMRI: Magnetic resonance imaging\n\nMW: Molecular weight\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\nZenodo: CARE checklist for “Polyvinylpyrrolidone deposition disease from repeated injection of opioid substitution drugs: report of a case with a fatal outcome”. https://doi.org/10.5281/zenodo.4667989.26\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Universal).\n\n\nConsent for publication\n\nWritten informed consent for publication of the patient’s clinical details and clinical images was obtained from the patient prior to his death and from the closest relative.", "appendix": "Acknowledgements\n\nWe appreciate the contributions of the patient and his family in allowing us to discuss his care and findings. We thank Ingrid Vallevik and Stine Kristoffersen for performing the autopsy and Anna Emilia Kozak for valuable comments on the radiologic images.\n\n\nReferences\n\nDegenhardt L, Larance BK, Bell JR, et al.: Injection of medications used in opioid substitution treatment in Australia after the introduction of a mixed partial agonist-antagonist formulation. Med J Aust. 2009; 191(3): 161–165. PubMed Abstract \| Publisher Full Text\n\nBretteville-Jensen AL, Lillehagen M, Gjersing L, et al.: Illicit use of opioid substitution drugs: prevalence, user characteristics, and the association with non-fatal overdoses. Drug Alcohol Depend. 2015; 147: 89–96. PubMed Abstract \| Publisher Full Text\n\nYeo AK, Chan CY, Chia KH: Complications relating to intravenous buprenorphine abuse: a single institution case series. Ann Acad Med Singap. 2006; 35(7): 487–91. PubMed Abstract\n\nEuropean Medicines Agency: Methadone medicinal products for oral use containing povidone.2014Reference Source Accessed 07.11.2015.\n\nBuehler V: Polyvinylpyrrolidone Excipients for Pharmaceuticals: Povidone, Crospovidone and Copovidone. Berlin: Springer; 2005.\n\nRobinson BV, Sullivan FM, Borzelleca JF, et al.: PVP - A Critical Review of the Kinetics and Toxicology of Polyvinylpyrrolidone (Povidone). Michigan: Lewis Publishers; 1990.\n\nSiber GR, Mayer RJ, Levin MJ: Increased gastrointestinal absorption of large molecules in patients after 5-fluorouracil therapy for metastatic colon carcinoma. Cancer Res. 1980; 40(10): 3430–6. PubMed Abstract\n\nRavin HA, Seligman AM, Fine J: Polyvinyl pyrrolidone as a plasma expander: studies on its excretion, distribution and metabolism. N Engl J Med. 1952; 247(24): 921–9. PubMed Abstract \| Publisher Full Text\n\nHonda K, Motoki R, Sakuma H, et al.: Complications following the use of plasma expander, especially polyvinylpyrrolidone. Int Surg. 1966; 45: 539–47.\n\nChristensen M, Johansen P, Hau C: Storage of polyvinylpyrrolidone (PVP) in tissues following long-term treatment with a PVP-containing vasopressin preparation. Acta Med Scand. 1978; 204(4): 295–8. PubMed Abstract \| Publisher Full Text\n\nWessel W, Schoog M, Winkler E: Polyvinylpyrrolidone (PVP), its diagnostic, therapeutic and technical application and consequences thereof. Arzneimittelforschung. 1971; 21(10): 1468–82. PubMed Abstract\n\nReske-Nielsen E, Bojsen-Moller M, Vetner M, et al.: Polyvinylpyrrolidone-storage disease. Light microscopical, ultrastructural and chemical verification. Acta Pathol Microbiol Scand A. 1976; 84(5): 397–405. PubMed Abstract\n\nWeiss SW, Goldblum JR: Benign fibrohistiocytic and histiocytic tumors. In: Enzinger and Weiss’s Soft Tissue Tumors. 6 ed. Philadelphia: Mosby Elsevier; 2014. p. 378–80.\n\nEuropean Medicines Agency: Assessment report for methadone medicinal products for oral use containing povidone.2014. Reference Source Accessed 07.11.2015\n\nKaur P, Bergrem A, Gerlyng P, et al.: A methadone user with anaemia, skeletal pain and altered appearance. Tidsskr Nor Laegeforen. 2016; 136(10): 925–9. PubMed Abstract \| Publisher Full Text\n\nKristoffersen AH, Bjanes TK, Jordal S, et al.: Polyvinylpyrrolidone induced artefactual prolongation of activated partial thromboplastin times in intravenous drug users with renal failure. J Thromb Haemost. 2016; 14(5): 936–9. PubMed Abstract \| Publisher Full Text\n\nKuo TT, Hsueh S: Mucicarminophilic histiocytosis. A polyvinylpyrrolidone (PVP) storage disease simulating signet-ring cell carcinoma. Am J Surg Pathol. 1984; 8(6): 419–28. PubMed Abstract \| Publisher Full Text\n\nKringsholm B, Christoffersen P: The nature and the occurrence of birefringent material in different organs in fatal drug addiction. Forensic Sci Int. 1987; 34(1-2): 53–62. PubMed Abstract \| Publisher Full Text\n\nAltemeier WA, Schiff L, Galle A, et al.: Physiological and pathological effects of long-term polyvinylpyrrolidone retention. AMA Arch Surg. 1954; 69(3): 308–14. PubMed Abstract\n\nKepes JJ, Chen WY, Jim YF: 'Mucoid dissolution' of bones and multiple pathologic fractures in a patient with past history of intravenous administration of polyvinylpyrrolidone (PVP). A case report. Bone Miner. 1993; 22(1): 33–41. PubMed Abstract \| Publisher Full Text\n\nKuo TT, Hu S, Huang CL, et al.: Cutaneous involvement in polyvinylpyrrolidone storage disease: a clinicopathologic study of five patients, including two patients with severe anemia. Am J Surg Pathol. 1997; 21(11): 1361–7. PubMed Abstract \| Publisher Full Text\n\nDunn P, Kuo T, Shih LY, et al.: Bone marrow failure and myelofibrosis in a case of PVP storage disease. Am J Hematol. 1998; 57(1): 68–71. PubMed Abstract \| Publisher Full Text\n\nHuang WC, Chang CH, Tsai CC: Polyvinylpyrrolidone storage disease presenting as pathologic fracture and anemia: report of a case with imprint cytology. Diagn Cytopathol. 2012; 40(1): 69–72. PubMed Abstract \| Publisher Full Text\n\nKojima M, Takahashi K, Honda K: Morphological study on the effect of polyvinyl pyrrolidone infusion upon the reticuloendothelial system. Tohoku J Exp Med. 1967; 92(1): 27–54. PubMed Abstract \| Publisher Full Text\n\nEdelmann U, Johansen P, Pedersen AB, et al.: PVP storage as the cause of specific organ symptoms. Ugeskr Laeger. 1977; 139(39): 2309–12. PubMed Abstract\n\nStalund IV, Riise GN, Leh F, et al.: Polyvinylpyrrolidone deposition disease from repeated injection of opioid substitution drugs: report of a case with a fatal outcome. Zenodo. 2021. Publisher Full Text" }	[ { "id": "83519", "date": "28 Apr 2021", "name": "Maike Büttner-Herold", "expertise": [ "Reviewer Expertise Nephropathology", "GvHD", "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 present case report illustrates a very impressive case of PVP deposition in histiocytes in a large number of organs in a patient who repeatedly injected oral opioid substitution drugs. The description of such a finding is very valuable as the identification of foreign material can be very difficult for a pathologist, if the histological picture has not previously been seen. The illustration of the findings is very nicely done.\nSome minor points could be addressed:\nBesides the reported anaemia did the patient also have thrombopenia and leukopenia, which could explain the infectious complications?\n\nWas the patient free of infectious complications when he died?\n\nCan a loss of pancreatic islets be shown by immunhohistochemistry? In Figure 4C one wonders whether remaining islets are depicted.\n\nAs PVP is filtered in the glomeruli, were histiocytes detecable in the glomeruli? Was the distribution of PVP-loaden histiocytes in the kidneys dependent on the renal compartment?\n\nDid the cardiac infiltration by histiocytes lead to functional impairment of the cardiac output or to arrhythmias?\n\nIs their a possible explanation why PVP should interfer with the structure of the bone in such a destructive way?\nDoes the lack of birefringence of PVP also apply to the Congo red staining?\n\nKeywords: It would maybe be helpful to include \"foreign material\" and \"histiocytic storage\" to facilitate search for a pathologist finding PVP in a patient without being able to assign the finding to the right cause.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6845", "date": "07 Jul 2021", "name": "Ida Viken Stalund", "role": "Author Response", "response": "We thank you for your thorough review of our manuscript. We have addressed your comments point by point. Besides the reported anemia, did the patient also have thrombocytopenia and leukopenia, which could explain the infectious complications? The patient had episodes of mild thrombocytopenia and leukopenia. In general, his leukocyte response to infection was weak. We added the following sentence in the section “Further development” to address this question: “He had short episodes of mild thrombocytopenia and leukocytopenia, and in general, his leukocyte response to infection was weak.” Was the patient free of infectious complications when he died? In the days and weeks prior to his death, he had stable, moderately increased CRP. Clinically, he showed no signs of acute infection and he did not receive antibiotic treatment. The autopsy revealed small foci of acute inflammation in the pancreas, but otherwise there were no signs of an acute infection. We added the following sentence in the section “Autopsy” to address this question: \"The autopsy revealed no evidence of infection besides minor foci of acute inflammation in the pancreas.\" Can a loss of pancreatic islets be shown by immunohistochemistry? In Figure 4C, one wonders whether remaining islets are depicted. Based on H&E morphology there was little preserved pancreatic parenchyma, both pancreatic acini and islets. Synaptophysin and Chromogranin A staining revealed a marked loss of pancreatic islets. CK-19 staining revealed areas of some preserved ductal structures, though the morphology was impaired by autolysis. We changed the following sentence in the section “Autopsy” to address this question: \"There was little preserved exocrine and endocrine pancreas parenchyma. The pancreatic tissue was dominated by dense fibrosis interspersed by heavy infiltrations of histiocytes (Figure 4c).\" Figure 4C depicts fibrosis, PVP-containing histiocytes and ductal structures impaired by autolysis. Synaptophysin and Chromogranin A staining could not detect islets in this area. We changed the legend to figure 4C accordingly: (c) Pancreas: Pronounced fibrosis with histiocytic infiltrates. Poorly preserved ductal structures. As PVP is filtered in the glomeruli, were histiocytes detecable in the glomeruli? Was the distribution of PVP-loaden histiocytes in the kidneys dependent on the renal compartment? In H&E, we observed occasional PVP-containing vacuoles in glomeruli. With CD68 staining, we detected histiocytes in most glomeruli. The number of histiocytes per glomerulus cross section was variable, but always less than ten. We added the following sentence in the section “Autopsy” to address this question: In glomeruli, we observed a slightly increased number of histiocytes and occasional PVP-containing vacuoles. The vast majority of PVP-containing histiocytes were seen in the tubulointerstitial compartment, as stated in the “Autopsy” section. Did the cardiac infiltration by histiocytes lead to functional impairment of the cardiac output or to arrhythmias? The autopsy revealed infiltrates of PVP-containing histiocytes in the myocardium. We also observed peri- and intraneural distribution of the histiocytes. It is possible that such infiltrates could induce arrhythmias or other forms of cardiac impairments. Our patient did not show signs of such organ dysfunction, neither clinically nor in echocardiographic and electrocardiographic evaluations. We added the following sentence in the section “Autopsy” to address this question: “Peri- and intra-neural distribution was seen in several organs, including the heart. However, there were never any clinical, echocardiographic or electrocardiographic evidence of impaired cardiac output or arrhythmias prior to his death.” Is there a possible explanation why PVP should interfere with the structure of the bone in such a destructive way? The only mentioning of a possible cause for the bone destruction is in in a case report from 1993: Kepes et al. suggested that cells containing engulfed PVP would undergo mucoid alterations as a result of interactions between the engulfed material and the cytoplasm. The authors argue that in bone, the PVP-induced mucoid changes cause “softening of the bone” and interfere with the structural integrity of the bone trabeculae. One could also speculate that the massive infiltration of histiocytes in the marrow space could affect the blood supply to the osseous tissue, causing a form of avascular necrosis. We think a discussion of this unresolved matter is out of focus for this case report. Does the lack of birefringence of PVP also apply to the Congo red staining? Yes, the lack of birefringence of PVP also apply to the Congo red. We changed the following sentence in the section “Biopsy findings” to address this question: “The vacuoles did not show birefringence in Congo red stain or any other stains.” Keywords: It would maybe be helpful to include \"foreign material\" and \"histiocytic storage\" to facilitate search for a pathologist finding PVP in a patient without being able to assign the finding to the right cause Good point. We added these keywords." } ] }, { "id": "85062", "date": "26 May 2021", "name": "Robert Barnes Colvin", "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 paper shows no new information, but the problem is a major one, with missed diagnoses and serious consequences. I think the paper will receive some attention with the major opioid crisis we have.\nI have no specific suggestions, other than a careful editing of the prose and the addition of at least on other major publication which has embolized material (EM) - e.g., Ganesan et al. (2003)1.\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": "6846", "date": "07 Jul 2021", "name": "Ida Viken Stalund", "role": "Author Response", "response": "We thank you for your thorough review of our manuscript. To address your comment, we have added the following sentence in the second paragraph of the discussion: “PVPs light blue color in H&E and the typical localization of the deposits differentiates it from the non-water soluble variant of PVP, crospovidone.” and added your suggested reference." } ] } ]	1	https://f1000research.com/articles/10-300
https://f1000research.com/articles/10-540/v1	06 Jul 21	{ "type": "Case Report", "title": "Case Report: An immunocompromised, vaccine breakthrough COVID-19 patient with pneumonia", "authors": [ "Antonios Patrinos", "Dimitris Komninos", "Katerina Dimouli", "Andreas Kartsonakis", "Dimitris Serpanos", "Georgios Kalogeropoulos", "Dimitra Dimitropoulou", "Dimitris Komninos", "Katerina Dimouli", "Andreas Kartsonakis", "Dimitris Serpanos", "Georgios Kalogeropoulos", "Dimitra Dimitropoulou" ], "abstract": "Background: The novel SARS-CoV-2 pandemic has inflicted a major blow on public health worldwide accounting for millions of deaths and subsequent socio-economic consequences. The main challenge for scientists and researchers has been to restrain transmission of the virus and prevent severe respiratory disease. Novel promising vaccines aim to fulfil these expectations, although new variants of the coronavirus have emerged. The present manuscript aims to add to the knowledge, through a case of an immunodeficient patient, who developed remarkably favorable recovery from SARS-CoV-2 pneumonia after having been fully vaccinated against the novel coronavirus. Case presentation: An 82-year-old Caucasian male with a history of metastatic pancreatic cancer was admitted with signs and symptoms of pneumonia. Workup revealed a positive SARS-CoV-2 real time – polymerase chain reaction (RT-PCR) and further investigation identified a B.1.1.7 variant. The imaging essays showed extensive lung disease. Interestingly, the patient had already received the second dose of the BNT162b2 (Pfizer) vaccine against the new coronavirus 16 days prior. After having been treated with appropriate antiviral and antibiotical agents the patient showed significantly favorable recovery and no need for high oxygen flows and no complications presented. The patient was discharged after six days of hospitalization in good condition, with no need for supplementary oxygen at home. Conclusions: Despite breakthrough cases, vaccination against COVID-19 is crucial for restraining the novel coronavirus. Further studies should be carried out in order to determine the optimal strategies for large-scale vaccination while minimizing the risk of further and faster evolution of the virus.", "keywords": [ "COVID-19", "vaccine breakthrough", "efficacy", "immunodeficiency", "case report" ], "content": "Introduction\n\nSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for approximately three million deaths, and subsequent vast financial and social burdens due to the current global pandemic.1 The rapid and wide utilization of effective, and safe vaccines against SARS-CoV-2 infection and COVID-19 that act against viral infection, disease, transmission, and death is essential for the establishment of public health internationally.1,2 Multiple clinical trials aim to investigate the efficacy and safety of novel vaccines, although, the most crucial efficacy endpoint, which is protection against severe disease and death, is difficult to estimate.1 With emphasis on the present efficacy endpoint, the present manuscript aims to add to the existing knowledge, while presenting the case of an immunodeficient patient, who developed remarkably favorable outcomes from SAR-CoV-2 pneumonia after having been fully vaccinated against the novel coronavirus.\n\n\nCase report\n\nAn 82-year-old Caucasian male presented to the emergency room of our institution with malaise, fever (max temperature 38°C), and productive cough. The patient’s vital signs were within normal range, his respiratory rate was 16/min and his mental status was normal. Clinical examination revealed reduced sounds and dry rales at lower auscultation. A laboratory workup was carried out. The complete blood count showed mild neutropenia (1450/μL, total white blood cells 3390/μL), D-Dimers 1260 mg/L and C-reactive protein 7,1 mg/L. Chest X-ray showed opacities in both lungs, especially in lower lobes consistent with the clinical findings (see Figure 1 and Figure 2). A nasopharyngeal and pharyngeal swab were sent for SARS-CoV-2 antigen and real-time polymerase chain reaction testing, respectively, the results of which were positive. Further investigation identified a B.1.1.7 variant.\n\nThe scan reveals ground glass opacities especially with onset of fibrotic lesions and concomitant vascular enlargement. CT findings suggest >50% extent of disease.\n\nThe patient’s medical history included pancreatic cancer with lung metastasis, prostate hyperplasia, and frequent RBC transfusions. His last chemotherapy session had taken place two weeks ago and he had received the second dose of the BNT162b2 (Pfizer) vaccine against COVID-19 16 days prior. Both doses had been administered without any complications. No symptoms indicative of acute infection (fever, chills, dyspnea, tachypnea, joint pain) were mentioned between the two doses. We preceded to admission of the patient for appropriate treatment. This included remdesivir 200 mg at first day of admission then 100 mg once daily, ceftriaxone 2 gr once daily, levofloxacin 500 mg once daily, N-acetylcysteine 600 mg bid p.os, inhaled agents (ipratropium and budesonide) and prophylactic dose of low-molecular-weight heparin (LMWH) enoxaparin 4000 once daily based on patient’s weight. Supplementary oxygen was administered with nasal cannula at a flow rate of two to three liters per minute. Meanwhile a chest computed tomography (CT) was performed which was indicative of diffuse COVID-19 lesions (>50% extent). The patient was closely monitored however he did not manifest clinical deterioration or respiratory distress. No changes were made regarding the therapeutic interventions. The inflammatory markers and PaO2/FiO2 ratio remained stable and gradually ameliorated. No other complications were presented whatsoever (thromboembolism, disseminated intravascular coagulation, arrhythmia, shock, sepsis or ARDS). The patient was discharged after six days of hospitalization with no need for supplementary oxygen at home.\n\n\nDiscussion\n\nThe global pandemic of COVID-19 seems to constitute the most challenging pandemic of the 21st century.3 A strongly efficacious vaccine is fundamental and crucial for the prevention of morbidity and severe mortality.1 Therefore, all clinically available vaccines against SARS-CoV-2 infection and COVID-19 are currently under evaluation through several multi-central clinical trials for their safety and efficacy.4\n\nIt is already documented that in comparison to unvaccinated patients, the probability of remaining asymptomatic after SARS-CoV-2 infection is lower among vaccinated persons at the period of more than 10 days after 1st dose of vaccine, and more than 0 days after 2nd dose.4\n\nAccording to the study of Polack et al. (2020), between the first and the second dose, the Pfizer COVID-19 vaccine provided an efficacy of 52%, with an incidence of 39 cases of infection in the vaccinated group, in comparison to 82 cases from the placebo group. Meanwhile the integration of two doses of the BNT162b2 mRNA COVID-19 vaccine offered 95% protection against the virus in patients older than 16 years of age.2\n\nMoreover, the study of Voysey et al. (2020) indicates that Oxford and AstraZeneca’s COVID-19 vaccine (AZD1222), presents an efficacy of 62.1% after the integration of two standard doses.5 Interestingly, for the persons that received a lower first dose combined with a standard second dose, an efficacy of 90% was observed.5\n\nIndeed, according to the literature there have been several documented cases of COVID-19 infections after vaccination.4,6 However, all the reported cases either remained asymptomatic or presented mild symptoms.4\n\nInterestingly our patient’s clinical course was remarkably favorable despite being immunodeficient. Herein it is implied that prior vaccination with BNT162b2 mRNA COVID-19 Vaccine had offered protection to some extent. This could be justified by the short period of hospitalization, the absence of septic and thromboembolic complications or severe respiratory distress, and finally by the fact that no high flow oxygen therapy was needed.\n\nAt this point we would like to address the issue of meticulous planning of vaccination campaigns. Given the rapid rate of COVID-19 evolution (presenting 1 × 10−3 mutations per genomic residue and year) alongside its genetic heterogeneity in every host, scientists are facing vaccination-driven virus evolution. As a result, even if massive vaccinations are implemented, there is still the risk of new infections by emerging variants. That is why authorities should take into consideration the epidemiological context of every country before vaccination campaigns are implemented.7\n\n\nConclusions\n\nThe current SARS-CoV-2 pandemic poses a real threat on public health due to the rapid and wide transmission of the virus. Despite the civil protection measures, the novel vaccines constitute a real game changer. To our knowledge this is the first reported vaccine breakthrough case with an extremely favorable clinical course indicating the efficacy of vaccination against COVID-19.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.", "appendix": "References\n\nHodgson SH, Mansatta K, Mallett G, et al.: What defines an efficacious COVID-19 vaccine? A review of the challenges assessing the clinical efficacy of vaccines against SARS-CoV-2. Lancet Infect Dis. 2021; 21(2): e26–e35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPolack FP, Thomas SJ, Kitchin N, et al.: Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 2020; 383(27): 2603–2615. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi YD, Chi WY, Su JH, et al.: Coronavirus vaccine development: from SARS and MERS to COVID-19. J Biomed Sci. 2020 Dec 20; 27(1): 104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTande AJ MD, Pollock BD PhD, MSPH, Shah ND PhD, et al.: Impact of the COVID-19 Vaccine on Asymptomatic Infection Among Patients Undergoing Pre-Procedural COVID-19 Molecular Screening. Clin Infect Dis. 2021. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVoysey M, Clemens SAC, Madhi SA, et al.: Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmit S, Beni SA, Biber A, et al.: Postvaccination COVID-19 among Healthcare Workers, Israel. Emerg Infect Dis. 2021; 27(4): 1220–1222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDomingo E, Perales C: The time for COVID-19 vaccination [published online ahead of print, 2021 Jan 19]. J Virol. 2021; JVI.02437-20. Publisher Full Text" }	[ { "id": "93330", "date": "23 Sep 2021", "name": "Yulia Desheva", "expertise": [ "Reviewer Expertise Infectious diseases", "virology", "immunology" ], "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' conclusions that the outcome of the disease is associated with vaccination are unfounded. The fact that the absence of septic and thromboembolic complications or severe respiratory distress, and finally by the fact that no high flow oxygen therapy was needed may not be the result of vaccination, it is not supported by anything other than a sequence of events. It is known that \"after something\" does not mean \"as a result of something\", you need to provide evidence.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "102324", "date": "01 Feb 2022", "name": "Thomas Tsaganos", "expertise": [ "Reviewer Expertise Infectious Diseases", "Sepsis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article, the authors present a male old patient with history of metastatic pancreatic cancer under current chemotherapy treatment who suffered from COVID-19 two weeks after his last chemotherapy and 16 days after his vaccination with the second dose of Pfizer vaccine. The patient was hospitalized with a remarkably mild clinical course, uncomplicated and without increased oxygen needs, who received both antiviral and antimicrobial treatment and prophylactic LMWH. They disclose that his mild disease and favorable outcome was attributed to the vaccination against COVID-19, despite being immunodeficient, and conclude that vaccines are real game changer in the fight against SARS-CoV-2.\nThis article is interesting, since it is one of the few presenting the protective role of vaccines against COVID-19 in immunodeficient patients. The presented case is helpful for clinicians, in order to urge their patients, especially immunodeficient ones, to get vaccinated promptly. This article merits careful examination in the era of pandemic that we all live.\nThe paper falls within the scope of the journal. There is a major issue with the rationale behind the paper. It can be hypothesized that vaccination contributed to the mild clinical course of the patient, but it cannot be proved that vaccination protected the patient against severe disease. The language in the manuscript is of superior quality; no ethical issues can be raised. In my opinion, the case report is also lacking in the justification of the treatment selected. The claims in discussion and conclusion are partly supported by the presented disease course.\nTechnically, the manuscript is very well written: the abstract is informative and the language used is high quality; the structure of the article is robust and helpful in the presentation of the findings and the manuscript is ordered in a meaningful way; in addition, the discussion has adequate length and conveys the messages of the authors; however, the limitations of the case report are not properly identified. The objective is clearly stated; the references strictly adhere to the guidelines of the journal. Lastly, the authors relate their study to previous studies (and also describe how their study is in line with previous ones) and explain the impact of their study and its contribution to current scientific knowledge. There is one minor issue that the authors should address regarding their study.\nMajor Comments:\nThe authors’ claim that vaccination of the patient against COVID-19 was protective against severe disease is unjustified. A few studies and extensive clinical experience have indicated that immunosuppressed patients frequently do not respond satisfactorily in the vaccines against COVID-19. In addition, the fact that vaccination was followed by chemotherapy two days afterwards further hinders the response of the host to the vaccine. Therefore, the authors should properly identify this limitation of their study in the discussion and conclusion sections.\n\nThere is no justification for the selection of the antimicrobial regimen. C-reactive protein was not markedly elevated and only ceftriaxone would have sufficed.\n\nMinor Comments:\nThe authors should discuss (and emphasize) that their case is only one (among many immunodeficient patients) who had a mild clinical course and favorable outcome and that their findings should be confirmed in the setting of a clinical study.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "102331", "date": "21 Mar 2022", "name": "Erni Nelwan", "expertise": [ "Reviewer Expertise Tropical medicine and infectious diseases", "immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case, I am not sure if we can say within 16 days of vaccination of Pfizer vaccine, it is sufficient enough to said \"a breakthrough\" of COVID-19. Among elderly potential the increase of antibody after vaccination is delay. The actual scenario may be the patient had an infection just before the antibody is high enough to protect him.\nOne important data to convince reader is actually if the level of antibody was measured against covid-19\nIf the patient had an history of lung metastasis and also could probably the hyper-coagulation state due to his primer illness, therefore a more detail information on the previous lab result or imaging is interesting to compare as part of the analysis of the case\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-540
https://f1000research.com/articles/10-221/v1	18 Mar 21	{ "type": "Review", "title": "Phytochemicals With Anti 5-alpha-reductase Activity: A Prospective For Prostate Cancer Treatment", "authors": [ "Aziemah Azizi", "Nuramalina H Mumin", "Naeem Shafqat", "Aziemah Azizi", "Nuramalina H Mumin" ], "abstract": "Prostate cancer (CaP) is one of the leading causes of death in men worldwide. Much attention has been given on its prevention and treatment strategies, including targeting the regulation of 5-alpha-Reductase (5αR) enzyme activity, aimed to limit the progression of CaP by inhibiting the conversion of potent androgen dihydrotestosterone from testosterone that is thought to play a role in pathogenesis of CaP, by using the 5-alpha-Reductase inhibitors (5αRis) such as finasteride and dutasteride. However, 5αRis are reported to exhibit numerous adverse side effects, for instance erectile dysfunction, ejaculatory dysfunction and loss of libido. This has led to a surge of interests on plant-derived alternatives that might offer favourable side effects and less toxic profiles. Phytochemicals from plants are shown to exhibit numerous medicinal properties in various studies targeting many major illnesses including CaP. Therefore, in this review, we aim to discuss on the use of phytochemicals namely phytosterols, polyphenols and fatty acids, found in various plants with proven anti-CaP properties, as an alternative herbal CaP medicines as well as to outline their inhibitory activities on 5αRs isozymes based on their structural similarities with current 5αRis as part of CaP treatment approaches.", "keywords": [ "5-alpha-reductase", "Testosterone", "Dihydrotestosterone", "Finasteride", "Dutasteride", "Phytochemicals", "Phytosterols", "Polyphenols", "Androgens", "Prostate cancer" ], "content": "Introduction\n\nProstate cancer (CaP) is the second most deadly malignancy in men after lung cancer and the fifth leading cause of death worldwide, accounting for 7.1% (1,276,106) of the new cases and 3.8% (358,989) of total death in males in 2018 (Rawla, 2019). According to the United Kingdom Cancer Research Centre, over 47,500 men are diagnosed with CaP each year, where one man dies from it every 45 minutes. CaP is also estimated to be the most common cancer by 2030, as one in eight men destined to be diagnosed with CaP in their lifetime. CaP is a malignant tumour that is caused by unregulated prostate cell division resulting in an abnormal cellular growth that leads to a potential spread of cancer to other body parts (Ochwang’i et al., 2014; Packer & Maitland, 2016). The current primary treatments for CaP are surgery, radiation therapy, proton beam therapy, chemotherapy, cryosurgery, high intensity focused ultra-sound and hormonal therapy, depending on the clinical conditions, outcomes and disease progression among others (Chen & Zhao, 2013). The latter strategy was largely anticipated, considering CaP as being hormones-driven disease especially during the initial stage (Taplin et al., 1995). Therefore, targeting the hormones involved in the CaP’s pathway mechanisms seems to be a potentially useful approach in developing CaP prevention and treatment strategies.\n\nThe physiologic functions and pathologic conditions of the prostate are regulated by numerous hormones and growth factors. For instance, androgens are essential for prostatic development and function as well as for cells’ proliferation and survival (Banerjee et al., 2018). Testosterone (T), synthesised by the Leydig cells of the testes under the control of hypothalamus and anterior pituitary gland, is the most abundant circulating androgen in males, where from it, more potent form dihydrotestosterone (DHT) is synthesised (Figure 1). The microsomal enzyme 5αR mediates a rapid and irreversible T conversion to DHT within the prostate where it then binds to androgen receptor (AR) to exert its biological function (Azzouni et al., 2012). 5αR exists in two isoforms, namely type 1 (5αR1) and type 2 (5αR2), which differ in their molecular genetics, structural and biochemical properties, and tissue localisation (Nahata, 2017). 5αR1 occurs predominantly in non-genital skin, the scalp, the sebaceous gland, the liver, the kidney and the brain, whereas 5αR2 is found extensively in the prostate, genital skin, seminal vesicles and in the dermal papilla (Nahata, 2017; Othonos & Tomlinson, 2019; Strauss & FitzGerald, 2019). Both isozymes are expressed at a much lower level in other peripheral tissues. Due to the tissue specific expression of 5αRs, DHT concentration is much higher than T concentration in the prostate. Androgens, although necessary for the development of prostate, could also allow CaP cells to grow. They promote the growth of cancerous prostate cells by binding to and activating the AR, resulting in the expression of specific genes responsible for the proliferation of CaP cells. Augmented levels of androgens, particularly DHT, are detrimental towards CaP later in life.\n\nThe figure is adapted and modified from National Center for Biotechnology Information (2020).\n\nProgression of cancer in prostate is typically dependent on the levels of androgens present during the initial stages of cancer growth (Taplin et al., 1995). Therefore, limiting the production of androgen provides a useful approach to androgen deprivation where it restricts the availability of T, allowing minimal conversion to DHT by 5αRs and androgen-receptor binding activity. The inhibition of 5αRs will subsequently limit the production of DHT and therefore represents a valid target for CaP risk prevention and reduction as well as treatment strategies as a whole.\n\nSynthetic 5αR inhibitors (5αRis) can be broadly classified into two categories, namely steroidal and nonsteroidal, where their development was aimed to bind to 5αR with little or no affinity for the androgen or other steroid receptors. The most promising and well-studied 5αRis by far are finasteride and dutasteride. Clinical treatment with finasteride and dutasteride have shown to decrease both mean serum and intraprostatic level of DHT in CaP patients (Andriole et al., 2004; Clark et al., 2004; McConnell et al., 1992; Span et al., 1999). Finasteride is the first synthetic steroidal 5αRi approved for the treatment of benign prostatic hyperplasia (BPH) and male pattern baldness (Aggarwal et al., 2010; Brough & Torgerson, 2017). Finasteride, a synthetic 4-azasteroid compound, is a potent competitive inhibitor of 5αR2 that also inhibits 5αR1 but less effectively (Figure 2a). Finasteride has been reported to decrease LNCaP cell growth rate in vitro in a dose dependent manner (Bologna et al., 1995). Meanwhile, dutasteride, also a synthetic 4-azasteroid compound and an approved drug for BPH treatment, is known as a dual 5αRi with a 45-fold more effective in inhibiting 5αR1 and 2-fold more effective in inhibiting 5αR2 than finasteride (Figure 2b). Dutasteride has been reported to inhibit T and DHT-induced LNCaP cell proliferation by targeting the 5aRs activity and displaying a more potent DHT inhibition than finasteride (Lazier et al., 2004). Dual inhibition of 5aRs is more beneficial than selective type 2 inhibition as it suppresses the DHT level to a great extent by also preventing the type 1 mediated synthesis of DHT production.\n\nThese observations, among others, provide a strong rationale for CaP risk reduction and prevention using 5αRis finasteride and dutasteride, although their use as a targeting therapeutic drug continues to be widely discussed. One of the main issues that halt the progression of 5αRis, considered as an effective CaP therapeutic agent, is the numerous undesirable side effects including erectile dysfunction, ejaculatory dysfunction and loss of libido (Erdemir et al., 2008). 5αRis, which are also commonly prescribed for women with hair loss, demonstrate headache, gastrointestinal discomfort and decreased libido as the most common reported side effects (Hirshburg et al., 2016). Other factors include the controversy that 5αRis appear to only preferentially prevent low-grade cancers and now concern lingers that 5αRis may induce or selectively promote growth of high-grade disease (Hamilton & Freedland, 2011).\n\nThe chemical structure of 5αRis; (a) Finasteride, and (b) Dustasteride. The figure is adapted and modified from National Center for Biotechnology Information (2020).\n\nIn contrast with synthetic drugs that are known to have various adverse side effects, safer alternative drugs, particularly those derived from plants, are often resorted to by many. Older people often use traditional plants as complementary and/or alternative remedies to sustain healthy life or cure diseases. The use of plants for medicine is not new, as traditional plants are known to be in medicinal practices for treatment of various diseases and ailments since ancient times (Falodun, 2010; Leroi-Gourhan, 1975; Pan et al., 2014) and the use of medicinal plants in the search of new drugs from nature has increased since (Savithramma et al., 2011). Indeed, plants have been considered as a valuable source of bioactive compounds for treatment of many conditions, including cancer, in almost all cultures and communities for thousands of years (Mohan et al., 2011). It has been reported that the plant kingdom is comprised of approximately 250,000 plant species and only around 10% have been studied for the treatment of different diseases (Iqbal et al., 2017). Approximately 25% of the modern drugs in clinical use are derived from plants, where the majority of these drugs were discovered as a direct result of studies that focused on the isolation of active compounds from plants used in traditional medicines (Calixto, 2019).\n\nWith regards to this, herbal drugs, which have been increasingly used in cancer treatment, represent a rich pool of new, interesting bioactive entities for the development of CaP therapeutic agents, with many exhibiting favourable side effects and toxicity profiles compared to conventional chemotherapeutic agents. Therefore, the aim of this review is to discuss the use of phytochemicals found in various plants that have been proven to exhibit anti-CaP as alternative herbal CaP medicines and to outline different types of phytochemical present in plants that exhibit inhibitory activities on 5αRs isozymes.\n\nPhytochemicals are the bioactive non-nutrient plant compounds that are found present in fruits, vegetables, grains and other plant foods, where its consumption has been linked to reduction on risk of many major chronic diseases (Sathishkumar & Baskar, 2014). Six major phytochemical categories that have been identified are phenolics, alkaloids, nitrogen-containing compounds, organosulfur compounds, phytosterols and carotenoids (Liu, 2013). The surge of interest in finding new natural bioactive entities as a template for new drug discovery and/or studying existing bioactive compounds for other biological and medicinal properties has kept scientists constantly conducting more chemical studies, particularly focusing on fractionating, isolating and identifying the active compounds. Phytochemicals offer a promising array of entities that can be further formulated into complementary or alternatives to conventional medicines that are less costly and have no/less harmful side effects. Many in vivo and in vitro studies have shown anti-CaP properties of various phytochemicals via numerous pathways as well as their ability to inhibit 5αR activity, particularly the phytosterols and phenolics, probably due to their structural similarity with the current inhibitors of 5αRs. Fatty acids, which differ in structure to any 5αRis, are also found to exhibit anti-5αR activity. Table 1 summarises the inhibitory action of various phytochemicals on 5αRs.\n\n1. Phytosterols\n\nPlant sterols or phytosterols (PS) are bioactive components in plants with 28- or 29-carbon alcohols and double bonds at the C-5 position of the ring that resemble cholesterol in vertebrates in terms of both of their structure and function (Zaloga, 2015). More than 200 different types of phytosterols have been reported, with β-sitosterol, campesterol and stigmasterol being the most abundant type of PS (Miras-Moreno et al., 2016). The toxicity profiles of PS have shown that there are no obvious side effects after long-term feeding of PS in both animals and humans (Ling & Jones, 1995). PS play essential roles in the reduction of cholesterol in blood that eventually decrease cardiovascular morbidity, therefore are well known for their beneficial effect on cardiovascular disease risk. Katan et al. (2003) reported that the intake of 1–2 g of PS daily can effectively lower low-density lipoprotein cholesterol levels by 8%-12%. However, little attention was received with regard to PS on their potential in cancer aetiology, although increasing evidence of biochemical and molecular effects of PS may make them strong candidates for cancer therapeutic agents.\n\nBeing structurally similar with four rings to synthetic 5αRis finasteride and dutasteride, PS could stand as the strongest promising candidate for plant-derived 5αRis. A study by Awad et al. (2001) showed that β-sitosterol inhibits the growth and migration of PC-3 human CaP and slows down the growth of prostate tumour in SCID mice, which suggests an involvement of androgenic mechanism of action as CaP is dependent on androgen. An in vitro metabolic study in hamster prostate by Marisa Cabeza and colleagues revealed that β-sitosterol inhibits the enzymatic activity of 5αRs in dose-dependent manner, which therefore confirms the ability of β-sitosterol as a 5αRi (Cabezal et al., 2003).\n\nAnother PS, stigmasterol, was reported to be associated with a reduction in common cancer risks including colon cancer, breast cancer and CaP (Bradford & Awad, 2007). Kamei et al. (2018) studied Phyllanthus urinaria where the extract was shown to suppress androgen activity of DHT in LNCaP cell lines and has inhibitory activity against 5αRs, of which the active bioactive compound responsible for the activity was identified as stigmasterol isolated from an activity-guided fractionation. An in vitro study of Serenoa repens extract (SPE) using baculovirus-directed insect cell expression system demonstrated the inhibition of both 5αR1 and 5αR2 in a non-competitive and uncompetitive manner, respectively (Iehlé et al., 1995). The major active compounds from PS of SPE includes β-sitosterol and stigmasterol (Suzuki et al., 2009). SPE, a well-known phytotherapeutic agent, most frequently used to treat lower urinary tract symptoms and as a BPH medicine, not only targets the regulation of 5αRs activity but also hampers the binding of DHT to androgenic receptors (Dawid-Pać et al., 2014). Pais (2010) reported in his study that in a cell-free test system, ethanolic extract of Serenoa repens was a potent inhibitor of 5αR2 with 61% inhibition. From these observations, β-sitosterol and stigmasterol are found to exhibit inhibitory activity on both isozymes of 5αRs. Various plants reported to have β-sitosterol as their major active compound include Hypoxis rooperi extract (Harzol®), Secale cereal (Rye Grass Pollen), Urtica dioica and Prunus Africana (Komakech et al., 2017; Madersbacher et al., 2007). A study by Nahata & Dixit (2014) analysing the inhibitory effects of different types of Urtica dioica extracts on the activity of 5αR2, demonstrated that ethanolic extracts were the best 5αRis, followed by petroleum ether and aqueous extracts. Stigmasterol, with known 5αR2 inhibitory activity, is also reported to be present in various medicinal plants including Croton sublyratus, Ficus hirta, Eclipta alba (L.) Hassk, Eclipta prostrate, Parkia speciosa, Gypsophila oldhamiana, Eucalyptus globules, Aralia cordata, Emilia sonchifolia, Akebia quinata, Desmodium styracifolium, Heracleum rapula (Chaudhary et al., 2011).\n\nLupeol, another PS, has also been shown to exhibit various pharmacological properties including anti-CaP activity (Siddique & Saleem, 2011). Siddique et al. (2011) demonstrated in their study that lupeol inhibited the growth of various CaP cells i.e LAPC4, LNCaP and CRPC cells, in vitro. Another in vivo study using implanted CaP cells as xenograft tumours in mice also revealed that lupeol treatment effectively halts tumour growth, which further suggests the ability of lupeol as an effective agent that can potentially inhibit the tumourigenenicity of CaP cells. Lupeol has also been observed to have a striking ability to preferentially kill CaP cells while sparing normal prostate epithelial cells (Saleem et al., 2005). SPE, which contains lupeol as its bioactive compound, has been shown to possess a dual 5αRs inhibition activity (Iehlé et al., 1995; Rainer et al., 2007), therefore confirming the ability of lupeol to inhibit both 5αR1 and 5αR2. Lupeol can also be found in other numerous medicinal plants such as American ginseng, Shea butter plant, Tamarindus indica, Allanblackia monticola, Himatanthus sucuuba, Celastrus paniculatus, Zanthoxylum riedelianum, Leptadenia hastata, Crataeva nurvala, Bombax ceiba and Sebastiania Adenophora (Siddique & Saleem, 2011). PS, being able to exhibit dual inhibition on both isoforms of 5αRs, further strengthens its potential as the most promising candidate as plant-derived 5αRis.\n\n2. Phenolics\n\na) Polyphenols\n\nPolyphenols (PP) are generally subdivided into two large groups: flavonoids and non-flavonoids. For centuries, preparation containing PP-flavonoids were applied as major active components in different remedies which were used to treat different human diseases (Salvamani et al., 2014). PP exert various pharmacological effects such as anti-oxidant, anti-hypertensive, anti-inflammatory and anti-thrombotic activity that can further help in promoting human health (Hollman et al., 1997; Kleemann et al., 2011; Manach et al., 2005; Vinson et al., 1995). The toxicity profiles have shown that PP exert their therapeutic effect in a dosage-dependent manner in animal studies, whereas moderate dosages of PP do not seem to elicit any adverse effects, hence indicating its beneficial effects and safe use. Conversely, at high dosages, PP might show parallel adverse effects and/or toxicity, particularly due to accumulation of high levels of PP (Silva & Pogačnik, 2020).\n\nPP, although lacking one ‘ring’, exhibit a chemical structure similar to the synthetic 5αRis, hence representing a potential plant-derived 5αRis candidate. Quercetin, one of the PP-flavonoids, has a 3-OH group on its pyrone ring and is abundant in many fruits and vegetables. It has been shown to be non-toxic and possesses an anti-cancer property in various human cancer cell lines both in vitro and in vivo including CaP (Piao et al., 2014). In vitro, quercetin exhibits significant arrest of cell cycle, decreases cell viability, inhibits proliferation, and induces cell apoptosis especially in PC-3, LNCaP and DU-145 cell lines, whereas when used in vivo, growth of a CaP cell xenograft tumour was effectively halted at a selective dosage (Yang et al., 2015). Another PP, myricetin, possesses an aglycone structure that has been thought to attribute strongest inhibitory effects on enzymes such as DNA polymerases and DNA topoisomerase II and hence interferes with cellular proliferation activities (Shiomi et al., 2013). Myricetin has been reported to exhibit anti-tumour activity in in vitro (DU-145 and PC-3 cell lines) and in vivo (thymic nude mice) models, by promoting cell apoptosis and inhibition of cell migration and invasion (Ye et al., 2018).\n\nAnother PP, fisetin, which has two aromatic rings linked via a 3-C oxygenated heterocyclic ring with four hydroxyl groups and one oxo group, has also shown remarkable anti-cancer effects in multiple in vitro and in vivo systems. Fisetin-promoted apoptotic activation was seen in DU-145, LNCaP, and PC-3 human CaP cells (Szliszka et al., 2011). Khan & colleagues (2008) conducted a study to determine whether fisetin inhibits cell growth and induce apoptosis in human CaP cells, where the study revealed fisetin treatment decrease the viability of LNCaP, CWR22Rupsilon1 and PC-3 cells while exerting only minimal effects on normal prostate epithelial cells. Fisetin arrested the G1-phase cell cycle activity in LnCAP cells and induced cell apoptosis (Khan et al., 2008). A study by Szliszka et al. (2011) has also demonstrated fisetin’s ability to enhance cytotoxicity and apoptosis in LNCaP, DU-145 and PC-3 cells. From all of the outcomes, the PP quercetin, myricetin, and fisetin present a significant role and impact towards CaP treatment strategies via numerous pathways and this includes targeting the inhibition of 5αRs activity. An extensive study conducted by Hiipakka et al. (2002) to determine inhibition of 5αRs using varieties of polyphenols in cell-free assay and whole-cell assay, showed that PP quercetin, myricetin and fisetin were more potent against 5αR1 than 5αR2 isozyme (IC50 < 100 µM) in cell-free assay but showed little or no activity in whole-cell assay. Structure-activity relationships were also examined where it appeared that the number and position of B-ring hydroxyl groups were important for inhibitory activity against 5αR1. Many plants are reported to contain PP like quercetin, myricetin and fisetin. For example, Camellia chinensis, Allium fistulosum, Calamus scipionum, Moringa oleifera, Centella asiatica, Hypericum hircinum and Hypericum perforatum have been reported to have high contents of quercetin (Salvamani et al., 2014). High contents of myricetin has also been reported in Myrica cerifera L, Calamus scipionum, Chrysobalanus icaco L, Moringa oleifera and Aloe vera (Salvamani et al., 2014). While plants like Butea frondosa, Gleditsia triacanthos, Quebracho colorado, Curcuma longa, Rhus verniciflua, Acacia greggii and Acacia berlandieri are rich sources of fisetin (Salvamani et al., 2014).\n\nSeveral other PP have also exhibited anti-CaP effects. The effect of the PP, genistein, daidzein, and biochanin A on the growth of LNCaP and DU-145 human CaP cell lines was studied where all except daidzein inhibited the cells growth (Peterson & Barnes, 1993). Wang & colleagues (2003) studied the PP reduction effect on CaP cell proliferation and apoptotic resistance in vitro using a AT6.3 rat CaP cell line and revealed that the PP kaempferol, biochanin A, and genistein were responsible for inhibited cell proliferation in a dose-dependent manner and induced apoptotic effects, except for daidzein, which counteracted the effect (Wang et al., 2003). Szliszka et al. (2013) in their study demonstrated that biochanin A remarkably augmented selective-cancer cell cytotoxicity and apoptosis in both LNCaP and DU-145 cell lines. Many in vivo and in vitro studies have demonstrated PP’s ability as 5αRis in combating CaP (Evans et al., 1995; Hiipakka et al., 2002; Park et al., 2003). Kaempferol, biochanin A and genistein were found to be more effective as inhibitors of 5αR2 than 5αR1 in a cell-free assay as well as significantly inhibit 5αR2 in a whole-cell assay (Hiipakka et al., 2002). A previous study has also demonstrated genistein and biochanin A as potent inhibitors of 5αRs, more specifically on type 2 in human genital skin fibroblasts and BPH tissue homogenates and on type 1 in prostate tissue homogenates (Evans et al., 1995). A study that used isolated kaempferol from Camellia sinensis showed good inhibition on 5αR2 in HEK-293 cells lines that expressed both 5αRs type 1 and 2 (Park et al., 2006). Park et al. (2003) revealed that Thujae occidentalis semen (TOS) extract showed high inhibition activity on 5αR2 that were expressed in HEK-293 cell lines. Previous studies have shown that TOS extracts contain PP flavonoids, which suggests a promising potential of PP as strong inhibitors of 5αRs (Hidehiko et al., 1996). Kaempferol has been identified in many other plants including Centella asiatica, Euonymus alatus, Kaempferia galanga L, Ginkgo biloba, Equisetum spp., Tilia spp., Sophora japonica and propolis (Salvamani et al., 2014). Genistein, daidzein and biochanin A which are the isoflavones that are mostly found in soybean (Glycine max), lupin (Lupinus) and red clover (Trifolium pratense L).\n\nb) Catechin\n\nCatechin is a type of PP that is found abundant especially in green tea. Two out of four major types of catechin are discussed herein, namely epigallocatechin-gallate (EGCG) and epicatechin-gallate (ECG). An in vivo study where PC-3 and LNCaP cell lines from tumour-induced mice was injected with EGCG revealed that within seven days the EGCG rapidly inhibited the growth and reduced the size of the CaP tumours (Liao et al., 1995). Kao et al. (2000) found that EGCG reduces blood levels of T as well as prostate growth. Stadlbauer et al. (2018) studied the anti-tumour effect of ECG in vitro and demonstrated that the treatment of LNCaP and PC-3 cell lines using ECG inhibited cell viability in a dose-dependent manner. Both EGCG and ECG were also reported to have significant inhibitory effects on cell proliferation and induced apoptosis in DU-145 cells (Agarwal, 2000; Chung et al., 2001). In regard to catechin as a 5αRi, a previous study using rat liver microsomes that expressed different types of 5αRs via retroviral expression vector pMV7 system has shown that ECG and EGCG are potent inhibitors of 5αR1 but not of 5αR2 (Liao & Hiipakka, 1995). A further extensive 5αRis study by Hiipakka et al. (2002) using a similar method as previous has demonstrated that ECG and EGCG were better inhibitors against 5αR1 than 5αR2. An in vitro study by Koseki et al. (2015) showed the reduction in DHT conversion from T in 5αRs enzymatic activity in rat liver microsomes using Quercus acutissima extract where both EGCG and ECG were identified as being amongst the major components in the extract. Catechins are found in other plants such as Betula pubescens, Betula pendula, Cocos nucifera, fruit pulp of Argania spinosa and Cassia fistula (Hiipakka et al., 2002).\n\n3. Fatty acids\n\nFatty acids (FA) are monocarboxylic acids containing long hydrocarbon chains found naturally in various plants and in general can either be saturated or unsaturated (Jóźwiak et al., 2020). Saturated FA includes myristic acid (MA) and lauric acid (LA), which are a long-chain fatty acid with a 14-carbon backbone and medium-chain fatty acid with a 12-cabon backbone, respectively. Oleic acid (OA) and linoleic acid (LNA) are mono-unsaturated omega-9 FA and poly-unsaturated omega-6 FA, respectively. Toxicity profiles of FA demonstrate positive impacts on various tissues as they generally pose no significant safety concern but have only low systemic toxicity potential (Burnett et al., 2017; Karacor & Cam, 2015).\n\nThere are various studies that showed a decreased incidence of CaP with consumption of a FA-rich diet, especially from marine-derived FA, although knowledge on the effect of plant-derived FA on CaP remains limited. A clinical study that aimed to investigate the association of FA with risk of CaP in a case-control study of 209 CaP patients and 224 cancer-free men revealed that FA reduced the risk of CaP (Jackson et al., 2012). In an in vivo study by de Lourdes Arruzazabala et al. (2007) that determined the effect of coconut oil (CO), which is rich in MA and LA, on uncontrolled growth of prostate gland using Sprague-Dawley rats, it was found that CO significantly reduced the prostate growth, suggesting that CO MA/LA-rich content could be attributed to the outcomes. This is further supported by a 14-day study by Babu et al. (2010) that showed MA/LA treatment in rats significantly inhibited prostate enlargement, and a four-week study by Patil & Yadav (2016) where treatment with MA and LA in rats led to significant reduction in prostate weight and DHT level in prostate.\n\nAn in vitro study showed that LA, OA and LNA showed proliferation inhibitory effect on LNCaP cell lines (Liu et al., 2009). Another study also demonstrated LNA effects on CaP cell proliferation where it inhibited cell viability in PC-3 and LNCaP cell lines (Eser et al., 2013). Prunus Africana bark extracts, where amongst the major compounds identified are MA, LA and LNA, exhibit a very strong anti-androgenic activity and can prevent proliferation and kill CaP tumour cells (Nyamai et al., 2015). Oils of Cocoz Nucifera and Helianthus annuus contains unsaturated FA, OA, and LNA as their major components (de Lourdes Arruzazabala et al., 2007; Sheeba et al., 2015). FA therefore represent a noteworthy contribution in both prevention and treatment of CaP through animal model and cell culture studies by mediating its effect in various pathways including via the inhibition of 5αRs enzymatic activity. Raynaud et al. (2002) conducted an extensive study on Serenoa repens lipido-sterolic extracts, which are mainly constituted of FA MA, LA, OA and LNA, for its inhibitory effects on 5αR enzymatic activity. The study determined the specificity of each FA inhibitory effect on both isozymes of 5αRs that have been cloned and expressed in the baculovirus-directed insect cell expression system Spodoptera frugiperda (Sf9). The results showed OA and LNA to be more potent against 5αR1 than 5αR2, while LA was found to be potent against both 5αR1 and 5αR2, whereas, the inhibitory effect of MA was found only active against type 2 and therefore, is a potent inhibitor of 5αR2.\n\n\nDiscussion and conclusions\n\nCaP is one of the leading causes of death in men worldwide (Daniyal et al., 2014). Until today, various preventive and treatment strategies have been carried out to tackle the disease (Tindall & Rittmaster, 2008). The androgens, which are the modulator of prostate growth, are also thought to contribute to the pathogenesis of CaP and this has led to a surge of interest in studies that aim to block the activity of 5aRs, which are the enzymes responsible on converting androgen T to its potent form DHT, using available synthetic inhibitors of 5aRs resulting in androgens deprivation as part of the strategies. The idea therefore represents a valid strategy for CaP prevention and treatment. However, the use of synthetic 5αRis such as finasteride and dutasteride as 5aR activity-targeting CaP medicines continues to be widely discussed. 5aRis have been reported to have numerous adverse side effects (Erdemir et al., 2008; Hirshburg et al., 2016). Due to this, study interests have switched to finding a safer remedy with no/less harmful side effects by means of natural-derived entities found in plants as an alternative to synthetic 5αRis. Plants are constituted of numerous bioactive compounds and are proven to have various powerful medicinal properties that could contribute significantly towards a healthier life (Mohan et al., 2011; Sathishkumar & Baskar, 2014). The phytochemical PS, PP and FA are discussed in this review for their potential as CaP medicines and 5αRis. Numerous in vitro studies using different type of CaP cell lines and in vivo studies using xenograft/tumour-induced animal models have revealed the ability of PS, PP and FA as potential CaP medicines targeting various mechanisms including inhibiting cell proliferation, migration and invasion, as well as promoting selective tumour cell apoptosis. In addition, the ability of PS, PP and FA as potential naturally-derived 5αRis is also demonstrated in many studies, which further validates their exhibition of anti-5αR enzymatic activity that can produce beneficial interference in androgen-dependent CaP progression. In terms of structural similarities to current synthetic 5αRis, PS that are characterised with four ‘rings’ stand as the most promising candidate for naturally-derived 5αRis and they are found to be potent against both 5αR1 and 5αR2. PP have also demonstrated anti-5αR activity on both 5αR1 and 5αR2 despite lacking one ‘ring’. FAs that exist in either saturated or unsaturated forms do not display any structural similarities to the synthetic 5αRis, but are also reported to have significant inhibitory effect against both 5αRs. All of these observations suggest a strong implication of various phytochemicals, especially PS, PP, and FA as potential CaP medicines targeting 5αR activity. In conclusion, plants represent a reservoir of novel phytochemicals that can further provide a promising line on the development of CaP therapeutic agents, especially in targeting the inhibition of 5αR enzymes.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nAgarwal R: Cell signaling and regulators of cell cycle as molecular targets for prostate cancer prevention by dietary agents. Biochem Pharmacol. 2000; 60(8): 1051–1059. PubMed Abstract \| Publisher Full Text\n\nAggarwal S, Thareja S, Verma A, et al.: An overview on 5alpha-reductase inhibitors. Steroids. 2010; 75(2): 109–153. PubMed Abstract \| Publisher Full Text\n\nAndriole GL, Humphrey P, Ray P, et al.: Effect of the dual 5alpha-reductase inhibitor dutasteride on markers of tumor regression in prostate cancer. J Urol. 2004; 172(3): 915–919. 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Oncol Rep. 2015; 33(6): 2659–2668. PubMed Abstract \| Publisher Full Text\n\nYe C, Zhang C, Huang H, et al.: The Natural Compound Myricetin Effectively Represses the Malignant Progression of Prostate Cancer by Inhibiting PIM1 and Disrupting the PIM1/CXCR4 Interaction. Cell Physiol Biochem. 2018; 48(3): 1230–1244. PubMed Abstract \| Publisher Full Text\n\nZaloga GP: Phytosterols, Lipid Administration, and Liver Disease During Parenteral Nutrition. JPEN J Parenter Enteral Nutr. 2015; 39(1 Suppl): 39S–60S. PubMed Abstract \| Publisher Full Text\n\nZhang HY, Cui J, Zhang Y, et al.: Isoflavones and prostate cancer: A review of some critical issues. Chin Med J (Engl). 2016; 129(3): 341–347. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "81614", "date": "18 May 2021", "name": "Binesh Shrestha", "expertise": [ "Reviewer Expertise Protein Biochemistry", "Microbiology and Biotechnology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction\nParagraph 3: This can be simplified - e.g. in the second sentence, what does “limiting the production of androgen” mean? It would be clearer to say reduction or increase.\n\nParagraph 4: Similar is true here. The content of the whole paragraph would be easier to follow if it is written in simple forms. One easy way would be to break down long sentences into short sentences.\n\nParagraph 5: Please correct the typo mistakes of genus and species of some names - e.g. Sebastiania (genus starting with upper case) adenophora (species starting with lower case). Please correct the same in Table 1 as well for Prunus africana (throughout the article 3 times in Table 1 and also in text) and Cocos nucifera. For Quercus acutissima please change it to italics.\nDiscussion\nThe third sentence starting with “The androgens, which are the.......as a part of the strategies” is very long (7 lines). Please simplify by breaking it down.\nThe overall content is good and comprehensive. I believe that the researchers in the field will have a short comprehensive overview reflection from this review. With the above-mentioned corrections, I would recommend this review for indexing.\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? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "6704", "date": "14 Jun 2021", "name": "Sheikh Naeem Shafqat", "role": "Author Response", "response": "Reply to the comments made by the Reviewer (Dr. Binesh Shrestha): Thank you for reviewing and suggesting some constructive amendments to the review article. Following are the actions taken in response to the reviewer’s comments. The reviewer should be able to see all the changes made to the article in response to the comments by downloading/reviewing the latest version of the review article. The comments made by the reviewer in Paragraph 3 and Paragraph 4 regarding the complexity of written text have been addressed and now it has been rewritten in a simple language suitable for the understanding of general readers. The comments made by the reviewer in Paragraph 5 regarding the few typo mistakes, while addressing some genus and species names, and the use of italic font has been addressed and all the corrections have been made. The comments made by the reviewer in the Discussion regarding the simplification of the written text by breaking down the text into smaller sentences, for the understanding of general readers have been addressed. Thank You, Regards, Dr. Naeem Shafqat (Author of the article)" } ] }, { "id": "85610", "date": "25 May 2021", "name": "Martin L. Read", "expertise": [ "Reviewer Expertise Endocrine Cancer", "Drug Discovery", "Bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n5-alpha-Reductase inhibitors (5αRis) used in the treatment of prostate cancer are reported to have numerous adverse side effects. Here, the authors report on plant-derived alternatives that might offer favourable side effects and less toxic profiles. In particular, the authors discuss the inhibitory activities of phytochemicals on 5αRs isozymes and their structural similarities with current 5αRis used in prostate cancer therapy. Overall, this review article is clear, follows a logical narrative, and is relatively well-written. I have a few minor suggestions:\nThere are a few minor edits below that need to be made in the abstract and introduction to improve clarity:\nAbstract: “This has led to a surge of interests on plant-derived alternatives” should be “This has led to a surge of interest on plant-derived alternatives”.\nAbstract: “Therefore, in this review, we aim to discuss on the use of phytochemicals namely phytosterols” should be “Therefore, in this review, we aim to discuss the use of phytochemicals namely phytosterols”.\nIntroduction: “The latter strategy was largely anticipated, considering CaP as being hormones-driven disease” should be “The latter strategy was largely anticipated, considering CaP is a hormones-driven disease”.\nIntroduction: The authors should clarify the sentence “Testosterone (T), synthesised by the Leydig cells of the testes under the control of hypothalamus and anterior pituitary gland, is the most abundant circulating androgen in males, where from it, more potent form dihydrotestosterone (DHT) is synthesised”.\n\nIt was unclear from Table 1 which type of phytochemical (i.e., PS, PP and FA) was the most effective at inhibiting the activity of 5αRs. Would it be possible for the authors to include some information (e.g., IC50 values or equivalent) to give an indication of the relative efficacies of the different phytochemicals in the inhibition of 5αRs (type I/II)? Information on the type of prostate cancer cell lines used in each study would also be helpful to include in Table 1.\n\nThe data shown in Table 1 is based on in vitro data. It would be useful to the readers if the authors included a second table giving an overview of how the different phytochemicals (i.e., PS, PP and FA) have been studied in vivo (i.e., mice models). In particular, the table should include information on the types of prostate cancer mouse models, the type of anti-cancer mechanisms investigated, outcomes, etc. The inclusion of this table would give important context on how these phytochemicals (i.e., PS, PP and FA) have been investigated in vivo and how much more work is needed.\n\nIt would be helpful if the authors provided additional context in the discussion and included a sentence on how many (if any) of the phytochemicals (i.e., PS, PP and FA) have been investigated in human clinical trials to inhibit 5αRs, and what still needs to be done in future studies.\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": "6861", "date": "01 Jul 2021", "name": "Sheikh Naeem Shafqat", "role": "Author Response", "response": "Reply to the comments made by the Reviewer: Thank you for reviewing and suggesting some constructive amendments to the review article. Following are the actions taken in response to the reviewer’s comments: The comments made by the reviewer for the Abstract and Introduction to improve the clarity of certain phrases have now been addressed and rewritten as suggested. Abstract: “This had led to a surge of interests on plant-derived alternatives” changed to “This had led to a surge of interest on plant-derived alternatives”. Abstract: “Therefore, in this review, we aim to discuss on the use of phytochemicals namely phytosterols” changed to “Therefore, in this review, we aim to discuss the use of phytochemicals namely phytosterols” Introduction: “The latter strategy was largely anticipated, considering CaP as being hormones-driven disease” changed to “The latter strategy was largely anticipated, considering CaP is a hormones-driven disease”. Introduction: “Testosterone (T), synthesised by the Leydig cells of the testes under the control of hypothalamus and anterior pituitary gland, is the most abundant circulating androgen in males, where from it, more potent form dihydrotestosterone (DHT) is synthesised” changed to “Testosterone (T), the most abundant circulating androgen in males, is synthesised by the Leydig cells of the testes under the control of hypothalamus and anterior pituitary gland, and can further be converted to more potent form dihydrotestosterone (DHT) by the action of enzyme 5αR” The comments for Table 1 (now should be referred as Table 2) regarding the unclarity due to missing of an indication of the phytochemicals’ relative efficacies has been addressed where the IC50 of each compound is included in the table and information on the type of cell lines used in each study is also added. The suggestions made by the reviewer for the inclusion of another table highlighting the in vivo studies are appreciated and have been taken into consideration. We hereby added another table (referred to now as Table 1) that summarise the findings of anti-CaP studies of various phytochemicals, which also includes the in vivo studies. The comments made by the reviewer in the Discussion section regarding the provision of additional context focusing on human clinical trials and future directions have now been addressed: Discussion: The sentences “These findings are hoped to assist in the next stage of human clinical trials, as to date, only synthetic 5aRis are investigated in such setting. However, further isolation of these phytochemicals needs to be done especially from the plant sources before it can be implied in human clinical setting” is added." } ] } ]	1	https://f1000research.com/articles/10-221
https://f1000research.com/articles/9-1466/v1	16 Dec 20	{ "type": "Research Article", "title": "Pneumococcal nasopharyngeal carriage and antimicrobial susceptibility profile in children under five in southern Ethiopia", "authors": [ "Siraj Hussen", "Solomon Asnake", "Demelash Wachamo", "Birkneh Tilahun Tadesse", "Solomon Asnake", "Demelash Wachamo", "Birkneh Tilahun Tadesse" ], "abstract": "Background: Streptococcus pneumonia causes high morbidity and mortality, particularly in children under five. Nasopharyngeal (NP) carriage predisposes individuals to pneumococcal infection and horizontal spread within the community. Overuse of antibiotics has been linked to increased risk of antimicrobial resistance to S. pneumonia. We investigated NP carriage rate and resistance to commonly prescribed antibiotics in under-five children visiting a public referral center in southern Ethiopia. Methods: In total, 413 under 5 children who visited the outpatient department for a health check-up, immunization or acute mild illnesses underwent NP sampling. Parent/caregiver surveys were administered at the clinic. Sterile plastic applicator rayon tipped swabs were used for NP sampling. Antimicrobial susceptibility testing was performed using modified the disk diffusion method. Results: S. pneumonia NP carriage was observed in 39% [95% confidence interval (CI): 34.4–43.8]. Living with one or more sibling (AOR (adjusted odds ratio) 1.95: 95% CI: 1.01, 3.76), age group of 3-23 months (AOR 2.31: 95% CI: 1.07, 4.98), co-sleeping with family (AOR 2.09, 95% CI: 1.16, 3.79), attendance at kindergarten/day-care (AOR 1.84: 95% CI: 1.09, 3.11) and malnutrition independently increased S. pneumonia carriage at the individual level. S. pneumonia was highly resistant to Oxacillin (38.5%), Tetracycline (37.3%), and Trimethoprim-sulfamethoxazole (34.2%). Multi-drug resistance was observed in 42.2% of isolates. Conclusions: A high streptococcal NP carriage rate was observed in under-five children. The high level of resistance to commonly used antibiotics calls for enhancing national surveillance of resistance patterns and enforce antibiotic stewardship efforts.", "keywords": [ "Nasopharyngeal carriage", "Streptococcus pneumonia", "antimicrobial susceptibility", "under-five children", "Ethiopia" ], "content": "Abbreviations:\n\nEDHS = Central Statistical Agency of Ethiopia, PCV = pneumococcal conjugate vaccine, NP = nasopharyngeal, STGG = skim milk tritons glucose glycerol, and S. pneumonia = Streptococcus pneumonia.\n\n\nIntroduction\n\nStreptococcus pneumonia (pneumococcus) is a Gram-positive extracellular pathogen associated with high morbidity and mortality in children all over the world, particularly in developing countries like Ethiopia1. S. pneumonia is the most important cause of bacterial pneumonia and meningitis worldwide. For instance, in 2010, it accounted for 33% of the deaths in children under 4 years old2. In Africa, pneumococcal disease is estimated to cause nearly half a million deaths among children under five years annually3. The World Health Organization (WHO) in 2010 estimated 541,000 global child deaths due to pneumococcal infections in under 5 years children3. Ethiopia is among the countries with the highest burden of pneumonia, especially in children under five4. In 2010, 312,857 cases community acquired pneumonia and 12,284 deaths caused by S. pneumonia were reported in children under five5.\n\nPneumococcal disease often follows nasopharyngeal (NP) colonization with homologous strains. The mucosal epithelium of the nasopharynx is the primary site of pneumococcal colonization6. S. pneumonia NP carriage, a necessity for the development of the disease, is considered to be an important source of horizontal spread of this pathogen within the community7.\n\nSeveral socio-demographic and clinical characteristics including young age, family size, low income, number of siblings, and malnutrition predicted NP pneumococcal colonization8. Household and environmental factors such as overcrowding, exposure to tobacco smoke9 and exposure to indoor air pollution also increased the risk of NP colonization8. The transmission of S. pneumonia occurs through respiratory droplets or more commonly from individuals who are asymptomatic carriers6. Pneumococcus susceptible individuals may become colonized upon exposure and can remain so for weeks to months. Acquisition of invasive serotypes could lead to pneumococcal disease, commonly after a 1 to 3 day incubation period10.\n\nThe increasing frequency and rapid spread of antimicrobial resistant pneumococcal strains is a global health threat. Antimicrobial resistance has made the choice of antimicrobial agents for treatment of pneumococcal infections more complicated and costly9,11. Nasopharyngeal colonization by antimicrobial resistant S. pneumonia had been increasing in different parts of the world including Ethiopia12,13. Minimizing NP carriage rate is an important step for prevention and control of pneumococcal disease. The variable risk factors in different populations and the risk factor differences necessitate generating evidence in various settings to better understand the factors that predispose to increased risk of exposure to S pneumoniae. We aimed to investigate the prevalence and predictors of NP pneumococcal colonization as well as antimicrobial susceptibility pattern of isolates in a setting where there is a high prevalence of undernutrition and low socioeconomic status.\n\n\nMethods\n\nEthical approval was obtained from the Institutional Review Board (IRB) of College of Medicine and Health Sciences, Hawassa University (IRB reference number: IRB/006/11). The purpose and importance of the study was explained to each study participants. To ensure confidentiality of participants, data collection tools were anonymous with no participant identifiers. Participants were interviewed alone to maintain privacy. All participants were not paid for the test. Informed written consent was obtained from a parent or guardian for children to participate in the study. The study incurs no cost to the study participants and were interviewed free of charge.\n\nThe study was conducted between November 2018 and March 2019 at outpatient departments (OPD) of two public Hospitals – Adare and Hawassa University comprehensive specialized Hospitals (HUCSH) in Hawassa City, which were purposively selected to represent primary healthcare and referral facilities in the region; Adare General Hospital is a primary care facility while HUCSH is the main referral hospital in southern Ethiopia. In the study district the coverage of three doses of pneumococcal conjugate vaccine (PCV) coverage was at 61% in 201914, which showed significant improvement from the 2016 reported coverage of 53%15.\n\nSample size (n) was calculated using single proportion formula (Equation 1) assuming a prevalence (p) of 43% based on data reported in North West Ethiopia (43.8%)16 with 95% confidence interval (z=1.96) and 5% precession (d), and 10% non-response rate which resulted in a sample size of 417. A systematic random sampling method was used to select participants – every kth child was selected from a total of OPD attendees every day. The list of all children who presented to the OPDs everyday was used as a sampling frame to decide the value of k. Parents or legal authorized representatives were invited to participate in the study. The informed consent process was administered to those who agreed to participate in the study.\n\n\n\nInclusion were all under-five children who visit OPDs of the two hospitals during the study period and who consent to participate in the study.\n\nExclusion criteria included subjects who had an illness that made nasal swabbing difficult, and those with severe respiratory problems (for example acute attack of bronchial asthma), had anatomical abnormalities of the nose (e.g. cleft palate) and who were on antibiotics in the two weeks prior to the start of the study.\n\nStructured questionnaires developed for the purpose of the study, pilot tested on 5% of the sample before implementation, were used to collect information on socio-demographics, clinical data, and associated factors. Based on the results of the pilot testing of whether questions were correctly understood by the interviewers and respondents or not, the questionnaires were revised to improve clarity. The pilot testing did not reveal significant errors in the questionnaires. The tools were first developed in English (see extended data17), translated to local language (see extended data18), and back translated to English by an independent translator to ensure internal validity. In addition to interviews of parents/guardians, medical records of participants were reviewed to abstract past medical history. Trained data collector healthcare professionals administered the questionnaires to the parents or LAR in a quiet room; data collectors also measured child’s weight to the nearest 0.1kg and height/length to the nearest 1cm using electronic weighing scale and length/height board. Anthropometrics were then interpreted using WHO Z scores, where a score of <-2 is considered to indicate undernutrition.\n\nNasopharyngeal specimens were collected using sterile swabs in two replicates. One NP specimen was collected per child by gentle insertion of sterile flexible plastic applicator rayon tipped swab (Copan, Brescia, Italy, catalogue number: 26061), which was done by tilting slightly backwards and immobilize child’s head while gently restraining the child’s body. Once in place, the swab was rotated and left in place for five seconds to saturate the tip before slowly removing it. After collection, the sample was immediately placed in 1ml skimmed milk tritons glucose glycerol (STGG) transport media in tubes. Any excess samples were cut off before inoculating in the transport medium in tubes, after which the caps were tightened securely. The NP specimen was processed within 8 hours of collection and in cases where delay was encountered, it was stored at -20°C. Culturing the NP swab-STGG specimens was done on tryptone soy agar base (Oxoid, Basingstoke, Hampshire, England; Catalogue number: 105459). Briefly, the NP swab-STGG specimens were mixed thoroughly by vortexing for 30 seconds, 10 µl of the sample was then used to inoculate the plates and streaked using a sterile wire loop. The streaked plates were incubated into 5% CO2 incubator at 37°C for 24 hours. Plates were fully examined for any growth and the plates displaying no growth were re-incubated before being reported as negative. Identification of positive culture results was performed based on the appearance of colonies and the hemolytic pattern – small and watery growth surrounded by a greenish zone of alpha-hemolysis on the media.\n\nWe employed similar microbiological methods to those reported by Gebre et al.16. Briefly, to isolate pneumococci, suggestive colonies were sub-cultured and tested for optochin susceptibility and bile solubility. Optochin susceptible strains with ≥14mm in diameter zone of inhibition were identified as S. pneumonia. Next, alpha hemolytic strains with zone of inhibition <14 mm underwent bile solubility test using 2% sodium deoxycholate or bile salt base (Oxoid, Basingstoke, Hampshire, England; Catalogue number: 89904).\n\nBacterial cell suspension samples were prepared from freshly streaked presumed positive colonies of S. pneumonia in sterile normal saline. An adjusted 1ml of suspension was divided into two equal amounts of 0.5 ml in each tube. Then 0.5 ml of normal saline was added to one tube and 0.5 ml of 2% bile salt to the other tube as a test followed by incubation in 5% CO2 incubator at 37°C for up to 2 hours. A loss of turbidity in the bile tube but not in the saline control tube was considered as a positive test.\n\nDisk diffusion (modified Kirbye-Bauer) method on Mueller Hinton agar (Oxoid, Basingstoke, Hampshire, England, Catalogue number: 105437) supplemented with 5% sheep blood was employed for AST19. Standard disks of commonly used antibiotics including Tetracycline – 30µg, Trimethoprim/sulfamethoxazole – 1.25+23.75µg, Oxacillin – 1µg, Chloramphenicol – 30µg, and Erythromycin – 15µg (Oxoid, Basingstoke, Hants RG24 8PW, UK) were used antimicrobial susceptibility testing of all the isolates.\n\nFollowing inoculation of the bacteria suspension on the Mueller-Hinton agar plate, which is supplemented with 5% sheep blood agar, and then air drying, the antibiotic disks were dispensed aseptically using an automatic disk dispenser. Next, the plates were incubated in a 5% CO2 incubator at 37°C for 24 hours. Finally, zone diameters of growth inhibition were measured to the nearest millimeters using a ruler and were interpreted using cut-off points for each antibiotic disk, which range from 0.5µg/mL for vancomycin to 8µg/mL for gentamicin and tetracycline, in the Clinical and Laboratory Standard Institute (CLSI) result interpretive standards19. Categorically, results were interpreted as susceptible, intermediate, or resistant20. S. pneumonia ATCC 49619 provided by the Ethiopian National Quality Assurance Directorate (Catalogue number: 0947L) was used as a positive quality control strain for all procedures.\n\nBivariate and multivariate binary logistic regression models containing sociodemographic and clinical variables to assess independent predictors of pneumococcal NP carriage were produced. Variables with p-value <0.2 in the bivariate model were included in the multivariate model. Odds ratios and 95% confidence intervals (CI) were used to measure the association between potential risk factors and occurrence of NP carriage at the individual level. Level of significance for the multivariate models was set at p-value < 0.005. Anthropometrics were assessed following standard procedures21, Z-score of <-2.0 was used as a cut off to define wasting, stunting, and underweight for weight-for-height, height/length-for-age and weight-for-age assessments respectively22. PCV vaccination status was assessed through interviews with parents/guardians and vaccination card. All the statistical tests were performed using Stata version 14.0 (StataCorp, Texas, USA).\n\n\nResults\n\nA total of 413 children participated in the study with 99.04% response rate; 226 (54.7%) were female. Age of the children ranged from 3–59 months with mean age (standard deviation – SD) of 36.63 (18.85) months. The majority, 308 (74.6%) of the children were from an urban setting; 157 (38.0%) of the parents/guardians attended primary education; and 230 (55.7%) of the parents/guardians were housewives. More than half, 215 (52.1%), of participants were from a family who had an average monthly income of USD 30 to 60 (Table 123).\n\nUSD – United states dollar; COR – Crude Odds Ratio; AOR – adjusted odds ratio; NP – nasopharyngeal; Ref – reference\n\nThe overall prevalence of pneumococcal NP carriage rate was 39% [95% confidence interval (CI): 34.4–43.8]. The highest prevalence of NP carriage was observed in those aged 3 – 23 months (49.8%). More boys than girls had NP colonization (42.0% in girls versus 35.3% in boys). Of the study participants who lived in urban settings, 46 (43.8%) were carrier for S. Pneumonia (Table 123).\n\nIn bivariate analysis, sociodemographic variables including sex, place of residence, and age of the child had statistically significant association with pneumococcal NP carriage. Similarly, family factors including larger family size, presence of other siblings, and co-sleeping with other family members were predictors of NP pneumococcal colonization. Attendance at day care centers and presence of acute and chronic malnutrition were associated with an increased probability of NP Streptococcal colonization. Interestingly, level of PCV vaccination and lack of any vaccination were not associated with probability of NP pneumococcal colonization.\n\nNext, we constructed a multivariable regression model including variables with a p-value < 0.2 in the bivariate analysis. Being under two years of age (AOR 2.31: 95% CI: 1.07, 4.98), those living with one or more siblings (AOR 1.95: 95% CI: 1.01, 3.76), history of co-sleeping with family members (AOR 2.09, 95% CI: 1.16, 3.79) and attendance at kindergarten/day care (AOR 1.84: 95% CI: 1.09, 3.11) were found to result in an increased probability of pneumococcal NP colonization (Table 123). Children who were stunted, 2.17(1.07,4.34); and wasting, 2.68(1.58,4.55) had a higher probability of pneumococcal colonization.\n\nAntimicrobial susceptibility was determined for all 161 isolates of S. pneumonia to six commonly prescribed antimicrobial agents: Oxacillin, tetracycline, Erythromycin, TMP-SMX, and chloramphenicol. Among tested antimicrobial agents, higher rates of S. pneumonia resistance was reported in Oxacillin, 62 (38.5%); Tetracycline, 60 (37.3%) and Trimethoprim-sulfamethoxazole, 55 (34.2%). Comparatively, the lowest resistance rate was exhibited by Erythromycin, 11 (6.8%); chloramphenicol, 117(10.6%); and Vancomycin, 13(8.1). multidrug resistance to two or more antimicrobials was identified in 68 (42.2%) isolates (Table 223).\n\nNo. – Number; OPD – Outpatient Department\n\n\nDiscussions\n\nIn the current study, we showed that streptococcal colonization is a common condition. The findings highlight the importance of pneumococcus as a common cause of bloodstream infections and cause septicemia, meningitis, and pneumonia24. Ethiopia introduced the PCV vaccine in the expanded program of immunization (EPI) schedule for under five children since 2011 and the coverage has since increased. However, carriage rate of S. pneumonia and pneumococcal invasive disease remains a public health problem. The prevalence of pneumococcal nasopharyngeal carriage among children < 5 years in Hawassa City was 39% [95% confidence interval (CI): 34.4–43.8], a finding similar to reports from Jimma (43.8%)16 and Gondar (41.03 %)25. However, higher prevalence (64.8%) was reported in the Wolayita Zone of southern Ethiopia26, which is similar to carriage rates reported in other African countries, for example 64.8%27 and 65.8% of NP colonization in Kenya28. The factors affecting the variabilities in the burden of streptococcal NP colonization have yet to be explored but could include differences in socioeconomic and population characteristics29.\n\nEven though the findings in this study represent health facility data in under-five children who visited the recruitment centers for mild health conditions, routine health check-up or vaccination, a community-based NP sample collection would have enabled inference in to the general population of under-five children. Furthermore, both recruitment facilities are urban hospitals which could limit representation of rural communities. However, the study included a significant proportion of rural residents since both hospitals have both urban and rural catchments.\n\nIn this study, NP carriage of S. pneumonia was significantly lower among children whose aged 24–41 months old, a finding similar to other studies in Arsi zone, South East Ethiopia30 and Gondar, North West Ethiopia25. The decline in S. pneumonia colonization rate with increasing age maybe due to the progressive acquisition of mucosal immunity as a result of repeated colonization by several serotypes and the potential reduction of exposure31. Supporting this argument, our study showed that co-sleeping with other family and living with one or more siblings is associated with increased odds of NP colonization. Similar findings were reported in elsewhere26,30.\n\nThe high resistance to Tetracycline (37.8%) was consistent with other studies which reported in Gondar (33.2%) and Hawassa (42.6%)16,31, but was lower than prevalence of tetracycline resistant of S. pneumonia isolates from Wolayta Sodo (48.9%) and Jima (53.2%)16,32. The high resistance to tetracycline maybe due to widespread inappropriate prescription, which exerts selection pressure for the presence of increased tetracycline-resistant bacterial isolates. S. pneumonia isolates were also resistant to Trimethoprim-sulfamethoxazole and Oxacillin, which is in agreement with other reports16,31, reflecting local and regional antimicrobial use practices. Oxacillin is often used for soft tissue infections and injuries, including for road traffic accidents.\n\nA worrying finding in our study was the high prevalence (42.2%) of multi-drug resistant S. pneumonia isolates (i.e. resistant to two or more drugs), which could be linked to mobile genetic units (including plasmids, gene cassettes in integrons and transposons)32, lack of effective medicines, inappropriate dispensing, medication sharing, counterfeit drugs, bacterial evolution, climate changes, lack of medical practitioner with proper training, poor-quality and unhygienic sanitary conditions33.\n\n\nConclusion and Recommendation\n\nThe prevalence of pneumococcal Nasopharyngeal Carriage in the study area was high. The proportion of drug resistance to Tetracycline, Trimethoprim-sulfamethoxazole and Oxacillin was very high. Younger age, co-sleeping with family and living with one or more sibling independently predicted the probability of pneumococcal Nasopharyngeal carriage. The results of the study will have critical input to enforce antimicrobial stewardship efforts in the study area and beyond. Furthermore, surveillance of carriage and antimicrobial resistance in different populations will help to formulate targeted interventions.\n\n\nData availability\n\nFigshare: Last Pneum Referal2.sav siraj Dem F.sav. https://doi.org/10.6084/m9.figshare.13297724.v123\n\nThis project contains the following underlying data:\n\nLast Pneum Referal2.sav siraj Dem F.sav (Deidentified nasopharyngeal colonization data)\n\nFigshare: Questionnaire (English Version).docx. https://doi.org/10.6084/m9.figshare.13297781.v117\n\nThis project contains the following extended data:\n\nQuestionnaire (English Version).docx\n\nFigshare: Amharic version of the questionnaire. https://doi.org/10.6084/m9.figshare.13356641.v118\n\nThis project contains the following extended data:\n\nQuestionnaire_Translated version (Amharic).docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nFirst and foremost we would like to express our deepest thanks and gratitude to Hawassa University College of Medicine and Health sciences for giving us the opportunity to do this research.\n\n\nReferences\n\nHenriques-Normark B, Tuomanen EI: The pneumococcus: epidemiology, microbiology, and pathogenesis. Cold Spring Harb Perspect Med. 2013; 3(7): a010215. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Introduction of pneumococcal vaccine PCV13: a handbook for district and health facility staff. In.: World Health Organization, 2013. Reference Source\n\nO'Brien KL, Wolfson LJ, Watt JP, et al.: Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet. 2009; 374(9693): 893–902. PubMed Abstract \| Publisher Full Text\n\nJohansson KA, Memirie ST, Pecenka C, et al.: Health gains and financial protection from pneumococcal vaccination and pneumonia treatment in Ethiopia: results from an extended cost-effectiveness analysis. PLoS One. 2015; 10(12): e0142691. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRudan I, O’Brien KL, Nair H, et al.: Epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries. J Glob Health. 2013; 3(1): 010401. PubMed Abstract \| Free Full Text\n\nSimell B, Auranen K, Käyhty H, et al.: The fundamental link between pneumococcal carriage and disease. Expert Rev Vaccines. 2012; 11(7): 841–55. PubMed Abstract \| Publisher Full Text\n\nDagan R, O'Brien KL: Modeling the association between pneumococcal carriage and child-care center attendance. The University of Chicago Press. Clin Infect Dis. 2005; 40(9): 1223–1226. PubMed Abstract \| Publisher Full Text\n\nBogaert D, de Groot R, Hermans PWM: Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis. 2004; 4(3): 144–54. PubMed Abstract \| Publisher Full Text\n\nFarida H, Severin JA, Gasem MH, et al.: Nasopharyngeal carriage of Streptococcus pneumonia in pneumonia-prone age groups in Semarang, Java Island, Indonesia. PLoS One. 2014; 9(1): e87431. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernatoniene J, Finn A: Advances in pneumococcal vaccines: advantages for infants and children. Drugs. 2005; 65(2): 229–55. PubMed Abstract \| Publisher Full Text\n\nCenters for Disease Control and Prevention: Antibiotic resistance threats in the United States, 2013. Centres for Disease Control and Prevention, US Department of Health, 2013. Reference Source\n\nBayer M, Aslan G, Emekdaş G, et al.: Nasopharyngeal carriage of Streptococcus pneumoniae in healthy children and multidrug resistance. Mikrobiyol Bul. 2008; 42(2): 223–30. PubMed Abstract\n\nTadesse BT, Ashley EA, Ongarello S, et al.: Antimicrobial resistance in Africa: a systematic review. BMC Infect Dis. 2017; 17(1): 616. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEMDHS: Mini Demographic and Health Survey. 2019; [Accessed: 22 August 2020]. Reference Source\n\nEDHS: Ethiopian Demographic and Health Survey. 2016; [Accessed on 22 August 2020]. Reference Source\n\nGebre T, Tadesse M, Aragaw D, et al.: Nasopharyngeal carriage and antimicrobial susceptibility patterns of Streptococcus pneumoniae among children under five in Southwest Ethiopia. Children (Basel). 2017; 4(4): 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTadesse BT: Questionnaire (English Version).docx. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.13297781.v1\n\nTadesse BT: Amharic version of the qustionnaire. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.13356641.v1\n\nWayne P: Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. CLSI Document M100-S24. Wayne (Pennsylvania): Clinical and Laboratory Standards Institute. 2014. In.: Clinical and Laboratory Standards Institute Washington, DC; 2014. Reference Source\n\nWorld Health Oranization: Manual for the laboratory identification and antimicrobial susceptibility testing of bacterial pathogens of public health importance in the developing world. In. Geneva, Switzerland: WHO Press; 2003. Reference Source\n\nWHO: WHO Child Growth Standards: Methods and development. [Accessed on 23 August 2020]. Reference Source\n\nWHO: Child growth standards. [Accessed on 24 August 2020]. Reference Source\n\nTadess BT: Last Pneum Referal2.sav siraj Dem F.sav. figshare. 2020. http://www.doi.org/10.6084/m9.figshare.13297724.v1\n\nÖzdemir H, Çiftçi E, Durmaz R, et al.: Risk factors for nasopharyngeal carriage of Streptococcus pneumoniae in healthy Turkish children after the addition of heptavalent pneumococcal conjugate vaccine (PCV7) to the national vaccine schedule. Turk J Pediatr. 2013; 55(6): 575–83. PubMed Abstract\n\nAssefa A, Gelaw B, Shiferaw Y, et al.: Nasopharyngeal carriage and antimicrobial susceptibility pattern of Streptococcus pneumoniae among pediatric outpatients at Gondar University Hospital, North West Ethiopia. Pediatr Neonatol. 2013; 54(5): 315–21. PubMed Abstract \| Publisher Full Text\n\nWada FW, Tufa EG, Berheto TM, et al.: Nasopharyngeal carriage of Streptococcus pneumoniae and antimicrobial susceptibility pattern among school children in South Ethiopia: post-vaccination era. BMC Res Notes. 2019; 12(1): 306. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdegbola RA, DeAntonio R, Hill PC, et al.: Carriage of Streptococcus pneumoniae and other respiratory bacterial pathogens in low and lower-middle income countries: a systematic review and meta-analysis. PLoS One. 2014; 9(8): e103293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdullahi O, Karani A, Tigoi CC, et al.: The prevalence and risk factors for pneumococcal colonization of the nasopharynx among children in Kilifi District, Kenya. PLoS One. 2012; 7(2): e30787. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBozio C: Risk factors for bacterial load of pneumococcal colonization among peruvian children. In.: Emory University. 2011.\n\nHaile AA, Gidebo DD, Ali MM: Colonization rate of Streptococcus pneumoniae, its associated factors and antimicrobial susceptibility pattern among children attending kindergarten school in Hawassa, southern Ethiopia. BMC Res Notes. 2019; 12(1): 344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Brien KL, Nohynek H; World Health Organization Pneumococcal Vaccine Trials Carriage Working Group: Report from a WHO Working Group: standard method for detecting upper respiratory carriage of Streptococcus pneumoniae. Pediatr Infect Dis J. 2003; 22(2): e1–e11. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Antimicrobial resistance: global report on surveillance. World Health Organization, 2014. Reference Source\n\nChurch DL: Major factors affecting the emergence and re-emergence of infectious diseases. Clin Lab Med. 2004; 24(3): 559–86. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "76205", "date": "21 Jan 2021", "name": "Abate Yeshidinber Weldetsadik", "expertise": [ "Reviewer Expertise Paediatric", "paediatric pulmonary and critical care", "quality of health care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHussein and colleagues reported a study on \"Pneumococcal nasopharyngeal carriage and antimicrobial susceptibility profile in children under five in southern Ethiopia\". They underwent nasopharyngal swab culture in 413 children and found out that streptococcal carriage is common and especially increased in selected group of children with risk factors. They also reported high level of bacterial resistance.\nWhile it is similar to previous studies from similar setting, the study adds to the current knowledge in the region. However, their culture was determined for a group of antibiotics which are not primarily used to treat strept infection including penicillin and cephalosporins. The conclusion of strept infection as a common cause of sepsis and other infection, while a possibility and a clear risk, is also over-calling of the result in this context. These two limitations should be mentioned and rephrasing would make the paper more acceptable.\nLanguage edit is also strongly recommended.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6660", "date": "29 Jun 2021", "name": "Birkneh Tilahun Tadesse", "role": "Author Response", "response": "We thank the reviewer for taking the time to review our paper. As suggested, copyediting was done by a native speaker. We have reworded the first sentence of the discussion to indicate the possibility of severe infections following Streptococcal colonization." } ] }, { "id": "79335", "date": "09 Mar 2021", "name": "Ritah F. Mutagonda", "expertise": [ "Reviewer Expertise Infectious disease particularly malaria and HIV", "pharmacokinetics and pharmacogenomics studies", "pharmacodynamics research." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work addresses a very pertinent issue which is the prevalence of pneumococcal nasopharyngeal carriage and antimicrobial susceptibility profile in children under five in southern Ethiopia. With the increase in antimicrobial resistance which is currently a global health concern the information obtained from antimicrobial surveillance studies give us the clue of the current trends in pathogen antimicrobial resistance in different populations which will enable the development of targeted approaches to help control antimicrobial resistance. There are recent similar studies conducted in Ethiopia that are not featured in this manuscript eg. Negash et al. 20191.\nThere are minor comments which need to be addressed to improve the quality of the manuscript as described below:\nAbstract:\nThe summarized methodology sub-section needs to be improved a bit by adding information with regards to the study design, the study site, study duration, sampling technique and how data was analysed so that the reader can easy grasp this information when reading the abstract.\n\nBe consistent with the way you present information. Example in the methodology it is written ‘under 5’ while in the conclusion it is written ‘under-five’.\nIntroduction:\nInconsistency of how you write under-five’ is also observed here.\n\nI do understand there is pneumococcal vaccine (PCV) which is given to children. This information is presented in the results section so I think it will be very informative to describe a bit in the introduction section as one of pneumococcal preventive measures. Could also describe the coverage which might have an impact on the pneumococcal carriage rate in children.\nMethodology:\nI suggest that the Ethics approval and consent to participate sub-section should come after the data analysis part.\n\nAdd subsection describing the study design --- analytical cross-sectional study.\n\nIn data analysis, describe how you were able to analyse antimicrobial susceptibility test results. State the dependent and independent variables. Please remove this sentence “PCV vaccination status was assessed through interviews with parents/guardians and vaccination card.” It does not fit under data analysis part.\nResults:\nRemove this sentence “Next, we constructed a multivariable regression model including variables with a p-value < 0.2 in the bivariate analysis.” Under the predictors of nasopharyngeal carriage part.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6661", "date": "29 Jun 2021", "name": "Birkneh Tilahun Tadesse", "role": "Author Response", "response": "We thank the reviewer very much for taking the time to review our paper and for the important suggestions. We agree with the importance of keeping consistency using such terms as “under-five” and we have revised throughout the paper to conform to that. We also appreciate the comment on including more information regarding the introduction of PCV in Ethiopia and we have included a paragraph in the introduction to address that. We have also made the requested formatting changes while trying to conform to the Journal's formatting requirements. Please note that the final formatting was based on the requirement of the Journal. The sentence regarding the regression models was included to ensure that there is a good flow." } ] } ]	1	https://f1000research.com/articles/9-1466
https://f1000research.com/articles/10-534/v1	05 Jul 21	{ "type": "Research Article", "title": "Fecal calprotectin as a predictor of gastrointestinal immune-related adverse events (CF-19): A prospective study.", "authors": [ "Ana Cardeña Gutiérrez", "Xabier Mielgo Rubio", "Manuel Ruiz Muñoz", "Ruth Martinez Cabañes", "Diana Moreno Muñoz", "Susana Hernando Polo", "Clara Olier Garate", "Alicia Hurtado Nuño", "Verónica Sotelo Peña", "Maria Virginia Sánchez Becerra", "Andrea María González López", "Mónica Esteban García", "Teresa Robles Bermejo", "Elia Pérez Fernández", "Juan Carlos Cámara Vicario", "Xabier Mielgo Rubio", "Manuel Ruiz Muñoz", "Ruth Martinez Cabañes", "Diana Moreno Muñoz", "Susana Hernando Polo", "Clara Olier Garate", "Alicia Hurtado Nuño", "Verónica Sotelo Peña", "Maria Virginia Sánchez Becerra", "Andrea María González López", "Mónica Esteban García", "Teresa Robles Bermejo", "Elia Pérez Fernández", "Juan Carlos Cámara Vicario" ], "abstract": "Background: Colitis is a frequent immune-related toxicity, without any biomarker that may predict its onset. It is endoscopically similar to intestinal inflammatory diseases, where fecal calprotectin (FC) is used as a biomarker to early-detect a relapse. We found contradictory evidence about FC and immunotherapy and no prospective study was already published.\n\nMethods: We present an analytical, observational and prospective study of one year’s duration. We analyzed FC basal, and then prior to each cycle until the sixth, ending with quarterly follow-up. For evaluating the predictive value of FC we estimated the area under the ROC curve for basal absolute values and for each cycle, and calculated its relative percentage change with respect to basal. We also planned to estimate sensitivity, specificity and predictive values indexes for different cut-off points. Because of lack of recruitment we did a preliminary analysis at the end of the initially estimated period before suggesting its prolongation.\n\nResults: 24 patients (19 male) were included in the study. This included n=15 diagnosed with lung cancer, head and neck, renal, bladder and colorectal cancer (n=2, each), and melanoma (n=1). They were treated with Anti PD-1/PDL-1 mono therapy (n=18), combo with chemo (n=2), or combo with anti-CTLA4 (n=2). Three patients had G1 colitis and two, >=G2, all treated with anti-PD1 and before 6th cycle, as described on literature. ROC curve presents AUC 0,559 (CI95%:0,32-0,798) and RR for colitis taking FC value is 1,001 for each 10 units (p=0,493).\n\nConclusion: Even though we must take into account the limitations of the study we cannot conclude that FC could be used as a predictor for detecting immune-mediated colitis.", "keywords": [ "Immunerelated adverse event (irae)", "Biomarker predictors", "Fecal calprotectin", "colitis", "immune checkpoint inhibitors" ], "content": "1. Introduction\n\nImmunotherapy has reached oncology to stay, and a new class of immune-mediated adverse effects (irAEs) has emerged, colitis being one of the most frequent.1,2 It is still unknown if any biomarker may predict irAEs onset, even though they resemble classic autoimmune diseases and some data suggests that some of the parameters that we use in this scenario could be useful in predicting this kind of toxicity.\n\nFor example, a phase I study found that adding BCG to ipilimumab in stage III-IV melanoma (n = 15), describes a remarkable increase of auto-antibodies (ImmunomeTM protein array) just before the development of severe irAEs.3 However, there are not prospective studies designed to validate this observation. Also, a retrospective study from a French group describes that CRP (C-reactive protein) increase and hemoglobin and albumin decrease are the main alterations that can be observed in patients diagnosed with immune-mediated colitis during anti-CTLA4 treatment (n = 27).4 Furthermore, during ESMO (European Society for Medical Oncology) 2018 Congress some groups showed that the increase in neutrophil/lymphocyte ratio decreases immune-related colitis risk. It was also found that decreasing absolute lymphocyte count and increasing monocytes and eosinophyles correlates with a irAEs risk increase (n = 130).5 However, all these parameters could be affected by other illnesses that may be associated with our oncological patients and for that reason, they do not seem sufficiently conclusive to change our daily-clinical practice.\n\nFocusing our attention on gastrointestinal toxicity and on the French study mentioned before,4 they concluded that endoscopic lessons from immune-related colitis were very similar in comparison with the ones that appear during inflammatory intestinal diseases like Ulcerative Colitis or Crohn’s disease, some also with extra-intestinal manifestation associated. In eight patients of the study, fecal calprotectin (FC) was analyzed, and it was remarkably high in all of them (1755 μg/g [299–12900]), just like in patients diagnosed with intestinal inflammatory diseases.6 Furthermore, their condition is improved if we use the same treatments that are approved for this illness (corticosteroids, infliximab, vedolizumab).7-9 Nevertheless, Berman et al.10 cast FC aside as a predictive tool based on the data of Weber’s study,12 where FC is measured in two groups (ipilimumab + placebo vs ipilimumab + rectal budesonide), but this data could have been affected by the use of corticosteroids in one of the groups. Thus the evidence in the literature is contradictory.\n\nWe know that the use of CF as a predictor for intestinal inflammatory disease relapse is globally accepted and may diagnose relapse 3 months before the onset of symptoms (S 78%, E 73%).6 However, there are no prospective studies that analyze FC value as gastrointestinal irAEs predictor.\n\nTaking into account that immune-mediated colitis could be endoscopically compared with inflammatory intestinal diseases, we hypothesize that FC may be useful to predict severe immune-related colitis.\n\n\n2. Methods\n\nBetween May 2019 and December 2019, recruitment was open to participate in a prospective, analytical, observational and multi-centric study, following approval by our institution’s Committee for Clinical Research (H. Universitario Fundación Alcorcón). None of these dropped out of the study. The follow-up was done by the investigators in every oncology consultation that the patient needed. The data manager of the study registered all clinical and analytical data in an excel database after each appointment. All the clinicians followed the regular clinical practice guidelines. This is an observational study to analyze if fecal calprotectin can be considered as a good biomarker to predict immune related colitis, and the control cases were those that did not developed the event (colitis). The monitoring ended on April 2020, assuring a minimum of 4 months follow-up for all subjects included, making sure that we covered the weeks of higher probability to develop immune-related colitis.\n\nPatients were recruited at the time of their first visit in medical oncology and were included if they were going to receive immunotherapy (both anti-CTCLA4 and anti-PD-1/PDL-1). All tumors and stages could be considered for inclusion. Also, signed informed consent was obtained from all participants.\n\nKey exclusion criterion were previous diagnosis of inflammatory intestinal disease and regular treatment with NSAIDs (Nonsteroidal anti-inflammatory drugs). NSAIDs could alter FC values because they could cause enteropathy themselves. Moreover, NSAIDs are related with higher risk of developing immune-mediated colitis – in one particular study healthy volunteers who took diclofenaco for two weeks experienced FC elevation, which normally occurs two weeks after stopping medication.4 For this reason, we only included patients who had not taken any NSAIDs in the two weeks prior to beginning the study.12 Furthermore, there is evidence that healthy volunteers who received acetylsalicylic acid (100 mg/day) present significant elevation of CF. Those levels were low (<60μg/g) and considering the importance of antitrombotic treatment in higher risk patients, stopping this treatment is not justified.13 However, we will take this into account.\n\nPatients who met the inclusion criteria and signed informed consent had a basal recording taken of their FC, CRP and absolute number of Hemoglobin, Neutrophils, Eosinophils, Lymphocytes and Monocytes. Then, sub-investigators included those parameters in every blood test required by standard clinical practice until cycle number 6. Afterwards, if patients had not developed immune-mediated colitis, they continued their normal follow-up in consultations, and had to repeat these parameters every three months until the end of the study or the development of immune-mediated colitis, as the purpose of this project was predicting the event. The main objective of this study was serial evaluation of FC in every patient who needs immunotherapy from the participant centers. If they developed immune-mediated gastrointestinal toxicity, we used this data in order to analyze if there was a previous elevation of this parameter before the development of any symptoms.\n\nOur hypothesis was that the determination of FC could be used as a predictive marker of immune mediated colitis. If this had been demonstrated, we might be able to propose an early treatment to avoid toxicity ≥ grade 2 that could force the suspension of the treatment, and as a result, deprive patients of its potential benefit.\n\nWe also took into account the administration of antibiotics during immunotherapy treatment because it is known that this affects the treatment’s efficiency, and could also affect gut mycrobiome and in consequence, the development of immune-mediated colitis.\n\nClinicians who participated in this study did not take the value of FC into consideration. This did not affect their standard clinical practice.\n\nOur data was described by absolute and relative frequency for qualitative variables and by mean, standard deviation or median and interquartilic range taking into account data distribution for quantitative variables.\n\nFor evaluating predictive value of FC we estimated the AUC [Area Under the ROC (receiver operating characteristic) curve] for basal absolute values and for each cycle, and calculate its relative percentage change with respect to basal (Cciclox-Cdx)/Cdx. Also, we estimated sensitivity, specificity and predictive values indexes for different cut-off points.\n\nIn order to study whether the change of FC during the first six cycles of treatment differed between groups with or without colitis, we used Mixed Models with time as repeated measure, group as fixed factor and their interaction. A statistically significant interaction effect indicates a different trend in the marker for each group. Analogous methodology was used to study the rest of the remaining laboratory markers.\n\nAll statistical tests were 2-sided and probability values of <0.05 were considered statistically significant. All tests were performed using the SPSS 17.0 statistical package for Windows (SPSS Inc, Chicago, IL, USA). The same análisis can be performed on the open-acess software R.\n\nWe calculated sample size using Epidat 4.2, July 2016. Consellería de Sanidade, Xunta de Galicia, España; Organización Panamericana de la Salud (OPS-OMS); Universidad CES, Colombia. ESMO guidelines estimate an incidence of immune-mediated colitis for anti-CTL4 ranging between 27-54%. So, to estimate a sensitivity about 80% with a precision of 10% and 95% confidence level, we need a sample size of 114 patients.\n\nUnfortunately, recruitment was harder than expected because of the large number of patients that had to be excluded for taking NSAIDs, and also, because of the lack of recruitment from the other centers that were included. Furthermore, some patients complained that having to bring stool samples was too disgusting, and sometimes, they missed the delivery. Other patients did not bring all the samples because of opioid-induced constipation. For this reason, we did a preliminary analysis at the end of the initially estimated period before suggesting its prolongation.\n\nEthical approval\n\nEthical approval for this study was granted by the ethical committee for medical research (Comité Ético de Investigación con Medicamentos) of the Hospital Universitario Fundación Alcorcón, and signed by the secretary of this committee.\n\nWe assured confidentiality in the data management with an anonymizated database with verified entry. Personal data was confidential and biological samples were identified with a unique code and only investigators will have access. We applied organic law on data protection and digital rights guarantee (Organic Law 3/2018, 5th December, BOE 2018;294, 6th December: 119788-119857). We asked participants to sign informed consent as established by on biomedical research Law 14/2007 (BOE 4-VII-2007). Also, there were either no benefits related with the participation in this study and no additional risk. The benefit of this study is just the data that could be useful for the scientific community to improve management of immune-related colitis.\n\nPatient consent\n\nWritten informed consent for publication of the patients’ details was obtained from the patients.\n\n\n3. Results\n\n24 patients were included, 19 of those were male. The primary origin of the tumors included were, from higher to lower frequency: lung (n = 15), head and neck, kidney and bladder (n = 2, each), and colorectal and melanoma (n = 1, each). Almost all of the patients received monotherapy with anti-PD-1/PDL-1 (n = 18) -nivolumab, pembrolizumab, atezolizumab, durvalumab-. Four of them, in combination with chemotherapy (carboplatin or cisplatin with pemetrexed and pembrolizumab). Two patients received anti-CTLA4, ipilimumab, in combination with nivolumab.\n\nFor 15 patients, immunotherapy was the first line of treatment for their pathology (2nd line n = 7, and 3rd line n = 2), so, for the majority of subjects included, previous treatments could not have disrupted our results by affecting their microbiome.\n\nOnly two patients stopped their treatment due to toxicity (one of them because of immune-mediated colitis). Other reasons to stop immunotherapy were progressive disease (n = 4), death (n = 2), COVID-19 (n = 1), and social issues (n = 1).\n\nFocusing our attention on the main objective of this study, we registered three grade 1 colitis and two ≥ grade 2 (one grade 2 and other, grade 3). All colitis observed appeared during anti-PD1 treatment, always before cycle number six, according to the already published temporality, that suggests immune-related colitis peaks between 8-12 weeks.\n\nNo patient was using NSAIDs before being included in this study (according to the exclusion criteria). However, four of them took NSAIDs during immunotherapy treatment, and one of them developed grade 1 colitis. The rest of the patients did not suffer from toxicity, but we registered modifications on their FC values during NSAIDs administration.\n\nThe ROC curve (Figure 1) presents an AUC 0,559 (IC95%:0,32-0,798) and colitis RR (Relative Risk) taking into account that FC value is 1,001 for each 10 units (p = 0,493). Furthermore, Figure 2 shows the FC value distribution comparing patients who have developed colitis with patients that have not. We did not find a statistical difference. In group 1 (the ones that did not develop the condition), FC median value was 63,5 mg/kg (31,9-172). In contrast, in the group of patients that developed colitis, FC median value was 124 mg/kg (5-1119). We could not find an explanation for the extreme value that was registered for one of the patients who, in addition, did not develop severe immune-related colitis.\n\n\n4. Discussion\n\nTaking into account the already published data about FC and the urgent need to find biomarkers that may predict immune-mediated toxicity, this study seemed promising. Also, it is the first prospective study that analyzes this issue, and we expected to find a biomarker that allowed us to not stop potentially effective treatments14–16 for patients with limited options. However, some factors may have contributed to not having reached our main objective.\n\nFirst of all, it is true that the small sample size recruited may reduce the power of the study, and having completed the estimated size and having included patients in all the centers that we planned, would have been extremely interesting to observe the number of immune-related colitis and to show stronger conclusions. However, if we analyze the figures above we cannot find any trend towards statistical significance, and we are afraid that increasing the number of patients included would not produce more significant results. Furthermore, if we analyze the only patient that was diagnosed with severe immune-related colitis we can see that FC was not only not increased before the development of the toxicity, but also, it was even decreasing until very close to the event (Figure 3).\n\nIn addition, and after analyzing Figure 3 we find one of the other big problems that we faced during the development of this study was lost values. Oncological patients, due to either their illness, related symptoms, or and palliative treatment as opioids, suffer from constipation. Usually, patients were not able to comply with the timeline, and sometimes, they decided not to bring us the sample because they found it excessively disgusting even though we tried to transmit its importance. These lost values could also have affected to the final results.\n\nSecondly, the exclusion criteria of “not being under NSAIDs” was very problematic during recruitment. The vast majority of potential candidates were under this kind of treatment and taking into account the complexity of oncological patients and that pain is usually their main symptom, NSAIDs are a useful treatment that we cannot easily avoid. FC value is easily altered in this context as we showed before and because of that reason, it was considered to be an exclusion criterion. However, and despite remembering its importance in every visit, some patients started NSAIDs during the course of this study. We registered and analyzed this fact, but luckily, it did not significantly affect our results.\n\nAnother fact that may be related to the high variability of FC in our study is the microbiome as well as inflammatory activity related with tumors and with immunotherapy,17 and its uncertain evolution. There are different ongoing studies that are trying to analyze if the microbiome can modulate or even improve the efficacy of some treatments.18,19\n\nTo sum up, and taking into account the huge complexity that we had to confront to include patients, the extensive variability of FC, especially in oncological patients, and also, the high number of lost values, we consider that extending this study would not contribute to clarifying results in favor of FC as a predictor biomarker for immune-related colitis.\n\n\n5. Conclusion\n\nEven though we must take into consideration our study limitations (unicentric, small sample size), we cannot conclude that FC would be useful as predictive biomarker for immune-related colitis development. Also, we consider that increasing our sample size and extending this study would not clarify results in favor of FC. Furthermore, and thanks to the expertise that this study brought to us, we consider that FC is not a good parameter to use in daily clinical practice in this context because of its enormous variability, unpredictability, and also the effort that it entails to measure in oncological patients. We will continue looking for predictive biomarkers able to predict immune-related toxicity in order to try to design studies that may offer early treatment to avoid stopping potentially effective treatments.\n\n\nAuthor contributions\n\nAna Cardeña was the beneficiary of the grant that covered this project and consequently, its main coordinator. She was the protocol designer, the informed consent writer, and an active participant during recruitment, data entry and final analysis. Also, she is the author of the complete manuscript. Xabier Mielgo contributed to the protocol design and was second supervisor of the study activity and during the writing of the manuscript. Manuel Ruiz helped to focus the initial idea and also, participated during designing of the protocol that was sent to apply for the grant and finally, contributor for designing of future studies where we could use FC as predictor. Ruth Martinez contributed during the data entry and designing of CRF. Elia Pérez helped with the statistical design of the protocol and final data interpretation. Juan Carlos Cámara is the head of our investigator team and supervised all the process and also, participated actively in the patient recruitment. The rest of the authors contributed equally during recruitment and management of our patients in consultations and oncology ward.\n\n\nData availability\n\nDryad: Fecal calprotectin as a predictor of gastrointestinal immune-related adverse events (CF-19): A prospective study - Excel data and supplementary tables, https://doi.org/10.5061/dryad.rbnzs7hbk.20\n\nThis project contains the following underlying data:\n\n• Excel data: raw data from each of the cycles that was given to each patient.\n\n• Supplementary tables: Summary tables with information about patients characteristics during our study.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nDougan M: Checkpoint Blockade Toxicity and Immune Homeostasis in the Gastrointestinal Tract. Front Immunol. 2017; 8: 1547. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaanen JBAG, Carbonnel F, Robert C, et al.: Management of toxicities from immunotherapy: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2017; 28(suppl_4): iv119–iv142. PubMed Abstract \| Publisher Full Text\n\nAndrews MC, Blackburn JM: Autoantibodies May Predict Immune-Related Toxicity: Results from a Phase I Study of Intralesional Bacillus Calmette–Guérin followed by Ipilimumab in Patients with Advanced Metastatic Melanoma. Front Immunol. 2018; Mar 2; 9: 411. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarthey L, Mateus C, Mussini C, et al.: Cancer Immunotherapy with Anti-CTLA-4 Monoclonal Antibodies Induces an Inflammatory Bowel Disease. J Crohns Colitis. 2016; 10(4): 395–401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIacono D, Vitale MG, Cortiula F, et al.: Serum markers as predictors of immune checkpoint inhibitors (ICI) related adverse events in a real- world scenario. En: ESMO 2018 Congress. Munich, Germany; 19-23 October 2018. 1198P. Publisher Full Text\n\nSipponen T, Kolho KL: Fecal calprotectin in diagnosis and clinical assessment of Inflammatory Bowel Disease. Scand J Gastroenterol. 2015 Jan; 50(1): 74–80. PubMed Abstract \| Publisher Full Text\n\nDai C, Jiang M, Sun M: Fecal markers in the management of inflammatory bowel disease. Postgrad Med. 2018 Oct 3; 130(7): 597–606. PubMed Abstract \| Publisher Full Text\n\nvon Roon AC, Karamountzos L, Purkayastha S, et al.: Diagnostic Precision of Fecal Calprotectin for Inflammatory Bowel Disease and Colorectal Malignancy. The Am J Gastroenterol. 2007 Apr; 102(4): 803–813. PubMed Abstract \| Publisher Full Text\n\nBergqvist V, Hertervig E, Gedeon P, et al.: Vedolizumab treatment for immune checkpoint inhibitor-induced enterocolitis. Cancer Immunol Immunother. 2017; 66(5): 581–592. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerman D, Parker SM, Siegel J, et al.: Blockade of cytotoxic T-lymphocyte antigen-4 by ipilimumab results in dysregulation of gastrointestinal immunity in patients with advanced melanoma. Cancer Immun. 2010 Nov 24; 10: 11. PubMed Abstract \| Free Full Text\n\nWeber JS, Kähler KC, Hauschild A: Management of Immune-Related Adverse Events and Kinetics of Response With Ipilimumab. J Clin Oncol . 2012; 30(21): 2691–2697. PubMed Abstract \| Publisher Full Text\n\nRendek Z, Falk M, Grodzinsky E, et al.: Effect of oral diclofenac intake on faecal calprotectin. Scand J Gastroenterol . 2016 Jan; 51(1): 28–32. PubMed Abstract \| Publisher Full Text\n\nGuardiola J, Lobatón T, Cerrillo E, et al.: Recomendaciones del Grupo Español de Trabajo en Enfermedad de Crohn y Colitis Ulcerosa (GETECCU) sobre la utilidad de la determinación de calprotectina fecal en la enfermedad inflamatoria intestinal. Gastroenterol Hepatol . 2018; 41(8): 514–529.\n\nBarnhill JW: DSM-5® Clinical Cases . Washington: American Psychiatric Publishing; 2014.\n\nDougan M: Gastrointestinal and Hepatic Complications of Immunotherapy: Current Management and Future Perspectives. Curr Gastroenterol Rep. 2020 Mar 17; 22(4): 15. PubMed Abstract \| Publisher Full Text\n\nNagasaka M, Sexton R, Alhasan R, et al.: Gut microbiome and response to checkpoint inhibitors in non-small cell lung cancer-A review. Crit Rev Oncol Hematol . 2020 Jan; 145: 102841. PubMed Abstract \| Publisher Full Text\n\nDougan M: Understanding and Overcoming the Inflammatory Toxicities of Immunotherapy. Cancer Immunol Res . 2020 Oct; 8(10): 1230–1235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElkrief A, Derosa L, Zitvogel L, et al.: The intimate relationship between gut microbiota and cancer immunotherapy. Gut Microbes . 2019; 10(3): 424–428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGori S, Inno A, Belluomini L, et al.: Gut microbiota and cancer: How gut microbiota modulates activity, efficacy and toxicity of antitumoral therapy. Crit Rev Oncol Hematol . 2019 Nov; 143: 139–147. PubMed Abstract \| Publisher Full Text\n\nCardeña A: Fecal calprotectin as a predictor of gastrointestinal immune-related adverse events (CF-19): A prospective study - Excel data and supplementary tables. Dryad, Dataset . 2021. Publisher Full Text" }	[ { "id": "121111", "date": "10 Feb 2022", "name": "Shaun Siong Chung Ho", "expertise": [ "Reviewer Expertise Biomarkers for gastrointestinal 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\nThis is a prospective, multicenter study that evaluated whether fecal calprotectin (FC) could predict severe immune-related colitis. A calculated sample size of 114 patients was required for this study; however, due to challenges in recruitment, authors presented their preliminary results based on 24 patients. Of these 24 patients commenced on immunotherapy, five developed immune-related colitis, including one with severe colitis.\nStudy design:\nAs this is a multicenter study, authors to list how many centers were involved and how patients were recruited per center.\n\nAuthors to provide details on how patients with colitis were assessed, including the extent of colitis and definition of colitis grading. What grade of colitis constituted as severe colitis in this study?\n\nIt is not very clear whether patients taking acetylsalicylic acid were allowed to participate in the study or not. If yes, authors to provide how many patients were taking acetylsalicylic acid.\n\nPatients with gastrointestinal tumor can cause elevation of FC. Should this group of patients be excluded from the study?\n\nAuthors to provide details of FC assay used in this study, including reference range.\n\nAuthors to clarify whether FC results were blinded to clinicians who managed studied patients.\n\nAuthors reported that collecting faecal samples from patients has been a challenge. Authors to provide details on how the missing FC values (when samples were not received) were managed in data analysis.\n\nResults:\nIt will be helpful for authors to provide the median (IQR) follow-up period and interval time between when colitis developed and the commencement of immunotherapy (instead of stating before cycle number six).\n\nIt will be helpful for authors to provide median (IQR) FC values at baseline and at subsequent time points.\n\nThe timing of the reported FC values in Figure 2 is not clear. Was there a fecal sample (for FC) collected at the time of the colitis diagnosis (outside of the regular sample collection timings)?\n\nIs the reported FC value in Figure 2 (patients who developed immune-related colitis) based on all severity?\n\nAuthors to elaborate on what is ‘colitis RR taking into account that FC value is 1001 for each 10 units’.\n\nDiscussion:\nPatients who developed immune-related colitis are likely to develop loose/watery diarrhea. Therefore, the variability of FC values may be compounded by diluted fecal samples. There was also no mention whether infection causes were excluded in those patients who developed immune-related colitis as FC can also be elevated in the presence of infection.\n\nFigures and Tables:\nFigure 2 and 3: Figure legend can be presented in a clearer manner; numbers in Figure 2 can be explained in the legend. Figure 3 is missing of X-axis title.\n\nMultiple supplementary tables can be merged into a single table as patients’ background characteristics.\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": "123581", "date": "01 Mar 2022", "name": "Yinghong Wang", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a multi-center prospective study investigating the utility of fecal calprotectin (FC) as a biomarker to predict the development and severity of immune-mediated colitis. A total of 24 patients started on immune checkpoint inhibitors were included, and serial FC measurements were taken with every cycle. Authors concluded that there was no association between FC and immune-mediated colitis and that it would not be a good biomarker to predict IMC in clinical practice.\nGiven the value of fecal calprotectin has been reported to correlate with colitis severity and predictive value for colitis relapse in both inflammatory bowel disease and IMC on retrospective studies previously, this prospective study to measure the predictive value of calprotectin on IMC development becomes very critical and will provide very meaningful impact to the clinical practice. However, the suboptimal study design and small sample size raises significant concerns regarding the validity of the conclusion.\nComments to consider:\n\nBackground literature review may need to be further expanded on the use of FC, especially for colitis severity measure and disease status monitoring. The FC level has been shown to be strongly correlated with the severity of IMC. Pls consider adding the following articles for the background review:\n\nSom et al. (20191)\n\nZou et al. (20212)\n\nGupta et al. (20153)\n\nThe abbreviation for fecal calprotectin has been changing back and forth between FC and CF. Pls be consistent.\n\nIt is mentioned that aspirin use and antibiotic use will be \"taken into account\" on pages 3 and 4 respectively. Can you elaborate on how this information affected your analysis?\n\nHas patient received education that NSAID use should be on hold during the study window? It is a surprise that 4 patients started NSAID after enrollment.\n\nStudy design needs to be explained more thoroughly:\nIt is unclear if education for the enrolled patient is adequate based on #4 concern. As some patients with mild symptoms may not have reported them and their immune-related colitis may have been overlooked.\n\nWere there standard timings before each cycle that labs were collected or did it differ between each patient as different ICI regimen may have different administration cycles?\n\nWill patient get additional FC tested if there is concern of new GI symptom? Was there follow-up with patients in between cycles?\n\nBaseline demographic information was missing in a table format, suggest to add.\n\nThere is no definition of severe IMC. Is this based on CTCAE grade? What are the symptoms included for IMC diagnosis? Diarrhea alone or diarrhea and colitis symptoms e.g. bleeding etc. It makes a huge difference between grade 3 diarrhea and grade 3 colitis.\n\nOne of the biggest flaws of this study design is the lack of objective confirmation of IMC. Clinical symptom alone is far less sensitive enough for detection of early IMC. Lower endoscopy with tissue biopsy is the gold standard to correlate with the calprotectin level and confirm the development of early IMC. The clinical manifestation may be delayed in those cases.\n\nThere is such a big variation in the FC level, it is unclear if the FC test is centralized in one specific lab for the same reference level and uniform calibration.\n\nThe other major concern is the predominant use of PD-1/L1 in this study group. The lower risk of IMC among PD-1/L1 monotherapy treated patients could have contributed to the small number of IMC patients identified.\n\nIt is unclear how the ROC curve was created, e.g. what was the cutoff value of FC used?\n\nRegarding the label of the group on the figure, intuitively, group 0 will be defaulted to no colitis group or control group, the current label is quite confusing. Recommend to clearly label the definition instead of using the number code.\n\nDate of the lab tests should not be listed in the public domain, as it is part of PHI (patient’s health information).\n\nDo you have additional analysis on the other lab work collected from patients (CRP, hemoglobin, neutrophils, eosinophils, lymphocytes, and monocytes)? Are they correlate with the IMC event?\n\nThe data does not support the conclusion made. The small sample size and suboptimal design will unlikely contribute to meaningful result.\n\nPlease considering utilizing a medical editing service to polish your manuscript. There are spelling and grammatical errors throughout the manuscript as well as poor word choices in some instances.\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-534
https://f1000research.com/articles/9-1278/v1	28 Oct 20	{ "type": "Research Article", "title": "TAPAS: Towards Automated Processing and Analysis of multi-dimensional bioimage data", "authors": [ "Jean-François Gilles", "Thomas Boudier", "Jean-François Gilles" ], "abstract": "Modern microscopy is based on reproducible quantitative analysis, image data should be batch-processed by a standardized system that can be shared and easily reused by others. Furthermore such system should require none or minimal programming from the users. We developed TAPAS (Towards an Automated Processing and Analysis System). The goal is to design an easy system for describing and exchanging processing workflows. The protocols are simple text files comprising a linear list of commands used to process and analyse the images. An extensive set of 60 modules is already available, mostly based on the tools proposed in the 3D ImageJ Suite. We propose a wizard, called TAPAS menu, to help the user design her protocol by listing the available modules and the parameters associated. Most modules will have default parameters values for most common tasks. Once the user has designed her protocol, she can apply the protocol to a set of images, that can be either stored locally or on a OMERO database. An extensive documentation including the list of modules, various tutorials and link to the source code is available at https://imagej.net/TAPAS.", "keywords": [ "Image processing", "image analysis", "automation", "OMERO", "ImageJ", "Fiji" ], "content": "Introduction\n\nModern microscopy, through new systems like light-sheet or high-throughput microscopes, is generating a vast amount of complex data that needs to be analysed. These data can be large in size or in number. Furthermore, for the purpose of reproducible quantitative analysis, these data should be batch-processed by a standardized system, that can be easily shared and reused. Some batch systems already exist such as CellProfiler1, ICY2 protocols or ImageJ macros3. However, these systems may require some programming knowledge or time to set up for inexperienced users or are not yet fully multi-dimensional.\n\nIn the last 10–20 years, and more recently with deep-learning methods, a lot has been accomplished in the field of image processing, especially for image segmentation. However, there is no real standardization for image analysis protocols. Arganda-Carreras and Andrey4 designed a first version of a systematic image analysis pipeline. Furthermore, due to the recent advances in fast volumetric microscopy, more and more data are produced, but it lacks a systematic way of organizing raw data and subsequent analysed data and results. With the spread of database systems such as OMERO5, more and more imaging facilities and labs are storing their data in a more organized fashion.\n\n\nMethods\n\nWe developed TAPAS (Towards an Automated Processing and Analysis System) as a system for describing and exchanging processing workflows. The protocols are simple text files comprising a linear sequence of commands. An extensive set of 60 commands is already available, mostly based on the 3D ImageJ Suite6,7. The design of the protocol allows simplified tracking of processed data and quantitative results, by using keywords to design the image data, such as ?image?.\n\nTAPAS is focusing on data organization rather than complex segmentation or analysis algorithms. TAPAS focused originally on data stored on an OMERO database, by allowing to retrieve, perform classical segmentation procedures and analysis, and push back the results, both images and tables, to the database. Data on OMERO are, by design, organized by user, then projects and datasets. In TAPAS the current analysed image is simply referred by the keyword ?image?, and the corresponding project and dataset the data belongs to by ?project? and ?dataset? respectively. Subsequent processed data are then referred as ?image?-processing, for instance ?image?-nucleus for the result of nucleus segmentation. Similarly, additional datasets can be created such ?dataset?-labels to store the results of segmentation. The results tables can be stored using the name of the image as reference such as ?image?-nucleus-volume.csv. Results tables will be linked to the original raw image using OMERO attachments.\n\nThe system is implemented in Java, with a core library, including OMERO and BioFormats input/output utilities, and a plugins library including a comprehensive set of modules. Each module is generic as it will process a generic Image class, and each class will get an Image as input and will return an Image as output. Parameters are managed as simple String files, allowing flexible management of parameters, even for inexperienced Java programmers. The current system uses the ImageJ ImagePlus class as implementation for the Image class, however any other class can be used, allowing the use of any Java library.\n\nThe processing pipeline is constructed as an ordered list of processing classes, with their corresponding parameters. Since the classes are generic, a processor is also specified as how to process the image data, by default an ImagePlus processor is built. An experimental version of processor with a set of processing modules using the clearCLBuffer class has been tested and validated using the CLIJ system8.\n\nTAPAS is java-based and works with the ImageJ/Fiji system, the use of a OMERO database is optional. TAPAS is designed to work with a database; either OMERO or a local database. A local database is a folder organized, not unlike OMERO, as projects, datasets, and finally images. We also add an attachments' folder to store the results tables. Having a completely similar organization between OMERO and a local database allows to have an exact same protocol to run using an OMERO database or a local database. A typical workflow of the system is presented in Figure 1.\n\n1) The data to be processed is input into the processing pipeline from the Database (either OMERO or local). ?image? is a keyword used to refer to the image name. The names in boxes refer to the module names. 2) The necessary data to be used later is saved locally, in a temporary folder (home folder, ImageJ/Fiji folder or system temporary folder). Here we saved the raw data for channel 1. 3) The data is processed, here a classical pipeline consisting of filtering, thresholding and labelling. 4) The resulting labelled data is output to the Database, here the labelled structure for channel 1 is the nucleus. 5) The previously saved raw data is used as parameter to quantify intensity inside the labelled nuclei. The results table is saved first in a file locally. 6) The results table file is then attached to the original processed image. The temporary saved data (raw data for channel 1 and results table file) can be then deleted within the pipeline or manually.\n\n\nUse case\n\nThe system separates the data to be processed from the processing pipeline. Firstly, the list of image data to be processed is built, each image data to be processed is identified by its project, dataset and name (either on OMERO or on a local DB), and by the channel and frame to be processed. Second, the processing pipeline file is to be selected. After clicking run, the system will process the images sequentially, displaying information for each module, and the final processing time per image. Raw data will be pulled from the database and processed and analysed data will be pushed back to the database.\n\nWe propose a simple TAPAS menu that will display in an organized manner the list of available modules with their corresponding category and documentation. After selecting a module, the list of parameters will be displayed, the user can then manually enter the parameters values, and the corresponding processing pipeline text will be created.\n\n\nConclusion\n\nTAPAS is a comprehensive system for data processing automation, relying on an extensive set of more than 60 modules for processing and analysis of multi-dimensional image data. An extensive documentation including the list of modules, various tutorials and links to the source code is available at https://imagej.net/TAPAS.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nSoftware available from: https://imagej.net/TAPAS.\n\nSource code available from: https://github.com/mcib3d/tapas-core/.\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.40911779.\n\nLicense: GPL 3.0", "appendix": "Acknowledgements\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology)\n\n\nReferences\n\nBray MA, Carpenter AE: CellProfiler Tracer: exploring and validating high-throughput, time-lapse microscopy image data. BMC Bioinformatics. 2015; 16(1): 368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Chaumont F, Dallongeville S, Chenouard N, et al.: Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods. 2012; 9(7): 690–6. PubMed Abstract \| Publisher Full Text\n\nSchneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7): 671–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArganda-Carreras I, Andrey P: Designing Image Analysis Pipelines in Light Microscopy: A Rational Approach. Methods Mol Biol. 2017; 1563: 185–207. PubMed Abstract \| Publisher Full Text\n\nAllan C, Burel JM, Moore J, et al.: OMERO: flexible, model-driven data management for experimental biology. Nat Methods. 2012; 9(3): 245–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoudier T: 3D ImageJ Suite [Internet]. 2012; [cited 2016 May 23]. Reference Source\n\nBoudier T: Introduction to 3D Analysis with 3D ImageJ Suite. F1000Res. 2020; [cited 2020 Jul 17] 9. Publisher Full Text\n\nHaase R, Royer LA, Steinbach P, et al.: CLIJ: GPU-accelerated image processing for everyone. Nat Methods. 2019; 17(1): 1–2. PubMed Abstract \| Publisher Full Text\n\nBoudier T: mcib3d/tapas-core 0.6.4 (Version 0.6.4). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4091177" }	[ { "id": "73799", "date": "02 Nov 2020", "name": "Robert Haase", "expertise": [ "Reviewer Expertise Bio-Image Analysis", "Computer Science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present TAPAS which is an approach Towards an Automated Processing and Analysis System. One can see it as a workflow-design tool, primarily, but not exclusively, to the ImageJ 3D Suite.\n\nThe authors' efforts are appreciated because TAPAS fills a gap in the ImageJ/Fiji ecosystem. Furthermore, TAPAS is extensible and user friendly. TAPAS allows the removal of technical implementation details from the process of designing an image processing workflow. Furthermore, users are suggested to organize their data in a structure similar to OMERO databases. Standardization in image data organization is also a field where every step forward is deeply acknowledged.\nFor the presented manuscript, I would suggest minor additions: First of all, a more detailed report of the state-of-the-art would be interesting. Differentiation of TAPAS against Icy Protocols1 and Knime2 could give new insights to the reader. Furthermore, also commercial systems such as Zen and Apeer (Zeiss, Jena, Germany) and Arivis (Arivis, Munich, Germany) come nowadays with workflow design user interfaces which might be worth exploring and distinguishing.\nFrom the user's perspective, I would love to see an example workflow and an explanation of how the user benefits from exploiting the capabilities of TAPAS in a practical context. The TAPAS website contains examples and tutorials and the manuscript could benefit from this amazing documentation.\nOn the implementation side, I would happy to read some insights about how plugins for TAPAS can be implemented and distributed to collaborators. I think if the involved procedures would be explained a bit more in detail, developers could estimate efforts to implement TAPAS wrappers for their tools and algorithms, e.g. for denoising and segmentation. This increases the chance of other developers picking up TAPAS as a distribution system of their tools.\nFurthermore, on the technical side, I wonder if it is possible to implement processing steps that take two images from two former processing steps as input. Figure 1 suggests TAPAS is a linear system where each operation has just one input and one output. I'm not sure if I interpret this correctly.\nThe fact that TAPAS is developed fully in the open endorses the authors as fantastic contributors to the bio-image analysis open-source community. The audience knows from former projects like the authors' famous ImageJ 3D Suite that long-term support is available for many years. I think potential future plans for TAPAS could be mentioned in the manuscript. To my experience, communicating long-term perspectives clearly to the community pays off, because if users know that long-term support is given, the chance is higher that they pick up a new tool and build their workflows with it.\nTo conclude, TAPAS is a great extension for ImageJ/Fiji and eases integration of workflows in the OMERO ecosystem. It is extensible and flexible in use giving a powerful toolbox in the hands of end-users.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "73800", "date": "18 Nov 2020", "name": "Lit-Hsin Loo", "expertise": [ "Reviewer Expertise computational and system biology", "image analysis", "high-throughput screening and imaging", "machine learning", "in vitro cell models" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: Gilles et al. report a new software tool called TAPAS for batch processing and analysis of microscopy images. The tool is based on ImageJ/Fiji and can load images from the local file system or remote OMERO database. A set of 60 analysis modules or commands are currently available, mostly come from a previously developed package, 3D ImageJ Suite. The ability to batch process images using a standardized system is critical for ensuring the reproducibility of published results, and also the continuity of the development and applications of established analysis pipelines. The authors correctly point out that there are many existing tools that can do batch processing, such as CellProfiler, Icy, or ImageJ macros. However, one of the key advantages of TAPAS is the simplification of the specifications of an analysis workflow using text files (which can be edited using any text editor) and intuitive command syntax (which require minimum effort to learn). Thus, TAPAS can still fill a gap currently unaddressed by these other tools in the field.\nOverall, the work is technically sound and the article is well written. However, insufficient details and examples or use cases have been given to demonstrate the applications of TAPAS. Also, the targeted applications and the current limitations of the software are not well explained or discussed.\nMajor comments:\nFor this reviewer, the syntax of TAPAS is one of the most interesting and novel parts of the software. However, insufficient details and examples have been provided to explain the syntax and rules of the “TAPAS language”. One suggestion is to provide an example for a typical image analysis pipeline (e.g., image import, preprocessing, segmentation, feature extraction, result export). The authors may include pseudo codes (or a block diagram) and the corresponding TAPAS code that can accomplish the tasks.\n\nIt is not very clear what types of microscopy images can TAPAS process. The authors kind of alluded that the tools is meant for 3D images, but this has not been clearly presented. For example, can TAPAS process bright-field or RGB images? Time-lapse images? Or 3D-stack images?\n\nCan TAPAS run any ImageJ commands/functions? Or is it restricted only to functions from the ~60 modules?\n\nThe authors may present some simple examples of the results and screenshots that can be obtained from TAPAS. For example, counting of cells or measurement of protein expression levels in 3D images. These examples will be very helpful to let the readers understand how TAPAS may be used.\n\nAnother major advantage of using text-file based configurations is that the configurations may be version-controlled using standard software such as Git. Perhaps the authors may consider adding this function in the future.\nMinor comments\nIn the abstract, “Once the user has designed her protocol, she can … “ may be changed to “he/she”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1278
https://f1000research.com/articles/10-529/v1	02 Jul 21	{ "type": "Research Article", "title": "Pancreatic cancer incidence trends in the United States from 2000-2017: Analysis of Surveillance, Epidemiology and End Results (SEER) database", "authors": [ "Hassam Ali", "Rahul Pamarthy", "Meghana Vallabhaneni", "Shiza Sarfraz", "Hadiqa Ali", "Hamza Rafique", "Rahul Pamarthy", "Meghana Vallabhaneni", "Shiza Sarfraz", "Hadiqa Ali", "Hamza Rafique" ], "abstract": "Background: Recent incidence trends of pancreatic cancers were reviewed by demographics and histologic type to observe any new findings. Methods: Data was used from the Surveillance, Epidemiology, and End Results (SEER) registry 18 (2000-2017) and it underwent temporal trend analysis. Pancreatic cancer incidence rates were reported based on histological subtype and demographics. Results: The incidence rate of white males increased significantly during 2000-2017 (annual percent change (APC) = 3.5%) compared to previously reported APCs. The incidence of white females grew from an APC of 1.29% to 2.9%. Rates among black ethnicity increased with an APC of 4.2%. Rates among Hispanics and other ethnicities also showed increment. The rates for ductal adenocarcinoma showed a positive trend in all races, with the APC ≥ 6 % for females and APC ≥ 6.5 % for males. The rates of non-secretory endocrine tumors showed a decline in both genders of all five races in recent years after showing an initial positive trend till 2010. Rates for pancreatic adenocarcinoma continued to rise in all ethnicities from 2000-2017. Interestingly, there was a rise in carcinoid type pancreatic neuroendocrine tumors (PNETs) in all ethnicities. Cumulatively, males had a higher incidence than females; male to female Incidence Risk Ratio (IRRs) was 1.32. The IRR was > 1 for age groups ≥ 35 years. The male to female IRRs was less than 1 for cystic adenocarcinoma, secretory endocrine, and solid pseudopapillary carcinomas (IRR = 0.5, 0.9, and 0.2 respectively, confidence intervals 0.4–0.6 and 0.9-1.3, 0.2–0.3, respectively). Conclusion: Pancreatic cancer incidence continued to rise in the years 2000-2017. However, incidence differed by demographics and histologic type. Interestingly, recent years discerned a rise in PNETs (carcinoid type) which has not been reported previously.", "keywords": [ "Pancreatic cancer", "SEER database", "PNETs", "pancreatic neuroendocrine tumors", "pancreatic cancer risk factors", "Pancreatic cancer incidence", "SEER 18" ], "content": "Introduction\n\nAs per global reports in 2018, pancreatic cancer remains the twelfth most common cancer in men and the eleventh in women1. Cancer-related deaths have pancreatic cancer as the seventh leading cause1. In the United States of America (USA), pancreatic cancer (PC) incidence has been increasing as previously reported1,2. PC mortality is estimated to rise as time progresses, and recent projections estimate that it might overpass colorectal cancer to become the second leading cancer-related cause of death after lung cancer3. In 2020 reports showed 57,600 new cases and 47,050 PC related deaths3. The PC risk increases with age, and the median age of diagnosis is 71 years4. Previously the increases were most significant for non-secretory endocrine cancers, followed by ductal adenocarcinomas and adenocarcinoma5.\n\n\nMethods\n\nThe results were obtained from the analysis of the Surveillance Epidemiology and End Results (SEER) cancer registry database. SEER 18 includes 28% of the U.S. population as of the 2010 census6. The rates are available by expanded race/ethnicity of cases diagnosed, including white, black, Asian/Pacific Islander, and American Indian/Alaskan Native and Hispanic ethnicity. SEER 18 also includes adjustments for areas impacted by hurricanes Katrina and Rita. It contains a record for each of 8,131,919 tumors. 19 age groups are available and can be classified as < 1 year, 1–4 years, 5–9 years to 85+ years, or unknown. The registries included in SEER 18 are San Francisco-Oakland SMSA, Connecticut, Detroit (Metropolitan), Hawaii, Iowa, New Mexico, Seattle (Puget Sound), Utah, Atlanta (Metropolitan, San Jose-Monterey, Los Angeles, Alaska Natives, Rural Georgia, California excluding San Francisco, San Jose Monterey, Los Angeles, Kentucky, Louisiana, New Jersey, Greater Georgia. SEER 18 encompasses the SEER 13, which itself includes seer 9 in addition to supplementary registries6.\n\nWithin the SEER database, International Classification of Diseases (ICD)-0–2 was used through 20007, and ICD-0–3 following 2001 for coding the primary site and histologic type of diagnosed pancreatic cancer cases8. Site recode ICD-0–3/WHO 2008: Pancreas (ICD-O-3 site C25) was used via SEERStat software to select cases pancreatic cancer9. Only cases that were malignant and had a microscopically confirmed diagnosis were included. The ages below 30 years were filtered out due to reportedly low incidence per previous literature5. Cases were characterized into exclusive groups based on morphological classification of pancreatic cancer in the ICD (Numbers given below) for Oncology and previous similar study5,8. In addition to the undermentioned cases, all other cases were classified as poorly specified types.\n\n• Adenocarcinoma, not otherwise specified (NOS) (8140)\n\n• Mucinous adenocarcinoma (8480, 8481)\n\n• Ductal adenocarcinoma excluding cystic or mucinous (8507, 8510, 8514, 8521, 8560, 8570, 8523, 8255, 8490, 8500)\n\n• Cystic adenocarcinoma (8440, 8470, 8504)\n\n• Intraductal papillary mucinous neoplasm (IPMN) (8453, 8471, 8503, 8144, 8450)\n\n• Non-secretory endocrine (8150, 8246)\n\n• Secretory endocrine (8151, 8152, 8156, 8153, 8155)\n\n• Carcinoid tumors (8243, 8244, 8245, 8240, 8241)\n\n• Acinar cell adenocarcinomas (8550, 8551),\n\n• Solid pseudopapillary tumors (8452),\n\n• Other adenocarcinoma (8145, 8260, 8441, 8154, 8574, 8460)\n\n• Non-carcinomas (8680–9999)\n\nSEERStat9 was utilized to calculate the incidence and age-adjusted rates, grouped by histologic subtype, gender, ethnicity, and age group. SEER 18 registry was used for analyzing tends with detailed racial group categories from 2000 to 2017 using yearly diagnosis. The period-specific rates were plotted and utilized temporal trend figures at two-year intervals and utilized a logarithmic scale with a base of 10 degrees portrayed rate per 100,000 person-years10. The annual percent changes (APCs) were quantified using annual rates for temporal trends (Table 1 & Table 2), and 95% confidence intervals (CIs) were included. SEERStat9 was used for all analyses6. Incidence Rate Ratios (IRRs) based on gender and ethnicity/race were calculated; these were further classified based on age groups and histological types. 95% CIs for the IRRs was calculated using the Tiwari method, and the figures were generated using Microsoft excel. Potential sources of bias include concern of patient migration across seer registries. Data is de-identified by SEER, and this potential for sample bias cannot be intervened but mentioned in this study's limitations.\n\nAPC = Annual percent change; UCI, LCI = Upper, Lower 95% confidence interval.\n\nConfidence intervals are 95% for trends.\n\nPercent changes were calculated using 1 year for each end point; APCs were calculated using weighted least squares method.\n\n~ Statistic could not be calculated due to lower number of cases or at least one year with no reported cases.\n\n The APC is significantly different from zero (p<0.05).\n\nCases and annual percentage changes were only mentioned if there were enough cases to yield results after temporal trends analysis.\n\nAPC = Annual percent change; UCI, LCI = Upper, Lower 95% confidence interval.\n\nConfidence intervals are 95% for trends.\n\nPercent changes were calculated using 1 year for each end point; APCs were calculated using weighted least squares method.\n\n~ Statistic could not be calculated due to lower number of cases or at least one year with no reported cases.\n\n* The APC is significantly different from zero (p<0.05).\n\nCases and annual percentage changes were only mentioned if there were enough cases to yield results after temporal trends analysis.\n\n\nResults\n\nSEER 18 allowed us to explore short-term, nonetheless, relatively recent and previously unreported trends in pancreatic cancer (Table 1) (Figure 1). The incidence rate of white males has increased significantly during 2000–2017 (APC = 3.5%) compared to previously reported APC of 0.95% from 1994–20135. The incidence of white females also increased from an APC of 1.29% (1999–2013) to 2.9% (2000–2017)5. Rates among black males and females have also increased in recent years, per our analysis, with an APC of 4.2% each, respectively. Previously, the rates among black males decreased, while rates remained unchanged among black females (1975–2013)5.\n\na: Females, b: Males.\n\nRates among Hispanic men and women have continued to increase (APC = 6.5% and 6.4%). Similarly, rates among American Indian/Alaska Native in men increased more than women (APC = 7.4% vs. 2.1%). In Asian or Pacific Islander, the APC was similar in both genders (6.1%) as compared to lower incidence rates of 0.16% for males and 1.23% for females (1992–2013)5.\n\nThere was a positive trend in rates for white males from ages 30–39 and ≥50 years for the 5-year age groups up to 85 and above as shown in Table 2 and Figure 2. White females showed a statistically significant positive trend from age ≥50. Interestingly, ages 40–49 showed a negative trend in white males, and age group 40–44 had a statistically significant negative APC of -2.3%.\n\na: Males, b: Females.\n\nBoth genders in the black population showed a positive trend after age ≥ 45 (Table 2) (Figure 2). It was impossible to calculate APCs among younger age groups for black females due to at least one year with zero cases. Age-specific rates also rose among Asians or pacific islanders and Hispanic men from ages ≥ 35 and ≥ 40, respectively (Figure 2). APCs for females of Asians or pacific islanders and Hispanic ethnicity also showed a positive trend after ages ≥ 40 and ≥ 35, respectively (Figure 2). Further analysis was performed to explore trends in American Indian/Alaskan native (Figure 2). However, APCs could not be calculated in either males or females for any age group due to at least one year with zero cases. The results are therefore not shown in tables. From 2000 to 2017, for ages, more than 30 years, 152,548 of 190,586 pancreatic cancer cases (80.04%) diagnosed had a microscopic confirmation. This represented an increase compared with the 77% microscopically confirmed cases from 1992–2013, including all ages. (Results are not shown). We did not report the analysis of the temporal trends by following histologic-type groups due to the number of cases being too low: cystic adenocarcinoma (n= 408), ductal specified as arising from an IPMN (n = 520), secretory endocrine (n = 203), other adenocarcinomas: acinar cell (n = 438), solid pseudopapillary tumors (n = 228), other adenocarcinomas (n = 448), other non-carcinomas (n = 6).\n\nRates for pancreatic adenocarcinoma, the most common histologic type of pancreatic cancer, continued to rise in all ethnicities from 2000–2017. The rates for ductal adenocarcinoma (excluding mucinous and cystic) showed a positive trend in all races (including American Indian/Alaska Native) with the APC ≥ 6 % for females and APC ≥ 6.5 % for males (Table 2) (Figure 3). The rates of non-secretory endocrine tumors showed a decline in both genders of all five races in recent years after showing an initial positive trend till 2010. However, the APC from 2000–2017 remained positive and ≥ 3.5% for males and females (Table 2). APC for American Indian/Alaska Native was not calculated due to at least one year with zero cases; however, trends are reported in Figure 3.\n\na: Males, b: Females.\n\nInterestingly, the rates for carcinoid endocrine tumors showed a positive trend and rise in rates from 2010 and onwards (Figure 3), which was not reported previously. The APC was highest in white males and females, and black females (32.5%, 30.3%, and 26.9%, respectively). APCs for the rest of the races were not reported as statistics were incalculable as at least one year had no reported cases from 2000 to 2017.\n\nIn SEER 18 (2000–2017), 152,548 cases were microscopically confirmed from ages ≥ 30; males: 79081 and females: 73,467. This was higher than previously reported rates, which was 69,049 in males and 69,548 in females (2000–2013)5. The IRR was greater in males’ total cases (IRR 1.32,95% CI 1.30-1.33) (Table 3). All age groups from 35 and above showed an increased incidence risk in males vs. females (IRRs >1: increased risk) (Table 3). Rates among males were higher compared to females for all histological subtypes except ductal cystic adenocarcinoma (0.5,95% CI 0.4-0.6), secretory endocrine tumors (0.9, 95% CI 0.9-1.3), solid pseudopapillary (0.2, 95% CI 0.2-0.3) and non-carcinomas (0.1, 95% CI 0.7-7.7). Carcinoid endocrine tumors also showed a greater IRR in males (1.4, 95% CI 1.3-1.5) (Table 3).\n\nRates are per 100 000 person-years, age-adjusted to the 2000 US Standard Population (19 age groups - Census P25–1130).\n\nIRR = Incidence Rate Ratios (based on unrounded rates); LCI, UCI = lower, upper 95% confidence interval.\n\nConfidence intervals (Tiwari mod) are 95% for the ratios.\n\nFor SEER 18 (2000–17), the highest rates for pancreatic cancer in ages greater than 30, were in both black males and females (IRR = 1.21 and 1.36, respectively) when compared to white (non-Hispanic) ethnicity. Incidence, when compared to white (non-Hispanic) ethnicity, was higher in both black males and females (IRR = 1.21 and 1.36, respectively). The rates for both genders in American Indian/Alaska, Native Asian, or Pacific Islander Hispanic (All Races) was lower as compared to the white(non-Hispanic) race (Table 4).\n\nRates are per 100 000 person-years, age-adjusted to the 2000 US Standard Population (19 age groups - Census P25–1130).\n\nIRR = Incidence Rate Ratios (based on unrounded rates); LCI, UCI = lower, upper 95% confidence interval.\n\nConfidence intervals (Tiwari mod) are 95% for the ratios.\n\nThe reference group is White non-Hispanic.\n\n\nDiscussion\n\nOur statistical analysis for SEER 18 registry revealed reportable findings by histologic subtype and demographic details. Incidence rates of pancreatic carcinomas increased amongst white males and females consistently. Incidence rates for black, Asian, or pacific inlander and Hispanics all rose in recent years for both genders. Rates for American Indian/Alaskan natives were inconsistent throughout 2000–2017; however, the final annual percent change remained positive for males (7.4%, CI 4.7-10.1) and females (2.7%, 0.7-4.7). Based on histological type, the overall increase in incidence remained secondary to pancreatic adenocarcinoma, as mentioned in a previous similar study5.\n\nPancreatic carcinoma rates more predominantly rose in males when compared to females. In white males and females, there was a female prominence noted for incidence rates of age group 30–34 (Table 2, Table 3). Rates for other ethnicities for ages <45 were comparable or could not be analyzed due to the lower number of cases. Rates of common histologic types were more in males, except for cystic adenocarcinoma, secretory endocrine cancers, solid pseudopapillary cancers, and non-carcinomas. This differs based on the histological type could be attributed to cystic adenocarcinoma and solid pseudopapillary cancers as they had the highest female predominance. Previous research comparing male to female incidence rate ratios also came to a similar conclusion for long-term rates from 1975–2003 and 2000 – 20135. A review of 718 patients in 2005 stated a female predominance of solid pseudopapillary pancreatic cancers; almost 90% occurred in younger females aged between 19 to 50, with a ratio of close to 10:1 for females to males11. A systemic review also showed an increased predominance of cystic adenocarcinomas in females, and our results were consistent with these studies12.\n\nAn interesting finding in our analysis was the recent rise in pancreatic endocrine carcinoid tumors in all ethnicities. There was a male predominance with (IRR 1.4%, CI 1.3-1.5). The rates were highest in white males and started to increase after 2010 in all races except American Indian/Alaska Native who showed sporadic rise (Figure 3). The black and Asian or pacific inlanders' incidence rates showed some plateau/decline in recent years (Figure 3). A previous study on pancreatic neuroendocrine tumors (PNETs) suggested that cigarette smoking, Type II Diabetes, and family history in a first-degree relative are independent risk factors for non-functional PNETs. Another independent risk factor for functional PNETs was found to be heavier consumption of alcohol13. No study currently comments solely on the risk factors of pancreatic carcinoid tumors, and it would be interesting to see associated risk factors for this recent rise. Our analysis revealed a rising rate of pancreatic carcinoid tumors with age, however, the results declined for ages >75 (results not shown).\n\nRates for intraductal papillary mucinous tumors and secretory endocrine carcinomas were similar in both genders. IPMN are malignancies of the pancreas that grow within the pancreatic ducts and produce mucin, hence the name. The risk factors of IPMN are not well understood. However, possible risk factors include insulin-dependent diabetes mellitus, chronic pancreatitis, positive family history, and smoking14. Our analysis revealed a decline in mucinous adenocarcinomas (Figure 3). This is consistent with the previously reported study5. This can be explained by the fact that improved diagnostic imaging has enabled the detection of cystic or mucinous precursor lesions at an earlier stage. Our analysis only included only malignant lesions15.\n\nNumerous risk factors exist for pancreatic carcinoma and could be the reason behind the rising incidence. Individuals with non-O blood groups (type A, AB, or B) are more prone to the risk of pancreatic cancer16. A possible link between Helicobacter pylori infection and non-O blood type may also exist; however, evidence is still lacking17. Cystic fibrosis patients are at a greater risk of pancreatic cancer, especially adenocarcinoma, as their life span increases. It was also two to five-fold higher in patients with a history of organ transplantation18. Up to 10% of pancreatic cancer patients have a family history19. Some studies, though not explicit, identified a germline mutation involving a known susceptibility gene for pancreatic cancer (including BRCA, ATM, CDKN2A and PALB2) in patients with familial etiology20.\n\nChronic pancreatitis is also a risk factor for pancreatic cancer21. This is hypothesized due to inflammation-induced metaplasia of the acinar cells into ductal cells by NF-kB and matrix metalloproteinases21. Patients with a type of neoplastic pancreatic cyst, intraductal papillary mucinous neoplasm of the pancreas (IPMN) can develop an invasive malignancy called IPMN-associated adenocarcinoma. These patients can also develop ductal adenocarcinoma22. Smoking is the most common cause of pancreatic cancer. Studies show a linear relationship with the number of cigarettes smoked23. However, the risk of pancreatic cancer decreases by 48% two years after smoking cessation. This is equivalent to that of non-smokers by 10 to 15 years after smoking cessation23. Individuals who struggle with body weight develop pancreatic cancer at a relatively younger age with shorter survival compared to individuals with normal bodyweight24. Data regarding the effects of alcohol and coffee consumption has always been conflicting. Pooled analysis data showed a slight increase in the risk of pancreatic cancer in heavy drinkers and a high intake of coffee. This could be due to the confounder, which is cigarette smoking25. An inverse relationship between consumption of a healthy diet and pancreatic cancer26. However, the protective advantages of a healthy diet were only significant in men who were overweight/obese26. Low levels of selenium and lycopene were found in subjects who developed pancreatic cancer in later life, however, the results are unclear27. Though studies have shown an association with Hepatitis B and C with pancreatic cancer, the magnitude of risk is still significantly less than hepatocellular cancer28. Numerous studies show an association between diabetes and pancreatic cancer. A meta-analysis showed the relative risk for pancreatic cancer in diabetes compared to the non-diabetic counterparts was 2.0829. Defects in metabolism of glucose, insulin resistance, and hyperinsulinemia are few etiologic factors for pancreatic cancer30–32. Increased risk of pancreatic cancer in patients with metabolic syndrome could be due to lower levels of adiponectin (a protein hormone linked to glucose level regulation and fatty acid breakdown), which results in a lack of insulin-sensitizing and anti-inflammatory capabilities33.\n\nOur study had its limitations, especially from an etiological point of view, as SEER does not report any risk factors, which can influence these trends. Comorbidities like obesity, diabetes, smoking are also not reported. There is a concern of migrating patients in and out of areas registered in the SEER database and selection bias. The data of the SEER registry holds high-grade quality, and statistics based on population can be informative of risk patterns and trends based on time. The critical strength of our studies was the large sample size, as SEER 18 covers more recent cases up to 2017 and covers almost 30% of the US population. It also allowed us to analyze histological types of pancreatic cancer.\n\n\nConclusion\n\nPancreatic cancer incidence rates have continued to rise according to SEER 18 (2000–2017) in the USA. Our analysis concludes incidence of pancreatic cancer patterns based on demographical data and histologic type. We report the new rising incidence of pancreatic carcinoid tumors and the continued rise of pancreatic adenocarcinoma. We also report an increased incidence of cystic adenocarcinoma and solid pseudopapillary cancers in females as compared to males. These trends could be secondary to known risk factors for pancreatic carcinomas. Further comparative data regarding exclusive risk factors and contributing factors for histological subtypes of pancreatic carcinomas, specially PNETs, are required to understand the recent increase in incidence.\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\nHassam Ali: Conceptualization, Methodology, Software, Validation, Writing- Reviewing and Editing. Meghana Vallabhaneni: Data curation, Writing- Original draft preparation. Rahul Pamarthy: Visualization, Project administration, Investigation. Shiza Sarfraz: Supervision, software, resources. Hadiqa Ali: Software, Validation. Hamza Rafique: Writing- Reviewing and Editing.", "appendix": "References\n\nBray F, Ferlay J, Soerjomataram I, et al.: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6): 394–424. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBao Y, Giovannucci EL, Kraft P, et al.: A prospective study of plasma adiponectin and pancreatic cancer risk in five US cohorts. J Natl Cancer Inst. 2013; 105(2): 95–103. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "88964", "date": "12 Jul 2021", "name": "Syed Hamza Bin Waqar", "expertise": [ "Reviewer Expertise Gastroenterology", "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\nInteresting article which is dealing with SEER database to study one of the most invasive pancreatic CA out there. I like the design of the study and it's a smart idea to build up a database from SEER. It is interesting to note that PNETs are now going up the trend, although the reason is uncertain. It might be something that scientists/doctors would like to look into moving forward - as to why the incidence is increasing now? Is it just coincidence or is there is an actual correlation and if so, with what? A very thorough epidemiological, smart, and short 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": "88966", "date": "01 Sep 2021", "name": "Harigopal Sandhyavenu", "expertise": [ "Reviewer Expertise General Internal Medicine", "Cardiology", "Geriatrics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting way of looking at the temporal trends of Pancreatic cancers. It is surprising to see increasing in the overall incidence, especially pancreatic endocrine carcinoid tumors among all ethnicities. Is it an actual rise in their number, or increasing positive tests with advanced diagnostic strategies? Either way this paper will allow the readers to look for possible correlation, if any. Overall this is an interesting article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7111", "date": "02 Sep 2021", "name": "Hassam Ali", "role": "Author Response", "response": "Thank you for your review. For your question \"Is it an actual rise in their number, or increasing positive tests with advanced diagnostic strategies?\": Our reported cases are biopsy confirmed and malignant therefore an actual rise instead of incidental findings." } ] } ]	1	https://f1000research.com/articles/10-529
https://f1000research.com/articles/10-20/v1	12 Jan 21	{ "type": "Research Article", "title": "Current market rates for scholarly publishing services", "authors": [ "Alexander Grossmann", "Björn Brembs", "Björn Brembs" ], "abstract": "For decades, the supra-inflation increase of subscription prices for scholarly journals has concerned scholarly institutions. After years of fruitless efforts to solve this “serials crisis”, open access has been proposed as the latest potential solution. However, the prices for open access publishing are also high and are rising well beyond inflation. What has been missing from the public discussion so far is a quantitative approach to determine the actual costs of efficiently publishing a scholarly article using state-of-the-art technologies, such that informed decisions can be made as to appropriate price levels. Here we provide a granular, step-by-step calculation of the costs associated with publishing primary research articles, from submission, through peer-review, to publication, indexing and archiving. We find that these costs range from less than US$200 per article in modern, large-scale publishing platforms using post-publication peer-review, to about US$1,000 per article in prestigious journals with rejection rates exceeding 90%. The publication costs for a representative scholarly article today come to lie at around US$400. We discuss the additional non-publication items that make up the difference between publication costs and final price.", "keywords": [ "publishing", "journals", "costs", "prices", "scholarly publishing", "scholarly communication", "publisher" ], "content": "Introduction\n\nThe affordability problem of scholarly publishing, i.e., the supra-inflationary price increases with stagnating library budgets, has been discussed for decades (see, e.g., 1–7). In recent years, perhaps precipitated by some so-called ‘gold’ open access (OA) journals charging article-processing charges (APCs; fees usually charged to authors or their institutions upon acceptance for publishing an article and making it openly available), the average cost of an article has emerged as a useful measure with which to compare different business models (but see 8 for a critique). However, most authors refer to the prices charged by the publisher, not the actual cost to the publisher (e.g., 9–12). One consequence of this mis-attribution is a potential overestimation of the actual costs of scholarly publishing due to the inclusion of the business models and pricing strategies of publishers into the calculation. To close this gap, here we provide a bottom-up calculation of the cost of efforts and services which are required to achieve a certain service level in order to publish an academic journal article. These calculations are analogous to what a new publisher would have to calculate before entering the publishing market. We compare our cost estimate with the current pricing schemes of publishers.\n\nIn this article, we assume the role of a newcomer to the academic publishing market and list the various steps and procedures for a representative publishing workflow according to current industry standards. Each step incurs a cost which can be determined by analyzing the market rates for each service or procedure. These costs comprise the direct costs. We also add several indirect (or fixed) cost items which do not accrue on a per article basis. The final per-article costs are then specified as a range depending on the number of articles published and the service level desired. These ranges denote current market rates at which customers can obtain publishing services.\n\n\nMethods\n\nTo arrive at a meaningful figure denoting how much the publication of an article costs on average, it is necessary to arrive at the exact cost for each step in the processing workflow of a manuscript being submitted for publication. These direct or variable costs then have to be combined with the indirect or fixed costs of running a publishing enterprise, such as staff costs, real estate, insurance and energy costs, etc. The former requires granular insight and expertise about the different service levels for the entire publishing workflow. The latter is commonly calculated as staff overhead. In this work, we have therefore calculated the cost for each step in the standard publication workflow under consideration of both fixed and variable costs. Both external and internal expenses have been taken into account as well as overhead costs to cover fixed non-direct company costs of the publishing venture.\n\nExpenses and fees for each individual service have been arrived at from two main sources. Some standard services have been taken from openly available price lists (Table 1).\n\nSecond, we requested quotes from vendors without publicly available fees, or turned to other sources13. For services such as manuscript submission and peer review management systems we considered vendors such as Manuscript Central (Clarivate) and Editorial Manager (ARIES).\n\nOther costs such as internal staff costs (including overhead, EU/US standard) were estimated taking into account not only current market costs we have requested ourselves, but also numbers from major publishing houses (MDPI, Wiley, Springer, DeGruyter, Frontiers, Ubiquity, SciELO, Open LIbrary of the Humanities). While some of these publishers have made their costs public (Table 2), others have either provided their numbers under the condition of confidentiality or the numbers were gained from internal sources.\n\nFor certain tasks, for example copyediting or typesetting, there are hundreds of individual companies worldwide providing those services on an industry-standard level. In our quote requests, we have considered only those with which we have collaborated in real business life so far or from which we know the performance and service level in detail from co-operations over two decades. Having compared the pricing of those service providers with others, we found only a very small variation of cost for such tasks, which justifies our practical approach. It was never our ambition to perform an exhaustive but always incomplete market study of service providers worldwide, but an attempt to provide an authoritative documentation of approximate current publishing costs as a valuable information tool for decision-makers and other stakeholders in policy drafting, contract negotiations or public discourse.\n\nThere are three main areas in which production steps have to be considered: content acquisition, content preparation (production) and content dissemination/archiving. Importantly, ‘content acquisition’ does not imply active acquisition of authors and/or manuscripts.\n\n1. Content acquisition\n\na. Searching and assigning reviewers\n\nb. Communication with reviewers\n\nc. Communication with authors\n\nd. Handling of re-submission process\n\ne. Plagiarism check\n\nf. Online submission system\n\ng. CrossRef Similarity Check\n\nh. CrossRef DOI for article\n\ni. CrossRef DOI for 2 or more reviews\n\nj. APC collection\n\n2. Content preparation (production)\n\na. Manuscript tracking system\n\nb. Production system check-in\n\nc. Technical checking of manuscript\n\nd. Copyediting\n\ne. Language editing\n\nf. Typesetting\n\ng. Formatting figures/graphs/tables\n\nh. Altmetric badge\n\ni. XML and metadata preparation\n\nj. Handling author corrections\n\n3. Content dissemination/archiving\n\na. Web OA platform and hosting\n\nb. CLOCKSS/Portico\n\nc. OAPEN\n\nd. Upload to Scopus, PMC, etc.\n\nPricing figures have been deducted by openly available price lists of vendors, as for example for Scholastica, Akron Aps, CrossRef, CLOCKSS (see Table 1, Table 2). In all other cases where pricing list or fees were not openly available on the web, prices were indicated after a direct request for proposal or communicated privately. For the latter we have checked with other partners to validate that information. Some service vendors have not split their services in a granular manner but offer a full service for more steps of the publishing workflow. In those cases, we have tried to split those costs or consider the full cost as part of one of the scenarios (see below) which cover the complete manuscript acquisition and article production process.\n\nThe calculation of per-article figures from costs that do not accrue on a per-article basis (e.g., salaries, annual fees, etc.) was based on the following assumptions: (i) The average STM article contains 12 printed pages12. (ii) We estimated an average STM article to contain 10 non-text items such as figures or tables. (iii) We also assumed an average rejection rate of 50% after conventional (pre-publication) peer-review with at least two reports and ten contact requests to secure one reviewer. (iv) We assume a desk-rejection rate of 10% after editorial review. (v) We also base our staff costs on the granular workload per article and not on full-time equivalents (FTE). These assumptions entail that all editorial duties (on average 7.5 person-hours per submitted manuscript) are handled by in-house staff and none by academic editors, while peer-review is still performed by volunteer academics. In this way, staff costs, including overhead expenses, are calculated on a per-article basis (i.e., per published article, not per submitted manuscript). Salary costs are based on industry standards in more economically developed countries for the different editorial tasks. Overhead expenses can vary significantly depending on the profit and loss structure of the publisher and include rent, repairs, depreciation, interest, insurance, travel expenditures, labor burden, telephone bills, supplies, taxes, accounting fees, etc. We have estimated an average 33% overhead on top of salary costs. The following publication tasks are commonly covered by annual (membership) fees plus an initial, one-time set-up or installment fee: Web OA platform and hosting, CLOCKSS/Portico, OAPEN, Altmetric Badge and Crossref. Because these costs accrue regardless of how many articles are published (i.e., fixed costs), we have calculated per-article costs for journals with different numbers of articles published per year.\n\nWhile some general fixed costs are covered by salary overheads (see above), we deliberately chose to not include certain fixed costs: Cost of sales have not been considered because for open access journals sales representatives are required no longer which have to negotiate renewals of subscriptions with libraries on an annual basis. We also excluded management costs as these are highly variable and in large publishers with many journals (and hence articles), per article costs of management are often negligible. We realize that this may be different for publishers which publish low-volume journals but with nevertheless highly paid executives (see Discussion). Because making an article public (i.e., ‘publishing’) is distinct from locking it behind a paywall, we have also not calculated the often very significant paywall costs. While innovation (or acquisition of innovative technologies) as well as branding and advertising/marketing are crucial for a company to succeed and thrive in a market in the long term, we have also not included these costs as they are not directly related to publishing scholarly articles. Such costs would include conference attendance, advertisement in print, online, social media and search platforms, as well as search engine optimization (SEO). Similarly, government relations (lobbying) may be considered a necessary expense for any business, but as it does not directly relate to the process of publishing academic papers, we did not include these costs in our calculations either. However, we do discuss the probable extent to which these non-publication costs may affect pricing.\n\nThe motivation for the above assumptions was to combine a robust cost estimate (i.e., sourced from measurable time efforts and industry salaries) with an upper bound cost estimate which would come to lie above most academic-run journals. We also calculated a cost estimate for articles handled exclusively by volunteer academics. Prices for journals where volunteer and compensated editors cooperate, will hence fall between these two extremes.\n\nWith a modern, decentralized/federated platform providing publishing functionalities without journals, some of these steps become obsolete, while others remain relevant. Steps that may become obsolete include DOIs, long-term archiving such as CLOCKSS or Portico, indices such as Scopus. Relevant steps remaining are typesetting/copyediting, XML preparation, format conversion, plagiarism checks.\n\nWe have grouped the various combinations of tasks and publication options into six broad scenarios, for which we have calculated all associated publication costs (Table 3). These scenarios correspond either to existing publishing options or to options that have been discussed in the literature. For each of the six scenarios, we have also calculated the same costs, but assuming a 90% rejection rate (see raw data file).\n\nImportantly, all costs are calculated per published article, i.e., a journal that publishes 1,000 articles per year has received 2,000 articles if their rejection rate is 50%. Our costs are calculated for the 1,000 published articles, not for the 2,000 submissions the journal has received.\n\nAll the data we have based our calculations on are available at Figshare (see Underlying data; DOI: 10.6084/m9.figshare.8118197)14.\n\nAn earlier, non-peer-reviewed version of this article can be found on PeerJ (DOI: 10.7287/peerj.preprints.27809v1).\n\n\nResults\n\nOne of the first findings of our calculations is that in order to employ at least one 50% FTE of an in-house editor, a journal has to publish approx. 100 articles per year or more. Hence, in the following, we will base our estimates on journals publishing at least 100 articles per year (corresponding to 50% FTE) or 1,000 articles (corresponding to 5 FTEs), to show the spread of fixed and indirect costs over the number of articles published.\n\nOur estimate of per-article publishing costs in a conventional pre-publication peer-review (50% rejection rate) scenario where all editorial duties are performed by in-house staff (Scenario B) ranges from US$643.61 for a journal that publishes 100 articles per year down to US$565.15 for such a journal that publishes 1,000 articles (or more, as the indirect costs become increasingly negligible around this value). These values consist of US$266.53 direct publishing costs (i.e., CrossRef Similarity Check, CrossRef DOI for an article, CrossRef DOI for two or more reviews, copyediting, typesetting, formatting figures/graphs/tables, altmetric badge, upload to Scopus and XML and metadata preparation), US$289.91 for editorial staff and US$8.72 to US$87.18 for 1,000 to 100 articles, respectively, in indirect costs (i.e., Web OA platform and hosting, CLOCKSS, OAPEN, Altmetric Badge and Crossref).\n\nThese numbers were calculated using generic, full-service providers (based in India), where applicable. There are open access service providers that provide packaged deals for the same services as these generic service providers. We have calculated the same steps using a well-known provider in this area, Scholastica (Scenario A). Interestingly, these figures are slightly higher: US$374.08 for direct publishing costs and US$5.92 to US$59.18 for 1,000 to 100 articles, respectively, for indirect costs (editorial staff costs remain the same).\n\nWhile these costs have been calculated for a generic journal with 50% rejection rate, per-article costs will increase with increased rejection rates and decrease with less rejections as in, e.g., a post-publication peer-review (PPPR) model. In a journal that uses generic service providers and publishes all submitted manuscripts as PDF preprints with a DOI before performing otherwise identical peer-review as described above (i.e., PPPR with in-house editors and volunteer reviewers), per article editorial services drop from US$289.91 to US$140.69 (Scenario A2/B2), with all other costs remaining nearly identical. Conversely, prestigious journals with rejection rates of around 90% see their costs rise to US$1053.87 for 100 articles per year or US$770.53 for the larger journals with about 1,000 articles per year (generic service providers).\n\nThese numbers also show that for a conventional journal today, where academics perform their editorial duties on a volunteer basis (i.e., Scenario B, but no editorial costs as editor salaries are paid for by their academic institutions), direct publication costs come to lie at US$266.53 with generic service providers and total costs depend on the scale at which the journal operates. Small journals with 100 articles would face average per article total publication costs of US$353.71, while journals with 1,000 or more articles would only face costs of US$275.25 or less per published article. Even at the highest convenience for a small, volunteer-run journal, costs come to lie at US$454.63 where a full-service provider (Scholastica) handles all of the technical aspects of the work (Scenario C2).\n\nThe above calculations (summarized in Table 4) demonstrate economies of scale. The more articles are being published, the lower the costs for each article, approaching the fixed costs for each article.\n\nThe scenarios are labeled with A, A2, B, B2, C, C2 (see Table 3).\n\nBecause of the economies of scale and recent calls for the replacement of journals with a modern publishing platform15–20, we have also calculated the cost of publishing the annual output of the STM community, approx. 3 million articles, on such a platform that facilitates PPPR organized by academic editors on a single, decentralized, federated platform running modern software solutions. Such a platform would dispense with several production steps which are necessitated by the current balkanization of the literature in different journals published by different publishers, but keep others (see Methods). In this scenario, the indirect and fixed costs per article approach zero due to the high number of published articles (but see Discussion), such that the only remaining costs would be the direct publishing costs of US$190.17 per published article.\n\nFinally, taking a ballpark cost figure of US$600 for a scholarly article with full editorial services (i.e., scenario A/B) and comparing it to the low end of the average price estimate for a subscription article of about US$4,000, it becomes clear that publication costs only cover 15% of the subscription price (Figure 1). Assuming a conservative profit margin of 30% (i.e., US$1,200 per article) for one of the large publishers21–24, there remains a sizeable gap of about US$2,200 in non-publication costs, or 55% of the price of a scholarly subscription article (Figure 1).\n\nAssuming the commonly accepted US$4,000 price tag for a subscription article, published profit margins of 30% and our calculation of US$600 in publication costs for a full-service subscription article (scenario A/B, see Table 4), there remain US$2,200 in non-publication costs per article.\n\n\nDiscussion\n\nSince the 1990s, it has been recognized that the prices of scholarly journals were escalating at unsustainable rates3. In the last 30 years, this “serials crisis” has never been coherently addressed, let alone solved. With this work, we aim to provide more financial evidence for future evidence-based policies addressing the affordability problem of scholarly communication1,2.\n\nNot only current discussions are addressing the affordability problem in the unit of cost per article9–12,25–28 and we follow this precedent. Drawing from publicly available price lists and industry-standard service costs, we find that publishing costs per article vary from US$194.89 to US$723.16, depending on the level of service and publishing volume (Table 4). It is important to note that these are conservative estimates, likely to constitute upper bounds, where innovation and changes in practice can be expected to decrease costs.\n\nPerhaps not surprisingly, the convenience of outsourcing the main publishing services to a specialized full-service provider comes with a small increase in cost (scenario A vs. scenario B), when compared to an itemized sourcing of publishing services. In our cost estimate, we have not factored in the management cost of sourcing the itemized services, as we have not included company management in our calculations. Any decision between these two options will thus have to be made after factoring in such costs as well.\n\nEven in the rare, most expensive case, these costs compare very favorably both to the current subscription pricing of around US$4,000–5,000 and current APCs (US$1,400–2,200). Our highest value encompasses conventional, journal-based pre-publication peer-review with a generic 50% rejection rate at a small journal (~100 articles per year) where all management of peer-review is performed by in-house editorial staff with no volunteer academic editors. Our data suggest that increasing only the rejection rate, for example from 50% to 90%, leads to an increase in publication costs of around 30–40% (e.g., in scenario B from US$565.15 to US$770.53 for 1,000 article journals or from US$643.61 to US$1,053.87 for 100 article journals). Apparently, this is a consequence of the respective increase of direct personnel expenses for managing the peer review process and communicating with both reviewers and authors for classical pre-publication peer review. As currently most highly selective journals publish on the order of 800–900 research articles per year about US$1,000 per article can be seen as an upper bound of total publication costs at such journals.\n\nThe workflow we model consists of verifiable, modular components, available to any entity with the desire to enter the academic publishing world. Several publishers are already on the record to operate at similar costs to the ones we have calculated. These publishers include, but are not limited to SciELO, Pensoft/arpha, Open Library of the Humanities, Ubiquity, PeerJ or Scholastica. In fact, the 2018 STM report cites survey-based data that arrive at only slightly higher average costs than our calculation (US$420–650, excluding overhead, i.e., about US$560–870 with overhead)12. Our calculations also fall in the same range as other methodologies29.\n\nOur calculations show that with publishing volumes exceeding 1,000 articles per year, fixed costs shrink below 1% of the direct article costs and become negligible. This was expected and already concluded in a previous analysis30. These insights are important for designing a transition towards a scholarly publishing platform instead of journals.\n\nDue to the limited possibility in dividing labor contracts into arbitrarily small portions, we find that journals with volumes below approx. 100 articles per year would be best served financially if they operated on the concept of volunteer academic editors handling the peer-review, instead of in-house staff.\n\nIn conclusion, given the congruence of the available data and the publicly available prices for the services required, the market rate ranges for publication services we arrive at here do not appear controversial. Perhaps more controversial is the number and amount of non-publication costs a scholarly article, funded by the taxpayer, ought to contain.\n\nIf the lowest publication costs for journals with volunteer editors constituted merely 5–10% of current subscription prices and publicly reported publisher profits only amount to an additional 30–40%, which non-publication costs are publishers currently facing and taxpayers paying for? While these costs are opaque and variable between publishers and, indeed, between journals, some estimates can be made from publicly available data. If one assumes revenue of about US$4,000 per subscription article (i.e., on the low end of the converging estimates), a conservative 30% profit margin (i.e., US$1,200 per article) for one of the large publishers21–24 and generous publication costs of US$600 per article (scenario A/B; Table 4), then there remains a sizeable gap of about US$2,200 in non-publication costs per article - more than the sum of publication costs and profits combined, or 55% of the subscription cost of a scholarly article (Figure 1). While some of these costs may be considered necessary for any business, none of them are associated with publishing primary research articles (see Methods).\n\nRunning a business: Management. While our cost calculations include generic running costs such as rent, repairs, depreciation, interest, insurance, travel expenditures, labor burden, telephone bills, supplies, taxes, accounting fees, etc., we have explicitly omitted some indirect costs such as management cost and paywalls. For instance, according to their 2016 tax statement, the New England Journal of Medicine spends 4% of its publication revenue on their top ten management staff alone (which would translate to about US$160 per article if applied to our example above; Figure 1).\n\nPreventing access: Paywalls. Subscription journals also face costs associated with paywalls. It’s difficult to estimate the cost of such technology for publishers, but the cost of a new paywall for the New York Times was reported to lie between US$25–50 million31,32. Alternatively, as the functional distinction between subscription articles and OA articles is precisely the missing paywall in OA articles, one could also assume that publishers arrive at their current APC pricing of around US$2,000 by subtracting paywall costs from their subscription price. This assumption would entail paywall costs of approx. US$2,000 per article (i.e., the difference between APC and subscription pricing).\n\nOn top of the technical costs of a paywall, one may also consider the legal fees for defending paywalls for this cost item. Publishers have a track record of litigation with regard to articles outside of their paywalls and regularly seek damages in court for actual or perceived threats to their subscription business model33–39. These costs accrue by seeking to enclose the scholarly literature within the paywalls of publisher via alternative routes in addition to the digital paywalls.\n\nNews, advertising, sales, marketing, public relations: branding. Another cost item is publishing non-research content. For instance, for 2017, PubMed lists a total of 1,595 articles published by the Lancet, while Clarivate Analytics only counts 302 articles for their Impact Factor. Assuming that only the latter articles amount to primary research publications, this journal’s revenue also pays for 1,293 non-research articles. Similar numbers also hold for other prestigious journals (e.g.: Nature: 837/2469, Science: 769/2629, New England Journal of Medicine: 327/1449; research/total), often with their own journalist and editorial staff commissioning articles and/or reporting themselves on research and policy news. However, the number of journals where this can constitute a significant fraction of their total costs is presumably small, likely restricted to the most prestigious journals.\n\nPrestigious journals also often practice active author or materials acquisition by traveling to conferences and laboratories, building networks in a strategy to entice the next exciting research finding to be published in their journals. Active author acquisition accrues costs both in terms of travel and time spent networking and communicating with authors that is not covered in our cost estimates (see Methods).\n\nSometimes, new journals also need to engage in such author acquisition practices, which, perhaps, can be best subsumed under general marketing or public relations costs required for building and maintaining a brand. These marketing costs also include, e.g., advertising in various venues targeting both authors and subscribers. For many publishers it is also common to promote their brand at conferences and institutions with, e.g., hosted speakers, travel grants or sponsored awards.\n\nBecause of the complex, time-consuming negotiations with libraries on ever tighter budgets due to the supra-inflationary subscription price increases, publishers also need to employ expert sales teams. The task of these sales teams is not only to find the most irresistible way to package and bundle subscription journals and/or databases, but also to device the most inexorable psychological strategy for their negotiations with librarians. These sales teams need to operate in close connections with the various advertising, marketing and public relations teams of the publisher to accomplish a coherent brand image. One may argue that in times of OA, these sales costs are not necessary expenses any more and more associated with paywall costs than with publication costs. On the other hand, in an OA world, one may argue that branding was never more important for author acquisition.\n\nNew technologies: innovation and acquisitions. Publishers also need to invest in innovation in order to stay current with their technologies and functionalities. While scholarly publishers have been quick to transition from print to web-based technologies in the past, the digital functionalities of most of the scholarly literature today lag at least a decade behind current functionalities of other digital objects outside of the scholarly literature. The level of investment in innovation thus remains unclear and its effects questionable. Instead of investments into their own technological innovation, publishers today appear to acquire companies that have invented desired functionalities around the scholarly workflow, with the goal to provide services beyond publications40–43.\n\nGovernment relations: Lobbying. Most international publishers, as any other corporation, also spend significant amounts of money on government relations (i.e., lobbying). Some of these corporations employ staff at the vice president level not only in the most important research nations, but also at the level of supra-national bodies such as the European Commission44. These staff, in turn, employ assistants and other members of their teams. Obviously, the task of these employees is to protect current revenue streams, e.g., subscription or APC income. For instance, one publisher, Elsevier, spends more than 400,000€ per year on lobbying at the level of the European Commission alone45. The consequences of such efforts have been observable, e.g., in the so-called “Finch Report” in the UK46, which surprised many commentators with its publisher-friendly recommendations (44, see, e.g., 47).\n\nWhich non-publication costs should remain bundled up with publishing? Regardless of all of these estimates necessarily remaining vague and imprecise, the fact remains that the scholarly community must eventually make a number of decisions if it is to tackle the affordability problem. Which of the above non-publication costs should remain bundled up with the process of publishing scholarly research articles? Which of these costs are avoidable, which necessary and which even desirable? Are profit margins of 30–40% on taxpayer funds tolerable?\n\nIn fact, one may even ask whether many of the services we list as part of the scholarly publishing standard are actually necessary for scholarly publishing. After all, journals such as the Journal of Machine Learning Research, Discrete Analysis or the Journal of Open Source Software publish their articles with internal costs below US$1048,49. Likewise, the preprint archive arXiv publishes their articles at similar costs50.\n\n\nData availability\n\nFigshare: Journal_Production_Cost_010519.xlsx. https://doi.org/10.6084/m9.figshare.8118197.v114.\n\nThis project contains the data used to calculate production costs for articles.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe are indebted to Michael Dowling, Jon Tennant, Isabell Welpe, Bernhard Mittermeier and Pete Binfield for critically reading and commenting on earlier versions of this document. We are also grateful to Abel Packer from SciELO and Brian Cody from Scholastica for privately sharing cost data from their organizations with us.\n\n\nReferences\n\nChan L: Supporting and Enhancing Scholarship in the Digital Age: The Role of Open Access Institutional Repository. CJC. 2004; 29(3). 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStern BM, O’Shea EK: A proposal for the future of scientific publishing in the life sciences. PLoS Biol. 2019; 17(2): e3000116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrossmann A: Publishing in transition – do we still need scientific journals? ScienceOpen Res. 2015 [cited 2019 Apr 2]. Publisher Full Text\n\nNosek BA, Bar-Anan Y: Scientific Utopia: I. Opening Scientific Communication. Psychol Inq. 2012; 23(3): 217–43. Publisher Full Text\n\nHartgerink C: Verified, Shared, Modular, and Provenance Based Research Communication with the Dat Protocol. Publications. 2019; 7(2): 40. Publisher Full Text\n\nPerakakis P, Taylor M, Mazza M, et al.: Natural selection of academic papers. Scientometrics. 2010; 85(2): 553–9. Publisher Full Text\n\nMcGuigan GS, Russel RD: The business of academic publishing: A strategic analysis of the academic journal publishing industry and its impact on the future of scholarly publishing. Electronic Journal of Academic and Special Librarianship. 2008 [cited 2019 May 14]; 9(3). Reference Source\n\nLarivière V, Haustein S, Mongeon P: The Oligopoly of Academic Publishers in the Digital Era. PLoS One. 2015; 10(6): e0127502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeverungen A, Böhm S: The poverty of journal publishing. Organization. 2012; 19(6): 929–38. Publisher Full Text\n\nHarvie D, Lightfoot G, Lilley S, et al.: What are we to do with feral publishers? Organization. 2012; 19(6): 905–14. Publisher Full Text\n\nOdlyzko AM: Tragic loss or good riddance? The impending demise of traditional scholarly journals. Int J Hum Comput Stud. 1995; 42(1): 71–122. Publisher Full Text\n\nJahn N, Tullney M: Data and code used from: A study of institutional spending on open access publication fees in Germany. 2016 [cited 2019 Apr 2]. Publisher Full Text\n\nSolomon D, Björk BC: Article processing charges for open access publication-the situation for research intensive universities in the USA and Canada. PeerJ. 2016; 4: e2264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorrison HG: Global OA APCs (APC) 2010-2017: Major Trends. In: 22nd International Conference on Electronic Publishing. OpenEdition Press; 2018. Reference Source\n\nPEER Economics Report. Centro ASK, Università Bocconi; 2011 [cited 2020 Oct 20]. Reference Source\n\nBogich TL, Ballesteros SP: On the Marginal Cost of Scholarly Communication. Research. 2016. Publisher Full Text\n\nPulley B: New York Times Fixes Paywall to Balance Free and Paid. Bloomberg. 2011 [cited 2019 Jun 7]. Reference Source\n\nKramer SD: New York Times Paywall Cost: More Like $25 Million. 2011 [cited 2019 Jun 7]. Reference Source\n\nHansen D: Giving the Authors a Voice in Litigation? An ACS v. ResearchGate Update - Scholarly Communications @ Duke. Scholarly Communications @ Duke. 2019 [cited 2019 May 9]. Reference Source\n\nChawla DS: Publishers take ResearchGate to court, alleging massive copyright infringement. Science. 2017. Reference Source\n\nVan Noorden R: Publishers threaten to remove millions of papers from ResearchGate. Nature. 2017; 112: 241. Publisher Full Text\n\nAssociation Of American Publishers: Statement on Sci-Hub Litigation. Statement on Sci-Hub Litigation. Association Of American Publishers; 2015 [cited 2019 May 9]. Reference Source\n\nCox K: Eleventh Circuit Reverses and Remands Georgia State E-Reserves Case (Again) \| ARL Policy Notes. 2018 [cited 2019 May 9]. Reference Source\n\nFlaherty C: An academic press sues a librarian, raising issues of academic freedom. 2013 [cited 2019 May 9]. Reference Source\n\nSchiermeier Q: US court grants Elsevier millions in damages from Sci-Hub. Nature. 2017; 6: 541. \|Publisher Full Text\n\nBosman J, Kramer B: Workflows. Innovations in Scholarly Communication. 2018 [cited 2019 May 7]. Reference Source\n\nCrunchbase: Website. 2019 [cited 2019 May 7]. Reference Source\n\nPosada A, Chen G: Inequality in Knowledge Production: The Integration of Academic Infrastructure by Big Publishers. In: 22nd International Conference on Electronic Publishing. OpenEdition Press; 2018. Reference Source\n\nCampfens Y: Market research report: What has become of new entrants in research workflows and scholarly communication? Open Science Framework. 2019. Publisher Full Text\n\nTennant J: Democratising Knowledge: a report on the scholarly publisher, Elsevier. Education International; 2018. Reference Source\n\nLobbyFacts: RELX. Lobbyfacts. 2018 [cited 2019 Jun 5]. Reference Source\n\nFinch J: Accessibility, sustainability, excellence: How to expand access to research publications: Report of the Working Group on Expanding Access to Published Research Findings [“The Finch Report”]. 2012 [cited 2019 May 7]. Reference Source\n\nPrior A: Key Issue - The “Finch Report”: the future is gold, but many challenges lie ahead. Insights: the UKSG journal. 2013; 26(1): 77-81. Publisher Full Text\n\nTennant J: Why the term “Article Processing Charge” (APC) is misleading - Green Tea and Velociraptors. Green Tea and Velociraptors. 2018 [cited 2019 Jun 7]. Reference Source\n\nKatz DS, Barba LA, Niemeyer KE, et al.: Cost models for running an online open journal \| Journal of Open Source Software Blog. 2019 [cited 2019 Jun 16]. Reference Source\n\nCornell University Library: arXiv Business Model White Paper \| arXiv e-print repository. arXiv. 2010 [cited 2019 Jun 7]. Reference Source" }	[ { "id": "77204", "date": "01 Feb 2021", "name": "Pandelis Perakakis", "expertise": [ "Reviewer Expertise Scholarly communication", "Open peer review" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reports a breakdown of article publication costs —from manuscript submission to indexing— for different publication scenarios. There are at least two fundamental reasons why analysing and reporting the realistic cost of publishing a research article is of paramount importance. First, given the exorbitant sums of public money spent to publish the world’s scholarly output it is imperative that we know where exactly this money goes. This information can perhaps mobilise governments, funders, individual scholars, and the general public to demand more sustainable publication models that provide only those essential services that truly add value to scholarly works. Second, a granular account of publishing costs can incentivise more scientific societies and academic groups to abandon their commercial publishers and establish alternative models that better serve the needs of scholarly communication. There is no doubt therefore that this is an extremely valuable report that should be widely disseminated. My review will mainly focus on some recommendations that in my opinion would improve the presentation and utility of the results.\nThe cost analysis provided in this report is based on a list of 24 services grouped in three distinct categories: content acquisition, preparation, and dissemination. A first minor observation is that in the category of content acquisition, it would probably be more intuitive to list “online submission system” as the first item (before “searching and assigning reviewers”) to match the publication workflow. A second and more important observation is that in this list, and throughout the manuscript, services are sometimes confounded with service providers. Since there are more than one providers for each of the services, I strongly recommend that the list only includes services, whereas in the manuscript different options for each service can be discussed. For example Crossref is not the only option for “DOI registration”, which I believe should be the title of this particular service. Similarly, the service provided by CLOCKSS/LOCKSS/Portico, could be called “long-term digital preservation”. Also, it is not clear what the service provided by OAPEN is, while “upload to Scopus…”, could be called “distribution to indexing services”. Should the authors decide to follow this recommendation I would advise to also modify the accompanying excel file accordingly.\nThe “Direct or variable costs” subsection in the methods section would be easier to follow if it was structured differently, starting with the list of services and then explaining how pricing information was obtained.\nFollowing the logic that the list of services is the nucleus of the cost analysis, and the report itself, I suggest that table 1 is modified so that the first column displays the services and the second column the providers. Also, it is important that the terms in the services list are used consistently throughout the manuscript. For example, in table 1 we find a service termed “peer review” and another called “peer review management”. It is not clear how these services correspond to the original list. When a service provider offers more than one service in the list, I would recommend to include all of these services in the table. If it is considered convenient to create a new grouping (e.g., peer review management), it should be clear —in the table and in the manuscript— which services from the original list are included in this group (e.g., 1a, 1b, 1c, etc.).\nThe “scenarios” section should include a more thorough description of the different scenarios, as well as the motivation for selecting each of these scenarios as a separate use case. This description of what each scenario refers to is attempted in the “results” section but in a rather anarchic and hard to follow manner. In the “scenarios” section the authors vaguely mention that these scenarios “...correspond either to existing publishing options or to options that have been discussed in the literature”, but neither are these options clearly delimited nor specific references are provided. I therefore found it very hard by reading the manuscript to understand exactly which publication model, combination of services, and choice of providers each scenario refers to. This was made possible only by carefully analysing the formulas for the different calculations in the excel file. However, even after this more careful analysis, the motivation behind each scenario remained unclear. For example, it seems that the only difference between scenarios A and B in terms of content acquisition is using one service provider instead of another. In scenario A scholastica is used for online submission management (cell L18), while Akron is used in scenario B (cell J19).\nI recommend that the different scenarios are not defined based on the choice of specific providers but according to different, clearly explained publication models, characterised, for example, by use of existing publishing infrastructure (e.g., institutional or disciplinary repositories as in the case of existing overlay journals), variable rejection rates, number of published articles, review policies, voluntary or hired editorial work, etc. The available choice of providers for specific services (or groups of services) and their impact on the costs can be discussed in the manuscript and even presented as a separate table. However, I consider it important that the initial scenarios are reported without any reference to specific providers but rather using average (or low/high) estimates. Otherwise, in their present form, tables 3 and 4 are very hard to follow. In both tables publication choices (number of articles, review models, voluntary editors) are mixed with providers (scholastica, generic providers, etc.) without a clear description (in the table or the manuscript) of the different publication models, or the services offered by each provider. For example, from the excel file I deduced that scenarios A2, B2 and C do not include costs for online submission, but it is not clear from the discussion in the manuscript or from the brief labels in table 3, which exact models allow the omission of these costs and how.\nInsisting on the necessity to adequately describe the different publication scenarios, in the “scenarios” section the authors refer to a “decentralized/federated platform providing publishing functionalities”. However, again it is not clear what are the foundations of this model (e.g., a reference could be provided to the next generation repositories initiative promoted by COAR [1], or other similar proposals in the literature, such as in reference “20” in the manuscript), and whether this model is represented in one of the scenarios. Similarly, the authors briefly mention a PPPR model in the “results” section but there is no clear description of what exactly this model entails and which services from the original list allows to omit or circumvent.\nTo summarise, I strongly recommend that the different publication scenarios refer to publication options (not choice of providers) and that they are concisely described in the corresponding section with references to the literature or to existing examples when possible. It should be clear which services from the list correspond to each scenario and how different scenarios allow the omission of certain services. The different options for service providers should be discussed separately. For example, it would be useful to report the impact on the publication costs of choosing scholastica or Akron as a provider for a specific list of services (drawn from the initial services list) in a given publication scenario.\nAs a minor comment, I suspect that J21 is missing from the formula used to calculate cell J26. I would recommend that the authors had another careful look at their formulas to avoid similar omissions or errors.\nOverall, I commend the authors for their work and invite them to consider my recommendations that I believe will significantly improve the uptake of this extremely valuable information.\nReferences: [1] https://www.coar-repositories.org/news-updates/what-we-do/next-generation-repositories/\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6716", "date": "01 Jul 2021", "name": "Björn Brembs", "role": "Author Response F1000Research Advisory Board Member", "response": "Response to Reviewer #1: Pandelis Perakakis: The cost analysis provided in this report is based on a list of 24 services grouped in three distinct categories: content acquisition, preparation, and dissemination. A first minor observation is that in the category of content acquisition, it would probably be more intuitive to list “online submission system” as the first item (before “searching and assigning reviewers”) to match the publication workflow. Thank you, corrected. A second and more important observation is that in this list, and throughout the manuscript, services are sometimes confounded with service providers. Since there are more than one providers for each of the services, I strongly recommend that the list only includes services, whereas in the manuscript different options for each service can be discussed. For example Crossref is not the only option for “DOI registration”, which I believe should be the title of this particular service. Similarly, the service provided by CLOCKSS/LOCKSS/Portico, could be called “long-term digital preservation”. Also, it is not clear what the service provided by OAPEN is, while “upload to Scopus…”, could be called “distribution to indexing services”. Should the authors decide to follow this recommendation I would advise to also modify the accompanying excel file accordingly. This was our oversight and it is an excellent suggestion. We are now following it 100%, also in the Excel spreadsheet. The “Direct or variable costs” subsection in the methods section would be easier to follow if it was structured differently, starting with the list of services and then explaining how pricing information was obtained. This is how it should be and we have no good explanation for why we did not write it that way. We have now re-ordered all components as suggested. Following the logic that the list of services is the nucleus of the cost analysis, and the report itself, I suggest that table 1 is modified so that the first column displays the services and the second column the providers. Corrected. Also, it is important that the terms in the services list are used consistently throughout the manuscript. For example, in table 1 we find a service termed “peer review” and another called “peer review management”. It is not clear how these services correspond to the original list. When a service provider offers more than one service in the list, I would recommend to include all of these services in the table. If it is considered convenient to create a new grouping (e.g., peer review management), it should be clear -in the table and in the manuscript- which services from the original list are included in this group (e.g., 1a, 1b, 1c, etc.). I see the point here, of course. We have been able to quickly fix the instances where it was just a matter of replacing a few words. However, it was not easy to address this very relevant point everywhere, as it is many times easier to refer to a series of steps with a shorthand rather than with a long list. Sometimes that shorthand can be a provider (e.g., Scholastica), sometimes it is a functionality such as, e.g., “OJS” for a system that manages submission, manuscript tracking and review management all at the same time. We have tried to rephrase as many of such instances as possible, but we are not sure if we were able to address this point in every single instance. The “scenarios” section should include a more thorough description of the different scenarios, as well as the motivation for selecting each of these scenarios as a separate use case. This description of what each scenario refers to is attempted in the “results” section but in a rather anarchic and hard to follow manner. In the “scenarios” section the authors vaguely mention that these scenarios “...correspond either to existing publishing options or to options that have been discussed in the literature”, but neither are these options clearly delimited nor specific references are provided. I therefore found it very hard by reading the manuscript to understand exactly which publication model, combination of services, and choice of providers each scenario refers to. This was made possible only by carefully analysing the formulas for the different calculations in the excel file. However, even after this more careful analysis, the motivation behind each scenario remained unclear. For example, it seems that the only difference between scenarios A and B in terms of content acquisition is using one service provider instead of another. In scenario A scholastica is used for online submission management (cell L18), while Akron is used in scenario B (cell J19). I recommend that the different scenarios are not defined based on the choice of specific providers but according to different, clearly explained publication models, characterised, for example, by use of existing publishing infrastructure (e.g., institutional or disciplinary repositories as in the case of existing overlay journals), variable rejection rates, number of published articles, review policies, voluntary or hired editorial work, etc. The available choice of providers for specific services (or groups of services) and their impact on the costs can be discussed in the manuscript and even presented as a separate table. However, I consider it important that the initial scenarios are reported without any reference to specific providers but rather using average (or low/high) estimates. Otherwise, in their present form, tables 3 and 4 are very hard to follow. In both tables publication choices (number of articles, review models, voluntary editors) are mixed with providers (scholastica, generic providers, etc.) without a clear description (in the table or the manuscript) of the different publication models, or the services offered by each provider. For example, from the excel file I deduced that scenarios A2, B2 and C do not include costs for online submission, but it is not clear from the discussion in the manuscript or from the brief labels in table 3, which exact models allow the omission of these costs and how. It is completely obvious how this section must be confusing for readers and, again, we do not have a good explanation for why we did not provide sufficient detail. Our lack of explanation could also be seen in some of Lisa Rose-Wiles’ comments. In attempting to follow these suggestions, we now have provided not only detailed explanations for each scenario in the text, but have also updated Table 3. In brief, scenario A and B differ in that B cases source multiple, generic publishing providers for the different steps (lower cost, more contracts, requires expertise), while A sources many steps from a single provider, specialized in scholarly publishing (more convenient, less expertise required, higher cost). Scenario C also covers all steps in principle, but does not count some costs such as editors (volunteers) or servers (institutional server) or a submission/tracking system (e.g., OJS). Post-publication peer-review decreases rejections and hence price (A2/B2). Scenario C2 replaces, e.g., servers and OJS with Scholastica. Insisting on the necessity to adequately describe the different publication scenarios, in the “scenarios” section the authors refer to a “decentralized/federated platform providing publishing functionalities”. However, again it is not clear what are the foundations of this model (e.g., a reference could be provided to the next generation repositories initiative promoted by COAR [1], or other similar proposals in the literature, such as in reference “20” in the manuscript), and whether this model is represented in one of the scenarios. Similarly, the authors briefly mention a PPPR model in the “results” section but there is no clear description of what exactly this model entails and which services from the original list allows to omit or circumvent. We now cite Perakakis et al. 2010 very prominently, as well as the COAR report, together with a brief explanation of the concept. Both are referenced with the name they gave these solutions, i.e., “global open archive” or ”next generation repository”, respectively. To summarise, I strongly recommend that the different publication scenarios refer to publication options (not choice of providers) and that they are concisely described in the corresponding section with references to the literature or to existing examples when possible. It should be clear which services from the list correspond to each scenario and how different scenarios allow the omission of certain services. The different options for service providers should be discussed separately. For example, it would be useful to report the impact on the publication costs of choosing scholastica or Akron as a provider for a specific list of services (drawn from the initial services list) in a given publication scenario. We are confident that the current version now addresses all the raised issues and that our choice of scenarios becomes clearer in the way we explain them now. As a minor comment, I suspect that J21 is missing from the formula used to calculate cell J26. I would recommend that the authors had another careful look at their formulas to avoid similar omissions or errors. We have triple-checked these cells in the spreadsheet and could not find any missing values. We have also checked all adjacent cells for possible errors and were not able to find any. We have gone over the entire spreadsheet both for the consistency of the calculations, the accuracy of the text descriptions and whether any of the costs needed to be updated due to change market rates. We have not been able to locate any problems. We suggest arranging an online call with video and screen sharing to identify and remove potential errors we may have missed or are unable to identify." }, { "c_id": "6909", "date": "13 Jul 2021", "name": "Pandelis Perakakis", "role": "Reviewer Response", "response": "I checked again and J21 is included in the calculation of J26. If it was my mistake in the original report, I apologise sincerely." } ] }, { "id": "77201", "date": "16 Feb 2021", "name": "Lisa Rose-Wiles", "expertise": [ "Reviewer Expertise Library science", "information literacy", "citation analyses", "circulation and journal/book pricing analyses." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 concur that the “supra-inflation increase of subscription prices for scholarly journals” is a critical issue, especially for academic libraries, and that this is a valuable contribution to the literature.\nI also concur with the previous reviewer that the “scenarios” and tables could be better explained and organized for greater clarity. Also further indicators of variance would be helpful - for example, there are surely significant differences (especially in “fixed or indirect costs”) based on geographic location. I also find myself wondering which (if any) of the costs to publishers are tax deductible, which would effectively increase the profit margin.\nWhile the main focus of the manuscript is the cost of producing articles, to place this in broader perspective It would be helpful to clarify more explicitly that there are two major price components to journal articles: (1) Journal subscription costs, which are typically borne by libraries (although no doubt some researchers still subscribe to their favorite journals themselves), and (2) article-processing charges (APC’s) which are in principle an open-access alternative to subscriptions, but in practice often simply shift the cost from an institution’s library to its faculty, research office and/or some other institutionally-funded entity. Both contribute directly or indirectly to the high price of student tuition and the chronic under-funding of many academic libraries, but subscription prices seem to be the greater issue since the average “per article price” (see below) is considerably higher.\n\nFor readers not necessarily familiar with the various journal pricing models, the authors might note that both journal prices and APC’s vary enormously by discipline, especially STEM vs. humanities, and that the practice of “Big Deal” bundling (especially by large commercial publishers) can make it very difficult for librarians to disentangle the actual subscription cost per journal. It would be helpful to include some discussion and more explicit data on the widely varying range of APC’s, which, in my experience, seem independent of actual subscription costs. I am more familiar with the pricing models for STEM journals (typically > US $2,500 APC regardless of journal subscription price), but it is my impression that when humanities journals do charge authors, the fee is much lower. This would be an interesting topic to explore in a follow-up article. Granted that STEM articles typically include multiple tables and figures that are costly to produce, but is the price differential really as substantial as the differences in subscription costs or APC’s?\nIn the final paragraph of the results (p.6) and in Figure 1, the authors compare the article cost with “the average price estimate for a subscription article of about US$4,000”. Where did this figure come from and what is it based on? Is this an across the board “per library” average, in which case the wide variation in prices paid by different libraries (at least in the US) is surely another confounding factor. Again, there should be some discussion of the vast difference in pricing among disciplines, or if this is based on STEM journals as I suspect, that should be clarified. Also the average APC price should be included in the comparison; it is interesting that it appears to be considerably less than the quoted US$4,000 subscription “per article” cost. This brings up a further question for the future: in hybrid journals, does charging APC’s result in lower subscription prices, or are APC’s effectively layered on top of subscription prices?\nAnother point that is only tangentially referenced (e.g. “volunteer academic editors”) is that many of the costs associated with publishing are actually borne by the institution and its academic faculty, who not only produce the scholarship but also review articles and often act as editors as well. This labor is typically unpaid. “Volunteer academic editors” in well-funded institutions may receive some form of course release, additional office space and/or clerical support, but again (see first paragraph) these costs are borne by the institution and to some degree passed on to its students; they are not borne by the publishers. Of course this point has been made many times before, but since the authors are discussing various costs involved in publication it is worth noting again.\nAs a final comment, it would be informative to have responses from publishers, especially small “non-profit” or “not-for-profit” publishers on this publication.\nVery thought provoking!\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6717", "date": "01 Jul 2021", "name": "Björn Brembs", "role": "Author Response F1000Research Advisory Board Member", "response": "Response to Reviewer #2: Lisa Rose-Wiles: I also concur with the previous reviewer that the “scenarios” and tables could be better explained and organized for greater clarity. We thank both reviewers for their suggestions and are confident to have now adequately addressed this very valid concern in the new, revised version of the manuscript. Also further indicators of variance would be helpful - for example, there are surely significant differences (especially in “fixed or indirect costs”) based on geographic location. I also find myself wondering which (if any) of the costs to publishers are tax deductible, which would effectively increase the profit margin. We have now further emphasized that our salary calculations are based on western (i.e., US/EU) industry standards and that countries with lower salary levels would hence face lower costs. This is to arrive at upper bound figures, describing the costliest way of publishing, from which one can always find ways to reduce costs. We have further emphasized this approach also at the very beginning of the Discussion section now. While the main focus of the manuscript is the cost of producing articles, to place this in broader perspective It would be helpful to clarify more explicitly that there are two major price components to journal articles: (1) Journal subscription costs, which are typically borne by libraries (although no doubt some researchers still subscribe to their favorite journals themselves), and (2) article-processing charges (APC's) which are in principle an open-access alternative to subscriptions, but in practice often simply shift the cost from an institution's library to its faculty, research office and/or some other institutionally-funded entity. Both contribute directly or indirectly to the high price of student tuition and the chronic under-funding of many academic libraries, but subscription prices seem to be the greater issue since the average “per article price” (see below) is considerably higher. For readers not necessarily familiar with the various journal pricing models, the authors might note that both journal prices and APC's vary enormously by discipline, especially STEM vs. humanities, and that the practice of “Big Deal” bundling (especially by large commercial publishers) can make it very difficult for librarians to disentangle the actual subscription cost per journal. It would be helpful to include some discussion and more explicit data on the widely varying range of APC's, which, in my experience, seem independent of actual subscription costs. I am more familiar with the pricing models for STEM journals (typically > US $2,500 APC regardless of journal subscription price), but it is my impression that when humanities journals do charge authors, the fee is much lower. This would be an interesting topic to explore in a follow-up article. Granted that STEM articles typically include multiple tables and figures that are costly to produce, but is the price differential really as substantial as the differences in subscription costs or APC's? We agree with all the arguments here. Therefore, we now more explicitly reference the longer, preprint version of our manuscript, where we not only discuss pricing strategies and business models, as suggested, but also policy options. However, in this more condensed version, we made a conscious effort to focus exclusively on the cost aspect, as we felt the original version was too long, unwieldy and difficult to read as it was covering too many topics at the same time. In the final paragraph of the results (p.6) and in Figure 1, the authors compare the article cost with “the average price estimate for a subscription article of about US$4,000”. Where did this figure come from and what is it based on? Is this an across the board “per library” average, in which case the wide variation in prices paid by different libraries (at least in the US) is surely another confounding factor. This explanation was indeed lost as we transitioned from our longer preprint version to this shorter one. We are very grateful for having this pointed out. We now reference the sources of these calculations. The most recent one is very simple: dividing the estimated US$10b in journal revenue by the number of published articles (2m): ~5k. As some other sources mention 4k, we went with the lower bound of these estimates in the literature. Again, there should be some discussion of the vast difference in pricing among disciplines, or if this is based on STEM journals as I suspect, that should be clarified. Also the average APC price should be included in the comparison; it is interesting that it appears to be considerably less than the quoted US$4,000 subscription “per article” cost. This brings up a further question for the future: in hybrid journals, does charging APC's result in lower subscription prices, or are APC's effectively layered on top of subscription prices? We now mention the APC price range of US$1,400-2,200 and the 8 references upon which we base this range. Another point that is only tangentially referenced (e.g. “volunteer academic editors”) is that many of the costs associated with publishing are actually borne by the institution and its academic faculty, who not only produce the scholarship but also review articles and often act as editors as well. This labor is typically unpaid. “Volunteer academic editors” in well-funded institutions may receive some form of course release, additional office space and/or clerical support, but again (see first paragraph) these costs are borne by the institution and to some degree passed on to its students; they are not borne by the publishers. Of course this point has been made many times before, but since the authors are discussing various costs involved in publication it is worth noting again. We now make it more explicit what is being paid in terms of salary and which work is being carried out by volunteers. We also explicitly mention the costs covered by institutions (e.g., salaries, servers) and relate them to per-article cost. As a final comment, it would be informative to have responses from publishers, especially small “non-profit” or “not-for-profit” publishers on this publication. We list numerous publishers which are already on the record for publishing in this cost range and cite journals that have much lower costs than our figures. In the acknowledgements, we mention that we have been made privy of internal cost structures to validate and test our figures. As we mention in the article, our numbers match those from other methodologies (e.g. surveys). The preprint version has been available since 2019 and has even received attention in trade organs such as the “Scholarly Kitchen”. In other words, publishers have already vetted our numbers and have had ample opportunity over a number of years to comment on our work and improve it by criticism. Given that our numbers match so well with the available evidence and that our article has received considerable attention from the industry since 2019, perhaps one may interpret the fact that the expensive legacy publishers so far have not publicly commented on these numbers, as evidence that there may not be much left to criticize? Of course, another reason may be that the more expensive publishers may hesitate to draw more attention to the fact that they are so much more expensive. In this case, we would appreciate any suggestions in how we may be able to force these publishers to acknowledge and comment on our calculations. F1000Research allows for comments on the manuscript and perhaps a drive to invite publishers of all ranges to contribute so such a comment may be instructive? Society publishers, so far, have refrained from public comments other than generally stating that they use publication income to finance society services. We allude to many such and related costs in the “non-publication costs” section of the manuscript." } ] } ]	1	https://f1000research.com/articles/10-20
https://f1000research.com/articles/10-370/v1	11 May 21	{ "type": "Research Article", "title": "Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology", "authors": [ "Mauro Petrillo", "Carlo Brogna", "Simone Cristoni", "Maddalena Querci", "Ornella Piazza", "Guy Van den Eede", "Carlo Brogna", "Simone Cristoni", "Maddalena Querci", "Ornella Piazza", "Guy Van den Eede" ], "abstract": "Background Scientific evidence for the involvement of human microbiota in the development of COVID-19 disease has been reported recently. SARS-CoV-2 RNA presence in human faecal samples and SARS-CoV-2 activity in faeces from COVID-19 patients have been observed. Methods Starting from these observations, an experimental design was developed to cultivate in vitro faecal microbiota from infected individuals, to monitor the presence of SARS-CoV-2, and to collect data on the relationship between faecal bacteria and the virus. Results Our results indicate that SARS-CoV-2 replicates in vitro in bacterial growth medium, that the viral replication follows bacterial growth and it is influenced by the administration of specific antibiotics. SARS-CoV-2-related peptides have been detected in 30-day bacterial cultures and characterised. Discussion Our observations are compatible with a ‘bacteriophage-like’ behaviour of SARS-CoV-2, which, to our knowledge has not been observed or described before. These results are unexpected and hint towards a novel hypothesis on the biology of SARS-CoV-2 and on the COVID-19 epidemiology. The discovery of possible new modes of action of SARS-CoV-2 has far-reaching implications for the prevention and the treatment of the disease.", "keywords": [ "SARS-CoV-2", "COVID-19", "gut microbiota" ], "content": "Introduction\n\nRecent scientific articles and reviews1–3 discuss the relationship between gastrointestinal microbiota and COVID-19 disease. In particular, the prolonged presence of SARS-CoV-2 RNA in human faecal samples from COVID-19 patients has been reported4 and the potential role of orofecal transmission of SARS-CoV-2 has been examined in systematic reviews5,6 and open evidence briefs.7,8 Cases of SARS-CoV-2 detection in faecal samples from patient with typical symptoms of COVID-19 but negative to multiple SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) tests on oropharyngeal and nasopharyngeal swabs have been reported.9 SARS-CoV-2 faecal viral activity was depicted in association with gut microbiota composition in patients with COVID-19,10 and the replicating virus was detected in faeces.11 At the same time, Wölfel et al.12 observed high viral RNA concentration in stool samples, but reported isolation of infectious virus only from throat- and lung-derived samples, while Yao et al.13 had indication of viable SARS-CoV-2 particles in stool samples, denoting that the detailed biology of SARS-CoV-2 is not yet fully elucidated. Our experiments further explored the relationship between COVID-19 disease and SARS-CoV-2 infected faeces to provide data relevant for pandemic understanding and disease management. The results however did not correspond with current thinking of the epidemiology of SARS-CoV-2 and, therefore, we believe a quick sharing with the scientific community of our findings is imperative.\n\n\nMethods\n\nThe experimental design included:\n\n\n\n1) the inoculation of NutriSelect™ Plus nutrient broth at 37°C, fit for the growth of more fastidious bacteria, with a faecal sample (stool) from one subject positive to SARS-CoV-2 and from a healthy individual (here called sample A and sample B, respectively) following written informed consent.\n\n2) The assessment of the presence of SARS-CoV-2 RNA in both samples, after seven days of culture, using the Luminex technology; with confirmation of the presence of SARS-CoV-2 RNA in sample A, and of its absence in sample B.\n\n3) Inoculation of sample B with the supernatant of sample A, obtained after centrifugation (hereafter called sample B(A+)) and resuspension of the formed pellet (sample C).\n\n4) Incubation of all samples (A, B, B(A+) and C) for 30 days under the same conditions in NutriSelect™ Plus nutrient broth at 37°C with measurement of the viral RNA load in each sample at days 1, 2, 3, 7, 14, 21, and 30, following the date of inoculation (day 0).\n\n5) Antibiotic treatment on 18 aliquots derived from sample B(A+) at day 21, consisting in the addition of a specific antibiotic (each of the following: metronidazole, clindamycin, lincomycin, piperacillin+tazobactam, vancomycin, amoxicillin, ampicillin, cefixime, ceftriaxone, meropenem, rifaximin, azithromycin, erythromycin, gentamicin, ciprofloxacin, colistin, levofloxacin, and teicoplanin) to each aliquot. SARS-CoV-2 RNA load was measured by Luminex technology in each aliquot before and 3 days after antibiotic administration.\n\n6) In parallel, additional analyses were performed to evaluate and monitor over time the bacterial growth and metabolic activity of all samples and all aliquots of sample B(A+), using SANIST Biotyper according to the method described by Cristoni et al.14\n\n7) Purification and analysis of the peptides present in sample B(A+) at day 30.\n\nThe details of the procedures and protocols used are presented in the extended data, together with a schematic representation of the experimental design (Graphical abstract).35\n\n\nResults\n\nThe experimental design included a series of analyses (performed on all samples A, B, B(A+) and C) aimed at verifying: 1) the permanence/survival over time and the eventual multiplication of SARS-CoV-2 RNA in vitro; 2) the presence/synthesis of SARS-CoV-2 peptides in the cultures having confirmed SARS-CoV-2 RNA presence; 3) the effect of antibiotics administration in sample B(A+); 4) the concomitant presence of other metabolites; and 5) the characterisation of the bacterial samples, including the verification of the presence of eukaryotic cells.\n\nThe results presented and discussed here, carried out over a period of 30 days, confirmed the extra-corporal multiplication of SARS-CoV-2 RNA: viral load highly increased over time in sample B(A+), slightly increased in sample A, decreased in sample C while, as expected, sample B was found constantly negative (Figure 1).\n\nSARS-CoV-2 RNA load measurements (reported as AU, see extended data) of samples A (blue bars), B (orange bars), B(A+) (red bars), and C (azure bars) grown, all under the same conditions for thirty days from inoculation (day 0). SARS-CoV-2 RNA load in sample B(A+) had a power increase trend over time (as shown in the small frame on top-left), slightly increased in sample A, and decreased in sample C. As expected, sample B was found constantly negative.\n\nIn order to verify the reproducibility of our results, the whole experiment was repeated independently three times using the same infected and healthy samples (with the exception that the repetition experiments were stopped at day 14 instead of day 30). The results of the SARS-CoV-2 RNA load measurements in the repetitions are reported in Figure 2, where results are depicted as the average of the measurements of the three repetitions, together with the calculated standard deviations. The trend was confirmed, with the increase over time of measured viral RNA load in sample A and sample B(A+). Decrease in sample C and no detection in sample B were also confirmed, but they are not reported in Figure 2.\n\nThe graph reports average results of three repetitions of the experiment conducted using the same starting material as described in Figure 1 (with the exception that the repetitions were stopped at day 14 instead of day 30). To normalise the measurements, all values at day 0 were used as denominator (at day 0 all values = 1), i.e. for each sample, at day X, the ratio between LuminexCountAtDayX/LuminexCountAtDay0 was calculated. Each bar represents the average of the SARS-CoV-2 RNA load ratio of samples A (blue bars) and B(A+) (red bars), together with the calculated standard deviations.\n\nResults of experiments combining samples from three “infected donor” sources (A1, A2 and A3) and from three “healthy recipient” sources (B1, B2 and B3). The graphs report results of nine combinations. To normalise the measurements, all values at day 0 were used as denominator (at day 0 all values = 1), i.e. for each sample, at day X, the ratio between LuminexCountAtDayX/LuminexCountAtDay0 was calculated. Each bar represents the SARS-CoV-2 RNA load ratio. Although with certain differences, the observed trends are similar, confirming the increase over time of SARS-CoV-2 RNA load in samples of type A and samples of type B(A+), independently from the sources of A and B.\n\nIn addition, three new couples of faecal samples from different “infected donors” (i.e. sources of A) and “healthy recipients” (i.e. sources of B) have been recruited, and subject to the same experimental procedure. Samples were collected from anonymous donors, and no information (i.e. age, sex, blood serotype, severity of the disease, time of the collection, fatality, etc.) is available. All combinations of “infected donors” sources (A1, A2 and A3) and “healthy recipients” donors (B1, B2 and B3) were subject to the same experimental procedure. Although with certain differences, the observed trends are similar, confirming the increase over time of SARS-CoV-2 RNA load in samples of type A and in samples of type B(A+), independently from the sources of A and B.\n\nAliquots of sample B(A+) tested after three days of culture in the presence of the single different antibiotics belonging to different classes were analysed and the SARS-CoV-2 RNA load measured in each of them. SARS-CoV-2 RNA load was found to be influenced by the presence of antibiotics in different ways (Figure 4):\n\n• SARS-CoV-2 RNA load was reduced to undetectable levels in the four aliquots treated with metronidazole, vancomycin, amoxicillin and azithromycin, respectively.\n\n• SARS-CoV-2 RNA load decreased by 20% to 85% in the aliquots treated with piperallicin+tazobactam, ampicillin, cefixime, ceftriaxone, meropenem, gentamicin, ciprofloxacin and teicoplanin. For example, cefixime induced a decrease of viral RNA load of 85%, ciprofloxacin of 61% and teicoplanin of 56%.\n\n• SARS-CoV-2 RNA load was not substantially affected by the presence of clindamycin, lincomycin, rifaximin, erythromycin, colistin and levofloxacin.\n\nSARS-CoV-2 RNA load measurements (reported as AU, see Supplementary Material) of eighteen aliquots pre- (red) and post- (three days, dashed) treatment with the following selection of antibiotics (ABX): Metronidazole (class: Azoles); Clindamycin, Lincomycin, Piperacillin+Tazobactam, Vancomycin (class: Carboxylic acids and derivatives); Amoxicillin, Ampicillin, Cefixime, Ceftriaxone, Meropenem (class: Lactams); Rifaximin (class: Macrolactams); Azithromycin, Erythromycin, Gentamicin (class: Organooxygen compounds); Ciprofloxacin, Colistin, Levofloxacin (class: Quinolines and derivatives); Teicoplanin (semisynthetic glycopeptide antibiotic). SARS-CoV-2 RNA load is reported as preABX-postABX variation in percentage.\n\nAfter 30 days of bacterial growth in culture, aliquots of samples B(A+) (from couple 0) were collected and tested for the presence of SARS-CoV-2-related peptides using mass spectrometry (details are described in extended data35). Several peptides found in the aliquot from sample B(A+) were assigned to SARS-CoV-2 proteins. As shown in Table 1, the sequence of some of the peptides (pep51 and pep121, matching on NSP3; pep199, matching on the spike protein; pep25 and pep68, matching on NS3 and N, respectively) have one or more amino acid (AA) changes (highlighted in red) with respect to the translations of CDS regions reported in the reference ‘Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome sequence’ (GenBank LOCUS: NC_045512.2). No SARS-CoV-2-related peptide was identified in the aliquot of sample B.\n\nAmino acids highlighted in red represent changes with respect to the translations of CDS regions reported in the reference severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome sequence (GenBank LOCUS: NC_045512.2). Pep51, pep121, and pep230 were found with a different mass spectrometry approach using the Q Exactive HF Hybrid Quadrupole-Orbitrap with an ultra-high-field analyser (Brogna, personal communication).\n\nThe identified AA changes have been checked for their existence among the observed variations in SARS-CoV-2 sequenced isolates available in GISAID15 at time of writing. As shown in Table 2, all of them except NSP3:A274K in pep51 have been already reported in humans; the majority of them have been never reported in the country of origin of the samples (Italy), the remaining ones have been observed in samples sequenced in Italy, but after the time of collection of the infected sample A (February 2020). Some of the found peptides mapping on the SARS-CoV-2 spike protein are shown in Figure 5.\n\nAll of them, except one, have been already reported in humans, and only two in Italy. For each amino acid change, the number of occurrences in GISAID isolates is reported, together with details of the first human isolate recorded in GISAID with reported collection date. AA change NSP3:A274K of pep51 has never been reported in human SARS-CoV-2 sequences, but it has been found in beta-CoV genome sequences from bats (isolate hCoV-19/bat/Yunnan/RmYN01/2019, collection date 25-06-2019).\n\nLocal alignments of peptides identified in sample B(A+) mapping on three different regions of SARS-CoV-2 reference spike protein (NCBI protein LOCUS YP_009724390.1). Amino acids highlighted in red correspond to changes described in Table 1 and Table 2.\n\nWe have already described the detection of toxin-like peptides in plasma, urine and faecal samples from COVID-19 affected individuals (Cristoni et al.,16 under review). The evaluation on the potential release of toxic-like peptides in aliquots from sample B(A+) has been assessed by performing the same analyses. Toxin-like peptides have been observed, but their presence was completely reduced to negligible levels in the aliquots treated with metronidazole and vancomycin administration (data not shown). These results need to be carefully interpreted, taking into account the different antimicrobials kinetics.\n\nSamples A and B(A+) were found to contain some bacterial genera particularly abundant and metabolically active during the whole experiment, as shown in Figure 6.\n\nThe presence of bacteria genera was monitored over time by looking at their metabolic activity as described by Cristoni et al.14 Measures on Y-axis are reported as “detection frequency” (range 0-10). The two charts report the most metabolically active genera identified together with the “generic bacterial gut flora” (representing other bacterial genera not classified by the instrument) at day 0, 1, 7, 14, 21, and 30 for samples A, and B(A+). Other microbial organisms were observed at low levels (2 or less, at day 7) and not reported in the figure: Mycobacterium, Actinobacteria, Bacteroidetes, Blautia, Brevibacterium, Brevundimonas, Candida (C. albicans), Collinsella, Enterococcus, Eubacterium, Klebsiella, Lactonifactor, Microbacterium, Porphyromonas, Propionibacterium, Sphingomonas, Stenotrophomonas, Streptococcus gordonii, Xanthomonas.\n\nAliquots of samples A, B, and B(A+), collected at different times, were analysed with both transmission electron microscope (TEM) and scanning electron microscope (SEM) to verify the presence of eukaryotic cells. More than 30 different preparations (including at 30 days of culture) have been observed: none was found to contain any structure resembling cells with nuclei, but only bacterial cells. Analyses of images on samples A and B(A+) revealed the presence of virus-like particles interacting with bacterial cells. Immune electron microscopy is ongoing for confirming that these particles are of SARS-CoV-2 origin (in preparation).\n\n\nDiscussion\n\nOur results indicate that the SARS-CoV-2 genome, in addition to its known interactions with eukaryotic cells, is additionally capable of replicating outside the human body, suggesting a possible ‘bacteriophage-like’ mode of action. It is not clear yet whether the SARS-CoV-2 genome could just be replicated by its RNA polymerase (which would correspond to a bacteriophage pseudo-lysogenic mechanism), or if the production of full-blown SARS-CoV-2 replicating particles within the bacteria occur (which would correspond to the typical lytic cycle of bacteriophages). In either case, according to our knowledge, this is a novel observation and has never described before for SARS-CoV-2.\n\nThe experiment here described was repeated three additional times using the same samples. In addition, independent replications were performed in a 3×3 design using different starting material. In all of them, very similar trends were observed and the increase of SARS-CoV-2 RNA load in sample A and sample B(A+) was confirmed in all experiments.\n\nIn the cases of replications with different starting material (i.e. faecal samples from different “infected donors” as sources of A, and “healthy recipients” as sources of B), the trends are similar and confirm that the experiment is reproducible. A whole-genome metagenomic sequencing on the samples is ongoing, aimed at characterising further which bacterial species are candidate target(s) of the observed behaviour of SARS-CoV-2.\n\nWhereas our experimental design was intended to grow bacterial cells, the possibility that SARS-CoV-2 RNA increase could be due to replication in human cells present in the original faecal samples, was considered. The human cells most abundantly present in faecal samples are colonic epithelial cells (colonocytes). Loktionov,16 reported that cell exfoliation events from colonic epithelium are rare under normal conditions, while they dramatically increase in cases of uncontrolled growth of cells not under physiologic control (like in neoplasia), when cell removal by apoptosis does not function properly. In addition, Iyengar et al.17 reported that colonic epithelial cells terminally differentiated are devoid of proliferative activity. More recently, Nair et al.18 and Chandel et al.19 developed specific methodologies to recover viable colonocytes from stool. In our case, both sample A and B originated from adult individuals with no diagnosis of cancer. In addition, it is unlikely that human cells potentially present in samples A and B are able to:\n\n• grow in a culture medium typically formulated for bacteria and not containing growth factors, serum, nor other important components for eukaryotic cell sustainment;\n\n• survive in such a medium for 30 days, and in co-occurrence with an event of SARS-CoV-2 infection;\n\n• multiply in the absence of specific CO2 concentration conditions.\n\nAlso, the possibility of interaction between SARS-CoV-2 and other eukaryotic organisms potentially present in the cultures, as e.g. parasitic nematodes and fungal cells, has been considered.\n\nDuring the whole experiment, parasitic nematodes were not noted at visual inspections by eye. In addition, stool of sample B was independently analysed and certified to be free of known parasites and microbial pathogens (certification provided by the Italian diagnostic laboratory Biomolecular Diagnostic Srl). Parasitic nematodes are usually not able to survive outside the host and many intestinal roundworms (like those of genus Ascaris) release antimicrobial factors that interfere with bacterial growth,20 in contrast with the found high increase of metabolic activities of some bacterial genera. In the used medium, chemical elements relevant for (parasitic and not) nematodes (e.g. cholesterol and traces of metals) are missing.\n\nThe possibility of involvement of the mycobiome fraction present in the stool was considered. As highlighted by Chin et al.,21 multifaceted and multidisciplinary approaches are necessary to identify uncultivatable, low-abundance, permanent and transient fungal species residing in the gut, confirming that the human mycobiome is not yet fully characterised. Accordingly, while the ability of unknown fungi to grow in the used culture medium cannot be excluded, no significant metabolic activity of Candida albicans, most commonly found in the microbiota, was observed.\n\nFinally, inspections of images from TEM and SEM on more than 30 different preparations did not reveal presence of eukaryotic cells (in preparation). If on the one hand, the possibility that a nematode or another unknown eukaryotic cell is able grow in the medium cannot be excluded, the used conditions make this possibility very unlikely. Anyhow, the ability of SARS-CoV-2 to interact either with nematodes or with fungal cells has never been observed before and would be a novel and surprising observation as well.\n\nAs indicated above, several peptides matching to SARS-CoV-2 proteins were found in the aliquot from sample B(A+). The identification of peptides with amino acid changes, compared to the translations of CDS regions of the reference SARS-CoV-2 genome, is intriguing but is compatible with the mechanism of viral replication in bacteria. RNA viruses such as SARS-CoV-2 inhabit the host as a population of variants called quasispecies, i.e. a group made of different variants that are genetically linked through mutation events, and contribute collectively to the characteristics of the whole (viral) population in the host.22 Recent studies highlighted the significant amount of intra-host genomic diversity in SARS-CoV-2 samples.23,24 In a ‘bacteriophage-like’ mode of action, as bacteria were grown for 30 days, it can’t be excluded that the observed amino acid changes represent viral quasispecies emerged through replication events in bacterial hosts. In relation to this, recent studies25–27 evidenced hypermutations occurrences in SARS-CoV-2 genomes and suggested APOBEC and ADAR deaminases as the possible responsible of these phenomena. The APOBEC family is related to bacterial, yeast, and plant deaminases all possessing highly conserved amino acid motifs responsible for coordination of zinc in the active site.28 As no sequencing was performed on the original infected stool sample, the presence of SARS-CoV-2 haplotypes in the initial SARS-CoV-2 population used to perform the experiments, therefore justifying the amino acid changes observed, cannot be excluded. However, all the amino acid changes found have been reported in sequences of SARS-CoV-2 found for the first time after the date of collection of the infected sample A (February 2020), and some of them have never been reported in Italy, the country of origin of the samples.\n\nOn the other hand, other mechanisms like those at the basis of diversity-generating retroelements (DGR) systems29 have that could contribute to SARS-CoV-2 hypermutation phenomena have recently been described in bacteria, and could therefore be responsible of the AA changes found.\n\nThese results can potentially provide new insights in the epidemiology of SARS-CoV-2. Considering the possible impact and implications that such relationship has on the manifestation, therapy and control of COVID-19 disease, some questions immediately arise like e.g.:\n\n• Can this ‘bacteriophage-like’ behaviour of SARS-CoV-2 explain the long-term presence of SARS-CoV-2 observed in some recovered patients?30\n\n• Can antibiotics and/or bacteriophage-based therapies play a role in the treatment of COVID-19 affected patients?31\n\n• How would the (antecedent) administration of antibiotics to patients, influencing the microbiota population, impact the clinical course of the disease?32\n\n• Can the involvement of bacteria in COVID-19 epidemiology help to explain clinical observations, like the elevated serum C-reactive protein, procalcitonin, D-dimer, and ferritin associated with poor outcomes in COVID-19?33\n\nThese questions are only examples of the plethora of questions to be addressed. Our results support the way to tackle COVID-19 pandemic proposed by Mushi,34 i.e. by using the One Health holistic approach. If individuals are considered not only human bodies, but as ‘holobionts’, i.e. discrete ecological units that need to be studied and treated as such, a deeper understanding of the role of the microbial community living in the human body is fundamental to tackle COVID-19 disease.\n\n\nConsent\n\nFaecal samples were collected and handled by CranioMed S.R.L. from anonymous donors who agreed to participate in this study by signing informed consent, as foreseen by Italian legislation. No personal information (i.e. age, sex, blood serotype, severity of the disease, time of the collection, fatality, etc.) were collected.\n\nThe study is compliant with the JRC Scientific Integrity and Research Ethics guidance.\n\n\nDeclarations\n\nThe scientific output expressed does not imply a policy position of the European Commission. Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use that might be made of this publication.\n\n\nData availability\n\nZenodo: Underlying data for ‘Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology’. https://doi.org/10.5281/zenodo.4723550.35\n\nThe project contains the following underlying data:\n\nMass spectrometry raw data of the peptides.\n\nNCBI Genome: Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome. Accession number: NC_045512.2; https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2\n\nNCBI Protein: Surface glycoprotein [Severe acute respiratory syndrome coronavirus 2]. Accession number: YP_009724390.1; https://www.ncbi.nlm.nih.gov/protein/1796318598\n\nZenodo: Extended data for ‘Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology’. https://doi.org/10.5281/zenodo.4723550.35\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe gratefully acknowledge the Authors and the Originating laboratories where the clinical specimen or virus isolate was first obtained and the Submitting laboratories, where sequence data have been generated and submitted to GISAID.\n\n\nAuthors’ notes\n\nThe efforts and commitment put into this research work are dedicated to all EU citizens who suddenly left us because of COVID-19, and to their families.\n\n\nReferences\n\nTrottein F, Sokol H: Potential Causes and Consequences of Gastrointestinal Disorders during a SARS-CoV-2 Infection. Cell Rep. 2020; 32: 107915. 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JAMA. 2020; PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWölfel R, et al.: Virological assessment of hospitalized patients with COVID-2019. Nature. 2020; 581: 465–469. PubMed Abstract \| Publisher Full Text\n\nYao H, et al.: Patient-derived SARS-CoV-2 mutations impact viral replication dynamics and infectivity in vitro and with clinical implications in vivo. Cell Discov. 2020; 6: 76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCristoni S, et al.: Predicting and preventing intestinal dysbiosis on the basis of pharmacological gut microbiota metabolism. Rapid Commun. Mass Spectrom. 2019; 33: 1221–1225. PubMed Abstract \| Publisher Full Text\n\nElbe S, Buckland-Merrett G: Data, disease and diplomacy: GISAID’s innovative contribution to global health. Glob. Challenges. 2017; 1: 33–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoktionov A: Cell exfoliation in the human colon: Myth, reality and implications for colorectal cancer screening. Int. J. Cancer. 2007; 120: 2281–2289. PubMed Abstract \| Publisher Full Text\n\nIyengar V, Albaugh GP, Lohani A, et al.: Human stools as a source of viable colonic epithelial cells. FASEB J. 1991; 5: 2856–2859. PubMed Abstract \| Publisher Full Text\n\nNair P, et al.: Coprocytobiology. J. Clin. Gastroenterol. 2003; 36: S84–S93. PubMed Abstract \| Publisher Full Text\n\nChandel DS, Braileanu GT, Chen J-HJ, et al.: Live Colonocytes in Newborn Stool: Surrogates for Evaluation of Gut Physiology and Disease Pathogenesis. Pediatr. Res. 2011; 70: 153–158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMidha A, et al.: The Intestinal Roundworm Ascaris suum Releases Antimicrobial Factors Which Interfere With Bacterial Growth and Biofilm Formation. Front. Cell. Infect. Microbiol. 2018: 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChin VK, et al.: Mycobiome in the Gut: A Multiperspective Review. Mediators Inflamm. 2020; 2020: 1–16. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlimczak LJ, Randall TA, Saini N, et al.: Similarity between mutation spectra in hypermutated genomes of rubella virus and in SARS-CoV-2 genomes accumulated during the COVID-19 pandemic. PLoS One. 2020; 15: e0237689. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimmonds P: Rampant C→U Hypermutation in the Genomes of SARS-CoV-2 and Other Coronaviruses: Causes and Consequences for Their Short- and Long-Term Evolutionary Trajectories. mSphere. 2020; 5: e00408–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRatcliff J, Simmonds P: Potential APOBEC-mediated RNA editing of the genomes of SARS-CoV-2 and other coronaviruses and its impact on their longer term evolution. Virology. 2021; 556: 62–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu L, et al.: Diversity-generating retroelements: natural variation, classification and evolution inferred from a large-scale genomic survey. Nucleic Acids Res. 2018; 46: 11–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nD’Ardes D, et al.: Long-term Positivity to SARS-CoV-2: A Clinical Case of COVID-19 with Persistent Evidence of Infection. Eur. J. Case Reports Intern. Med. 2020; 7: 1707. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoncalves Mendes Neto A, et al.: Bacterial infections and patterns of antibiotic use in patients with COVID-19. J. Med. Virol. 2020; jmv.26441. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang L, et al.: Retrospective analysis of clinical features in 134 coronavirus disease 2019 cases. Epidemiol. Infect. 2020; 148: e199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang I, Pranata R, Lim MA, et al.: C-reactive protein, procalcitonin, D-dimer, and ferritin in severe coronavirus disease-2019: a meta-analysis. Ther. Adv. Respir. Dis. 2020; 14: 175346662093717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMushi V: The holistic way of tackling the COVID-19 pandemic: the one health approach. Trop. Med. Health. 2020; 48: 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPetrillo M, Brogna C, Cristoni S, et al.: Underlying and extended data for 'Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology'.Publisher Full Text" }	[ { "id": "85393", "date": "17 May 2021", "name": "Kourosh Honarmand Ebrahimi", "expertise": [ "Reviewer Expertise Virology and immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPetrillo and colleagues report that SARS-CoV-2 could have a bacteriophage activity in faecal samples. They measure the presence of viral RNA using Luminex technology and viral proteins using mass spectrometry. The experimental design is sound and their findings are exciting, which support the publication of this work. However, I have few concerns that must be addressed before publication. Therefore, I recommend publication after minor revisions.\nSARS-CoV-2 is a respiratory virus. Many bacteria reside in the upper respiratory tract (URT) and interact with different viruses like influenza (Schenk et al., 20161). Moreover, recent reports suggest an interaction of URT microbiota with SARS-CoV-2 (e.g. (Ebrahimi, 20202); (Budding et al., 20203)). The authors should cite these literatures and discuss their findings with respect to a similar bacteriophage behaviour of SARS-CoV-2 in URT.\n\nIn Figure 3, the graph for donor A2: Why in B2(A2+) sample the amount of RNA is hugely different than the other samples? The authors need to explain.\n\nThe authors suggest that the presence of SARS-CoV-2 peptides is compatible with the mechanism of viral replication in bacteria. If this is true, shouldn't the authors observe an increase in viral peptides similar to viral RNA?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6679", "date": "18 May 2021", "name": "Mauro Petrillo", "role": "Author Response", "response": "Dear Dr Kourosh Honarmand Ebrahimi, Thanks a lot for your valuable comments and suggestions that you have provided in the report. We will address all of them, and wait for those of other reviewers, in order to provide a fully revised version of the manuscript. Best regards, Mauro Petrillo, on behalf of the authors." }, { "c_id": "6774", "date": "23 Jun 2021", "name": "Mauro Petrillo", "role": "Author Response", "response": "Dear Dr Kourosh Honarmand Ebrahimi, Thanks a lot for your valuable comments and suggestions that you have provided in the report. As anticipated, we have addressed all your points, and provided a new version of the manuscript: SARS-CoV-2 is a respiratory virus. Many bacteria reside in the upper respiratory tract (URT) and interact with different viruses like influenza (Schenk et al., 2016). Moreover, recent reports suggest an interaction of URT microbiota with SARS-CoV-2 (e.g. (Ebrahimi, 2020); (Budding et al., 2020). The authors should cite these literatures and discuss their findings with respect to a similar bacteriophage behaviour of SARS-CoV-2 in URT. Response: Your point is relevant and we modified the Discussion section accordingly: 'SARS-CoV-2 is considered as a respiratory virus, and many bacteria reside in the upper respiratory tract (URT) interacting with different viruses like influenza (see Schenk et al., 2016 for an overview). With this respect, our observations are in line with the hypothesis formulated by Shah, who has recently proposed the existence of a gut-lung equilibrium mediated by multiple mechanisms of action, including the abundance of certain microorganisms in the gut microbiota as responsible for determining the sensitivity and severity of SARS-CoV-2 infections. Moreover, recent reports suggest an interaction of URT microbiota with SARS-CoV-2 (Ebrahimi, 2020, Budding et al., 2020). In particular, Ebrahimi identified in silico a series of serine protease TMPRSS2 and peptidyl peptidases with high similarity to the ACE2 peptidase domain (ACE2-PD) in members of Proteobacteria phylum. It can’t be excluded that these or other similar proteins act as the cellular receptors for SARS-CoV-2 in bacteria. Looking for taxa, species or consortia that can be prone to act as receptor of virus is imperative, and, with this respect, a whole-genome metagenomic sequencing on the samples is ongoing, aimed at characterising further which bacterial species are candidate target(s) of the observed behaviour of SARS-CoV-2.' In Figure 3, the graph for donor A2: Why in B2(A2+) sample the amount of RNA is hugely different than the other samples? The authors need to explain. Response: We have modified Figure 3, by adding an additional panel. In addition, we modified the Discussion section accordingly: 'In terms of relevance, we noticed that SARS-CoV-2 RNA load was particularly high in one combination (A2×B2). It is thus plausible that viral RNA load depends on the gut eubiotic/dysbiotic condition met by the virus. This is also a plausible explanation of why in the initial couple of A-B sources (Figure 1) the difference between viral RNA load measurements in A and B(A+) is notable.' The authors suggest that the presence of SARS-CoV-2 peptides is compatible with the mechanism of viral replication in bacteria. If this is true, shouldn't the authors observe an increase in viral peptides similar to viral RNA? Response: Your point is relevant and we modified the Discussion section accordingly: 'Additional experiments aimed to verify increase of viral peptides similar to viral RNA over time are planned.' We hope that the quality of the manuscript, thanks to your comments, has been improved and you consider it suitable for publication. Best regards, Mauro Petrillo, on behalf of the authors." } ] }, { "id": "85802", "date": "07 Jun 2021", "name": "Margarita Aguilera", "expertise": [ "Reviewer Expertise Microbiota", "Probiotics", "taxonomy", "Microbiology", "Molecular biology", "Biotechnology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 titled “Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology” has been well designed and soundly performed.\nAfter the urgent needs to invest efforts on clinical research for handling the COVID-19, new questions should be posed and the present work has done innovatively. The hypothesis is relevant due to the high differential effects within SARS-CoV-2 patients, based on the individual health statuses that determine variable host responses. The Authors have shown experimental data to demonstrate that SARS-CoV-2 could have a bacteriophage activity through evaluating SARS-CoV-2 long-term survival, proliferation and the potential interaction between the virus and gut microbiota taxa. Multiple methodologies and experimental data support appropriately the results and conclusions: viral RNA by Luminex technology; impact of antibiotic treatments; Biotyper for cultured microbiota predominant taxa over the time, eukaryotic cells, peptides, other metabolites molecular alignments, etc.\nTherefore, I consider that Petrillo et al. have carried out an excellent and holistic approach; I would suggest its publication after minor corrections/explanations:\nPlease use the term gut microbiota instead gut flora along the document.\n\nPlease add the underlined phrase at the end of this paragraph: At the same time, Wölfel et al.12 observed high viral RNA concentration in stool samples, but reported isolation of infectious virus only from throat- and lung-derived samples, while Yao et al. had indication of viable SARS-CoV-2 particles in stool samples, denoting that the detailed biology of SARS-CoV-2 is not yet fully elucidated. Moreover, the interaction between SARS-CoV-2 and individual variable microbiota composition could drive the differential pathophysiological effects and severity of symptoms.\n\nPlease explain if the medium NutriSelect™Plus nutrient used for the inoculation and cultivation of microorganisms could have an impact on the specific taxa cultured as well as metabolites measured. Have you tried other media?\n\nPlease explain if you have been used only aerobic conditions for all experimental culturing data.\n\nPlease specify the concentration of antibiotics added to the experiment.\n\nFigure 1: Please give a plausible explanation for the data shown in this figure - Could it be that microbiota of healthy individuals contain eubiotic taxa that allow a better proliferation of SARS-CoV-2?\n\nFigure 3: Please explain the differences observed between the three graphics. The microbiota composition of the sample receptors seems to have an impact. Please give a plausible explanation.\n\nFuture research is directly derived from these results: to look for the taxa, species or consortia that can be prone to act as receptor of virus. Please comment on that.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6771", "date": "10 Jun 2021", "name": "Mauro Petrillo", "role": "Author Response", "response": "Dear Prof. Aguilera, Thanks a lot for your valuable comments and suggestions that you have provided in the report. We will address all of them, and will provide a revised version of the manuscript. Best regards, Mauro Petrillo, on behalf of the authors." }, { "c_id": "6775", "date": "23 Jun 2021", "name": "Mauro Petrillo", "role": "Author Response", "response": "Dear Dr Aguilera, Thanks a lot for your valuable comments and suggestions that you have provided in the report. As anticipated, we have addressed all your points, and provided a new version of the manuscript: Please use the term gut microbiota instead gut flora along the document. Response: Done, thanks. Please add the underlined phrase at the end of this paragraph: 'At the same time, Wölfel et al.12 observed high viral RNA concentration in stool samples, but reported isolation of infectious virus only from throat- and lung-derived samples, while Yao et al. had indication of viable SARS-CoV-2 particles in stool samples, denoting that the detailed biology of SARS-CoV-2 is not yet fully elucidated. Moreover, the interaction between SARS-CoV-2 and individual variable microbiota composition could drive the differential pathophysiological effects and severity of symptoms.' Response: Very relevant point. We added it, together with references. Please explain if the medium NutriSelect™Plus nutrient used for the inoculation and cultivation of microorganisms could have an impact on the specific taxa cultured as well as metabolites measured. Have you tried other media? Please explain if you have been used only aerobic conditions for all experimental culturing data. Please specify the concentration of antibiotics added to the experiment. Response: With respect to these points, the information has been added in the supplementary material, points 1, 3, 8). The new link for get the supplementary material is https://doi.org/10.5281/zenodo.4723549 Figure 1: Please give a plausible explanation for the data shown in this figure - Could it be that microbiota of healthy individuals contain eubiotic taxa that allow a better proliferation of SARS-CoV-2? Figure 3: Please explain the differences observed between the three graphics. The microbiota composition of the sample receptors seems to have an impact. Please give a plausible explanation. Future research is directly derived from these results: to look for the taxa, species or consortia that can be prone to act as receptor of virus. Please comment on that. Response: Your points are relevant and we modified the Discussion section accordingly: 'In terms of relevance, we noticed that SARS-CoV-2 RNA load was particularly high in one combination (A2×B2). It is thus plausible that viral RNA load depends on the gut eubiotic/dysbiotic condition met by the virus. This is also a plausible explanation of why in the initial couple of A-B sources (Figure 1) the difference between viral RNA load measurements in A and B(A+) is notable.SARS-CoV-2 is considered as a respiratory virus, and many bacteria reside in the upper respiratory tract (URT) interacting with different viruses like influenza (see Schenk et al., 2016 for an overview). With this respect, our observations are in line with the hypothesis formulated by Shah, who has recently proposed the existence of a gut-lung equilibrium mediated by multiple mechanisms of action, including the abundance of certain microorganisms in the gut microbiota as responsible for determining the sensitivity and severity of SARS-CoV-2 infections. Moreover, recent reports suggest an interaction of URT microbiota with SARS-CoV-2 (Ebrahimi, 2020, Budding et al., 2020). In particular, Ebrahimi identified in silico a series of serine protease TMPRSS2 and peptidyl peptidases with high similarity to the ACE2 peptidase domain (ACE2-PD) in members of Proteobacteria phylum. It can’t be excluded that these or other similar proteins act as the cellular receptors for SARS-CoV-2 in bacteria. Looking for taxa, species or consortia that can be prone to act as receptor of virus is imperative, and, with this respect, a whole-genome metagenomic sequencing on the samples is ongoing, aimed at characterising further which bacterial species are candidate target(s) of the observed behaviour of SARS-CoV-2.' We believe that, thanks to your comments, the quality of the manuscript has improved a lot. Best regards, Mauro Petrillo, on behalf of the authors." } ] } ]	1	https://f1000research.com/articles/10-370
https://f1000research.com/articles/10-523/v1	01 Jul 21	{ "type": "Systematic Review", "title": "Cinnamon extract effects on insulin resistance, metabolic factors, and menstrual cyclicity of women with polycystic ovary syndrome: A systematic review and meta-analysis", "authors": [ "Riska Wahyuningtyas", "Ashon Sa'adi" ], "abstract": "Background: Cinnamon is a herbal medicine that is supposed to improve the metabolic disorder polycystic ovary syndrome (PCOS), but there is still no data about the effectiveness and efficacy of this herbal medicine for the metabolic parameters of PCOS. This study aims to systematically evaluate the effects of cinnamon extract on improving insulin resistance, lipid profile and regularity of menstruation in PCOS women. Methods: This is a systematic review and meta-analysis of randomized controlled trials (RCTs) studies. We searched the MEDLINE, Cochrane, Google Scholar, and PubMed databases to identify relevant studies using cinnamon extract effects on insulin resistance, metabolic factors, and menstrual cyclicity of PCOS women. Results: Five RCTs consisting of 206 women were included in the meta-analysis. Significant differences were found in fasting blood glucose (FBG) (mean difference (MD)= -4.8 mg/dL, 95% CI: -8.04 to -1.57, p=0.004; 143 participants), High-Density Lipoprotein (HDL-cholesterol, HDL-C) (MD= -27.24 mg/dL, 95% CI: -32.62 to -21.85, p< 0.00001; 143 participants, Insulin level (MD = -2.20 mIU/dL, 95% CI: -4.17 to -0.23, p=0.03; 143 participants), and menstrual cyclicity in six months (MD= 2.28, 95% CI: 1.83 to 2.73, p< 0.00001; 33 participants) were obtained. Conclusion: Cinnamon can be a potential supplementary therapy agent for PCOS women as it improves fasting blood glucose, insulin level, HDL–cholesterol and menstrual cyclicity in PCOS women.", "keywords": [ "Cinnamon", "insulin resistance", "metabolic factors", "menstrual cyclicity", "polycystic ovary syndrome" ], "content": "Introduction\n\nPolycystic ovary syndrome (PCOS) is one of the most common and complex endocrine dysfunctions affecting 5-10% of fertile women.1 Manifestations of PCOS include hyperandrogenism (hirsutism, acne, obesity), reproductive dysfunction (infertility, menstrual irregularity, miscarriage, pregnancy complications), and metabolic complications (dyslipidemia, insulin resistance).2,3 PCOS is a combination of a reproductive and metabolic condition with psychological consequences. Patients suffer from anxiety, depression and distress due to its clinical manifestation.\n\nInsulin resistance, dyslipidemia, and diabetes are some hyperandrogenic manifestations of PCOS.4 Insulin resistance presents in 60-80% of PCOS women, as well as 95% in overweight women.5,6 Insulin resistance has a major role in the development of cardiovascular and metabolic disturbances. Cells fail to respond to insulin thus excess glucose is not completely absorbed by cells and increases the level of blood sugar.4 Dyslipidemia is one of the metabolic consequences of PCOS. The prevalence of dyslipidemia is three times more likely in patients with PCOS than in normal women.7 The other manifestation of hyperinsulinemia is irregular menstrual cyclicity because of the disruption of folliculogenesis and abnormal androgen secretion.6\n\nThere are some therapies for PCOS. Nonpharmacologic therapy consists of physical activities for weight reduction and managing diet intake. Two pharmacological drugs used for treating insulin resistance of PCOS are metformin and thiazolidines. But the gastrointestinal effects (61.5% suffer from nausea and vomiting, 59.1% abdominal pain as well as cramping and 64.9% diarrhoea) of these drugs make patients feel uncomfortable.5,8 Thus, alternative herbal medicine is a good choice with less adverse effects.\n\nCinnamon (Cinnamomum zeylanicum) is a natural medicine recently used as a complementary medicine for decreasing hyperglycemia, dyslipidemia, and other metabolic disorders of PCOS.5,8 A procyanidin substance from cinnamon extract stimulates autophosphorylation of the insulin receptor and inhibits protein tyrosine phosphate thus increasing insulin-stimulated glucose uptake and glycogen synthesis. It also reduces fasting glucose, triglycerides, low-density lipoprotein (LDL) and total cholesterols.5,8 Some studies prove that cinnamon can reduce hemostatic model assessment for insulin resistance (HOMA-IR) in PCOS patients.4,5 It also improves menstrual cyclicity in PCOS patients. However, no studies have evaluated the effectiveness of these extracts on insulin resistance, lipid profile, and regularity of menstruation in PCOS women. Thus, this study was conducted to systematically evaluate the effects of cinnamon extract on improving insulin resistance, lipid profile and regularity of menstruation in PCOS women.\n\n\nMethods\n\nThe search strategy was carried out by two independent authors (R.W., A.A.) separately from January 2019 to November 2020. To be included the study design had to be a randomized controlled trial in humans.\n\nA systematic review of published articles was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. The systematic review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; identification number CRD42021214007 on 21/01/2021).\n\nThe first step, a bibliographic search was performed on MEDLINE, Cochrane, Google Scholar, PubMed database using search terms; ((((((((“Polycystic ovarian syndrome” OR “PCOS”) AND (“Cinnamon” OR “Cinnamomum zeylanicum”) AND (“Insulin resistance” OR “HOMA IR”) AND (“Body mass index” OR “BMI”) AND (“Trygliceride”) AND (“Cholesterol”) AND (“Insulin level” OR “Blood insulin”) AND (“Menstrual cyclicity OR “Regularity of menstruation”)))))))), from January 2019 to September 2020. In order to eliminate bias, we do not limit the language. Literature reviews, book chapters, animal experimental articles were excluded. The related articles were then screened one by one manually in order to select the appropriate topics.\n\nSecondly, we screened all duplicated articles based on the title and abstracts. Then, the third step was to assess the full text articles for eligibility. Only studies focusing on the effect of cinnamon based on parameter lipid profile, insulin resistance and regularity of menstruation were included in this meta-analysis. We used the PICOS format to examine the study eligibility criteria (Participants, Intervention, Comparison, Outcomes, Study Design) (Table 1). The final step was to combine all selected articles in the meta-analysis.\n\nTwo authors (R.W., A.A.) independently assessed the quality of included studies using the Cochrane ‘Risk of Bias Assessment Tool’ involving the random sequence generation (selection bias), allocation concealment (selection bias), masking of participants and personnel (performance bias), masking of outcome assessment (detection bias), incomplete outcome data (attrition bias), and selective reporting (reporting bias). Risk of bias is evaluated as low, high, or unclear.\n\nTo perform the meta-analysis, Review Manager (RevMan version 5.3, The Cochrane Collaboration, Oxford, UK) was used. The statistical heterogeneity across trials were assessed by I2 statistic, which describes the percentage of variation, and a value of I2 greater than 50% was considered heterogeneous. All results were presented as a random-effect model when the detected heterogeneity was significant. The results of this study are displayed by forest plot diagrams.\n\n\nResults\n\nFigure 1 shows details of the study selection. The primary search identified 103 articles. We screened title and abstract and found five eligible studies with a full text format. All five articles were included in this review. The five articles were RCTs comparing cinnamon extract and a placebo in PCOS women based on changes in insulin resistance, lipid profile and changes in regularity in menstruation (Table 1). Only two RCTs analyzed showed changes in regularity of menstruation.9,10\n\nThe characteristics of included studies are described in Table 1. Two studies focused on Iranian women,5,11 two others on Caucasian women from Columbia9,10 and the last focused on New York, USA.6 Of the five studies, one study was conducted as a pilot study. Diagnosis criteria of PCOS was based on the Rotterdam criteria for all studies, including: hyperandrogenism (elevated serum T level ≥ 80 ng/dL, intermittent or absence of menstrual cycles (ovarian dysfunction), and/or a polycystic ovary in ultrasound with the exclusion of adrenal, ovary, and pituitary disorder.5\n\nAll studies used the same exclusion criteria for PCOS women who participated in the research. These were diabetes mellitus, hyperprolactinemia, thyroid disorders, liver or kidney disease, Cushing syndrome, cardiovascular disease, seizure, cerebrovascular disorder, hypertension, smoking, current treatment of infertility, allergy to cinnamon, and the use of oral hypoglycemic, insulin-sensitizing drugs, oral contraceptive drugs, and β-blocker in the two to three months prior to the study enrolment.\n\nThere were no specific criteria for body mass index as inclusion criteria in the study. Only three studies made a specific range of body mass index (BMI) criteria for enrolling the patient in the study.5,10,11 The range of BMI was 25-40 kg/m2, 20-50 kg/m2 and > 18 kg/m2.\n\nThe mean age for each group was reported in each study, but not for one study9 (mean age for all groups was 31.1 ± 2 years old). The mean age in each group reported by Hajimonfarednejad et al.,5 was not significantly different. The mean age for the cinnamon group and the placebo group in Hajimonfarednejad et al. and Kort and Borzoei et al. was respectively 28.62 ± 5.74 vs 26.53 ± 6.53 years old (p = 0.19); 26.95 (18-34) vs 27.86 (18-38) years old (p = no data); and 29.26 ± 6.14 vs 30.17 ± 6.69 years old (p = no data).5,10,11\n\nThe dose of cinnamon given for intervention in four studies was the same (1500 mg cinnamon daily). One pilot study, Wang et al., administered 1000 mg cinnamon daily.6 The length of the observational studies ranged from eight to 24 weeks.\n\nThe risk of bias in randomized trials included in the meta-analysis is shown in Figures 2 and 3.\n\n\nQualitative data synthesis and meta-analysis\n\nHajimonfarednejad et al.5 and Borzoei et al.11 studied the changes in BMI and weight between the cinnamon and control groups. Hajimonfarednejad et al. found no significant difference in BMI changes in the control group (p = 0.168) and cinnamon group (p = 0.054).5 But there was a significant difference in weight changes in the control group (p = 0.023) than in the cinnamon group (p = 0.058).5 Borzoei et al. analysed BMI changes in the cinnamon group (p = 0.002) and found it was significantly different to the control group (p = 0.89).11 This result was the same with weight changes in the cinnamon group (p = 0.00) compared to the control group (p = 0.85), which showed no significant result.\n\nWe found there was no significant mean difference in BMI and weight after intervention with cinnamon as well as the placebo group, respectively (−0.21[−1.80,1.38]; p = 0.79 and 1.08[−2.79,4.96]; p = 0.58) (Figures 4A and 4B).\n\nB. Forest plot showing the impact of cinnamon extract on weight. C. Forest plot showing the impact of cinnamon extract on insulin resistance (HOMA-IR). D. Forest plot showing the impact of cinnamon extract on fasting blood sugar level* E. Forest plot showing the impact of cinnamon extract on trygliceride level. F. Forest plot showing the impact of cinnamon extract on LDL-cholesterol level. G. Forest plot showing the impact of cinnamon extract on HDL-cholesterol level. H. Forest plot showing the impact of cinnamon extract on total cholesterol level. I. Forest plot showing the impact of cinnamon extract on insulin level. J. Forest plot showing the impact of cinnamon extract on DHEA level. K. Forest plot showing the impact of cinnamon extract on menstrual cyclicity in 6 months*.\n\nOf the five studies, four studies compared insulin resistance after cinnamon intervention by homeostasis model assessment insulin resistance (HOMA-IR) methods. But only three studies showed significant results in changes of insulin resistance after intervention. There were significant changes in HOMA-IR after cinnamon intervention in the Borzoei et al. study (p = 0.00), Wang et al. study (p ≤ 0.03), and Hajimonfarednejad et al. study (p = 0.001).5,6,11 Kort found no significant difference in the changes of insulin resistance.10\n\nIn this meta-analysis, we analyze mean difference of insulin resistance post cinnamon intervention. The result showed no significant difference between insulin resistance post cinnamon and post placebo intervention (−0.19 [−0.54, 0.15], p = 0.27) (Figure 4C).\n\nData from three studies were available for fasting blood sugar level, but Wang et al. did not show complete data thus we could not analyze the results in this meta-analysis. Borzoei et al. found there was a significant difference of fasting blood sugar changes in the cinnamon group (p = 0.00) than in the control group (p = 0.15).11 But Hajimonfarednejad et al. found no significant difference of fasting blood sugar changes in the cinnamon and control groups (respectively, p = 0.63; p = 0.148).5\n\nIn this meta-analysis we analyze the mean difference of post intervention fasting blood sugar level between the cinnamon and control groups. There was a significant difference between fasting blood sugar level post intervention in the cinnamon group and control group (−4.8[−8.04, −1.57]; p = 0.004) (Figure 4D).\n\n\nLipid profile\n\nData from two studies were available for triglyceride level. Borzoei et al. defined that there was a significant difference in triglyceride changes in the cinnamon group (p = 0.001) than in the control group (p = 0.29).11 But Hajimonfarednejad et al. found no significant difference in triglyceride changes in the cinnamon and control groups (respectively, p = 0.105; p = 0.935).5\n\nIn this meta-analysis, we analyze the mean difference of post intervention triglyceride level between the cinnamon and control groups. There was no significant difference between triglyceride level post intervention in the cinnamon group and control group (−10.73[−26.35, 4.88]; p = 0.18) (Figure 4E).\n\nData from two studies were available for LDL-cholesterol level. Borzoei et al. defined that there was a significant difference in LDL-cholesterol changes in the cinnamon group (p = 0.001) than in the control group (p = 0.85).11 Hajimonfarednejad et al. found the same results as Borzoei et al., they showed a significant difference in LDL-cholesterol changes in cinnamon than in control group (respectively, p = 0.004; p = 0.212).5,11\n\nIn this meta-analysis, we analyze the mean difference of post intervention LDL-cholesterol level between the cinnamon and control groups. There was no significant difference between LDL-cholesterol level post intervention in the cinnamon group and control group (−3.39[−12.27, 5.50]; p = 0.46) (Figure 4F).\n\nData from two studies were available for HDL-cholesterol level. Hajimonfarednejad et al. defined that there was no significant difference in HDL-cholesterol changes in the cinnamon group (p = 0.238) but there was a significant difference in the control group (p = 0.007).5 Borzoei et al. found the opposite result to Hajimonfarednejad et al. and showed a significant difference in HDL-cholesterol changes in the cinnamon group than in the control group (respectively, p = 0.0001; p = 0.06).11\n\nIn this meta-analysis we analyze the mean difference of post intervention HDL-cholesterol level between the cinnamon and control groups. There was a significant difference between LDL-cholesterol level post intervention in the cinnamon group and control group (−27.24[−14.25, −0.87]; p < 0.00001) (Figure 4G).\n\nData from two studies were available for total cholesterol level. Borzoei et al. defined that there was a significant difference in total cholesterol changes in the cinnamon group (p = 0.001) than in the control group (p = 0.3).11 Hajimonfarednejad et al. found the same results as Borzoei et al., they showed a significant difference in total cholesterol changes in the cinnamon than in the control group (respectively, p = 0.001; p = 0.685).5\n\nIn this meta-analysis we analyse mean difference of post intervention total cholesterol level between the cinnamon and control groups. There was no significant difference between total cholesterol level post intervention in the cinnamon group and control group (−7.8[−17.48, 1.89]; p = 0.11) (Figure 4H).\n\nData from two studies were available for insulin level. Borzoei et al. defined that there was a significant difference in insulin level changes in the cinnamon group (p = 0.01) than in the control group (p = 0.19).11 Hajimonfarednejad et al. found that there was a significant difference in insulin level changes in the cinnamon and control groups (respectively, p = 0.001; p = 0.002).5\n\nIn this meta-analysis, we analyze mean difference of post intervention insulin level between the cinnamon and control groups. There was a significant difference between insulin level post intervention in the cinnamon group and control group (−4.17[−4.17, −0.23]; p = 0.03) (Figure 4I).\n\nData from two studies were available for dehydroepiandrosterone (DHEA) level. Hajimonfarednejad et al. defined that there was no significant difference in DHEA changes in the cinnamon group and control group (respectively, p = 0.751; p = 0.237).5 Kort found the same results as Hajimonfarednejad et al. and showed no significant difference in DHEA changes in cinnamon than in the control group (p = no data).10\n\nIn this meta-analysis, we analyze mean difference of post intervention DHEA level between the cinnamon and control groups. There was no significant difference between DHEA level post intervention in the cinnamon group and control group (-0.10[-0.62, 0.41]; p = 0.70) (Figure 4J).\n\nTwo preliminary studies examined the effect of cinnamon in menstrual cyclicity. Kort et al. showed that cinnamon can be a useful adjunct in the treatment of menstrual dysfunction in PCOS women. They showed significant changes in the menstruation cycle after intervention for 24 weeks (p = 0.0076).10 The results were the same in the Kort et al. study, who concluded that there were significant changes in menstrual cyclicity after 24 weeks (p = 0.047).9\n\nIn this meta-analysis, we analyze mean difference of post intervention menstrual cyclicity between the cinnamon and control groups. There was a significant difference between menstrual cyclicity post intervention in the cinnamon group and control group (2.28[1.83, 2.73]; p < 0.00001) (Figure 4K).\n\n\nDiscussion\n\nThis is the first systematic review and meta-analysis to evaluate the effect of cinnamon extract on insulin resistance, lipid profile, and menstrual regularity in PCOS women. We also assess change of BMI, weight and DHEA level as a secondary outcome. This review evaluated the included studies using recommended methodology (PRISMA guidelines). We only analyzed randomized controlled trial studies, which are the most reliable method to test new treatment. Our meta-analysis including five RCTs and within these 190 PCOS women showed an insulin resistance change after cinnamon treatment, and 143 PCOS women showed the lipid profile change, BMI, weight, and menstrual cyclicity changes after cinnamon treatment.\n\nCinnamon has been used in traditional herb and food industry. The component of cinnamon was extracted from the bark of Cinnamoni cassia. Nowadays, cinnamon has been reported to improve insulin sensitivity and increase glucose uptake in 3T3-L1 adipocyte, thus it improves lipid profile.14\n\nObesity changes the phenotypic expression of PCOS, exacerbating metabolic, reproductive, and psychological features. Obesity itself effects ovulatory function, regularity of menstruation, and also infertility status. Thus, prevention of weight gain is recommended for controlling PCOS.15 The last guideline regarding obesity suggested an initial weight loss of 5-10% within six months which can generate an improvement in metabolic factors.16\n\nIn this meta-analysis, we found that there was a reduction in BMI after intervention of 1500 mg cinnamon extract within 8-12 weeks. The mean BMI difference between the cinnamon and placebo groups was 0.21 kg/m2, but it was not significantly different according to the analysis. This result could be caused by different including criteria for PCOS women in the two studies. Borzoei et al. declared a BMI 25-40 kg/m2 as including criteria, but Hajimonfarednejad et al. declared a BMI ≥ 18 kg/m2 as including criteria.5,11 But Hajimonfarednejad et al. did not compare a multivariate of patients’ characteristics, thus the risk of selection bias cannot be minimalized. Hence, we did not assess for daily energy and macronutrient intake between the two groups.\n\nWeight reduction could be associated with BMI itself. In this study, we showed a different result in weight reduction from BMI reduction in the two studies. Weight after treatment in the cinnamon group was higher than in the placebo group (average = 1.08 kg), and it was not significant statistically. Although, the Borzoei et al. study showed a significant weight change in the cinnamon group (average 0.45 kg) after intervention, Hajimonfarednejad et al. did not show the same results due to different BMI criteria as including factors.5,11 Hence, the different study criteria caused the different results.\n\nInsulin resistance has an impact on the symptoms of PCOS such as increasing androgen level, menstrual irregularity, hyperglycemia and hyperlipidemia. Thus, controlling insulin resistance was recommended as one of the management targets of PCOS.12 Cinnamon extract has been found to mitigate insulin resistance induced by high fructose diets and benefit glucose utilization by enhancing the insulin signalling pathway. Cinnamon extract increases glycogen synthesis by activating glycogen synthase and regulating insulin signalling thus it can improve insulin sensitivity. It can decrease absorption of glucose in the small intestine epithelium by enhancing glucosidase enzymes and inhibiting intestinal ATPase.2,17 The polyphenolic component of cinnamon has insulin-like activity, such as quercetin, rutin, catechin, and kaempferol.17 Significant heterogeneity was found for the four trials evaluating cinnamon for HOMA-IR (I2 = 84%, p = 0.0003) and effects were adjusted using the random effect model. Of 190 patients, there was 0.19 reduction in HOMA-IR after cinnamon intervention for eight to 24 weeks. This result was not significant statistically. No significant results can be caused when not all trials are adjusted for the characteristics of the participants.\n\nCinnamon regulates the blood sugar by stimulating insulin secretion from pancreas islets.17 Cinnamon has a potential for reducing post-prandial intestinal glucose absorption by inhibiting the activity of enzymes involved in carbohydrate metabolism (pancreatic a-amylase and a-glycosidase).12 It stimulates glucose metabolism and glycogen synthesis, inhibits gluconeogenesis by effects on key regulatory enzymes and motivates insulin release and insulin receptor activity, and increases Glucose transporter type 4 (GLUT-4) receptor synthesis. In animal studies, aqueous cinnamon extracts have been shown to increase the expression of peroxisome proliferator-activated receptors (PPARs), which are transcriptional factors involved in the regulation of HOMA-IR.12 In this meta-analysis, we found no significant difference between fasting glucose in the cinnamon group and placebo. Administering 1500 mg cinnamon for 8-12 weeks can reduce glucose level by 4.8 mg/dl. We have controlled bias such as diabetes mellitus comorbid disease in this analysis, all diabetes mellitus and other metabolic diseases were excluded from the study.\n\nAnderson et al. concluded that by consuming 500 mg cinnamon a day for two months, the level of fasting blood sugar, fasting insulin, and LDL-cholesterol increased in randomized controlled trials, however, HDL-cholesterol decreased in both groups (cinnamon and placebo groups).18 In our systematic review, there were different results, in which only HDL-cholesterol and fasting blood sugar were significantly different between the two groups. This discrepancy is due to the different criteria of included studies. Khan et al. studied diabetes mellitus patients who had a metabolic disorder before the start of the study, while in our study most of the patients had a normal level of glucose and lipid profile. Besides, there was no dietary control for the subjects.19\n\nApproximately 70% of women with recently diagnosed PCOS were shown to have borderline or high lipid levels, this included an increase in total cholesterol, triglyceride, LDL or a decrease in HDL levels. In a population study with median BMI 27 kg/m2, 41% of patients with PCOS had HDL <1.29 mmol/L and 21% patients had triglyceride >1.68 mmol/L.7 In registered studies, in patients with PCOS versus controls, a dyslipidemia diagnosis increased three times and anti-lipid prescription was two times higher in PCOS. In patients with PCOS the percentage treated with anti-lipids was, however, only 1.5%. Most women seek help for PCOS at a young age for irregular menses, hirsutism and concerns about infertility. It was previously reported that in those with a higher prevalence of smoking in PCOS versus controls that smoking in women with PCOS was related to increased adrenal responsiveness, a more adverse lipid profile and insulin resistance.20-23 In this study we lacked any data for smoking habits and other exercise, which could potentially effect lipid profile.\n\nDehydroepiandrosterone sulfate (DHEA), is the most abundant steroid hormone in the circulation and has been used to induce PCOS in a mouse model.2 The DHEA induced PCOS, with similar characteristics to humans such as hyperandrogenism, abnormal maturation of ovarian follicles and anovulation.2 In this study, we showed that oral administration of cinnamon extract would reduce DHEA level (0.1 mcg/mL) after eight to 12 weeks. But this result was not significant.\n\n\nSafety\n\nFrom the latest systematic review about cinnamon adverse effects, it was concluded that cinnamon was safe to be used in a routine diet as a spice and/or flavoring agent. It is also well tolerated in controlled clinical settings. However, its use for medicinal purposes, in large doses or long duration, may lead to some adverse effects and it should be clinically monitored. There are no data for adverse effects in PCOS women. But, in diabetes mellitus and healthy subjects it can induce acute hepatitis, edema, stomatitis, and dermatitis, which can be relieved by symptomatic drugs or the cessation of cinnamon administration.24-27 It was reported that cinnamon extract equivalent to doses of 5-14 g of cinnamon powder did not alter alanine aminotransferase (ALT) and aspartate aminotransferase (AST), bilirubin and alkaline phosphatase.28 The use of 1500 mg/dL of cinnamon powder for 12 weeks improved liver profile markers in patients with non-alcoholic fatty liver disease (NAFLD). ALT decreased by 26.6 IU/L, AST by 25.6 IU/L, gamma-glutamyltransferase (GGT) by 22.8 U/L and high sensitivity C-reactive protein (hs-CRP) by 2.9 mg/dL.28,29 It also showed hepatoprotection against liver injury and lower liver fat in animal studies.30,31 A phase I clinical trial of cinnamon showed no side effect including hepatotoxicity.32\n\n\nStrength and limitation\n\nThis is the first systematic review and meta-analysis of double-blind RCTs. We analyzed the data based on PICO (using the same PCOS Rotterdam criteria) and found the same analysis to be reviewed. Authors analyzed the articles separately then discussed any discrepancies. However, our study had some limitations including different study duration and cinnamon dose in RCTs, different BMI of patients included in the study with some studies not adjusted for these discrepancies. Therefore, we need further studies to evaluate the effect of cinnamon for the same duration and dose, and thus we can provide better evidence for the efficacy of cinnamon in routine use for PCOS.\n\n\nConclusion\n\nIn conclusion, the present data suggest that cinnamon can improve fasting blood glucose, insulin level, HDL–cholesterol and menstrual cyclicity in PCOS women, but it did not affect weight, BMI, insulin resistance, triglyceride, LDL-cholesterol, total cholesterol and DHEA level. The effect of this herbal extract can be shown after 1000-1500 mg dose, administered for a minimum of eight weeks. Cinnamon can be a potential supplementary therapy agent for PCOS women.\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 ‘Cinnamon extract effects on insulin resistance, metabolic factors, and menstrual cyclicity of women with polycystic ovary syndrome: A systematic review and meta-analysis’, https://doi.org/10.6084/m9.figshare.14716470.v1.33\n\nData are available under the terms of the Creative Commons Attributions 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nFauser BC, Tarlatzis BC, Rebar RW, et al.: Consensus on women’s health aspects of polycystic ovary syndrome (PCOS): the Amsterdam ESHRE/ASRM-sponsored 3rd PCOS consensus workshop group. Fertil Steril. 2012; 97: 28–38. e25. PubMed Abstract \| Publisher Full Text\n\nDou L, Zheng Y, Li L, et al.: The effect of cinnamon on polycystic ovary syndrome in a mouse model. Reprod Biol Endocrinol . 2018; 16: 99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodgers RJ, Avery JC, Moore VM, et al.: Complex diseases and co-morbidities: polycystic ovary syndrome and type 2 diabetes mellitus. PCOS and type 2 diabetes . 2019; 8(3):R71–R75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohammed ZH, Ridha MAS, Al- Hakeim HK: Insulin resistance in polycystic ovarian syndrome women: impact of regularity and hyperandrogenemia. J Pharm Sci and Res . 2019; 11(2): 471–476.\n\nHajimonfarednejad M, Nimrouzi M, Heydari M, et al.: Insulin resistance improvement by cinnamon powder in polycystic ovary syndrome: A randomized double-blind placebo controlled clinical trial. Phytother Res . 2017: 1–8. PubMed Abstract \| Publisher Full Text\n\nWang J: The effect of cinnamon extract on insulin resistance parameters in polycystic ovary syndrome: a pilot study. Fertil Steril. . 2007; 88: 240–243. PubMed Abstract \| Publisher Full Text\n\nGlintborg D, Andersen M: Management of endocrine disease morbidity in polycystic ovary syndrome. Eur J Endocrinol. 2017; 176(2): R53–R64. PubMed Abstract \| Publisher Full Text\n\nLi XJ, Yu YX, Liu CQ, et al.: Metformin vs thiazolidinediones for treatment of clinical hormonal and metabolic characteristics of polycystic ovary syndrome: a meta-analysis. Clin Endocrinol . 2011; 74(3): 332–339. PubMed Abstract \| Publisher Full Text\n\nKort DH, Sullivan C, Kostolias A, et al.: Cinnamon supplementation improves menstrual cyclicity in women with polycystic ovary syndrome. Fertil Steril . 2013; P696. Publisher Full Text\n\nKort DH, Lobo RA: Preliminary evidence that cinnamon improves menstrual cyclicity in women with polycystic ovarian syndrome: a randomized controlled trial. Am J Obstet Gynecol . 2014; S0002-9378(14): 00446–00443. PubMed Abstract \| Publisher Full Text\n\nBorzoei A, Rafraf M, Jafarabadi MA: Cinnamon improves metabolic factors without detectable effects on adiponection in women with polycystic ovary syndrome. Asia Pac J Clin Nutr . 2018; 27(3): 556–563. PubMed Abstract \| Publisher Full Text\n\nBorzoei A, Rafraf M, Niromanesh S, et al.: Effects of cinnamon supplementation on antioxidant status and serum lipids in women with polycystic ovary syndrome. J Tradit Complement Med. 2017: 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHugar AL, Kanjikar AP, Londonkar RL: Polycystic Ovary Syndrome (PCOS)-A Mini Review. J Gynecol . 2018; 2(1): 000148.\n\nArentz S, Smith CA, Abbott J, et al.: Nutritional supplements and herbal medicines for women with polycystic ovary syndrome; a systematic review and meta-analysis. BMC Complement Altern Med. 2017; 17: 500–514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeede H, Misso M, Costello M, et al.: International evidence-based guideline for the assessment and management of polycystic ovary syndrome 2018 . Melbourne Australia: Monash University; 2018.\n\nJensen MD, Ryan DH, Apovian CM, et al.: AHA/ACC/TOS guideline for the management of overweight and obesity in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and The Obesity Society. Circulation . 2014; 129(25 Suppl 2): S102–S138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAinehchi N, Khalili AF, Ghasemzadeh A, et al.: The Effect of Herbal Medicine Supplementation on Clinical and Para-clinical Outcomes in Women With PCOS: A Systematic Review and Meta-analysis. Int J Women’s Health Reproduction Sci . 2019; 7(4): 423–433. Publisher Full Text\n\nAnderson RA, Zhan Z, Luo R, et al.: Cinnamon extract lowers glucose, insulin and cholesterol in people with elevated serum glucose. J Tradit Complement Med. 2016; 6(4): 332–336. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan A, Safdar M, Khan MMA, et al.: Cinnamon improves glucose and lipids of people with type 2 diabetes. Diabetes Care . 2003; 26(12): 3215–3218. PubMed Abstract \| Publisher Full Text\n\nRanasinghe P, Jayawardana R, Galappaththy P, et al.: Efficacy and safety of ‘true’ cinnamon (Cinnamomum zeylanicum) as a pharmaceutical agent in diabetes: a systematic review and meta-analysis. Diabet Med . 2012: 1480–1492. PubMed Abstract \| Publisher Full Text\n\nGlintborg D, Mumm H, Hougaard DM, et al.: Smoking is associated with increased adrenal responsiveness, decreased prolactin levels and a more adverse lipid profile in 650 white patients with polycystic ovary syndrome. Gynecol Endocrinol . 2012; 28(3): 170–174. PubMed Abstract \| Publisher Full Text\n\nXirofotos D, Trakakis E, Peppa M, et al.: The amount and duration of smoking is associated with aggravation of hormone and biochemical profile in women with PCOS. Gynecol Endocrinol. 2015; 32(2): 143–146. PubMed Abstract \| Publisher Full Text\n\nStepto NK, Hiam D, Helm GB, et al.: Exercise and insulin resistance in PCOS: muscle insulin signaling and fibrosis. Endocr Connect. 2020; 9: 346–359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHajimonfarednejad M, Ostovar M, Raee MJ, et al.: Cinnamon: A systematic review of adverse events. Clin Nutr. 2019; 38(2): 594–602. PubMed Abstract \| Publisher Full Text\n\nCrawford P, Crawford AJ: Edema from taking Cinnamon for treatment of Diabetes: similar biochemistry and pathophysiology to Thiazolidinedione medications. J Am Board Fam Med . 2018; 31(5): 809–811. PubMed Abstract \| Publisher Full Text\n\nUlbricht C, Seamon E, Windsor RC, et al.: An evidence-based systematic review of Cinnamon (Cinnamomum spp.) by the natural standard research collaboration. J Diet Suppl. 2011; 8(4): 378–454. PubMed Abstract \| Publisher Full Text\n\nAdmani S, Hill H, Jacob SE: Cinnamon sugar scrub dermatitis: “natural” is not always best. Pediatr Dermatol. 2016; 34(1): e42–e43. PubMed Abstract \| Publisher Full Text\n\nSantos HO, Silva GAR: To what extent does cinnamon administration improve the glycemic and lipid profiles? Clin Nutr. 2018: 1–9. PubMed Abstract \| Publisher Full Text\n\nAskari F, Rashidkhani B, Hekmatdoost A: Cinnamon may have therapeutic benefits on lipid profile, liver enzymes, insulin resistance, and high-sensitivity C-reactive protein in nonalcoholic fatty liver disease patients. Nutr Res . 2014; 34(2): 143e8. PubMed Abstract \| Publisher Full Text\n\nEidi A, Mortazavi P, Bazargan M, et al.: Hepatoprotective activity of cinnamon ethanolic extract against CCL4-induced liver injury in rats. Excli J . 2012; 11: 495–507. PubMed Abstract \| Free Full Text\n\nSartorius T, Peter A, Schulz N, et al.: Cinnamon extract improves insulin sensitivity in the brain and lowers liver fat in mouse models of obesity. Plos One . 2014; 9(3): e92358. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRanasinghe P, Jayawardena R, Pigera S, et al.: Evaluation of pharmacodynamic properties and safety of Cinnamon zeylanicum (Ceylon cinnamon) in healthy adults: a phase I clinical trial. BMC Complement Altern Med. 2017; 17: 550–559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWahyuningtyas R, Sa’adi A: PRISMA Checklist 2020 for ‘Cinnamon extract effects on insulin resistance, metabolic factors, and menstrual cyclicity of women with polycystic ovary syndrome: A systematic review and meta-analysis’. Figshare. 2021. Publisher Full Text" }	[ { "id": "137740", "date": "05 Jul 2024", "name": "Makan Pourmasoumi", "expertise": [ "Reviewer Expertise Nutrition 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\nI read the article entitled\" Cinnamon extract effects on insulin resistance, metabolic factors, and menstrual cyclicity of women with polycystic ovary syndrome: A systematic review and meta-analysis\" with interest.\nThe methodology is well-conducted. The main concern is the small number of available studies for each outcome of interest which makes the result inconclusive.\n\nI also have two suggestions. As I said before, the included articles were really small in number, therefore, the conclusion must be written with caution. This small number of studies is not sufficient to render a reliable conclusion. In addition, the limitations are quite short. There is a range of limitations that should be mentioned.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "119188", "date": "05 Jul 2024", "name": "Mohsen Akbaribazm", "expertise": [ "Reviewer Expertise Fertility and Infertility" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWith respect, the following can be considered after reviewing \"Cinnamon extract effects on insulin resistance, metabolic factors, and menstrual cyclicity of women with polycystic ovary syndrome: A systematic review and meta-analysis\":\n\"Funnel plot\", \"Egger publication bias test\" and \"/ Linear/ Non-linear dose-response analysis\" - diagrams would help to provide better results.\n\nThe conclusion does not well reflect the comparison of the effects of \"Cinnamon\".\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] } ]	1	https://f1000research.com/articles/10-523
https://f1000research.com/articles/10-148/v1	25 Feb 21	{ "type": "Case Report", "title": "Case Report: Mosaicism of a novel nonsense variant in the neurofibromin gene underlies a mosaic generalized NF1 phenotype", "authors": [ "Hui Li Kwong", "Yong-Kwang Tay", "Ene-Choo Tan", "Hui Li Kwong", "Yong-Kwang Tay" ], "abstract": "Neurofibromatosis 1 (NF1) is a neurocutaneous syndrome characterized by multiple café-au-lait macules, cutaneous neurofibromas or plexiform neurofibromas, iris Lisch nodules, axillary and inguinal freckling. Mosaicism in NF1 can either present as a generalized disease, or in a localized (segmental) manner. Mosaic generalized NF1 may have presentations that are similar to generalized NF1 or have a milder phenotype and hence may be under-recognised in clinical practice. We report a nonsense mutation in the NF1 gene in a 55-year old Chinese male with the mosaic generalized phenotype. He reported noticing increasing numbers of skin-colored papules over his face, neck, back and abdomen when he was about 40 years old. From both next-generation and Sanger sequencing data, the variant appeared to be mosaic and present at about 24%. It is in exon 39 and has not been reported in any database or published literature.", "keywords": [ "mosaicism", "mutation", "neurofibromatosis 1", "sequencing" ], "content": "Introduction\n\nNeurofibromatosis 1 (NF1, OMIM#162200) is a neurocutaneous syndrome characterized by café-au-lait macules, cutaneous neurofibromas or plexiform neurofibromas, iris Lisch nodules, axillary and inguinal freckling1. Other features include optic nerve gliomas, central nervous system tumours, and long bone deformities. The autosomal dominant condition is caused by mutations or deletions involving the neurofibromin 1 gene (NF1, OMIM*613113) at chromosome 17q11.2, one of the largest human genes (282 kb) with 57 exons and three alternatively spliced transcripts2. Over 3600 clinically significant NF1 variants are documented in the Human Gene Mutation Database (HGMD). They are distributed over the whole gene from the first to the last exon.\n\nThe estimated incidence of germline NF1 is 1:2500–3000, with approximately half due to de novo mutations and the other half inherited from an affected parent. The disease is highly penetrant and although the offspring are at 50% risk of inheriting a mutation, the manifestations are extremely variable even for family members who share the same mutation1. Non-germline or mosaic NF1 is very rare at 1:36,000–40,000, although it is probably under-reported as most cases are diagnosed clinically and molecular confirmation is not always performed3.\n\n\nCase report\n\nWe report a patient with adult-onset NF1 presentations. The 55-year-old male engineer of Chinese ancestry presented in 2015 with increasing numbers of skin-colored papules over the past 13 years. He did not recall having these lesions earlier in life. Excision of one such representative lesion measuring 0.5 mm × 0.5 mm from the right lateral neck was consistent with a neurofibroma. He was referred to dermatology and neurology departments on suspicion of neurofibromatosis.\n\nClinically the patient had more than 20 small, soft, flesh-coloured papules and small nodules (ranging between 4 and 10 mm) on his face, neck, trunk and limbs. There was no axillary or inguinal freckling. He also had multiple scattered café-au-lait macules on his trunk. There were no lesions to suggest plexiform neurofibromas and no clinical evidence of long bone deformities. Slit-lamp examination revealed the presence of Lisch nodules in both irises. He had three older siblings, none of whom had similar skin lesions. Neither his parents nor members of his extended family had any features of neurofibromatosis. His two daughters (aged 17 and 21 years) were well with no features of neurofibromatosis. His other medical history was only significant for hyperlipidaemia for which he was on simvastatin (20 mg nightly) and cervical spondylosis which did not require intervention and he is no longer on active follow-up with the orthopaedics department.\n\nApproval for sequencing analysis was obtained from the SingHealth Institutional Review Board (CIRB Ref 2014/207/F). Venous blood was collected from the patient after written informed consent and genetic counselling. Next-generation sequencing on genomic DNA was performed using a customised targeted panel (which covered all NF1 exons and NF1-related genes) with the Agilent Haloplex Target Enrichment System (Agilent Technologies, Santa Clara, USA) and the MiSeq System (Illumina, San Diego, USA). Output reads were processed using the MiSeq Reporter pipeline and variants were annotated with ANNOVAR (2019Oct24)4. A heterozygous single nucleotide substitution in the NF1 gene that is predicted to result in premature termination: NM_000267.3: 5612T>A (p.L1871X) was identified. The number of reads for the variant was 28 out of 117. The presence of the variant was confirmed in our laboratory by Sanger sequencing using BigDye Terminator v3.1 in a volume of 10 µl. Amplification conditions included initial denaturation at 96°C for 1 min, followed by 25 cycles of 96°C for 10 sec, 50°C for 5 sec; and 60°C for 4 min. Following ethanol purification, the products were resuspended in Hi-Di™ formamide and loaded onto the ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA). The peak height of the variant was found to be approximately one-quarter to one-third that of the wild-type for both forward and reverse primers, confirming the mosaicism (Figure 1).\n\nShown is the wildtype T allele with a small peak for the variant allele A (rectangular box).\n\nThe variant identified has not been reported in any population databases and is not found in ClinVar or the Human Gene Mutation Database. However, a missense variant involving the same codon changing it to a phenylalanine residue has been reported in a patient with NF15. A search of the ClinVar database found several variants within 10 nucleotides identified from patients with NF1 or hereditary cancer-predisposing syndrome, indicating that this small region might be more prone to spontaneous mutations (Table 1).\n\nHCPS, Hereditary cancer-predisposing syndrome; NF1, Neurofibromatosis Type 1.\n\n#Interpretation is based on submission in ClinVar.\n\n\nDiscussion\n\nMosaic NF1 was previously known as segmental NF1. It was initially defined as clinical features of NF1 limited to one or more segments of the body. The mosaicism which is caused by postzygotic mutation in the NF1 gene in somatic cells can present as a generalized disease or in a localized (segmental) manner6,7. Mosaic generalized patients may be clinically indistinguishable from generalized NF1 patients or have a milder phenotype8. In contrast, the cutaneous manifestations of mosaic localized NF1 reflects the pattern of skin mosaicism and can vary from a narrow strip, one-quadrant, or half the body, unilaterally or bilaterally, in a symmetrical or asymmetrical fashion. Mosaic-localized NF1 patients may develop only pigmentary changes or neurofibromas, have a combination of pigmentary and neurofibromas, or have plexiform neurofibromas only7,9,10.\n\nLike generalized NF1, mosaic NF1 presents with pigmentary features and plexiform neurofibroma arising in childhood, and dermal neurofibromas usually in adulthood. However, not all patients with mosaic NF1 with pigmentary changes develop neurofibromas. Patients may also develop learning difficulties, pseudoarthrosis and malignancy, despite having a milder phenotype3,10,11. Our patient is cognitively normal and had noticed the development of neurofibromas while in his late thirties. He also had Lisch nodules, which were not frequently observed regardless of the patient's age in other cohorts3,6,12. He is currently on annual follow-up for his cutaneous presentations.\n\nMost cases of mosaic NF1 did not have molecular confirmation of the presence of an NF1 mutation, but genetic analysis was performed in a case series of eight patients with segmental NF1 from Canada. Sequencing of DNA from skin lesions revealed a pathogenic loss-of-function variant in the NF1 gene in all eight: six were single or multi-nucleotide variants, while the other two were microdeletions. Seven out of the eight patients had pigmentary changes (cafe-au-lait macules and axillary freckling) ˗ three had non-localized lesions and one had lesions crossing the midline. The eighth patient had cafe-au-lait macules, a plexiform neurofibroma and sphenoid wing dysplasia but no freckling. Only one patient had Lisch nodules12.\n\nThe risk of passing on full-blown or localized disease to future generations is 50% for those with germline mutations. Although gonadal or gonosomal mosaicism is rare; individuals with mosaic NF1 mutation may transmit the mutation to their offspring in such cases3,13. Thus, patients with mosaic NF1 should be counselled regarding the possibility of gonadal NF1 mosaicism and the risk of transmission to the next generation, especially when the mutation is also present in non-dermal tissues. Our patient had 24% mosaicism based on the number of reads from next-generation sequencing results of DNA from his peripheral blood. He was not evaluated for somatic or gonosomal mosaicism. His two daughters appear to have no features of NF1, although he is considering genetic testing for his children.\n\nIn summary, we report a novel nonsense mutation in the NF1 gene, resulting in a mosaic generalized phenotype of NF1. Studies have sought to examine the genotype-phenotype correlation in mosaic localized NF1, although there is little in the literature regarding mosaic generalized NF1, which may be under-recognized given the clinical similarities with classic NF1. Future genotype-phenotype correlation studies in mosaic generalized NF1 would lead to better understanding of the disease development process and long-term outcomes. With better sequencing technology that can detect low-level mosaicism, molecular confirmation should be carried out to enable healthcare professionals to provide more accurate prognosis and genetic counselling.\n\n\nData availability\n\nClinVar accession number: SCV001450721.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details was obtained from the patient.", "appendix": "References\n\nFriedman JM: Neurofibromatosis 1. In: GeneReviews® (eds. Adam, M.P. et al.) University of Washington, Seattle, Seattle (WA), 2019. PubMed Abstract\n\nHinman MN, Sharma A, Luo G, et al.: Neurofibromatosis type 1 alternative splicing is a key regulator of Ras signaling in neurons. Mol Cell Biol. 2014; 34(12): 2188–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcía-Romero MT, Parkin P, Lara-Corrales I: Mosaic Neurofibromatosis Type 1: A Systematic Review. Pediatr Dermatol. 2016; 33(1): 9–17. PubMed Abstract \| Publisher Full Text\n\nChang X, Wang K: wANNOVAR: annotating genetic variants for personal genomes via the web. J Med Genet. 2012; 49(7): 433–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuat Rodríguez A, Moreno GAM, Santo-Domingo YM, et al.: [Phenotypic and genetic features in neurofibromatosis type 1 in children]. An Pediatr (Barc). 2015; 83(3): 173–82. PubMed Abstract \| Publisher Full Text\n\nRuggieri M, Huson SM: The clinical and diagnostic implications of mosaicism in the neurofibromatoses. Neurology. 2001; 56(11): 1433–43. PubMed Abstract \| Publisher Full Text\n\nHuson S, Hughes R: Chapter 41, The Neurofibromatoses. Textbook of Paediatric Dermatology. Oxford: Wiley-Blackwell, Oxford, 2006.\n\nJouhilahti EM, Peltonen S, Heape AM, et al.: The pathoetiology of neurofibromatosis 1. Am J Pathol. 2011; 178(5): 1932–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoss C, Green SH: What is segmental neurofibromatosis? Br J Dermatol. 1994; 130(1): 106–10. PubMed Abstract \| Publisher Full Text\n\nRuggieri M: Mosaic (segmental) neurofibromatosis type 1 (NF1) and type 2 (NF2): no longer neurofibromatosis type 5 (NF5). Am J Med Genet. 2001; 101(2): 178–80. PubMed Abstract \| Publisher Full Text\n\nVázquez-Osorio I, Duat-Rodríguez A, García-Martínez FJ, et al.: Cutaneous and Systemic Findings in Mosaic Neurofibromatosis Type 1. Pediatr Dermatol. 2017; 34(3): 271–276. PubMed Abstract \| Publisher Full Text\n\nMarwaha A, Malach J, Shugar A, et al.: Genotype-phenotype data from a case series of patients with mosaic neurofibromatosis type 1. Br J Dermatol. 2018; 179(5): 1216–1217. PubMed Abstract \| Publisher Full Text\n\nConsoli C, Moss C, Green S, et al.: Gonosomal mosaicism for a nonsense mutation (R1947X) in the NF1 gene in segmental neurofibromatosis type 1. J Invest Dermatol. 2005; 125(3): 463–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "80275", "date": "15 Mar 2021", "name": "Richard J. Antaya", "expertise": [ "Reviewer Expertise I am an academic pediatric dermatologist with a cursory understanding of the details regarding the methodology of genetic mutation analysis but am very familiar with the cutaneous manifestations of NF1 and segmental NF1." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 enlightening and well written case report that brings to light a variant of a common genetic syndrome.\nIn the introduction the authors state that that non-germline or mosaic NF-1 is very rare, however they report that it is about 10 times less common than germline NF-1, which is actually relatively common. I would agree it is rare, but not very rare. The statement that many cases of generalized mosaic NF1 are likely undiagnosed is likely true and would further decrease the scarcity of this disorder.\n\nIn the description of the case, the authors state that the patient had café au lait macules (CALMs) on his trunk, but do not state the size, number, and especially the duration of these lesions. In germline NF-1 CALMs generally precede the development of neurofibromas by often a decade or more. I think this is important information to include for the readership.\n\nI feel that only gonadal mosaicism should be used in this discussion since the term gonosomal is relating primarily to sex chromosomes, and ch17, the chromosome with the mutation of interest, is an autosome.\n\nWhile not clear from the discussion, have any studies evaluated the percent mutated cells in somatic tissue, skin versus blood, in patients with either generalized or segmental mosaic NF1. Since patients with cutaneous “segmental” NF1 are known to harbor the mutation in other tissues and pass on the disease to offspring, do these individuals have a form or generalized mosaic NF1 or is it tissue/area specific? I understand if this info is not available.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6829", "date": "30 Jun 2021", "name": "Ene Choo Tan", "role": "Author Response", "response": "We have deleted the word \"very\" from \"very rare\" in the Introduction. We also added more details about the patient, including his ethnicity and that his parents are not consanguineous, and the size and duration of his lesions. We also changed \"gonosomal\" to \"gonadal\" as suggested." } ] }, { "id": "83797", "date": "18 Jun 2021", "name": "Joshua Tay", "expertise": [ "Reviewer Expertise I am an Otolaryngology surgeon with a PhD in Cancer Biology", "focusing on cancer genomics and molecular subtypes of 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 well written article demonstrating a thorough genomic evaluation of the NF1 gene in an adult-onset NF1 patient. The NF1 mutation identified was present on both NGS (23.9%) and Sanger sequencing (1/3 to 1/4) at similar variant allele frequencies, supporting the mosaicism.\nMosaic generalized NF1 is less commonly seen in the clinic compared to its well-known, germline NF1 counterpart. I agree with the authors that this could also be a result of an under-recognition of mosaic cases, given their clinical similarities. NGS diagnostics and recognizing the importance of variant allele frequencies, like those presented here, provides an opportunity to close this gap.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-148
https://f1000research.com/articles/10-520/v1	30 Jun 21	{ "type": "Research Article", "title": "Social groupwork for promoting psychological well-being of adolescents enrolled in sponsorship programs", "authors": [ "Shinto Joseph", "Dr. Sheeja Remani B Karalam", "Dr. Sheeja Remani B Karalam" ], "abstract": "Background: The dearth of data on adolescents highlighted in the UN’s data disaggregation against the agenda ‘no one left behind’ calls for research on ‘the second decade’. Moreover, India is a country with the world’s largest adolescent population, and as such, studies and policies for developing competencies of adolescents are crucial to the country’s development; interventions instilling confidence to aspire to a better future in underprivileged adolescents are vital to mitigate inequity. Methods: This intervention study adopted a quasi-experimental design to measure the effectiveness of social groupwork in raising the psychological well-being of adolescents in child sponsorship programs in Kerala. Forty adolescents from a Child Sponsorship Program (CSP) center in Kochi were recruited for the study. Those suggested by the CSP center considering their poor academic performance and behavior problems were allocated to the intervention group and the rest to the comparison group. The intervention was designed in response to the information garnered through a preliminary study and administered to the intervention group (n=20). We conducted pre-test and post-test for both the intervention group and comparison group (n=20). Results: Comparison between pre- and post-measurements carried out using paired sample t-test for the intervention group and comparison group separately gave a p-value of <0.05 for the intervention group and >0.05 for the comparison group. Thus, it was proved that psychological well-being of participants in the intervention group was raised significantly due to the social group work intervention. Conclusions: Applying refined granularity, this research adds data specifically on adolescents enrolled in child sponsorship programs and sets a blueprint for social groupwork to improve their psychological well-being. Proposing a conceptual framework for child sponsorship programs, this study recommends further research in all aspects of its functioning, and interventions at group, family, and community levels, for the well-being and empowerment of marginalized adolescents.", "keywords": [ "Intervention Research", "social group work", "marginalized adolescents", "psychological well-being", "empowerment" ], "content": "Introduction\n\nAssigning a “combined focus on the individual and the society” (Alissi, 1982:10), social group work (SGW) stands out as a quintessential method in the social work domain. SGW is developed to assist individuals as well as the group in meeting their needs and solving their problems through group experiences enabled by group workers (Benson, 2001; Konopka, 1983; Lindsay and Orton, 2014). Facilitating “learning from each other” (Teater, 2014:86), SGW affirms individual’s strengths and encourages mutual aid (Gitterman and Salmon, 2009). In contrast to methods which are primarily therapeutic, SGW engages in enhancing behavioral, emotional, and social well-being of individuals through group processes (Zastrow, 2015).\n\nThe core tenets of social work are enrichment of psychosocial functioning of humans and enhancement of their living environments (Northen and Kurland, 2001). Therefore, social workers are primarily concerned with specialized intervention accomplishing well-being of individuals. Since human needs and satisfaction vary in the flux of time, the concept of well-being assimilates new constructs while redefining the existing ones. A broad classification of well-being assigns it into two categories: internal or subjective well-being (SWB), and external or objective well-being (OWB) (Alatartseva and Barysheva, 2015; Schueller and Seligman, 2010). The latter defines well-being in terms of social indicators such as accessibility to resources and social networks, and attainability of food, shelter, education, employment, and health care (Schueller and Seligman, 2010; Western, Tomaszewski and Zeeb, 2016), whereas the former conceptualizes the notion of well-being on different philosophical frameworks and redesigns the models, incorporating nuances evolved through scientific research. Two predominant models of SWB are Diener’s (1984) hedonia model expressed by communicative emotions, and Ryff’s (1989) eudaimonia model concerning the inner experience of contentment and resilience. However, by involving a goal-directedness towards personal growth, tapping one’s potential, Ryff’s model of psychological well-being (PWB) gains distinctiveness (Disabato, et al., 2016). An extant investigation into well-being trajectories; “pleasure, engagement and meaning, suggests that engaging and meaningful activities may have stronger influences on well-being than pursuing pleasure” (Schueller and Seligman, 2010:253). These conceptions second researchers’ stand in adopting Ryff’s PWB model for intervention studies with specific objectives such as finding true self, gaining mastery over environment, creating meaningful relationships, achieving autonomy, finding meaning in life, and attaining personal growth in promoting one’s well-being (Schlegel et al., 2009). Ryff’s revised PWB model probes into six dimensions of well-being:\n\n(1) the extent to which respondents felt their lives had meaning, purpose and direction (purpose in life); (2) whether they viewed themselves to be living in accord with their own personal convictions (autonomy); (3) the extent to which they were making use of their personal talents and potential (personal growth); (4) how well they were managing their life situations (environmental mastery); (5) the depth of connection they had in ties with significant others (positive relationships) and (6) the knowledge and acceptance they had of themselves, including awareness of personal limitations (self-acceptance) (Ryff, 2014:11)\n\nAdolescence is a “stormy developmental stage” (Zastrow, 2015, p.450) characterized with emotional upheaval associated with physical, cognitive, psychological, and social growth as adolescents internalizes many traits of emotional instability (Asendorpf and Van Aken, 2003; Rosenberg, 1979). The capabilities that adolescents acquire serve as a base for their well-being through their life; conversely, their failure in acquiring capabilities in adolescence adversely affects not only their personal life, but their family and community as well (Sheehan et al., 2017). A heuristic model that correlates psychosocial functioning of adolescents with the supportive direction of groups, relationships, and interactions, exemplifies the role of SGW in fostering PWB (van Aken et al., 1999).\n\nStudies among adolescents posit adverse effects of social stratification on their developmental outcomes (Benner et al., 2018; Wheeler et al., 2020), which mandates social workers’ attention on adolescents from lower socio-economic strata or low-income families. While child sponsorship programs by international, national, and local sponsoring agencies and organizations improve OWB of adolescence by enabling accessibility to education and employment, their SWB remains under-researched (Wydick et al., 2013). There exists a correlation between children’s psychosocial competencies and material poverty of their family, so that the adolescent development dimensions such as self-efficacy, self-esteem, sense of inclusion, and educational aspirations score lower than that of children with a privileged family environment (Dercon and Krishnan, 2009). Studies from economic streams draw the conclusion that the poverty trap originates from internal constraints such as failure to aspire, lack of self-efficacy, and absence of hope, rather than external constraints (Bernard et al., 2011; Dalton et al., 2010). The strategies that they put forth for social mobility of people from lower strata by raising their aspirations are; behavior modification, observing role models, choosing cognitive windows to look at people with higher levels of self-confidence, and establishing social links to transfer aspirations. Heeding these scientific study recommendations, a few sponsoring agencies initiated after-school programs aiming at the raising aspirations and self-esteem of students (Glewwe et al., 2014; Wydick et al., 2013).\n\nThe strategic move of sponsorship agencies to nurture PWB of students provides an opening for social workers to work with groups. Concomitant research in SGW is imperative owing to paucity of research in this area (Breton, 2006). Research in social work involves understanding the problems that people are experiencing within society, and the effect and implications of social policies and expert interventions on individuals and society as well (Wilson et al., 2008). Regardless of the growing attention, there exists a dearth of intervention research in social work (Mishna et al., 2012). In the study outlined in this article, we analyzed the impact of an SGW intervention on adolescents enrolled in a sponsorship program by measuring their PWB statistically. Agile construction and administration of SGW tools targeting enhancement of PWB of participants brought about the desired outcome and created a framework for SGW with adolescents enrolled in sponsorship programs.\n\nEspousing a transformative world view (Mertens, 2008; Creswell and Creswell, 2017) we designed an interventional study for uplifting the PWB of a socially marginalized group of adolescents from low-income families. A transformative paradigm facilitates change in the lives of participants, addressing specific issues such as inequality, oppression, and alienation. In this study, our focal point was social stratification perpetuating the low PWB of adolescents. The SGW intervention was tailored to raise participants’ PWB to make them feel equal to their counterparts from more advantaged stratas of society.\n\nThe theoretical conceptualization of an intervention research study involves two stages: delineation of a problem theory, and of a program theory (Fraser and Galinsky, 2010). Delineation of a problem theory provides a clear understanding of the psycho-social conditions that produce problems, whereas, program theory underpins the intervention for achieving expected outcome.\n\nWith a person-in-environment perspective (Hare, 2004; Weiss-Gal, 2008), this study comprehended the psycho-social problems of adolescents induced by the low-income family environment. Ryff’s six-factor PWB model (Ryff, 2014, 1995), which combines different perspectives such as ‘fully functioning person’ (Rogers, 1961:183), ‘executive processes of personality’ (Neugarten, 1973:312), ‘self-actualization’ (Maslow, 1968), ‘individuation’ (Jung, 1958), ‘will to meaning’ (Frankl,1959), ‘personal development’ (Erikson, 1959), ‘basic life tendencies’ (Buhler,1935) and ‘maturity’ (Allport, 1961), provided a theoretical base for our SGW intervention program to augment the PSW of participants. The group was formed within the framework of empowerment theory (Zimmerman, 2000) in general, and particularly its personal construct, psychological empowerment (PE) (Zimmerman, 1995; Zimmerman et al., 1992).\n\n\nMethods\n\nThis research was conducted with a general objective to raise the PWB of adolescents in sponsorship programs, and specific objectives; i) to raise autonomy of adolescents in sponsorship programs, ii) to raise environmental mastery of adolescents in sponsorship programs, iii) to raise personal growth of adolescents in sponsorship programs, iv) to raise positive relations of adolescents in sponsorship programs, v) to raise purpose in life of adolescents in sponsorship programs, vi) to raise self-acceptance of adolescents in sponsorship programs. Alternative hypothesis set was; H(a): There is a significant difference between the PWB levels of participants before and after the SWG intervention. Effect of SGW intervention on all the six components of PWB were tested separately against hypotheses; H(1) : There is a significant difference between autonomy of participants before and after the SGW intervention; H(2) : There is a significant difference between environmental mastery of participants before and after the SGW intervention; H(3) : There is a significant difference between personal growth of participants before and after the SGW intervention; H(4) : There is a significant difference between positive relations of participants before and after the SGW intervention; H(5) : There is a significant difference between purpose in life of participants before and after the SGW intervention; H(6) : There is a significant difference between self-acceptance of participants before and after the SGW intervention.\n\nThis study was approved by the Center for Research of the CHRIST (Deemed to be) University, Bengaluru, which constitutes the Institutional Review Board/Ethics Committee that approved this study, (approval number: CU:RCEC/00037/l/19).\n\nThe Child Sponsorship Program (CSP) of Rajagiri OutREACH gave consent to conduct this study and the same was approved by the Institutional Review Board/Ethics Committee of CHRIST (Deemed to be) University. Objectives of intervention were set in line with visions and missions of the center. The purpose of the study was well explained and written informed consent for participation and publication data was obtained from participants and their parents, prior to the study. Pseudonyms were assigned to all participants for deidentification in the recording and reporting.\n\nThis trial is registered at the Centre for Research/IRB of CHRIST (deemed to be) University. The trial registration number is CU.CFR.PhD.CWCL.REG.No.1730087.2017.\n\nOur research design focused on the research question ‘is SGW effective in improving PSW of adolescents in sponsorship programs?’ (Kate, et al., 2018). We adopted a quasi-experimental, non- randomized control group design (Campbell and Stanley, 1963; Creswell and Creswell, 2017; Shadish et al., 2001) for the study. In this design both the intervention group (IG) and the comparison group (CG) took pre-test and post-test for measuring PWB before and after intervention while only the IG received the SGW intervention for raising their PWB. Inferences were made comparing scores of both groups.\n\nA shorthand notation for this design is:\n\nI - intervention group;\n\nC - comparison group;\n\nY1 - measurement of PWB in pre-test;\n\nY2 - measurement of PWB in post-test;\n\nX - social groupwork (intervention).\n\nRajagiri OutREACH, a professional service wing of Rajagiri College of Social Sciences, Kochi, reaches out to the community at large through various programs including the child sponsorship program (CSP). Across the state of Kerala, there are 30 CSP centers of Rajagiri OutREACH, which mainly provide economic support to children (11-18 years) from lower socio-economic stratas, and promote studies and services aiming at uplifting marginalized children. Currently, there are 2,862 children enrolled with Rajagiri OutREACH, and among them 1,086 belong to the13-16 years age group.\n\nFrom the 16 total CSP centers in Kochi run by Rajagiri OutREACH, one center at Kalamassery was selected as the study setting with two criteria; first, the study excluded the centers with less than 40 adolescents in them, and second, one center was selected using random sampling (Levy and Lemeshow, 2013); by assigning unique numbers to each center and randomly picking one from them. Entry to and consent for conducting this study at the center were negotiated through proper channels (Høyland et al., 2015). Recruitment, intervention, and data collection with participants were done in this CSP center. The study was conducted over a period of two years from June 2018 to July 2020.\n\nSecondary data was garnered from the selected CSP center records that revealed the poor academic performance, behavior problems, and issue coping with family of registered adolescents. For an in-depth exploration of the problems of participants and for identifying risk factors and protective factors, we employed case study as a method of inquiry (Yin, 2018), and conducted twenty case studies among participants and their parents, with prior consent and appointment.\n\nBased on the conclusion drawn from the preliminary study the first author under the guidance of second author developed questionnaires and conducted a ‘Delphi survey’ (Hasson et al.,2000) among selected (purposive sampling) social work professors in South India. We e-mailed professors in three leading social work colleges in South India at Rajagiri College, Kalamassery, Marian College, Kuttikkanam and Christu Jayanthi College, Bangaluru, and nine out of the fifteen we contacted expressed their willingness for participation in the survey by returning the electronically signed consent form which was attached in the mail. The panelists reached consensus after three rounds, on the most suitable method to enhance the psychological well-being of adolescence enrolled with CSP (See Figure 1 for details).\n\nWe did a two-stage sampling for this study. 1) Forty adolescents from the selected CSP center were selected for the study using the following inclusion/exclusion criteria.\n\nInclusion criteria\n\n• Adolescents (13-16 years.) with the sponsorship program for two years or more.\n\n• Regular attendance at the center\n\n• Willing to participate in the study\n\nExclusion criteria\n\n• Report of mental illness\n\n• Physical inability to be involved in activities\n\nThe pretest was conducted for all the 40 participants.\n\n2) Delphi survey results clearly stated that the ideal group size for SGW intervention is twenty. Therefore, twenty adolescents suggested by the CSP center considering the urgency of a solution to their problems such as poor academic performance and behavior problems, were assigned to the IG and the rest to CG, for ethical reasons outlined in the blinding section that the objectives of a SGW must go in line with that of the research settings, which in turn assist them to better handle the issues they are currently dealing with.\n\nWe found that blinding of intervention to IG is impossible in SGW intervention as the objectives of group has to be described to group members in the group forming stage itself. Blinding of researcher was also impossible in this study since the researcher was administering the intervention which was developed by him. However, data collection was done by blinded research assistants and all participants were anonymized during data analysis. Further, the bilingual translators were also blinded in this study. This research kept CG unaware of the study objectives to reduce the risk of bias.\n\nSocio-demographic details of participants were collected using a self-administered questionnaire (Joseph and Karalam, 2021). The 42-item version of Ryff’s PWB scale was administered for pre- and post-test, for collecting statistical data and estimating the effectiveness of intervention in raising PWB of participants. The scale is a six-point Likert scale ranging from strongly disagree to strongly agree. Ryff’s PSW scale has six subscales which measure “autonomy, environmental mastery, personal growth, positive relations, purpose in life, and self-acceptance” (Gao and McLellan, 2018:2). All items were translated into Malayalam (local language) and back translated into English once completed, ruling out discrepancies. The translation was done by a bilingual person and the accuracy is checked by the researcher. Pretesting was done by administering the scale to five adolescents and internal consistency was calculated statistically for each subscale after reversing the negatively phrased items. The average value obtained for Cronbach alpha coefficients was 0.79, which is > 0.7, indicating reliability of scale (Pallant, 2020).\n\nSecondary outcomes such as improvement in academic performance, personal competence and social competence were measured through follow up, which was carried out using in-depth interviewing of parent (father/mother) of each participant received intervention. In addition, three FGD’s were conducted with teachers of three local schools that participants were attending.\n\nSocio-demographic data collection and pretests were done immediately after the selection process. Socio-demographic questionnaire and translated version of Ryff’s 42 item PWB scale (Joseph and Karalam, 2021) were distributed to participants, one after the other. Research assistants facilitated data collection which was taken place at the research setting. As it was done in the pretest, post-test to both IG and CG for assessing their PWB after the intervention was carried out in the research setting. In the preliminary study, qualitative data was collected using case study method in which the participants and parents were interviewed by the researcher at their dwellings. Secondary data was collected through reviewing records in the CSP center. Qualitative data on intervention was gathered by audio recording the sessions and participant reflections, noting down participant observations, writing researcher reflections, and collecting vignettes of group experience from participants. Follow-up interviews with parents were conducted at their houses and FGD’s in the respective schools.\n\nAll the quantitative data collected was analyzed statistically using IBM SPSS statistics 25, keeping a group as the smallest unit of analysis. Paired sample t-test was employed to test hypotheses. Concurrently, qualitative data was analyzed at individual level since the effect of a SGW needs to assessed at both group and individual level. Qualitative data was transcribed verbatim and translated from Malayalam into the target language English by a bilingual expert and tested accuracy using back translation of three transcripts. Content analysis of qualitative data was done using Atlas.ti8. We did not face an issue of missing data since we could keep data collection intact with the small sample size.\n\nA social work intervention entails designing, testing, refining, administering, evaluating, and disseminating of a customized package. In this study we designed a social groupwork intervention in response to the concerns and needs assessed through the preliminary study among the selected adolescents enrolled in CSP, their family, and the sponsoring agency. The PE perspective contoured the groupwork program where PE refers to “empowerment at the individual level of analysis” (Zimmerman, 1995:581). The specifically designed SGW intervention was administered to the IG.\n\nThe primary purpose of the social groupwork intervention was ‘enhancement of PWB of group members.’ The expected secondary outcomes of the intervention were set as improved i) personal competence, ii) academic competence, and iii) social competence of participants, bearing in mind the fact that the group objectives and activities “must be compatible with the mission and purpose of the sponsoring organization” (Gitterman and Salmon, 2009, p.93).\n\nThis SGW intervention was carried out in 16 sessions with a duration of two hours/session, over a period of two months.\n\nPlacing the locus of intervention at psychological upliftment of adolescents from lower socio-economic strata, we selected the empowerment model of social groups. “The empowerment model evolved in response to the plights of marginalized and oppressed individuals of our society” (Gitterman and Salmon, 2009:73). The empowerment model adapts a strengths perspective ((Zastrow, 2015) to counter helplessness and hopelessness. “A strengths-based approach operates on the assumption that people have strengths and resources for their own empowerment” (Pulla, 2012:3). The significance of PE surfaces here, as it holds intrapersonal, interactional, and behavioral aspects (Zimmerman,1995). In this SGW intervention study, we framed the group activities in line with the three strategies of empowerment: collective action, consciousness raising, and capacity building.\n\nCollective action (CA) is a joint action by a group of individuals in the pursuit of enriching their lives. Personal empowerment remains impossible in the absence of collective action for marginalized groups because they need a collective empowerment as a deprived section of the society (Cislaghi et al., 2016). These SGW intervention activities were framed around the ideology of “collective action for a collective solution” (Hanish, 2000:114). The IG of adolescents from the disadvantaged strata of society was formed to act together to envision a better future, assuring their social mobility. Every group activity focused on instilling an altered perception of self; a transformation with an enlightened beliefs and attitudes in articulating aspirations.\n\nConsciousness raising (CR) is the centrality of group process with marginalized groups (Bruley, 2013). CR activates introspection to examine the extent of suppression within individuals and aids them to relate with others so that they can comprehend the reasons for their oppression and can think of ways to step out of it as a group. Inspiring stories of successful personalities from the same social strata were narrated for encouraging participants to dream high and to make resolutions for a bright future. In addition, an awareness on the constitutional rights, concepts of equality, and a basic lesson on the laws of the residing country was provided to implant hopes in them to set life goals, and to lessen vulnerabilities.\n\nCapacity building (CB) is the key strategy to alleviate social disparities through empowering the weaker section (Kaplan, 2000). This SGW intervention formulated activities to assist participants in their studies. Special tuition for those who had difficulty with particular subjects was arranged and peer learning promoted with members good at certain subjects. Training in sports and other leisure activities were also part of this intervention program. In addition, a session on kitchen gardening was organized to capacitate them in producing vegetables on their own.\n\nWe adopted a four-stage model of group development proposed by “Northen and Kurland” (Zastrow, 2015:22; Northen and Kurland, 2001), which highlights socio-emotional themes: a) inclusion–orientation, b) uncertainty–exploration, c) mutuality–goal achievement, and d) separation–termination, Activities grounded on the requirements of the group assisted group development through the stages towards realization of its goals (Lindsay and Orton, 2014).\n\nInclusion–orientation\n\nPost intake of members, the intervention sessions commenced as scheduled. Icebreaking techniques gave a head start by opening up the self-introduction of members, which helped them to get acquainted with other members, to reduce anxiety, and to facilitate group interaction. The GWR elucidated the objectives of the group; have faith in self, make academic progress, articulate aspirations, avoid vulnerabilities, improve familial and social relationships, and strive for a bright future.\n\nGaining group members’ trust was a major task for GWR. People from disadvantaged groups have enough reasons to distrust a GWR from upper stratas because of the presence of overt as well as covert discrimination in society (Northern and Kurland, 2001). Due to this, we demystified the SGW intervention, explaining it is aimed at their well-being and empowerment. We deliberately maintained sensitivity to their feelings, treated them with respect, and behaved courteously by shaking hands, greeting warmly etc.\n\nInterdependence is imperative in a group as it steers interactions, leading to the formation of group behavior (Johnson and Johnson, 2005). Group games demand interdependence, which catalyzes reciprocal interactions (Evans et al., 2012). We conducted group games such as hula hoop games, relay races, and scavenger hunts to expediate ‘group cohesion’ (Carron and Brawley, 2012). We awarded prizes for enticing members to participate actively and for motivating them to continue in the group.\n\nWe considered developing group norms in fostering helping behavior among members (Gonzalez-Mulé et al., 2014), and we shared certain norms such as mutual support, mutual respect, respect to privacy, self- disclosure stays harmless, give every task a try, it is alright to fail the tasks, open communication, conflict constructively, commitment to group and procedural norms.\n\nUncertainty–exploration\n\nThe first theme here is “group members’ uncertainty regarding issues of power and control” (Zastrow, 2015:22), which is inherent but not an obstacle to overcome. We employed an ‘I am good at’ strategy for assigning roles.\n\nAnticipating raising the consciousness of members to realize their value and growth potential, we administered bibliotherapy. Reading and listening to literature analogous with life experiences of individuals can constructively affect their attitudes and beliefs so that they alter and reshape their thoughts and behavior for an upgraded lifestyle (Hynes, 2019; Lenkowsky, 1987). We started this session reading out the Malayalam (native language) version of “King’s reiterated affirmation of ‘I have a dream’” speech (Sundquist, 2009:1). Resource people including social work professors and catholic priests narrated them stories of APJ Abdul Kalam, Abraham Lincoln, Nelson Mandela, and K. R. Narayanan. The speakers explained to them their constitutional rights, government schemes and reservations for higher studies and employment, the need for performing well in academics, staying away from illicit activities, and keeping personal integrity. In addition, members recited popular poems describing the notion of ‘equality’.\n\nAt this point, group members were asked to write down their ambition and ways to achieve it.\n\nThis stage was characterized by greater mutual acceptance, mutual aid, and self-disclosure. Group members were encouraged to work with unity to attain the immediate objective; better the academic performance that they set in the previous stage. Knowledge acquisition that activates self-determination was the opening process here, since an individual’s “knowledge, skills, and beliefs … lead to self-determination” (Field and Hoffman, 2002:114). Towards CB, the group was provided with academic supporting grammar sessions, mathematics, and science tuition. Members good at certain topics led peer learning sessions, motivating them to focus on studies regardless of their situations. Additionally, professional training in football and swimming were given to boost their morale through sustained reciprocal interaction and interpersonal relations in the group. They benefitted from a skill training session on kitchen gardens, which covered modules on planting vegetables in limited space, selecting seeds, organic pesticides and fertilizers, and effective ways of domestic waste management. This aimed to enable them to contribute to their household by growing vegetables, which is expected to improve their connectedness with family, and to mitigate coping issues with unpleasant family environments.\n\nThe ending of a group encompasses three aspects such as relationship between members, relationship with GWR and group as a whole ((Kurland and Salmon, 1998). “A successful termination is not an emotionally traumatic event for the members” (Gitterman and Salmon, 2009:168). We informed the group that it is about to end and encouraged members to reminisce about their group experience. GWR recapitulated the objectives and the group’s efforts to attain them. They made an affirmation on the requirement of keeping up the momentum they achieved through the intervention..\n\nA procedural review was made using the records we kept, that included the day, date, and time of sessions, name of attendees, list of activities, name of resource person and designation, summary of the session, participants’ reflections, GWR’s reflections, and plan for the next session.\n\nGroup members prepared vignettes on their group experiences and handed them over to the GWR as a token of their gratitude.\n\nFor a statistical analysis of the effectiveness of SGW intervention, post-test using Ryff’s PSW scale was carried out among both the IG and CG. After the completion of analysis, this SGW intervention program was administered to the CG for ethical reasons that every participant has an equal right to receive the intervention. It was carried out by the same resource persons, in the same research setting.\n\n“Greater attention needs to be given to fidelity both in the development of social, psychological, and behavioral interventions and in their execution. Fidelity is imperative in all stages and phases of intervention research, design, and implementation” (Gearing et al., 2011:83). Though assessing fidelity in intervention research is important, it is rarely done (Hardeman et al., 2008). In this study we assessed fidelity through three phases: delving, designing, and delivering. An intervention must be tailor-made to meet the concerns of the population; therefore, a precise exploration of their problems is crucial. We maintained fidelity using a detailed preliminary study in the first phase, and in the second phase we designed an SGW intervention with well-defined objectives, after an extensive literature review, and consulting with experts using the Delphi survey. In the third phase, we implemented the intervention for the target group with no deviation from the pre-defined protocol. All sessions were conducted under the supervision of social work experts.\n\n\nResults\n\nThe process of SGW intervention started with recruitment of participants in July, 2019, ended with final data collection and analysis by March, 2020.\n\nData collected from IG and CG using Ryff’s 42-item PWB scale before and after the intervention were analyzed statically using IBM SPSS Statistics 25. The tests were conducted against the null hypothesis, H(o): There is no significant difference between the PWB levels of participants before and after the intervention; and alternative hypothesis, H(a): There is a significant difference between the PWB levels of participants before and after the intervention.\n\nFor comparing scores obtained for PWB of participants before and after the intervention, we employed a paired sample t-test for both IG (n = 20) and CG (n = 20) separately. Tests results are shown in Tables 3 and 4.\n\nFor IG (Table 3), the p value obtained was <0.05 for all the six dimensions of PWB and for the total PWB; therefore, null hypothesis was rejected and alternative hypothesis was accepted. Whereas, for the CG (Table 4), the p value obtained was >0.05. Therefore, we accepted the null hypothesis. Hence, it was concluded that PWB of members of IG was raised through the intervention, while CG members’ PWB remained unchanged significantly. Thus, this analysis indicates that the social group work intervention successfully raised PWB of adolescents in the IG.\n\nContent analysis (Drisko and Maschi, 2016) of the preliminary case studies unfurled the needs and concerns of adolescents in the CSP. The risk factors identified were: low family income, stressful life events, addiction of parents, parental conflicts, single parenting, absence of social support, and absence of self-esteem and aspirations.\n\nHe always hangs out with his friends in the neighborhood. He doesn’t like studying … none in his gang is good at studies … I wish if he could study well and support our family … if things are going like this, he will become a daily wager like me … who will make him understand? (C1:M)\n\nMy husband doesn’t provide for us. Whatever he earns, he spends in the bar … he abuses us … she doesn’t even look at him … now a days she is not talking much to me also … she scores very less in exams … she doesn’t even bother that … I don’t know what would be her future … (C3: M)\n\nI am a daily wager … I can’t spend much for her studies because I need to save something for her wedding … she is also not that interested in studies, but ready to marry someone … (C5: F)\n\nI can’t dream high because we are poor. I just want to get through 10th grade exam to get a driving license … I don’t have friends in my neighborhood. They all study in private schools, but I go to a government school. (C8: B)\n\nMy father is no more and my mother is doing tailoring. I help her in that and I will be continuing with that … so, there is no point in studying more … (C10: G)\n\nBased on these results, we postulated that adolescents in the CSP need psychological support, family support, and community support along with financial support, and this study developed a program for providing them an enhanced PSW.\n\nA concomitant analysis of qualitative data collected through the intervention process enabled refining of the intervention.\n\nThe first session was characterized with members’ anxiety about an unknown situation. Based on this observation, more group games included in the next two sessions in order to reduce their anxiety and to improve group interaction. Further, the objectives of the intervention were explained well to make participants understand that the intervention is aiming at their development.\n\nAfter the first session I found a few members skeptical about the goals of the group. I overheard one boy saying that they are dubious goals. Their mind is bounded with unattainability of life goals … I was confused that what was hindering a few, particularly girls, from interacting well, fear or timidity or something else? (GWR reflections)\n\nThere were two members good at singing and they sang their favorite songs. They both were assigned to start the following sessions with a song alternatively.\n\nGWR: You’re an excellent singer … why do you want to become a driver?\n\nMember (M): I am not learning music … it’s a very competitive field … it will be a big failure, if I choose singing as my profession.\n\nGWR: Not just music lessons, but talent matters in singing … You’ve got that talent, that you must cherish …\n\nM: Ok … but … no one supports me at home …\n\nGWR: (To the group) What you want him to be? A driver or singer?\n\nGroup (G): A singer.\n\nAnother M: He is the winner in district level youth festival.\n\n(GWR starts clapping and the whole group follows)\n\nGWR: We all are your supporters.\n\nM: (Smiling) Thank you.\n\nGWR: Now … what do you choose? To be a Driver or Singer?\n\nM: Both … I will try for chances to sing … but I am skeptical if it can provide me enough …\n\n… … ……………………………………… … ..\n\nAnother member was good at drawing, and he was assigned to create the welcome board for the resource people. Establishing specific roles and developing mutual trust, the group advanced further.\n\nIn the ‘goal setting’ session, all the participants shared the goals that they wrote down in the group with a lessened inhibition. Nonetheless, all were encouraging others well with cheers and applause.\n\nAn analysis of vignettes provided a real picture about the impact of this SGW intervention program.\n\nI am very thankful to (GWR’s name). I am attending such a helpful program for the first time in my life. I came to know that I am not alone, there are many children like me. I understood the need for studying well using the aid from CSP. I want to get a decent job to support my family and I hope to end our struggles (Group member 2).\n\nI got real friends. They won’t look down on me. Now I feel that I am a valued person. I want to study well and reach a good position (Group member 13)\n\nThis qualitative outcome in congruence with the results of statistical tests that showed a significant growth in the PWB of participants, proved the effectiveness of SGW intervention in raising PWB of under privileged adolescents.\n\nThematic analysis of qualitative data collected from IG members’ parents, and their school teachers post intervention confirmed the secondary outcomes of SGW. Themes identified were gaining confidence to open up, articulating aspirations, showing academic progress, reducing truancy and involving in family matters.\n\nI had never seen him before talking to others in the class, but now he interacts … not much, but … and he answers my questions too … (FGD1:02).\n\nThey are regular to school now … and they are showing some interest in studies (FGD3:04).\n\nNow a days he goes to school every day … I don’t see him hanging out with friends … (M1).\n\nShe helps me in doing chores … she was so lazy before (M3).\n\nThese results ascertained the effectiveness of SGW in meeting the projected secondary outcomes. Being the best fit for raising PWB of CSP enrollees in the social and cultural context of research setting, these results defined the external validity and generalizability of the SGW developed and administered.\n\n\nDiscussion\n\nThe outcome of this study supports the view that SGW is instrumental in equipping adolescents with “a sense of competence and a feeling of hope” (Malekoff, 2015:20), for setting and reaching goals that upgrade their social status. This SGW intervention study not only provided participants a platform, but empowered them also to share their experiences, voice their views, and to propose a way to a better future as a transformation affords them aspirations.\n\nIn fact, for the realization of well-being and social mobility of underprivileged adolescents, we need scientific social interventions addressing the “intergenerational perpetuation of inequalities” (Hopenhayn, 2012:1). In congruence with UNICEF guidelines for Adolescent Development and Participation (Adolescent development and participation \| UNICEF India), India has several policies and programs for the empowerment of ‘the second decade’. Nonetheless, the top-down policies have to be complemented by grassroot level interventions with a strong theoretical base and a clear conceptual framework (Crescenzi and Rodríguez-Pose, 2011).\n\nConsidering the positive outcome this intervention research achieved, we recommend the SGW intervention to adolescents in similar contexts, with needed refining. Although this study took all possible efforts to consider expert opinions in all stages, it was limited to the professors in South India. Consulting with experts all around the world may draw wide range of opinions contributing to the fine-tuning of intervention. Moreover, this study endorses use of qualitative data in all stages of an intervention research.\n\nWhile extending the literature and contributing to the research method repertoire, this article proposes a conceptual framework for the CSP, for the sustainability of well-being that participants acquired through the SGW intervention. CSPs must be inclusive to reach the over-arching goal of integrating disadvantaged adolescents into the mainstream of society. The proposed framework delineates a set of services including; a) monitoring government support systems; b) overseeing school support systems; c) providing financial aid for schooling and nutrition; d) staging community interventions; e) executing family interventions; f) conducting social groupwork; g) promoting research; h) initiating policies.\n\nWe suggest that a CSP must ensure that the adolescents enrolled in it avail the government services as well as adequate support from their schools. It must continue with sufficient financial aid to ensure accessibility to education and health care, and availability of nutritious food regardless of their socio-economic origin. Devising and carrying out community interventions is another role of CSPs to facilitate the meaningful participation of disadvantaged adolescents in their communities, with an emphasize on equity. Family interventions are crucial in making their growing environment a conducive one, whereas group interventions with adolescents are decisive in securing their PWB which triggers aspirations. Additionally, we recommend CSP to encourage scientific studies that delve into the problems of poor adolescents, formulating strategies for resolving their issues, inventing, and reinventing tools for their empowerment, and preparing policies for their social mobility.\n\nIn a quasi-experimental design, a potential pitfall is the imperfect matching of groups due to the heterogeneity within groups because of the variations in living environments and family histories of members (Butler and Hickman, 2011). Another limitation is possible selection bias as this design lacks random selection of participants (White and Sabarwal, 2014), though it offers a practical option for avoiding ethical concerns in conducting impact evaluations of an intervention with a group that is already a part of another program or agency. Besides, an interference of confounding factors is likely to occur in any experimental or interventional study. Moreover, a possible shortcoming surfaces while administering Ryff’s inventory to adolescents in that they may respond in a socially desirable way rather than their real response to the statements (Seifert, T.A., 2005).\n\n\nConclusion\n\nAdvocacy for marginalized adolescents is an advancing area in the realm of social work practices, which gains an additional attention apropos ‘invisibility of adolescents’ within the UN’s Sustainable Development Goals. The ‘leave no one behind’ agenda calls for research leading to policies and programs for the well-being of adolescents. This social groupwork intervention research sets forth a method for social researchers to refer to, and an effective tool for groupworkers working with adolescents in CSPs. In addition, the framework developed for CSPs is expected to be adopted by agencies to secure well-being of adolescents for capacitating them to articulate aspirations that set the stage for their transformation. Finally, we assume this research will go a long way in relegating the vicious circle of disparity, and in promoting the virtuous circle of equity.\n\n\nData availability\n\nHarvard Dataverse: Social Groupwork for Promoting Psychological Well-being of Adolescents Enrolled in Sponsorship Programs. https://doi.org/10.7910/DVN/V6EBAX (Joseph, 2021).\n\nThe project contains the following underlying data:\n\n- Pre-Post Data for Well-being of Intervention Group (IG) and Comparison Group (CG).tab (SPSS file).\n\n- Socio-economic details of the comparison group. (Anonymized details of CG).\n\n- Socio-economic details of the Intervention group. (Anonymized details of IG).\n\nZenodo. Social Group Work for Promoting Psychological Well-being of Adolescents Enrolled in Sponsorship Programs. https://doi.org/10.5281/zenodo.4962858 (Joseph and Karalam, 2021).\n\nThis project contains the following extended data:\n\n- Socio-demographic details.docx (Questionnaire of socio-demographic details for participants).\n\n- Ryff’s Psychological well-being scale.pdf (Socio-demographic questionnaire).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nZenodo: TREND checklist for ‘Social Group Work for Promoting Psychological Well-being of Adolescents Enrolled in Sponsorship Programs’. https://doi.org/10.5281/zenodo.4962858.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgement\n\nThis research was carried out as part of a PhD at CHRIST (Deemed to be) University, Bengaluru. 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Sage; 2018.\n\nZastrow CH: Social Work with Groups: A Comprehensive Workbook. Cengage Learning; 2015.\n\nZimmerman MA: Empowerment theory. In: Rappaport J, Seidman E (eds), Handbook of Community Psychology. New York: Springer; 2000.\n\nZimmerman MA: Psychological empowerment: Issues and illustrations. Am J Comm Psychol. 1995; 23(5): 581–599. Publisher Full Text\n\nZimmerman MA, Israel BA, Schulz A, et al.: Further explorations in empowerment theory: An empirical analysis of psychological empowerment. Am J Comm Psychol. 1992; 20(6): 707–727. Publisher Full Text" }	[ { "id": "88773", "date": "08 Jul 2021", "name": "Jed W. Metzger", "expertise": [ "Reviewer Expertise Clinical intervention with youth" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStrong research. This study should be indexed.\nOne question I have is why were the adolescents with a mental health concern not included in the sample? With mental health being such a key driver, and so much more common today, it is hard to understand the exclusion. I would like a little more information about the actual intervention.\nThe study is mostly quantitative, but perhaps the qualitative data is richer given the small sample size.\nNot entirely clear where the group that did not receive the intervention came from.\nIn a few places (the first sentence in the Abstract for instance) the writing is so dense that it could be reworked for flow.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "88776", "date": "08 Jul 2021", "name": "Kiran Thampi", "expertise": [ "Reviewer Expertise Mental Health", "International Social Work", "social work education", "Development communication", "participatory community appraisal", "NGO management" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have enjoyed reading this article. The article presented an intervention study adopting a quasi-experimental, non-randomized control group design. The study explores the possibility of conducting social group work with adolescents enrolled in child sponsorship programmes to improve their psychological well-being. It is conveyed from the paper that the psychological well-being of participants in the intervention group was raised significantly due to the social group work intervention. Theoretically and methodologically, this is a well-written piece of work where the researchers were able to demonstrate considerable standards in research formulation, ethical consideration, tool selection, intervention protocols, data analysis, result presentation, and possibility for replication. Adoption of the empowerment model with strengths perspective and categorisation of group activities for IG into CA, CR, and CB is impressive. Efforts made to assess the secondary outcomes have also added to the credibility and logical connection to the findings.\nHowever, the following things could be taken care of/clarified by the authors to make this work more communicative to the readers.\nResearch setting: Could mention how many CSP centres are there with more than 40 respondents.\n\nThe authors could also mention, how you avoided subjective bias in selecting the 20 cases, or else, were these 20 respondents in IG?\n\nFigure 2: What is the attendance cut-off percentage in curtailing the number of respondents from 58 to 49 (84.48% of the registered children show up in CSP)? This could be mentioned. This is significant as the impact of CSP centres on the children is evident from this percentage and also your secondary outcomes show the impact of intervention in motivating them to be regular in their schools as well.\n\nIntervention: What is the time gap between the sessions you followed? This will have a considerable impact on the outcomes. This will allow practitioners to replicate the session with a similar impact.\n\nTools for data collection: In the last sentence, consider avoiding apostrophe for \"FGD\".\n\nThe authors have mentioned GWR in “Inclusion–orientation” in group development. You can give the expansion there as it is introduced for the first time.\n\nThe authors may consider adding a bit on cultural context, especially for developing group norms as the SGW is much reliant on setting culturally appropriate ground rules. This will root your intervention and practitioners could be vigilant in respecting cultural implications. You may add this in the Group Development portion (four-stage model).\n\nUnder “uncertainty-exploration” the authors mention the key personalities and their stories. Rather than mentioning their names, it would be impressive to communicate the broad areas/disciplines from which the selection of such personalities could be made. It would be good to introduce a common model template that could be adopted on the same.\n\nQualitative outcomes: You have identified risk factors (themes). Since the number is 20, thematic analysis would be better. You may elaborate on the identified themes which could be supported by the respondents’ comments (from the comments you have presented now). It is always good to have literature support for those findings since you have not used your qualitative assessment to support the quantitative findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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/10-520
https://f1000research.com/articles/10-517/v1	30 Jun 21	{ "type": "Software Tool Article", "title": "CHIPS: A Snakemake pipeline for quality control and reproducible processing of chromatin profiling data", "authors": [ "Len Taing", "Gali Bai", "Clara Cousins", "Paloma Cejas", "Xintao Qiu", "Zachary T. Herbert", "Myles Brown", "Clifford A. Meyer", "X. Shirley Liu", "Henry W. Long", "Ming Tang", "Len Taing", "Gali Bai", "Clara Cousins", "Paloma Cejas", "Xintao Qiu", "Zachary T. Herbert", "Myles Brown", "Clifford A. Meyer", "X. Shirley Liu" ], "abstract": "Motivation: The chromatin profile measured by ATAC-seq, ChIP-seq, or DNase-seq experiments can identify genomic regions critical in regulating gene expression and provide insights on biological processes such as diseases and development. However, quality control and processing chromatin profiling data involves many steps, and different bioinformatics tools are used at each step. It can be challenging to manage the analysis. Results: We developed a Snakemake pipeline called CHIPS (CHromatin enrIchment ProcesSor) to streamline the processing of ChIP-seq, ATAC-seq, and DNase-seq data. The pipeline supports single- and paired-end data and is flexible to start with FASTQ or BAM files. It includes basic steps such as read trimming, mapping, and peak calling. In addition, it calculates quality control metrics such as contamination profiles, polymerase chain reaction bottleneck coefficient, the fraction of reads in peaks, percentage of peaks overlapping with the union of public DNaseI hypersensitivity sites, and conservation profile of the peaks. For downstream analysis, it carries out peak annotations, motif finding, and regulatory potential calculation for all genes. The pipeline ensures that the processing is robust and reproducible. Availability: CHIPS is available at https://github.com/liulab-dfci/CHIPS.", "keywords": [ "chromatin profiling", "snakemake", "ChIP-seq", "ATAC-seq" ], "content": "Introduction\n\nProtein-DNA binding interactions are fundamental to gene regulation and are involved in regulating disease processes. However, the methods of investigating these interactions through ATAC-seq, ChIP-seq, and DNase-seq experiments generate data that require extensive processing before biological interpretation (Furey 2012). Chromatin profiling using sequencing technology can also generate bias (Meyer and Liu 2014), which needs to be mitigated before interpreting the biological significance. Therefore, consistent and reproducible processing of the chromatin profiling data is essential in deriving meaningful information from the experimental data. Moreover, experiments can fail due to technical complexities. Comprehensive quality control will help to identify failed samples, and robust processing can facilitate reproducible analysis.\n\nThere are other pipelines available for processing ChIP-seq data. For example, ENCODE has its own pipeline written in Workflow Description Language (WDL) (The ENCODE Project Consortium 2012). Recently, Snakemake (Köster and Rahmann 2012) workflow language becomes popular in the bioinformatics field partly because it is python-based. Using Snakemake v5.4.5 we developed CHromatin enrIchment ProcesSor (CHIPS) to standardize processing and quality control evaluation for ATAC-seq, ChIP-seq, and DNase-seq data following best practice (Bailey et al. 2013). Furthermore, CHIPS generates a comprehensive interactive HTML report using Plotly for the users to easily inspect the quality of the samples. Encapsulated in a Conda environment, it can be executed in the local computing cluster engine or in the cloud computing settings such as Amazon AWS and Google Cloud. CHIPS has been used to analyze >1500 samples since 2016 within Dana-Farber Cancer Institute, and now serves as the standard processing pipeline for tumor ATAC-seq data from the Cancer immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC) trials (H. X. Chen et al. 2021).\n\n\nMethods\n\nAlignment and basic quality control\n\nThe workflow of CHIPS is described in Figure 1. CHIPS takes FASTQ or BAM files as input and supports both single-end and paired-end data. To save time and resources, CHIPS subsamples 100,000 reads and uses them in the FASTQC module for basic quality control analysis. For aligning reads to the reference genome, FASTQ files are trimmed to remove adaptors and low-quality sequences using fastp (S. Chen et al. 2018) and then aligned by BWA-MEM (Li 2013) to generate sorted and deduplicated BAM files. After alignment, the mapping statistics, including the number of mapped and uniquely mapped reads, are then reported.\n\nThe CHIPS pipeline is designed to perform robust quality control and reproducible processing of chromatin profiling data derived from ChIP-seq, ATAC-seq, and DNase-seq. The CHIPS pipeline includes basic steps of read trimming, read alignment, and peak calling. For quality control, it calculates metrics such as contamination profile, mapping statistics, the fraction of reads in peaks (FRIP) score, PCR bottleneck coefficient (PBC), overlap with union DNaseI hypersensitive sites (DHS), and peak evolutionary conservation. For downstream analysis, CHIPS carries out peak annotation, motif finding, and putative target prediction. The inputs to the pipeline are FASTQ/BAM format DNA sequence read files.\n\nCHIPS carries out other basic quality control. The contamination profile reports the percentage of 100,000 reads that map to a contamination panel’s reference genomes. The contamination panel, specified by the user in a configuration file, includes dm3, Saccharomyces cerevisiae, E. coli, and mycoplasma of different types in addition to hg38, hg19, mm10, and mm9 genomes. We provide static reference files along with the installation of CHIPS. Users may add new assemblies to the contamination panel by adding the BWA index files. In addition, 4,000,000 reads are downsampled for calculating the PCR bottleneck coefficient (PBC). The PBC is the number of locations with exactly one uniquely mapped read divided by the number of uniquely mapped genomic locations. PBC ranges from 0-1, and a higher number indicates higher library complexity.\n\nParticularly useful in the setting of ATAC-seq experiments, CHIPS also provides a fragment lengths distribution plot. ATAC-seq data with high quality should have fragment length peaks at < 100 bp nucleosome-free regions and show periodical enrichment at the 1- and 2-nucleosome lengths.\n\nPeak calling and peak characteristics for quality control\n\nPeaks represent regions of the genome that are enriched with aligned reads. The MACS2 (Zhang et al. 2008) algorithm is used to call peaks from uniquely sorted BAM files. The minimum false discovery rate (FDR) cutoff for defining peak confidence is set to 0.01 by default but can be changed in the config.yaml file. A summary of the number of peaks, including those with a > 10 or > 20-fold increase relative to the background, is also reported describing the data quality. More peaks and a higher fraction of >10X peaks tend to indicate higher quality. Moreover, a read per million (RPM) normalized BedGraph signal track file generated by MACS2 is further converted to a BigWig file for visualization in the genome browsers more efficiently. A qualitative assessment of peak quality can be determined by static genome browser track views in the CHIPS output.\n\nAfter peak calling, the fraction of reads in peaks (FRIP) scores is calculated to assess the samples’ quality. The FRIP score is the fraction of 4,000,000 subsampled reads that fall within the peak regions. FRIP score increases with sequencing depth, so a subsample of reads is used. The FRIP score indicates data’s signal-to-noise ratio, and a higher FRIP score indicates higher quality.\n\nCertain characteristics of the peaks can be used to describe further the quality of the data. Peaks from a high-quality sample should have a high percentage of overlap with the known DNaseI sites. CHIPS overlaps the peaks with the union of the public DNaseI hypersensitive sites to determine the data’s quality. Moreover, high-quality peaks tend to be evolutionarily conserved across species. CHIPS plots the conservation plot across all peaks. The conservation plots of transcription factors typically show a high focal point around the peak summits, while histone modifications show bimodal peaks with a dip in the center.\n\nDownstream analysis\n\nPeak annotation is performed to describe how the peaks distribute across the genome. Specifically, CHIPS determines the proportions of peaks that overlap with promoters, exons, introns, or intergenic regions. Motif identification is carried out using HOMER v4.11 (Heinz et al. 2010). The top 5,000 most significant peak summits (ranked by the MACS P-value) are used for motif analysis. Finally, to determine which genes may be regulated by the peaks, a regulatory potential score is calculated for each gene using an exponential decay model implemented in LISA (Qin et al. 2020). LISA calculates regulatory potential scores that represent the cumulative influence of nearby peaks associated with each gene.\n\nOutput\n\nCHIPS provides results files in.txt and.png forms inside well-structured folders and a dynamic HTML report summarizing quality control metrics at the sample level. An example report for the Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) ATAC-seq data is available at here.\n\nCHIPS (Tang, 2021) can be executed in any Linux-based operating system. All the tools can be installed through Conda. Documentation accompanying the CHIPS software describes the installation process and the structure of the analysis results and report directories. Due to the modular nature of the Snakemake workflow, the report can be customized to meet individual needs and easily expanded if new metrics are added. Furthermore, the same metrics are reported in the CistromeDB (Zheng et al. 2019) which facilitates comparisons of results with that resource.\n\n\nUse case\n\nThere are three steps to run CHIPS. Step1: Install CHIPS Conda environment and download reference files. Step2: Set up CHIPS project folder. Step3: Run the Snakemake pipeline. All work is done within a single project folder. Four core components are required within the project directory: CHIPS/, config.yaml, metasheet.csv, and ref_files/. Each core component is indispensable to run CHIPS, and their relative paths are restricted. Optionally, we recommend soft linking the data folder to the project directory. If the analysis uses data from species other than human or mouse, a ref.yaml file must also be attached to indicate where the references are. In the following examples, we will use human TCGA and ENCODE ATAC-seq data to illustrate how to run the CHIPS in detail.\n\nIn this use case, we will demonstrate how to set up and run a CHIPS pipeline using 22 TCGA ATAC-seq datasets with bam files as input. Data are available in Genomic Data Commons Data Portal (see Underlying data). A recommended way to download the data is to use the manifest file.\n\n\n\nHere, we modify config.yaml and metasheet.csv according to the samples. The config.yaml is where CHIPS run parameters are defined. Each parameter is listed in Figure 2. Unused parameters are not shown. The metasheet.csv is where the samples are grouped to each run. Detailed settings can be found in Figure 3.\n\n\n\nWhen an entire run is completed, an analysis folder with all the output and report will be generated within the project directory.\n\nIn use case 2, we will process ENCODE ATAC-seq samples with two replicates of each. We will use paired-end fastq.gz files as input to demonstrate the workflow. The steps in running ENCODE data are the same as running the TCGA data. The main difference is that we need to group the replicates in config.yaml and metasheet.csv. One only needs to install and configure CHIPS once on a computer. Thus, in the second example, we will only use the soft link to set up the project directory. This is also the best practice to follow when using CHIPS multiple times for different data sets.\n\n\n\nThe two ENCODE datasets used in this example are ENCSR591PIX (ATAC-seq of Panc1) and ENCSR200OML (ATAC-seq of IMR-90) which are available in Underlying data. Four pairs of fastq.gz files are downloaded, renamed, and saved in the ENCODE_data/folder within your home directory with the following format:\n\n\n\nThen, we edit config,yaml to give each pair of fastq.gz files a sample name (Figure 4). In metasheet.csv file, we group replicates within the same run and leave control group empty (Figure 5).\n\n\n\nCheck the run.out and when the pipeline prints out 100% complete, an analysis folder with all the output and report will be generated within the project directory.\n\n\nConclusion\n\nTaken together, CHIPS is a scalable and reproducible pipeline written in Snakemake. It performs quality control and reproducible processing of the chromatin profiling data generated from ATAC-seq, ChIP-seq, and DNase-seq experiments. CHIPS does not explicitly label samples as being “low” or “high” quality overall. We rely on the users to interpret information from multiple quality control features to determine which samples to include for further downstream analyses. CHIPS also does not provide downstream analyses comparing cases and controls. Downstream analyses depend on the biological context of the experiments and may consist of differential binding, motif analysis, and pathway analysis in the setting of chromatin profiling experiments. An independent Snakemake pipeline COBRA (Qiu et al. 2020) is designed for this purpose.\n\n\n\n- The TCGA LUAD ATACseq data for Use Case 1 are available from Genomic Data Commons Data Portal: https://bit.ly/3bPytgG.\n\n- The ENCODE ATACseq data for Use Case 2 are available from ENCODE data portal: https://www.encodeproject.org/experiments/ENCSR200OML/ (ATAC-seq of Panc1). https://www.encodeproject.org/experiments/ENCSR591PIX/ (ATAC-seq of IMR-90).\n\nSource code available from: https://github.com/liulab-dfci/CHIPS.\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.4782801 (Tang, 2021).\n\nLicense: MIT.", "appendix": "Acknowledgements\n\nWe thank the Center for Functional Cancer Epigenetics and Molecular Biology Core Facilities at Dana-Farber Cancer Institute for valuable feedback on CHIPS.\n\n\nReferences\n\nBailey T, Krajewski P, Ladunga I, et al.: Practical Guidelines for the Comprehensive Analysis of ChIP-Seq Data. PLoS Comput Biol . 2013; 9(11): e1003326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen HX, Song M, Maecker HT, et al.: Network for Biomarker Immunoprofiling for Cancer Immunotherapy: Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC). Clin Cancer Res: An Official Journal of the American Association for Cancer Research, January, clincanres. 2021; 3241: 2020. PubMed Abstract \| Publisher Full Text\n\nChen S, Zhou Y, Chen Y, et al.: Fastp: An Ultra-Fast All-in-One FASTQ Preprocessor. Bioinformatics. 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFurey TS: ChIP–Seq and beyond: New and Improved Methodologies to Detect and Characterize Protein–DNA Interactions. Nat Rev Genet. 2012; PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeinz S, Benner C, Spann N, et al.: Simple Combinations of Lineage-Determining Transcription Factors Prime Cis-Regulatory Elements Required for Macrophage and B Cell Identities. Mol Cell. 2010; PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKöster J, Rahmann S: Snakemake—a Scalable Bioinformatics Workflow Engine. Bioinformatics . 2012; 28(19): 2520–2522. PubMed Abstract \| Publisher Full Text\n\nLi H: Aligning Sequence Reads, Clone Sequences and Assembly Contigs with BWA-MEM. ArXiv [q-Bio.GN]. arXiv. 2013. Reference Source\n\nMeyer CA, Liu XS: Identifying and Mitigating Bias in Next-Generation Sequencing Methods for Chromatin Biology. Nat Rev Genet. 2014; 15(11): 709–721. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQin Q, Fan F, Zheng R, et al.: Lisa: Inferring Transcriptional Regulators through Integrative Modeling of Public Chromatin Accessibility and ChIP-Seq Data. Genome Biol. 2020: 21 (1): 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQiu X, Feit AS, Feiglin A, et al.: CoBRA: Containerized Bioinformatics Workflow for Reproducible ChIP/ATAC-Seq Analysis - from Differential Peak Calling to Pathway Analysis. Cold Spring Harbor Laboratory. 2020. Publisher Full Text\n\nTang M: liulab-dfci/CHIPS: Release v1.0.0 (Version v1.0.0). Zenodo. 2021, May 24.Publisher Full Text\n\nThe-ENCODE Project Consortium: An Integrated Encyclopedia of DNA Elements in the Human Genome. Nature . 2012; 489(7414): 57–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng R, Changxin W, Shenglin M, et al.: Cistrome Data Browser: Expanded Datasets and New Tools for Gene Regulatory Analysis. Nucleic Acids Res. 2019; 47 (D1): D729–35. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "98897", "date": "16 Nov 2021", "name": "Anqi Zhu", "expertise": [ "Reviewer Expertise Statistical genomics", "Bulk and single-cell RNA-seq data", "Statistical software development" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Taing et al. introduced a pipeline that they developed using the Snakemake software to streamline the steps of processing chromatin profiling data, which includes the raw data processing, quality control and downstream analysis tasks. The pipeline is useful, however the manuscript could benefit from adding more description and discussion of the innovation and implementation details of the pipeline. The detailed comments are as below.\nWhat are the needs to develop the proposed pipeline after investigating the existing pipelines that process and analyze the chromatin profiling data? What are the differences between the developed pipeline and the existing pipelines that benefit the users?\n\nIn the introduction, the authors discussed the importance of removing the bias and the reproducibility of the data processing. The authors could highlight the steps where such goals are accomplished.\n\nHow were the softwares integrated into this pipeline. E.g: any specific steps needed to make sure the output/input files are compatible, or to reduce I/O burden?\n\nIs batch correction step part of the pipeline? If not, can you briefly explain the reason?\n\nWhat are the reasons of picking these selected softwares/packages in the pipeline? Can you briefly describe the methods, and comment on them?\n\nIn the use cases, I would be more interested to read how differently (or more efficiently) CHIPS handles the data from the existing pipelines, and the interpretation of the outputs from CHIPS with these two data, rather than how to set up and run the pipeline, which are also super important, but more suitable for a README or vignette but not a publication.\nSome minor things:\n“In addition, 4,000,000 reads are down sampled for calculating the PCR bottleneck coefficient (PBC)” It’s not clear to me where does the 4,000,000 reads sampled from?\n\nSome of the bash codes could be removed, so that the manuscript is more succinct.\n\nIt can be a little confusing to the reader that in the conclusion, it is said “CHIPS also does not provide downstream analyses comparing cases and controls.” While I understand this is not the same set of downstream analysis as provided in CHIPS, you could make it a little clearer.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] }, { "id": "202834", "date": "13 Sep 2023", "name": "Yaqiang Cao", "expertise": [ "Reviewer Expertise Bioinformatics", "Epigenetics", "Chromatin Structures" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTaing, Bai, and Cousins present CHIPS, a Snakemake pipeline that implements the necessary analysis steps for chromatin profiling sequencing data. CHIPS aims to improve the reproducibility and efficacy of chromatin profiling analyses. The reviewer finds the tool to be useful and implemented with state-of-the-art analysis techniques. It is also great to know that CHIPS has been stably running since 2016 and has processed more than 1500 samples, demonstrating its practical utility. It could be a great help to both wet and dry labs, especially those that are just starting out.\nHowever, the reviewer finds that the manuscript could be improved by better presentation of the figures and writing. The reviewer suggests that the authors refer to Bioinformatics two- to three-page Applications Note style manuscripts for guidance on the organization of the manuscript. The following suggestions may be helpful for the authors to improve the tool and manuscript:\nIt would be helpful to include a mini-review of other chromatin profiling sequencing data pipelines in the introduction, in addition to the ENCODE pipeline that is currently mentioned, and to highlight CHIPS' unique features.\n\nIt would be helpful to summarize the potential users of the tool.\n\nIt would be helpful to mention the hardware requirements and estimated time for running CHIPS on different cases.\n\nBash code for cases is not necessary in a manuscript. It would be better to present or summarize two example scripts in the README file on GitHub or in the documentation. I also suggest including report results for the cases to be a reference for users running the examples.\n\nIt is not clear whether the number of reads sampled for metric calculation can be adjusted by the user. It seems that 100,000 or 4 million reads are always sampled.\n\nI suggest that fragment length distribution plots be an option for all input data, not just ATAC-seq experiments.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? 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": [] }, { "id": "202840", "date": "12 Oct 2023", "name": "Robin H van der Weide", "expertise": [ "Reviewer Expertise Epigenetics", "bioinformatics", "scientific software." ], "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 manuscript, Taing et al. describe the CHIPS-pipeline for epigenomics data using the Snakemake pipeine-software. This enables scalable and streamlined processing of several bioinformatics steps that are usually done piece by piece. The pipeline also reports quality metrics which could be used to assess sample-quality. Overall, the manuscript shows promise but requires substantial improvements to meet the standards of clarity, completeness, and relevance.\nMajor Comments:\nContext and Significance: The authors introduce the CHIPS pipeline without adequately situating it within the broader landscape of existing epigenomics pipelines. They mention ENCODE but do not provide a comprehensive overview of how CHIPS compares to other pipelines for ChIP-seq/ATAC-seq/DNase-seq data analysis. This context is essential for readers to understand the unique advantages and contributions of CHIPS. The authors should expand on this aspect to highlight CHIPS' value in the field.\n\nComparative Analysis: The manuscript lacks a meaningful comparison of CHIPS' results with the original TCGA- and ENCODE-pipelines. To demonstrate the utility of CHIPS, it is crucial to assess its performance against existing standards. The authors should include a comparative analysis to showcase the advantages of using CHIPS.\n\nDocumentation Quality: The documentation on GitHub is incomplete and lacks essential explanations, particularly in Appendix E and the absence of Appendices A, B, and C. Comprehensive and user-friendly documentation is crucial for the adoption of a new tool. It would be beneficial if the authors improve the documentation by following the example set by their other pipeline, CoBRA, which features a well-structured ReadTheDocs manual.\n\nInterpretation of Quality Metrics: The manuscript mentions quality metrics without specifying the criteria for sample failure or success. It is crucial to clarify the significance of these metrics and establish thresholds for user guidance. For example, what does a 4% dm3 value indicate? The manuscript should provide clear guidelines on interpreting these metrics.\n\nReport Portability and Transparency: The portable report's visualization limitations are a concern, as it only displays the highest values, potentially misleading users (e.g., mm9 in the contamination-barplot). The authors should address this issue by improving report comprehensiveness, especially in the motif-page section (where only one motifs is shown per sample). Users should have access to all relevant data and results within the report to avoid confusion or misinterpretation.\n\nTool and Setting Rationale: The manuscript should offer more detailed explanations for the choice of tools and settings used in the pipeline. This information will help users understand the rationale behind these decisions and provide insights into potential adjustments for specific research scenarios.\n\nHandling Spike-In Experiments: The manuscript should clarify whether the CHIPS pipeline can handle spike-in experiments and explain any potential effects on contamination reports. Providing this information is important for users interested in spike-in experiments.\nMinor Comments:\nThe phrase \"Protein-DNA binding interactions\" needs revision, as it may not accurately describe the purpose of using this pipeline for ATAC-seq and DNase-seq data, which do not measure interactions per se.\n\nThe reasons for Snakemake's popularity as a workflow tool should be more comprehensively explained beyond its Python-based nature.\n\nThe description of BWA-MEM's role in alignment should be expanded to clarify its role in sorting and deduplication, as this process typically involves tools like Samtools or Picard.\n\nThe manuscript should acknowledge the existence of the PCR bottleneck coefficient (PBC) before CHIPS and provide a reference to the original paper and pipeline.\n\nThe manuscript's extensive code content may be more suitable for inclusion in a manual rather than the manuscript itself. This would allow for more comprehensive discussions and comparisons of CHIPS' niche in the field.\nOverall, this manuscript has potential but requires substantial revisions to address the major and minor concerns raised above. Improvements in clarity, completeness, and relevance are essential for the successful publication of this work.\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? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] }, { "id": "202831", "date": "12 Oct 2023", "name": "Chenxu Zhu", "expertise": [ "Reviewer Expertise Single-cell genomics", "epigenomics", "chemical 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\nIn this manuscript, Taing and Bai et al. present a Snakemake pipeline for streamlined ATAC-seq, DNase-seq and ChIP-seq analysis from raw sequencing data to downstream functional analyses. The pipeline is helpful for bioinformatics and biologists to perform analysis of epigenomics data. However, the manuscript (and the associated software) could be further improved on the following points:\nThe authors provided FRIP and overlap with Dnase peaks as quality control. However, peak calling pe se might be affected by the library quality and sequencing depth. Although union peaks from public DNaseI seq was also included, the overlap scores for different samples could vary. A more unbiased measurement is to include the TSS enrichment score, at least for ATAC-seq analysis.\n\nIn addition to the PBC, could the author include peak calling downsampling analysis? Which could better guide if sufficient sequencing depth has been achieved.\n\nWhat is the rationale for using 4,000,000 downsampled reads for PBC analysis? ATAC and ChIP-seq (of different histone modifications) may vary in library saturation due to their distinct genomic distribution patterns.\n\nLISA is for active marks only. The authors should clearly state this analysis is not suitable for ChIP-seq targeting repressive histone marks, i.e., H3K27me3 and H3K9me3.\n\nThe “Appendix A: System requirements” and “Appendix B: Recommended requirements” sections on the GitHub README.md page is missing.\nMinor:\nCould the authors add a feature to parse additional parameters for bigwig generation? For example, for ATAC-seq/DNase-seq, which has narrow peaks, and for H3K9me3, which has broad peaks: they need different smoothing lengths for optimal visualization.\n\nBesides ChIP-seq, CUT&Tag became a widely used assay for chromatin profiling. Extending this pipeline for CUT&Tag analysis should be straightforward, but finetuning the parameters may be needed if the authors provide some guidelines (or preassembled parameter sets), making it more accessible to biologists with limited bioinformatics skills.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-517
https://f1000research.com/articles/10-516/v1	30 Jun 21	{ "type": "Research Article", "title": "The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts", "authors": [ "Sinta Murlistyarini", "Lulus Putri Aninda", "Ufida Aini Afridafaz", "Sri Widyarti", "Agustina Tri Endharti", "Teguh Wahju Sardjono", "Lulus Putri Aninda", "Ufida Aini Afridafaz", "Sri Widyarti", "Agustina Tri Endharti", "Teguh Wahju Sardjono" ], "abstract": "Background: The senescence process in human dermal fibroblasts (HDFs) is caused by cell cycle withdrawal processes, one of which is the result of the retinoblastoma (Rb) protein being in a hypo-phosphorylated state. Since adipose-derived stem cells (ADSCs) have a paracrine effect, ADSCs were utilized to improve the senescence process of HDFs. The use of non-autologous cell culture media to grow ADSCs can be legally problematic; therefore, platelet-rich plasma (PRP) can be considered as an alternative medium. PRP contains various growth factors that can be used to process the reversal of senescent HDFs. The combination of ADSCs and PRP is expected to increase the expression of Rb protein in HDFs that have undergone the senescence process. Methods: This study was performed in vitro with a randomized sample, and non-blinded pre-and post-test control group. The primary culture of senescent HDFs was transfected with a combination of ADSCs and 10% PRP. The effect on migration was observed through the scratch test, while the effect of PRP on reversal senescence was observed through Sa-β-gal analysis and the expression of protein Rb with ELISA. Results: The senescent HDFs that received a combined transfection of ADSCs and 10% PRP proliferated rapidly in the scratch test. Based on the Sa-β-gal assay, they showed fewer senescent HDFs cells. The combination of ADSCs and 10% PRP elevated the expression of Rb protein significantly (P < 0.001). Conclusions: The combination of ADSCs and 10% PRP was shown to have a reversal effect on the senescence process of HDFs in vitro.", "keywords": [ "Cellular senescence", "human dermal fibroblasts", "adipose-derived stem cells", "platelet-rich plasma", "protein retinoblastoma" ], "content": "Introduction\n\nAdipose-derived stem cells (ADSCs) are mesenchymal stem cells that reside in fat tissue and were first identified by Zuk et al.1 ADSCs with a unique secretome have a paracrine effect on surrounding cells giving rise to the theory that the efficacy of stem cell therapy is more due to the paracrine effect of cell secretion than the effect of cell differentiation.2 The paracrine effect of ADSCs is widely used in regenerative medicine such as tissue repair, wound healing, and as an anti-aging therapy.3 Conditioned media from adipose-derived stem cells (ADSC-CM) contains all the bioactive components of ADSCs, and is easy to store and safer during application.4 The cells are often cultured using a basal medium containing foetal bovine serum (FBS). FBS is important in the cell culture process. However, when it is used for regenerative medicine, this serum is less safe because it contains xenoproteins, which can cause rejection or transmission of infectious products. As a result, nowadays, the use of non-autologous cell culture media, like FBS in ADSCs for clinical applications is legally challenged. Thus, to reduce the use of animal products in ADSC cultures, a study was conducted using platelet-rich plasma (PRP) instead of FBS.25\n\nThe use of PRP is intended as a safer alternative supplement medium for ADSC culture. PRP is blood plasma that contains many platelets which are often used for wound healing.5 Platelets are a natural source of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), and epidermal growth factor (EGF). They are efficient at triggering cell proliferation, differentiation, and tissue regeneration.6,7 There are many protocols for PRP preparation, from conventional blood centrifugation to other innovative methods, and the PRP can be activated by adding other materials or methods, for example, calcium, collagen, and/thrombin, or by glass contact or freeze-thaw cycles.8,9\n\nCellular senescence is an arrest of the cell cycle with a phenotypic change in which the cell will lose its replicative ability.10 In the past, cellular senescence was considered an irreversible process as protection against cancer, however, recent findings have shown its role in aging, tissue repair and development of senescent fibroblasts.11 In vitro, cells that experience senescence will undergo morphological changes such as cell flattening and enlargement, the apparition of vacuoles, and sometimes multiple nuclei; these contribute significantly to the aging process.12-14\n\nCells that experience senescence will lower the expression of genes involved in the cell cycle and several components of the extracellular matrix, and increase the expression of cell cycle inhibitor genes and matrix-degrading enzymes.15 Cell cycle withdrawal associated with cellular senescence is triggered by the p53-p21 and p16-Rb pathways.16,17 In senescent human dermal fibroblasts (HDFs), retinoblastoma (Rb) proteins accumulate in an active state; a hypo-phosphorylation state. In this condition, this protein is unable to phosphorylate in the middle or the end of the G1 phase and thus it is unable to enter the S phase.18 Research by Song et al. showed that ADSCs decreased p16 expression in senescent fibroblasts, thus it appears that ADSCs can reverse the aging process.19\n\nSeveral extensive studies on PRP have been conducted, however, the biological mechanisms and clinical effects of PRP on HDFs are unclear. Besides, there has been no research on the combination of ADSC-CM and 10% PRP on Rb protein in senescent HDFs.\n\n\nMethods\n\nOur research has been done at the Biomedical and Parasitology laboratory, Faculty of Medicine, Universitas Brawijaya, Malang Indonesia. The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). The HDF cells were an explant culture from human skin and were put into a 6-well plate until the explant attached. The cells were grown in a solution containing 20% FBS and Dulbecco’s Modified Eagle Medium (FBS-DMEM, ThermoFisher). The method used was a modified version of the method used by Takashima (1998), while the senescent HDF method used was that of Radiono et al. (2016).20,21 The HDFs were shifted into a senescent state by replacing the medium with 10% PRP in DMEM (ThermoFisher).\n\nADSCs from the pregnant woman were cultivated in 6-well plates and cultured in a complete culture medium (10% PRP in DMEM). The PRP preparation mentioned above was adopted from Amable et al.22 Briefly, whole blood was placed in tubes containing anticoagulants, and was centrifuged for 20 min (3,000 × g). After obtaining three layers in the whole blood, the upper layer was transferred into 10 ml empty sterile micro centrifuge tubes. The PRP was activated with 20mM CaCl2.\n\nThe expression of senescence-associated β-galactosidase (Sa-β-Gal) was analysed in HDFs using purchased Sa-β-Gal staining kit (MyBioSource, San Diego, CA, CAT# MBS168501) referring to manufacturer’s procedures. Concisely, the cells were fixed with 25% glutaraldehyde for 5 min, then they were washed with phosphate-buffered saline (PBS). Subsequently, the cells were incubated in cell staining solution overnight. The stained HDFs were washed with PBS and examined using an IX71 inverted microscope (Olympus) at 200× magnification.\n\nThe migration of HDF cells was investigated using a scratch assay. HDFs were grown to 80% confluence in 24-well plates and scraped with a 200 μl pipette tip. Next, the media were replaced to reduce the debris. HDF cells were photographed at 0 and 72h thereafter. After that, the HDFs were washed with PBS and detached by trypsin. The cell suspension was collected and centrifuged for 5 min (1000×g). The measurement of Rb protein levels was done using an enzyme-linked immunosorbent assay kit (ELISA) according to the protocol issued by the manufacturer’s instruction (MyBioSource, Cat#MBS2509425, RRID: AB_10568804). In brief, 100 μl of standard or sample were added to each well and incubated for 90 min at 37°C. The liquid was replaced with the provided biotinylated monoclonal antibody (60 min, 37°C) and followed by washing 3 times. The biotinylated detection antibody is specific to Human Rb1 and Avidin-Horseradish Peroxidase (HRP) conjugate. A 100 μl HRP conjugate was added (30 min, 37°C). After washing 5 times, 90 μl of substrate reagent was added and incubated for 15 min at 37°C. The reaction was stopped with 50 μl stop solution and the optical density (OD) was directly determined at 450 nm.\n\nThe data obtained was analysed using SPSS software version 25 (IBM). The normality test was carried out by the Shapiro Wilk test. If the P-value >0.05, then the normality assumption was fulfilled so an independent t-test was carried out. However, if the normality test was not fulfilled, the Mann-Whitney test was performed.\n\n\nResults\n\nThe 10% PRP - DMEM media was used to culture ADSCs and these media were used to shift the senescence state in HDFs. HDF explant culture began to grow on the seventh day. After the seventh passage, the HDF culture medium was replaced with 0.5% FBS for 72 hours to allow HDF cells to undergo senescence. The HDF media was then removed and the cells were transfected with the ADSCs - 10% PRP for another 72 h and subsequently examined by ELISA to determine the Rb protein level.\n\nThe scratch test showed that the senescent HDF cell group that received the ADSCs – 10% PRP medium proliferated faster than the control group (Figure 1). The Sa-β-Gal investigation showed that cells with ADSCs – 10% PRP medium had a lower number of senescent cells than the control group (Figure 2).\n\nScratch test of HDFs in the control group on day 0 (A) and after 72 hours (B). Scratch test of HDFs in the ADSCs – 10% PRP group on day 0 (C) and after 72 hours (D).\n\nADSCs: adipose-derived stem cells; HDF: human dermal fibroblasts; PRP: platelet-rich plasma.\n\nADSCs, adipose-derived stem cells; PRP, platelet-rich plasma.\n\nBased on the Shapiro Wilk normality test, the P-value obtained for both groups was > 0.05, thus the independent t-test was carried out and gave a statistically significant result (P < 0.001) (Table 1,38).\n\n\nDiscussion\n\nBesides being easy to develop in vitro, ADSCs have similar capabilities to bone marrow stem cells, but they can be obtained easily from subcutaneous adipose tissue without invasive action, can maintain concentrations and proliferation consistently, and represent a more biologically relevant model for studying aging mechanisms when compared to other stem cells.23,24\n\nPRP is a fraction of autologous blood plasma with a high platelet concentration.26 PRP also produces various cytokines, chemokines, and growth factors that can trigger recruitment, adhesion, proliferation, and maintain the differentiation of ADSCs.25,27\n\nResearch by Jia et al. (2017), which examined the effect of PRP in an in vitro photo-aging model, showed that PRP reduced the number of aging cells by 1% based on Sa-β-Gal positive cell tests. Furthermore, PRP prevents cell cycle arrest caused by irradiation by reducing the expression of p53 and p21, and decreasing the expression of matrix metalloproteinase (MMP) -1, -3, and -9 but not MMP-2 at the mRNA level.28\n\nResearch conducted by Kocaoemer et al. (2007) stated that 10% of non-autologous PRP activated by shock freezing or thrombin after being cultured for 11 days will increase ADSC proliferation faster than 10% FBS.29 Meanwhile, Pham et al. (2013) found that 15% PRP is the optimal concentration that will stimulate ADSC proliferation.30 Also, using the supernatant of the 10% activated PRP increases the proliferation of mesenchymal stem cells compared to other supplementary media such as whole blood, non-activated PRP, and non-activated platelet-poor plasma.31\n\nHigher PRP concentrations will interfere with cell growth in vitro because the platelets will release protein complexes that negatively affect the action of the PRP growth factor.32 Besides, the concentration of proteolytic enzymes such as collagenase, cathepsin, elastase, and acid phosphatase is very high in PRP; therefore, it causes inhibition of cell growth.33\n\nStessuk et al. (2016) studied the combination of PRP with ADSC-CM in vitro and found significant proliferation stimulation in fibroblasts cultured with 25% PRP combined with ADSC-CM after 48 hours, while keratinocyte proliferation also increased insignificantly after 24 hours.34 Park et al. (2011) found that 10% PRP stimulates fibroblast cell migration and proliferation within 24 hours, presumably because PRP releases the PDGF-AA growth factor. However, PRP did not have a significant effect on the activity of MMP-1, MMP-2, and MMP-9 on HDFs cells. This is appropriate because fibroblast cells will be strongly reactive to PDGF, b-FGF, and EGF.35\n\nLiu et al. (2014) examined the ability of PRP to improve cellular conditions from the senescence process and subsequently be able to slow down aging in animals. The research showed that PRP can change the expression of senescence markers such as p16, p19, and p53 on transgenic mice. In addition, PRP also can trigger cell growth, proliferation, and colony formation, and increase osteogenesis, reduce adipogenesis, and fight oxidative stress in stem cells from aged mice.36\n\nCho et al. (2012) examined the effects of PRP on cell migration, proliferation, and expression of various cell cycle regulatory proteins in HDFs cells. From this study, it was found that low concentrations of PRP (0.05 – 0.5%) would increase the expression of cyclin-A in HDFs cells, and 5% PRP would enhance the expression of proteins involved in the cell cycle, for example, Rb protein, cyclin-E, cyclin-A, and Cdk2. It seems that PRP through various growth factors will promote the progression of the cell cycle back into the G1 phase by regulating the expression level and activation of cyclin-Cdks.37\n\nIn summary, this study found an increase in Rb protein expression on senescent HDFs which were given a combination of ADSCs and 10% PRP. However, further studies are still needed to determine the effect of the combination of ADSCs and PRP on other senescence markers.\n\n\nData availability\n\nFigshare: Supplementary Data - The combination of ADSCs and 10% PRP increase Rb protein expression on senescent human dermal fibroblasts, https://doi.org/10.6084/m9.figshare.14716314.v1.38\n\nThis project contains the following underlying data:\n\n- The combination of ADSCs and 10% PRP increase Rb protein expression on senescent human dermal fibroblasts.xlsx\n\n- Statistic Result The combination of ADSCs and 10% PRP increase Rb protein expression on senescent human dermal fibroblasts.docx\n\n- Picture 1A. Scratch test of HDFs in the control group on day 0.jpeg\n\n- Picture 1B. Scratch test of HDFs in the control group after 72 h.jpeg\n\n- Picture 1C. Scratch test of HDFs in the ADSCs – 10% PRP group on day 0.jpeg\n\n- Picture 1D. Scratch test of HDFs in the ADSCs – 10% PRP group after 72 h.jpeg\n\n- Picture 2A. SaBGal staining of fibroblasts in the control group.jpeg\n\n- Picture 2B. SaBGal staining of senescent fibroblasts treated with ADSCs – 10% PRP.jpeg\n\n- Figure 1.jpeg\n\n- Figure 2.jpeg\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe authors would like to thank RSIA Melati Husada Malang, Indonesia, for providing the fat and dermal tissue. The authors would like to thank Dr. Tinny Endang Hernowati, SpPK (K), for her kindness and support during the research, may she Rest In Peace.\n\n\nReferences\n\nZuk P: Adipose-derived stem cells in tissue regeneration: a review, ISRN Stem Cells.2013 Article ID 713959. Publisher Full Text\n\nLai RC, Yeo RWY, Lim SK: Mesenchymal stem cell exosomes. Semin Cell Dev Biol. 2015; 40: 82–88. PubMed Abstract \| Publisher Full Text\n\nShen X, Du Y, Shen W, et al.: Adipose-derived stem cells promote human dermal fibroblast function and increase senescence-associated-β-galactosidase mRNA expression through paracrine effects. Mol Med Rep. 2014; 10: 3068–3072. PubMed Abstract \| Publisher Full Text\n\nWang T, Guo S, Liu X, et al.: Protective effects of adipose-derived stem cells secretome on human dermal fibroblasts from ageing damages. Int J Clin Exp Pathol. 2015; 8(12): 15739–15748. PubMed Abstract \| Free Full Text\n\nJia C, Lu Y, Bi B, et al.: Platelet-rich plasma ameliorates senescence-like phenotypes in a cellular photoaging model. RSC Adv. 2017; 7: 3152–3160. Publisher Full Text\n\nPham PV, Bui KH, Ngo DQ, et al.: Activated platelet-rich plasma improves adipose-derived stem cell transplantation efficiency in injured articular cartilage. Stem Cell Res Ther. 2013; 4(4): 91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtashi F, Serre-Beinier V, Nayernia Z, et al.: Platelet rich plasma promotes proliferation of adipose derived mesenchymal stem cells via activation of AKT and Smad2 signaling pathways. Stem Cell Res Ther. 2015; 5(8): 1–10. Publisher Full Text\n\nOudelaar BW, Peerbooms JC, Veld RHI, et al.: Concentrations of blood components in commercial platelet-rich plasma separation systems: a review of the literature. Am J Sports Med. 2019; 47(2): 279–287. PubMed Abstract \| Publisher Full Text\n\nCharles-de-Sa L, Gontijo-de-Amorim N, Sbarbati A, et al.: Photoaging skin therapy with PRP and ADSC: a comparative study. Stem Cells Int. 2020; Article ID 2032359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarthandan S, Priebe S, Hemmerich P, et al.: Long-term quiescent fibroblast cells transit into senescence. Plos ONE. 2014; 9(12): e115597. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan-Deursen JM: The role of senescent cells in aging. Nature. 2014; 509: 439–446. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchauble S, Klement K, Marthandan S, et al.: Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts. Plos ONE. 2012; 7(8): e42150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMunoz-Espin D, Serrano M: Cellular senescence: from physiology to pathology. Mol Cell Biol. 2014; 15: 482–496. PubMed Abstract \| Publisher Full Text\n\nHernandez-Segura A, Nehme J, Demaria M: Hallmarks of cellular senescence. Trends Cell Biol. 2018; 28(6): 436–453. PubMed Abstract \| Publisher Full Text\n\nBanito A, Lowe SW: A new development in senescence. Cell. 2013; 155(5): 977–978. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGu Z, Jiang J, Tan W, et al.: p53/p21 pathway involved in mediating cellular senescence of bone marrow-derived mesenchymal stem cells from systemic lupus erythematosus patients. Clin Dev Immunol. 2013: 134243. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMijit M, Caracciolo V, Melillo A, et al.: Role of p53 in the regulation of Cellular Senescence. Biomolecules. 2020; 10: 420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStein GH, Dulic V: Origins of G1 arrest in senescent human fibroblasts. BioEssays. 1995; 17(6): 537–543. PubMed Abstract \| Publisher Full Text\n\nSong SY, Jung JE, Jeon YR, et al.: Determination of adipose-derived stem cell application on photo-aged fibroblasts, based on paracrine function. Cytotherapy. 2011; 13: 378–384. PubMed Abstract \| Publisher Full Text\n\nTakashima A: Establishment of fibroblast culture. Curr Protoc Cell Biol. 1998; 00(1): 2.2.1–2.2.12. PubMed Abstract \| Publisher Full Text\n\nRadiono S, Wirohadidjojo YW, Budiyanto A: The effect of platelet-rich fibrin (PRF) on serum-starved human dermal fibroblast. J med Sci. 2016; 48(2): 110–118. Publisher Full Text\n\nAmable PR, Carias RB, Teixeira MVT, et al.: Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors. Stem Cell Res Ther. 2013; 4: 67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Y, Wu J, Chou P, et al.: Characterization and Evaluation Of The Differentiation Ability Of Human Adipose-Derived Stem Cells Growing In Scaffold-Free Suspension Culture. Cytotherapy. 2014; 16: 485–495. PubMed Abstract \| Publisher Full Text\n\nShan X, Roberts C, Kim EJ, et al.: Transcriptional and cell cycle alterations mark aging of primary human adipose-derived stem cells. Stem Cells. 2017; 35: 1392–1401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarbone A, Rucci M, Annacontini L, et al.: Adipose-derived stem cells and platelet-rich plasma: inputs for regenerative medicine. Med Res Arch. 2017; 5(10): 1–12. Reference Source\n\nHildner F, Albrecht C, Gabriel C, et al.: State of the art and future perspectives of articular cartilage regeneration: a focus on adipose-derived stem cells and platelet-derived products. J Tissue Eng Regen Med. 2011; 5: e36–e51. PubMed Abstract \| Publisher Full Text\n\nGawaz M, Vogel S: Platelets in tissue repair: control of apoptosis and interactions with regenerative cells. Blood. 2013; 122: 2550–2554. PubMed Abstract \| Publisher Full Text\n\nJia C, Lu Y, Bi B, et al.: Platelet-rich plasma ameliorates senescence-like phenotypes in a cellular photoaging model. RSC Advances. 2017; 7(6): 3152–3160. Publisher Full Text\n\nKocaoemer A, Kern S, Kluter H, et al.: Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem cells. 2007; 25: 1270–1278. PubMed Abstract \| Publisher Full Text\n\nPham PV, Bui KH, Ngo DQ, et al.: Activated platelet-rich plasma improves adipose-derived stem cell transplantation efficiency in injured articular cartilage. Stem Cell Res Ther. 2013; 4(4): 91. Publisher Full Text\n\nKakudo N, Minakata T, Mitsui T, et al.: Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts. Plast Reconstr Surg. 2008; 122(5): 1352–1360. PubMed Abstract \| Publisher Full Text\n\nHsu CW, Yuan K, Tseng CC: The negative effect of platelet-rich plasma on the growth of human cells is associated with secreted thrombospondin-1. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 107: 85–92. PubMed Abstract \| Publisher Full Text\n\nMills DC, Robb IA, Roberts GC: The release of nucleotides, 5-hydroxytryptamine, and enzymes from human blood platelets during aggregation. J Physiol. 1969; 195: 715–729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStessuk T, Puzzi MB, Chaim EA, et al.: Platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cells: stimulatory effects on proliferation and migration of fibroblasts and keratinocytes in vitro. Arch Dermatol Res. 2016; 308: 511–520. PubMed Abstract \| Publisher Full Text\n\nPark B, Yang J, Chung K: Characterization of the cytokine profile of platelet-rich plasma (PRP) and PRP-induced cell proliferation and migration: upregulation of matrix metalloproteinase-1 and -9 in HaCaT cells. Korean J Hematol. 2011; 46: 265–273. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu H, Huang C, Lin T, et al.: Delayed animal aging through the recovery of stem cell senescence by platelet-rich plasma. Biomaterials. 2014; 35: 9767–9776. PubMed Abstract \| Publisher Full Text\n\nCho J, Kim S, Lee K: Platelet-rich plasma induces increased expression of G1 cell cycle regulators, type 1 collagen, and matrix metalloproteinase-1 in human skin fibroblasts. Int J Mol Med. 2012; 29: 32–36. PubMed Abstract \| Publisher Full Text\n\nMurlistyarini S, Aninda LP, Aini Afridafaz U, et al.: Supplementary Data - The combination of ADSCs and 10% PRP increase Rb protein expression on senescent human dermal fibroblasts. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "90976", "date": "20 Aug 2021", "name": "PRASETYADI MAWARDI", "expertise": [ "Reviewer Expertise Clinical dermatology", "venereology", "skin cancer", "biomolecular", "dermatopathology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study presents the results of how the senescence process is not an irreversible process, which will spur other studies in the mystery of the cellular senescence process. The role of PRP and ADSC in increasing the migration process of human dermal fibroblast cells has proven that cellular senescence is a dynamic, continuous process that will be useful in aging therapy in the future. This research is also carefully structured, coherent, methodological, and has adequate analysis. In some respects, Sa-β-Gal, has its own characteristics as a parameter of cellular senescence. Determination of the Rb protein is an important biomarker in the cell cycle and is the right choice to support the research excellence.\nI suggest adding the following statement so that there is a relationship between the biomarker senescence-associated β-galactosidase (Sa-β-Gal) activity and Rb protein in the introduction:\n\"Cellular senescence is a cell cycle arrest process that accompanies depletion of replicative potential in normal somatic cell cultures. The senescent cells are characterized by several molecular and cytological markers, such as an enlarged and flattened morphology and the expression of senescence-associated β-galactosidase (Sa-β-Gal) activity. A large body of evidence suggests that tumor suppressor pathways Rb and p53 are key regulators of cellular senescence.\"\nMy suggestion can be included in the introduction before the last paragraph, which can certainly add to the strength of this research, because I noticed in the introductory section the problem of Sa-β-Gal and Rb protein was very little explained; whereas Sa-β-Gal and Rb proteins are the main objects of research. To support this statement I also include the reference that I use (Campisi J: Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell. 2005; 120 (4): 513-221).\nFor keywords: 'Cellular senescence, human dermal fibroblasts, adipose-derived stem cells, platelet-rich plasma, protein retinoblastoma' .. protein retinoblastoma can be replaced by retinoblastoma protein.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "279795", "date": "06 Jun 2024", "name": "Troy Harkness", "expertise": [ "Reviewer Expertise Yeast and human aging and cancer", "cell cycle regulation", "and ubiquitin signalling." ], "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 addresses the issue of cellular senescence, and the role it plays in processes such as aging. The authors test whether treatment with adipose-derived stem cells (ADSC) in combination with 10% platelet rich plasma (PRP) can reverse the senescent phenotype in human dermal fibroblasts (HDF). While the basic study design shows promise, there are several issues that need addressing to ensure validity of the research, and the correct interpretation of results. Likewise, improvement of the article organization and grammar, as well as the inclusion of additional details within the text, will ensure the readers ability to understand the article, methods, and analysis. Please address the following points. A transcript of the text with suggested edits for improving its spelling/grammar and readability is available upon request.\n\nAbstract: “The primary culture of senescent HDFs was transfected with a combination of ADSCs and 10% PRP.” Transfection refers to transferring DNA into a mammalian cell. I believe the authors meant that the HDFs and ADSCs were combined in a single culture with the addition of PRP. This was observed again in the Results section of the abstract. Also, some grammar issues in the abstract were observed. In the first paragraph of the Results section, it is again stated that “The HDF media was then removed and the cells were transfected with the ADSCs - 10% PRP for another 72 h”. Transfected is not correct. If the term transfected is being used for another purpose, then please explain. It is explained that this was done in order to use ELISA to determine Rb levels. The authors then move on to the scratch test and never state what levels the Rb protein were at. Why use ELISA and not simply a Western? It seems a western would be easier, quicker and less expensive. As the introduction is quite short, I believe the authors could easily include a sentence or two of background regarding the relationship between p16 and RB. Simply stating that p16 is an inhibitor of CDKs responsible for Rb phosphorylation will frame the role of p16, allowing better understanding for readers unfamiliar with the pathway when the authors state that “Research by Song et al. showed that ADSCs decreased p16 expression in senescent fibroblasts, thus it appears that ADSCs can reverse the aging process” in paragraph 4 of the introduction. I believe that paragraphs 3 and 4 of the introduction should be moved to the beginning, ahead of paragraphs 1 and 2. Paragraphs 3 and 4 introduce the issue that the authors are trying to address, and as such should be discussed first to provide context for the reader. The ELISA conducted measured total Rb protein, an increase in which is interpreted by the authors as evidence of a reduction in senescence. Would measurement of phospho-Rb, and potentially a comparison of the amount of phospho-Rb to total Rb, be a more appropriate indicator? In relation to point 5 the authors presented only quantification of Rb protein, but there was no normalization to account for variance in total protein expression, or cell density. I would suggest that the authors use western blots instead of ELISA. Using western blotting the authors will be able to more easily compare the expression of total and phosphorylated Rb, as well as normalize the Rb data to housekeeping genes and/or total protein amount. Furthermore, by resolving the lysates by size it will help confirm specificity of the antibody signal. It was unclear to me what the control cell treatment was. Please clarify this in the methods and/or figure legends. Was the control maintenance of serum starvation, or return to 20% FBS? I believe it is important for the authors to include a control of 20% FBS to show that the effect seen in the treatment is not simply a reversal of serum starvation when cells are given an alternate to serum (PRP). Similarly, the authors should include treatments where cells are treated with PRP alone, or ADSC conditioned media with FBS instead of PRP. This will allow the authors to show whether the combination of PRP and ADSC conditioned media has an additive effect, and how much of the effect is resultant from which component, or if the total effect is reliant on only one of the two. Building on point 7, I believe it would provide much better support for the authors finding if they were to replicate them using a model of senescence other than serum starvation. Induction of senescence using UV or H2O2 are potential techniques that have been used in human HDFs (Gerasymchuk et al., 2022; Yao et al., 2022). The methods for lysate preparation were not clear. Please clarify. It is hard for the reader to interpret figure one. Could the contrast be improved? Results should also be quantified by either measuring the portion of the wound that has closed, or by quantifying the migration distance of each cell in time lapsed images (Cory, 2011). The scratch test measures how fast a void in a cell lawn can be filled by cells. Typically, a time course over 24-48 hours is conducted with images taken every 12 hours or so. It isn’t a measure of proliferation, but a measure of migration. By 72 hours, migration is typically complete, so this experiment, which images cells at 0 and 72 hours, simply measures how many cells entered the void and says nothing regarding cell proliferation. The images shown appear to have more cells in the void in the control cells, so their conclusions are not supported by the images. The images should be quantified showing the numbers of cells that enter the void from at least 3 independent experiments that show a time course. It is not stated how many times this was repeated, so it is likely it was only done once. The data should be shown in graph form complete with statistical analyses, by either measuring the portion of the wound that has closed, or by quantifying the migration distance of each cell in time lapsed images (Cory, 2011). Quantification should also be done on Sa-β-Gal staining. Counting the ratio of stained to total cells would be a simple method of this. When discussing Sa-β-Gal staining in the results the author should make it clear that this is being used as a senescence marker. The b-gal assay appears to show that there is more staining in the control group, but the image is low resolution and there is a lot of background, with nothing labelled, so it is unclear what is being shown in this image. This really isn’t a clean experiment. As mentioned above, a western should also be done to validate the ELISA. We do not know the quality of the antibody used, so have no idea what is really being bound. Paragraph 3 of the results should be moved to follow paragraph 1, as this would improve the flow of the article. The authors could also comment more on the observed differences between treatment and control groups rather than just the significance of said differences. What was the number of replicates for the ELISA? State this in methods and figure legend. The discussion reads as a listing of previous findings by other research groups. The authors should use the discussion to address and draw conclusions from their own results and use previous findings from the literature to frame, support, or contrast their own conclusions.\n\nIn conclusion, this is an incredibly simple paper with very little experimentation that lacks complete controls and validation experiments. The conclusions drawn are not easily seen from the poor quality of the images shown. This paper is not suitable for indexing in its present form.\n1) Cory, G. (2011 [Ref -1]). Scratch-Wound Assay. Methods in Molecular Biology, 769, 25–30. https://doi.org/10.1007/978-1-61779-207-6_2\n2) Gerasymchuk, M., et al. (2022 [Ref - 2]). Modeling of the Senescence-Associated Phenotype in Human Skin Fibroblasts. International Journal of Molecular Sciences, 23(13). https://doi.org/10.3390/IJMS23137124\n3) Yao, X., et al. (2022 [Ref -3]). UV-induced senescence of human dermal fibroblasts restrained by low-stiffness matrix by inhibiting NF-í µí¼ B activation. Engineered Regeneration, 3, 365–373. https://doi.org/10.1016/j.engreg.2022.08.002\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-516
https://f1000research.com/articles/10-515/v1	29 Jun 21	{ "type": "Data Note", "title": "Occupational therapy and Beery VMI scores of children with autism spectrum disorder, brachial plexus injury, and cerebral palsy", "authors": [ "Blair Carsone", "Katherine Green", "William Torrence", "Bridgett Henry", "Katherine Green", "William Torrence", "Bridgett Henry" ], "abstract": "Data of 107 children (84 males and 23 females) who received occupational therapy services were collected. Data collected included age, gender, prescribed frequency of occupational therapy, number of sessions attended within the six-month timeframe, pre and post Beery Visual Motor Integration scores.", "keywords": [ "Autism Spectrum Disorder", "Brachial Plexus Injury", "Cerebral Palsy", "Occupational Therapy", "Beery-Buktenica Developmental Test of Visual Motor Integration" ], "content": "Introduction\n\nThe existing research is at a consensus that autism spectrum disorder (ASD), brachial plexus injury (BPI), and cerebral palsy (CP) affect visual motor integration (VMI) and that VMI may be positively impacted by occupational therapy intervention (Bumin & Kavak, 2008; Cho et al., 2015; Desai & Rege, 2005; Duff & DeMatteo, 2015; Green et al., 2016; Lu et al., 2016; Miller et al., 2014; Wadsworth et al., 2017). However, the research does not adequately address the potential relationship between occupational therapy intervention and the Beery VMI scores of children with ASD, BPI, and CP. This secondary data analysis cohort study addressed the current gap in knowledge by providing data that can be used to investigate the relationship between occupational therapy (prescribed frequency and total sessions attended), age, gender, and pre-Beery VMI scores on post-Beery VMI scores of children with ASD, BPI, and CP.\n\n\nMethods\n\nPatients with a formal diagnosis of ASD, BPI, and CP designated in the electronic medical record (EMR) who were between the ages of two years, zero months, and five years 11 months during the initial assessment (occupational therapy evaluation or re-evaluation) were considered for inclusion in the study. Subjects with pre- and post- Beery VMI scores who attended at least one therapy session (excluding the initial assessment and reassessment) were included in the study. Subjects’ identifying information was not reported, meeting Human Subject regulations and standards (Breault, 2006). Table 1 below provides the information of each subject included.\n\nThe inclusion criteria for the study was designed based on the findings from the literature. Patients with the diagnosis of ASD, BPI, and CP were selected as a focus for this study because these three conditions represent populations commonly affected by VMI deficits (Bonifacci, 2004; Dowd et al., 2011; Englund et al., 2014; Green et al., 2016). The age range accounted for the potential peak in VMI scores, while the six-month intervention period accounted for the confounding variable of maturation (Ercan et al., 2011; Fang et al., 2017; Heiz & Barisnikov, 2016). Limiting the study to one county accounted for the potential effects of location while focusing on one specific clinic increased the feasibility of the study (Cui et al., 2012; Ng et al., 2015; Visser et al., 2017).\n\nData for both questions were obtained from one pediatric outpatient clinic in South Florida. Subject data were collected from the pediatric outpatient electronic medical record system, RainTree, based on the inclusion and exclusion criteria. The data reviewed spanned from June 1, 2017 to March 13, 2020. Limiters of age (two years zero months to five years 11 months) and diagnoses (ASD, BPI, or CP) were then employed. After the limiters were applied each individual chart was analyzed for pre- and six-month post- Beery VMI scores. Patient charts with pre- and six-month post Beery VMI scores who attended at least one therapy treatment session were included in the study. Prescribed frequency, therapy attendance, and changes in Beery VMI scores were recorded with relevant demographic information (e.g., age and gender). A summary of this process can be seen in Figure 1.\n\nGender (male = 1, female = 0), age at initial assessment (24 months to 71 months), prescribed frequency (1 time per week =1, 2 times per week = 0), sessions attended (0 to 52 sessions), pre-Beery VMI raw score (0 to 30 points), post-Beery VMI raw score (0 to 30 points), and change in Beery VMI raw score (0 to 30 points).\n\nThis data set is part of a publicly defended dissertation and the study was approved by Concordia University Chicago’s IRB (study number 1698245-1). The study was exempt from review as it was determined to be a secondary data analysis. The research involves only information collection and analysis involving the investigator’s use of identifiable health information when that use is regulated under 45 CFR parts 160 and 164, subparts A and E, for the purposes of “health care operations” or “research” as those terms are defined at 45 CFR 164.501 or for “public health activities and purposes” as described under 45 CFR 164.512(b). Consent from participants was therefore not required.\n\n\nDataset description\n\nOverwhelmingly, 97% of subjects were diagnosed with ASD while 1% were diagnosed with BPI and 2% were diagnosed with CP. The majority of subjects were male (N = 84, 78.5%; female N = 23, 21.5%). The subjects’ mean age was 49.21 months or approximately 4 years of age. Table 2 below provides an overview of the descriptive statistics.\n\n\nData availability\n\nAll data underlying the results are available as part of the article (Table 1) and no additional source data are required.", "appendix": "References\n\nBonifacci P: Children with low motor ability have lower visual-motor integration ability but unaffected perceptual skills. Hum Mov Sci. 2004; 23(2): 157–168. PubMed Abstract \| Publisher Full Text\n\nBreault JL: Protecting human research subjects: The past defines the future. Ochsner J. 2006; 6(1): 15–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBumin G, Kavak ST: An investigation of the factors affecting handwriting performance in children with hemiplegic cerebral palsy. Disabil Rehabil. 2008; 30(18): 1374–1385. PubMed Abstract \| Publisher Full Text\n\nCho M, Kim D, Yang Y: Effects of visual perceptual intervention on visual-motor integration and activities of daily living performance of children with cerebral palsy. J Phys Ther Sci. 2015; 27(2): 411–413. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCui Y, Zhu Y, Laukkanen H, et al.: Evaluation of visual-motor integration skills in preschool and elementary school-aged Chinese children. J Behav Optometry. 2012; 23(5-6): 123–128.\n\nDesai AS, Rege PV: Correlation between developmental test of visual motor integration (VMI) and handwriting in cerebral palsy children. Indian J Occupational Therapy. 2005; 37(2): 27–32.\n\nDowd A, McGinley J, Taffe J, et al.: Do planning and visual integration difficulties underpin motor dysfunction in autism? A kinematic study of young children with autism. J Autism Dev Disord. 2011; 42(8): 1539–1548. PubMed Abstract \| Publisher Full Text\n\nDuff SV, DeMatteo C: Clinical assessment of the infant and child following perinatal brachial plexus injury. J Hand Ther. 2015; 28(2): 126–133. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEnglund JA, Decker SL, Allen RA, et al.: Common cognitive deficits in children with attention-deficit/hyperactivity disorder and autism: Working memory and visual-motor integration. J Psychoeducational Assessment. 2014; 32(2): 95–106. Publisher Full Text\n\nErcan Z, Ahmetoğlu E, Aral N: Investigating the visual-motor integration skills of 60-72 month children from high and low socio-economic status as regard age factor. Int Education Studies. 2011; 4(3): 100–104. Publisher Full Text\n\nFang Y, Wang J, Zhang Y, et al.: The relationship of motor coordination, visual perception, and executive function to the development of 4-6-year-old Chinese preschoolers’ visual motor integration skills. Biomed Res Int. 2017; 2017: 6264254. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFaul F, Erdfelder E, Buchner A, et al.: Statistical power analyses using G*Power 3.1: Tests for correlation and regression analyses. Behav Res Methods. 2009; 41: 1149–1160. PubMed Abstract \| Publisher Full Text\n\nGreen RR, Bigler ED, Froehlich A, et al.: Beery VMI performance in autism spectrum disorder. Child Neuropsychol. 2016; 22(7): 795–817. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeiz J, Barisnikov K: Visual-motor integration, visual perception and motor coordination in a population with Williams syndrome and in typically developing children. J Intellect Disabil Res. 2016; 60(10): 945–955. PubMed Abstract \| Publisher Full Text\n\nIBM Corp: IBM SPSS Statistics for Macintosh, Version 26.0. Armonk, NY: IBM Corp; 2019.\n\nLu YC, Liu HQ, Hua XY, et al.: Supplementary motor area deactivation impacts the recovery of hand function from severe peripheral nerve injury. Neural Regen Res. 2016; 11(4): 670–675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller M, Chukoskie L, Zinni M, et al.: Dyspraxia, motor function and visual-motor integration in autism. Behav Brain Res. 2014; 269(1): 95–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNg M, Chui M, Lin L, et al.: Performance of the visual-motor integration of preschool children in Hong Kong. Hong Kong J Occupational Therapy. 2015; 25(1): 7–14. Publisher Full Text\n\nVisser M, Nel R, Jansen T, et al.: Visual perception of five-year-old English-speaking children in Bloemfontein using the Beery VMI-6, DTVP-3 and TVPS-3. South African J Occupational Therapy. 2017; 47(2): 17–26. Publisher Full Text\n\nWadsworth HM, Maximo JO, Lemelman AR, et al.: The Action Imitation network and motor imitation in children and adolescents with autism. Neuroscience. 2017; 343: 147–156. PubMed Abstract \| Publisher Full Text\n\nWagner WE: Using IBM SPSS statistics for research methods and social science statistics. 7th ed. Sage; 2020." }	[ { "id": "153646", "date": "28 Oct 2022", "name": "Caroline J. Mills", "expertise": [ "Reviewer Expertise Children's occupational therapy", "autism" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 data note which explores VMI scores pre and post for children who attended occupational therapy. The VMI is a commonly used tool in children’s occupational therapy and publication of this dataset may assist in collaboration and production of high quality research. There is however, some more context required in order to ensure that the data set is used to its maximum potential.\n\nIntroduction:\nThe term ‘autism spectrum disorder (ASD)' is not preferred by the autistic community. Consider saying ‘autism spectrum disorder’, hereafter ‘autism’ rather than ASD.\n\nMethods: Participants:\nBeery VMI- this data note may be improved by providing more information about the VMI- you mentioned ‘peak scores’. It’s not clear what this is if the reader does not have the context of the VMI. You should offer more context about where the score came from. Was it from the whole VMI assessment (including visual perception and motor coordination) or just the main VMI test?\n\nDataset description:\nYou should include actual numbers of participants here. How many in total, how many had autism, CP, BPI etc.\n\nThe data is presented clearly. I see that majority of the children are autistic. There’s only 1 child with BPI and 2 children with CP. It may appear that these children are outliers. It would not be possible for any researcher to use this data to ‘determine a relationship’ between OT attendance, VMI scores based on these low numbers. I suggest that these outliers are excluded from the dataset unless you can present a more firm justification for why they should be left in there.\n\nIt is not clear whether any of these children had any co-occurring diagnoses (in particular global developmental delay or intellectual disability). These may impact a child’s participation in occupational therapy and their VMI scores. Consider including this information or stating if/why it is absent.\nContext is needed around what the children worked on in therapy. Were they focused on visual motor integration/visual perception/hand skills in therapy sessions? If a researcher were to use this data to determine a relationship between OT attendance and VMI scores, they would need to context around what type of OT. With a dataset like this, this could be achieved through a process of categorisation, where therapy activities are grouped into categories (fine motor, play etc.). If this data is not available- you should provide a justification as to why.\nTable 2:\n\nDon’t use Mean and SD for nominal variables like Gender- use number and percentage of total. You could also offer some more descriptive information such as score range (min-max).\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] }, { "id": "120500", "date": "28 Nov 2023", "name": "Livia Taverna", "expertise": [ "Reviewer Expertise Developmental 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\nResearch question: Formulate the research hypotheses: for example that you want to see if there are differences in the response to occupational therapy treatment in the various disorders, taking into account age, gender, and the duration of the sessions.\n\nParticipants:\nYou can maybe include some socio-demographic information: do these children attend any formal educational institution (kindergarten, pre-kindergarten)? I assume that those attending to any form of school will be exposed to eye-hand coordination activities.\n\nSocio-demographic information about families: parent's age, education and economic status.\n\nMethod:\n\nInstrument: A brief description of the VMI would be appreciated. Even if it is a well known instrument, I think that for an article it is necessary to have a description. Include also information about scoring; scales and scores.\n\nData Analysis:\n\nDescribe the statistical analysis you will run, which variables are the independent and the dependent and for what purpose you choose this statistical analysis.\n\nThese are some suggestion to information that would be interesting.\n\nYou can provide some further analysis comparing children with respect to diagnosis accounting for age, gender or for treatment duration; qualitative analysis giving information about the target figures that were more difficult to realize (obtaining 0 to the VMI).\n\nYou can also provide detailed information about VMI scores (VMI; Visual Perception and Motor Coordination) and analyze in which areas the improvement occurs more frequently accounting for treatment, gender, age and type of diagnosis.\n\nResults: Report the results and related tables.\n\nDiscussion Discuss the abovementioned results.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-515
https://f1000research.com/articles/10-513/v1	29 Jun 21	{ "type": "Review", "title": "Approaches for containerized scientific workflows in cloud environments with applications in life science", "authors": [ "Ola Spjuth", "Marco Capuccini", "Matteo Carone", "Anders Larsson", "Wesley Schaal", "Jon Ander Novella", "Oliver Stein", "Morgan Ekmefjord", "Paolo Di Tommaso", "Evan Floden", "Cedric Notredame", "Pablo Moreno", "Andreas Hellander", "Payam Emami Khoonsari", "Stephanie Herman", "Kim Kultima", "Samuel Lampa", "Marco Capuccini", "Matteo Carone", "Anders Larsson", "Wesley Schaal", "Jon Ander Novella", "Oliver Stein", "Morgan Ekmefjord", "Paolo Di Tommaso", "Evan Floden", "Cedric Notredame", "Pablo Moreno", "Andreas Hellander", "Payam Emami Khoonsari", "Stephanie Herman", "Kim Kultima", "Samuel Lampa" ], "abstract": "Containers are gaining popularity in life science research as they provide a solution for encompassing dependencies of provisioned tools, simplify software installations for end users and offer a form of isolation between processes. Scientific workflows are ideal for chaining containers into data analysis pipelines to aid in creating reproducible analyses. In this article, we review a number of approaches to using containers as implemented in the workflow tools Nextflow, Galaxy, Pachyderm, Argo, Kubeflow, Luigi and SciPipe, when deployed in cloud environments. A particular focus is placed on the workflow tool’s interaction with the Kubernetes container orchestration framework.", "keywords": [ "Workflows", "Containers", "Cloud computing", "Reproducibility", "Automation", "Big Data" ], "content": "List of abbreviations\n\nAPI: Application programming interface\n\nAWS: Amazon web services\n\nCRD: Custom resource description\n\nGUI: Graphical user interface\n\nHPC: High-performance computing\n\nNGS: Next-generation sequencing\n\nPV: Persistent volume\n\nPVC: Persistent volume claim\n\nSFTP: SSH file transfer protocol\n\nUI: User interface\n\nVM: Virtual machine\n\nVMI: Virtual machine image\n\nWMS: Workflow management system\n\n\nIntroduction\n\nThe life sciences have become data-intensive, driven largely by the massive increase in throughput and resolution of molecular data generating technologies. Massively parallel sequencing (also known as next-generation sequencing or NGS) is where the largest increase has been seen in recent years, but other domains are also increasing dramatically, including proteomics, metabolomics, systems biology and biological imaging.1-3 Consequently, the need for computational and storage resources has continued to grow, but the focus has also changed towards the downstream steps of biological experiments and the need to carry out efficient and reproducible data analysis. While high-performance computing (HPC) and high-throughput computing clusters remain the main e-infrastructure resource used in biological data analysis, cloud computing is emerging as an appealing alternative where, in the infrastructure-as-a-service case, scientists are able to spawn virtual instances or infrastructure on demand to facilitate their analysis, after which the resources are released.4,5 One area that has traditionally often caused headaches for users is the installation of software tools and their inclusion into workflow tools or other computing frameworks.6 This is an area where cloud resources provide an advantage over HPC, in that scientists are not dependent on system administrators to install software, but can handle the installation themselves. However, software for biological analyses can be quite challenging to install due to sometimes complex dependencies. Virtual machines (VMs) offer the benefit of instantiating ready-made environments with data and software, including all dependencies for specific tasks or analyses and constitute a big step towards making computational reproducibility easier and more realistic to achieve in daily work. VMs form the backbone of traditional cloud-based infrastructures. Such images can easily be shared between users and can be deposited and indexed by one of the available virtual machine image (VMI) catalogs.7 VMIs, however, can easily become large and take considerable time to instantiate, transfer or rebuild when software in the image needs to be updated.\n\nSoftware container technology has emerged as a complement to VMs. While they offer a bit less isolation between processes, they are on the other hand more lightweight in terms of storage and memory requirements and are thus easier to share. By sharing the operating system kernel with the host computer, they are also much faster to launch and terminate.8 Containers encompass all dependencies of the provisioned tools and greatly simplify software installations for end users. The most widely used containerization solution is Docker (www.docker.com), but Singularity,9 uDocker10 and Shifter,11 are recent alternatives that prevent users from running containers with root privileges, thus addressing the most common security issue when deploying containers in multi-tenant computing clusters such as on HPC clusters. Docker containers are commonly shared via Docker Hub.12 There are also initiatives for standardizing containers in the life sciences such as BioContainers.13 Containers have seen an increased uptake in the life sciences, both for delivering software tools and for facilitating data analysis in various ways.14-18\n\nWhen running more than just a few containers, an orchestration system is needed to coordinate and manage their execution and handle issues related to e.g. load balancing, health checks and scaling. Kubernetes19 has over the last couple of years become the de facto standard container orchestration system, but Docker Swarm20 and Apache Mesos21 are other well-known systems. A key objective for these systems is to allow the users to treat a cluster of compute nodes as a single deployment target and handle the packaging of containers on compute nodes behind the scenes.\n\nWhile orchestration tools such as Kubernetes enable scientists to run many containers in a distributed environment (such as a virtual infrastructure on a cloud provider), the problem remains how to orchestrate the scheduling and dependencies between containers and being able to chain (define data dependencies between) them into an analysis pipeline. This is where scientific workflow management systems (WMSs) can be very useful; in fact, it can be difficult to carry out more complex analyses without such a system. Traditionally in data-intensive bioinformatics, an important task for WMSs has been to support analyses consisting of several components by running a set of command-line tools in a sequential fashion, commonly on a computer cluster. The main benefits of using a WMS include: i) making automation of multi-step computations easier to create, more robust and easy to change; ii) providing more transparency as to what the pipeline does through its more high-level workflow description and better reporting and visualization facilities; and iii) providing more reliable policies to handle transient or persistent error conditions, including strategies to recover from interrupted computations while re-using any partially finished data.\n\nUsing containerized components as the nodes in a scientific workflow has the advantage of adding isolation between processes and the complete encapsulation of tool dependencies within a container, and reduces the burden to administrate the host system where the workflow is scheduled to run. This means the system can be tested on a local computer and executed on remote servers and clusters without modification, using the exact same containers. Naturally, this opens up for execution on virtual infrastructures, even those provisioned on-demand with features such as auto-scaling. Apart from greatly simplifying access to the needed distributed compute resources, a positive side-effect is that the entire analysis becomes portable and reproducible.\n\nData ingestion and access to reference data constitute a key step when using cloud-based resources. When spawning a virtual infrastructure, data used in the analysis either needs to be available on storage connected to the compute nodes, or ingested into a locally provisioned file system. At the end of the analysis, the resulting data needs to either be put into persistent storage on the cloud or downloaded to local storage. However, the overhead with this can also be turned around. If data resides on storage connected to a cloud infrastructure, it is possible to simply move the workflow and containers there and “bring compute to the data”.\n\nOne key difference with running workflows locally on a desktop computer or server, or on a single HPC node, is that when running containerized workflows, the workflow engine often sits on another compute node than the executing containers. Also, the storage system is also typically separated, and accessible from any container, regardless of the exact compute node in a cluster where it is executed. Figure 1 illustrates this.\n\nThe tools pass data between each other by reading and writing to a shared storage system.\n\nThere are also multiple possibilities on how to run a workflow engine in relation to the containers, as the workflow engine can either be run on a separate system from the container cluster, such as a researcher’s local computer, or the workflow engine can itself run in a container on the cluster. Figure 2 illustrates this.\n\nIt can run either a) on the user’s computer, or b) in a pod in the Kubernetes cluster.\n\n\nWorkflow systems\n\nDue to their simplicity, containers are supported out of the box by most workflow tools. However, the approach for interacting with containers differs, especially when using virtual infrastructures on cloud resources. This section describes the approaches taken by a set of workflow management systems to interacting with containers in cloud environments, with varying degree of uptake in the life science community.\n\nNextflow22 is a workflow framework that aims to ease the implementation of scientific workflows in a portable and reproducible manner across heterogeneous computing platforms and to enable the smooth migration of these applications to the cloud. The framework is based on the dataflow paradigm, a functional/reactive programming model in which tasks are isolated from each other and are expected to be executed in a stateless manner. This paradigm simplifies the deployment of complex workflows in distributed computing environments and matches the immutable execution model of containers.\n\nNextflow provides built-in support for the most used container runtimes i.e. Docker, Singularity, Shifter and uDocker (though the last two are undocumented because of still being in an experimental state). The use of containers is defined in a declarative manner, i.e. by specifying the container image that a workflow needs to use for the task executions. Once annotated in this way, the Nextflow runtime takes care of transparently running each task in its own container instance while mounting the required input and output files as needed. This approach allows a user that needs to target the requirements of a specific execution platform to quickly switch from one containerization technology to another with just a few changes in the workflow configuration file (e.g. the same application can be deployed in an HPC cluster using Singularity or in the cloud with Docker).\n\nGreat flexibility is given for container image configuration. Nextflow supports both the image-per-workflow pattern (the same image for all executed tasks) and the image-per-task pattern (each task uses a different container image). The container image used by a task can even be specified at runtime by a given input configuration.\n\nNextflow allows the deployment of containerized workloads in a cloud-native manner using different strategies, as briefly discussed in the following paragraphs.\n\nCloud-managed computing service: in this approach workflow tasks are submitted to a cloud provider batch execution service. The containerization and auto-scaling are delegated to the cloud service. The workflow inputs/outputs and intermediate result files need to be stored using the provider object storage service. Nextflow adapts the task definition, resource requests and container image to the cloud provider format, submitting the corresponding API requests and properly staging the task data. Currently, Nextflow has built-in support for the Amazon Web Services (AWS) Batch service and the Google Genomics Pipelines service.\n\nCloud unmanaged computing service: when using this deployment strategy, Nextflow takes care of the provisioning of a set of cloud VM instances in which it automatically sets up its own clustering engine for the distributed execution of the workflow. The VM instances only require the availability of a Docker runtime and the Java virtual machine. Workflow tasks exchange data via a shared file system or by using a cloud object storage service. The workflow execution is managed by Nextflow which also handles the cluster auto-scaling, i.e. adds or removes VM instances on-demand to adapt the actual needs of a workload at any point in time and optimizes the overall computing cost when applicable. At the time of writing, this feature supports the use of the AWS EC2 cloud service and Google Compute Engine service.\n\nFigure 3 provides an overview of the components involved when executing a containerized Nextflow workflow.\n\nThe tools pass data between each other by reading and writing to a shared storage system.\n\nGalaxy (Galaxy, RRID:SCR_006281) is a data-analysis workflow platform that has probably the most active development community in the field of workflow environments in Bioinformatics. This has enabled it to persist as an active project and to continue to evolve since 2005, enabling reproducible research through active channels for sharing tools, workflows and datasets.23 Galaxy is currently used by tens of thousands of scientists across the globe.24 Besides offering a modern user interface (UI) for end users, Galaxy is accessible through its REST API or through the BioBlend Python binding25 (that talks to this REST API).\n\nFor execution versatility, Galaxy has adapters to offload jobs in a wide variety of systems, from local Docker containers to different batch scheduler systems. In Galaxy, there is a complete separation of concerns between tools, workflows and execution environments, meaning that a workflow definition is not bound to a particular execution environment. Within the last seven years Galaxy gained the ability to launch on AWS through the CloudMan Launcher Galaxy sub-project.25 This initial setup followed a relative VM centric approach, which has natural limitations for scaling in the number of tools included as they all need to be provisioned inside the VM used for the provisioned cluster.\n\nMore recently, through efforts within the PhenoMeNal H2020 Project,26 and later contributions by the Genomics Virtual Lab (GVL) initiative, Galaxy gained the ability to deploy inside Kubernetes in an automated fashion through the use of Helm Charts.27 Since then, the Galaxy Helm chart has been adopted as a project by the Galaxy community (https://github.com/galaxyproject/galaxy-helm), and two major versions have been released, making v3.x of the Galaxy Helm Chart a mature product relying on a minimal Galaxy container for fast deployment. Galaxy Helm Chart v3 allows the deployment of multiple web and jobs handlers, making the setup scalable from any perspective (number of user and number of jobs). Newer versions of the CloudMan Galaxy launcher now rely on this setup instead of VM deployments and recently a Single Cell RNA-Seq Galaxy setup28 was released based on the Galaxy Helm chart.\n\nThe Kubernetes job runner, also contributed to the Galaxy community by the PhenoMeNal H2020 Project and then further maintained by the Galaxy community, allows Galaxy to offload jobs to Kubernetes, either when Galaxy runs inside the container orchestrator or from the outside (provided that the shared file system is accessible to both the container orchestrator and Galaxy). The Kubernetes runner for Galaxy will take care of resource requirement settings, recovery of jobs in Kubernetes if there is a restart, auto-upgrading of resource utilization, Persistent Volume Claim mounting both for Galaxy and in each jobs pod, as well as adequate supplementary groups handling for network filesystem and other shared file systems. This integration requires the use of a shared file system, which needs to be mountable in Kubernetes in a read-write-many configuration, as both the main Galaxy job handler pod and the job pods need to mount it at the same time (through the PV/PVC abstraction). The Kubernetes runner for Galaxy adheres to the principles of resilience and redundancy commonly considered in cloud computing, providing ways to recover from jobs being lost by cluster nodes going down in the middle of an execution.\n\nThe Kubernetes runner for Galaxy can benefit from explicit tool-to-containers mapping through Galaxy dynamic destinations, or take advantage of dynamic bioconda-to-containers mapping thanks to built-in functionality in Galaxy for this purpose. This means that if a Galaxy tool declares a Bioconda package as the dependency to be resolved, it will bring an automatically built container for that Bioconda package29 from BioContainers.13 For tools with multiple conda packages as dependencies, the Galaxy/Biocontainers community builds automatically multi-tool “mulled” containers (https://github.com/BioContainers/multi-package-containers) through Planemo-Monitor (https://github.com/galaxyproject/planemo), and these are as well resolved automatically when running on the Kubernetes setup.\n\nThe Galaxy-Kubernetes integration, which includes all the aspects related to Kubernetes mentioned in the previous paragraphs, has been battle tested by the PhenoMeNal H2020 Project, having its public instance receiving in the order of tens of thousands of jobs during the past 4 years, having tens of deployments in production environments and possibly hundreds of deployments in development settings. It has also been used as part of GVL’s CloudMan for the past year and for running training courses with more than 30 concurrent users in the context of Single Cell RNA-Seq. This integration has been tested locally on Minikube, on OpenStack deployments, on Google Cloud Platform and AWS turn-key Kubernetes services. In these two last cases, the setup can behave elastically, deploying more Kubernetes worker nodes automatically for increased capacity on demand, as the number of jobs or users increment, and then automatically reducing the number of nodes and containers deployed when load diminishes. It is expected that in the next few years the Galaxy-Kubernetes integration is embraced in the areas of proteomics and transcriptomics, and becomes one of the main routes of deploying Galaxy installations.\n\nPachyderm30 is a large-scale data processing tool built natively on top of Kubernetes. This platform offers: i) a data management system based on Git semantics and ii) a workflow system for creating distributed and reproducible pipelines based on application containers. In order to create workflows with Pachyderm, users supply a JSON pipeline specification including a Docker image, an entrypoint command to execute in the user containers and one or more data input(s). Afterward, Pachyderm will ensure that the corresponding pods are created in Kubernetes, and will share the input data across them and collect the corresponding outputs. Thanks to using Kubernetes for container orchestration, Pachyderm is able to, among other things: i) optimize cluster resource utilization, ii) run seamlessly on multiple cloud and on-premise environments, iii) self-heal pipeline jobs and related resources.\n\nThis workflow tool provides an easy mechanism to distribute computations over a collection of containers. In other words, it offers similar capabilities to frameworks such as Apache Spark, but replaces MapReduce syntax with legacy code. More specifically, Pachyderm is able to distribute workloads over collections of containers by partitioning the data into minimal data units of computation called “datums”. The contents of these datums are defined by glob patterns such as “/” or “/*”, which instruct Pachyderm how to process the input data: all together as a single datum, or in isolation (as separate datums). Users can define the number of pipeline workers to process these datums, which will be processed one at a time in isolation. When the datums have been successfully processed the Pachyderm daemon gathers the results corresponding to each datum, combines them and versions the complete output of the pipeline. The scheduling of pipeline workers is determined by the resource utilization status of the workers’ nodes and the resource requests that are made for each pipeline, allowing for efficiently distributing workloads. Recently, work to integrate and demonstrate Pachyderm in bioinformatics was published.31\n\nLuigi32 is an open-source Python module for defining and running batch-like workflows. The system is strongly oriented towards Big Data, and it comes as a convenience tool to pipe jobs from well-established analytics frameworks (e.g. Hadoop, Spark, Pig and Hive). Hence, instead of trying to substitute any of the available analytics systems, Luigi provides the means to stitch together jobs from different frameworks in a single batch-oriented workflow. In doing so, Luigi seamlessly handles many boilerplate operations such as: dependency resolution, visualization, failure tolerance and atomic file system operations.\n\nThe Kubernetes integration was developed by the PhenoMeNal consortium, and it is now part of the official Luigi project and thus being supported and maintained by the community. The integration enables to include pipeline processing steps as Kubernetes Jobs, which represent batch-like application containers that run until command completion. Hence, Luigi mainly uses Kubernetes as a cluster-resource manager where multiple parallel jobs can be run concurrently. To this extent, it is important to point out that Luigi is not meant to be used as a substitute for parallel processing frameworks (such as Spark and Hadoop), as it has limited support for concurrent tasks. In fact, in our experience Luigi can handle up to 300 parallel tasks before it starts to break down, or 64 parallel tasks if running on a single node. A project to adapt Luigi for scientific applications in general and bioinformatics in particular resulted in a tool named SciLuigi.33\n\nWe benchmarked Luigi on Kubernetes by reproducing a large-scale metabolomics study.34 The analysis was carried out on a cloud-based Kubernetes cluster, provisioned via KubeNow and hosted by EMBL-EBI, showing good scalability up to 40 concurrent jobs.35 Workflow definition, and instructions to reproduce the experiment, are publicly available on GitHub.36\n\nSciPipe37 (SciPipe, RRID:SCR_017086) is a workflow library based on Flow-based programming principles, which enables building workflows from component libraries of predefined workflow components. SciPipe is implemented as a programming library in Go, which enables using the full power of the Go programming language to define workflows, as well as to compile workflows to executable files, for maximum ease of deployment in cloud scenarios.\n\nSciPipe has been used to orchestrate machine learning pipelines to build predictive models for off-target binding in pharmacology.38 SciPipe currently provides experimental support for container based workloads via Kubernetes, implemented through the official Kubernetes Go client library.39 The Go client library enables transparent access to a Kubernetes cluster regardless of whether the SciPipe workflow runs outside the cluster or inside, in a pod. When running inside the cluster, it automatically detects cluster settings. When not inside a cluster, the connection to a cluster can be configured by setting the appropriate environment variables. The Kubernetes integration in SciPipe is demonstrated through the implementation of a use case workflow consisting of a set of data preparation steps for mass-spectrometry data, using the OpenMS software suite.40 It contains a Go file (with the .go extension) with the workflow, and a Kubernetes job-spec file in YAML format (with the .yml extension) for starting SciPipe inside a Kubernetes cluster is together with the workflow.41\n\nSingularity containers9 can already today be used from SciPipe on a simple level, by configuring the appropriate shell commands when creating new processes through the shell-command process constructor (the scipipe.NewProc() function in SciPipe). Development of more integrated support for Singularity containers is planned.\n\nArgo workflow42 originates from a corporate application with the need of a competent continuous integration/continuous deployment solution but matured into a generalized workflow engine. It was open sourced by Intuit and is now adopted across several industries and companies among them life science and biopharma. A big differentiator of Argo is that no domain specific language knowledge is required to build workflows. The workflows are instead constructed by a clearly defined declarative approach manifested as Kubernetes custom resource definitions (CRDs), which is a templated approach to enable composability and extendability to the original Kubernetes resource orchestration. Argo is often referred to as a cloud native workflow orchestration tool and is designed for container native execution without the overhead of other non-native container solutions. Argo provides a robust way to declare and run workflows as arguments and artifact objects can be declared as input and output and containers run as state transitions merely passing objects and arguments through the pipeline. In each workflow step the state is stored in redundant etcd43 memory for resilience and reproducibility. Argo is complemented by an artifacts concept that enables data and parameter versioning through complementing Argo with an S3 compatible storage backend. Furthermore Argo events enable signals and events for triggering workflows on webhooks, resource changes, GitHub or Docker image changes etc enabling additional automation scenarios.\n\nKubeflow44 is another open-source platform for Kubernetes, specifically aimed at building and running machine learning workflows and is developed by Google. As a framework, Kubeflow is intended to provide a set of tools for various tasks in the process of machine learning workflows, from data preparation and training to model serving and maintenance. Many of the tools are existing tools with a user base in the industry, such as Jupyter Notebook and Seldon, while others are customized wrappers or endpoints to frameworks like TensorFlow, PyTorch.\n\nIn particular, Kubeflow provides a workflow tool called Kubeflow Pipelines, which allows users to create workflows of varying complexity, chaining together series of container operations. The tool is based on the workflow engine Argo, which has been integrated with the Kubeflow ecosystem with a different UI than the original Argo UI. In essence, a user declares a workflow in the Kubeflow Pipelines software development kit, defining the different steps in terms of container operations, i.e. what container to use for the step, Kubernetes resource allocations, commands to execute in the container and so on. The user also declares the order of these container operations and any desired input parameters to the workflow, and from this definition a pipeline can be compiled. This pipeline is a portable configuration which in theory can be uploaded and used in any Kubeflow environment. With the pipeline deployed in Kubeflow, a user can start a run where available pipeline parameters can be provided and the progress of the run tracked in the Kubeflow Pipelines UI. Here, logs of the container’s standard output, output artifacts and various Kubernetes information can be tracked for each step, along with a visualization of the workflow graph.\n\nAs an evaluation of the Kubeflow Pipelines system, a simple pipeline was developed, based on previous work45 where a convolutional neural network is trained to “… predict cell mechanisms of action in response to chemical perturbations...”, based on a dataset of cell microscopy images from the Broad Bioimage Benchmark Collection. The pipeline covers the following steps of the machine learning process: data pre-processing, model training, model evaluation and model serving preparation. The result is a workflow that handles the entire process end-to-end, building a servable machine learning model and publishing this as a Docker container that can be deployed for serving predictions. The pipeline source code, while partly tailored to a specific environment, is available in.46\n\n\nDiscussion\n\nThe tools covered in this study have taken slightly different approaches to work with containers, with different implications. Most tools are designed to work with general command-line tools while also supporting containers, except Pachyderm, Argo and Kubeflow that are Kubernetes-native and only support containers as means of processing.\n\nWorkflow tools can almost always work with containers instead of command-line tools, using the docker run command, and taking advantage of an external scheduling system such as a batch scheduler (e.g. SLURM). When deployed in cloud environments, Kubernetes is the most widely used orchestration framework by the tools, though Galaxy also supports Docker Swarm as well and provides a convenient mapping from the Conda package manager to containers. For Kubernetes, tools differ in the way they interact with its API; Pachyderm, Argo, Kubeflow Pipelines and SciPipe interact via the Go API whereas Nextflow, Galaxy and Luigi communicate via the REST API. While these APIs are not identical, the API designs are very similar in terms of data structures and in effect allow for the same level of control. In more detail, the structure of the JSON documents sent to the REST API is closely matched by the hierarchic structure of the ‘spec’ structs in the Go API. The workflow tools also differ somewhat in the level of flexibility they allow for utilizing the API features, where Luigi is the most versatile as it allows for passing a raw JSON string with full flexibility but also requires the most skills from workflow authors.\n\nError management for containerized workflows is an important concept. Kubernetes is designed to restart pods that fail, which is not particularly useful if there is an error reported from the containerized tool. The cloud paradigm includes fault-tolerance, and the tools differ in their strategies. Galaxy, Luigi and SciPipe use Kubernetes Jobs, which allows restarting the pods N times before reporting as failure. Argo and Kubeflow Pipelines run workflow steps as pods that are governed by Argo’s custom Kubernetes resource workflow; as such errors for individual containers are handled as for normal Kubernetes pods, including error messages detailing reasons for failure etc. Nextflow offers the possibility to automatically tune the job, e.g. to increase the available memory for a job before reporting it as failed.\n\nThe way in which workflow systems schedule containers on Kubernetes is one factor where they differ from each other. Nextflow implements an actor-like model to handle job requests. Jobs are submitted in a manner similar to an internal job queue. Then a separate thread submits a request to the Kubernetes cluster for each of them and periodically checks their status until the job completion is detected. Galaxy can deploy an unlimited number of job handlers, that can each iterate over thousands of jobs, and are separate processes to the web server handlers. It keeps no separate threads or processes per Kubernetes job. Pachyderm makes direct use of Kubernetes scheduling and uses etcd for storing metadata for data versioning and status of all jobs. It is also the only system that uses the Kubernetes feature of “parallel jobs”. Argo and Kubeflow Pipelines similarly utilize etcd for storing metadata of workflows and executions, and employ a workflow controller component to schedule containers based on the state of deployed workflows. Luigi keeps a thread open and continuously polls the job status. SciPipe keeps a lightweight thread (go-routine) alive for each created job, as the Go API call for creating jobs do block the calling go-routine until the job finishes or is stopped for other reasons.\n\nHandling many concurrent jobs can become a problem in Luigi/SciLuigi, where a separate Python process is started for each job which imposes a practical limit on the number of concurrent jobs; in the authors’ experience 64 is a rule of thumb for an upper limit if running on a single node, or else 300. Going above this tends to result in HTTP timeouts since workers are talking to the central scheduler via HTTP calls over the network.\n\nSo what are the main differences between the systems when running containers in cloud environments? Galaxy has a graphical user interface and might be more attractive for users with less expertise in programming/scripting, although it still provides programmatic access (REST and Python bindings) for workflow/tool execution and data/metadata retrieval. It can be run on both HPC batch schedulers and on cloud. Workflows in Argo can be submitted and controlled both from multi platform command-line interfaces as well as monitored through a graphical UI (GUI) but can also be controlled by other Kubernetes cluster resources since workflows are standard CRD resources. Kubeflow also provides a GUI as the main method for running pipelines, in addition to being a very portable platform in Kubernetes environments. Kubeflow Pipelines does however have a somewhat steep learning curve as the Python-based domain-specific language requires a good grasp of Kubernetes concepts. Nextflow, just like Galaxy (and Snakemake47), provides some versatility when working with containers, as being able to operate both on cloud or HPC batch schedulers. Pachyderm, Argo and Kubeflow are built specifically for running on Kubernetes and Pachyderm has a data-versioning file system built-in. Argo and Kubeflow Pipelines support artifacts in workflows and versioning of input and output artifacts through interfacing with an optional S3 storage backend. Luigi/SciLuigi supports many data sources out of the box, including HDFS, S3, etc and integrates seamlessly with Apache Hadoop and Spark. SciPipe implements an agile workflow programming API that smoothly integrates with the fast growing Go programming language ecosystem.\n\nFinally, perhaps somewhat surprisingly, we would like to end with a word of caution regarding the use of containers. While containers have many advantages from a technical perspective and alleviate many of the problems with dependency management, it also comes with certain implications, as it can make tools more opaque and discourage accessing and inspecting the inner workings of the code inside the container. This could potentially lead to some lack of understanding of what a specific tool does. Thus, when wiring together containers in a scientific workflow, proper care needs to be taken that the observed output matches what would be expected from using the tool directly.48 Also for this reason we strongly suggest to follow community best practices for packaging and containerization of bioinformatics software.49\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Competing interests\n\n\n\nCompeting interests: PDT and EF are founders of Seqera Labs, a company based in Barcelona, Spain, offering commercial support for the (open source) Nextflow software. SL is involved in RIL Partner AB, a Sweden based company offering commercial support for the (open source) SciPipe software.\n\n\nGrant information\n\nThis research was supported by The European Commission’s Horizon 2020 programme funded under grant agreement number 654241 (PhenoMeNal) and grant agreement number 731075 (OpenRiskNet), the Swedish Foundation for Strategic Research, the Swedish Research Council FORMAS, the Swedish e-Science Research Centre (SeRC), Åke Wiberg Foundation, and the Nordic e-Infrastructure Collaboration (NeIC) via the Glenna2 project.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nA previous version of this article is available from: https://doi.org/10.7287/peerj.preprints.27141v1.\n\n\nReferences\n\nMarx V: Biology: The big challenges of big data. Nature. Jun 2013; 498(7453): 255–260. 1476-4687. 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July 2018; 4: e158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGruening B, Sallou O, Moreno P, et al.: Recommendations for the packaging and containerizing of bioinformatics software. F1000Res. March 2019; 7: 742. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "88649", "date": "28 Jul 2021", "name": "Maciej Malawski", "expertise": [ "Reviewer Expertise Cloud computing", "scientific workflows" ], "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 provides a review of approaches for using containers in scientific workflows in clouds, with the particular focus on life-science applications. The topic is timely and relevant for the computational life science research community, as workflow systems and container technologies are state of the art approaches and still under active development. The paper gives a short introduction to cloud computing, containers, and workflow management systems (WMS). Subsequently, it shortly describes how containers are supported in Nextflow, Galaxy, Pachyderm, Argo, Kubeflow, Luigi and SciPipe systems. From the description we can learn that all the systems use Kubernetes to manage containers, and what are the main features of WMS-Kubernetes integration. The paper is generally well written and of good quality, but there are several items that are worth improving.\n\nIn general, the paper provides a nice high-level introduction and overview of the solutions, but I would expect more rigorous and systematic description, with definitions of key concepts, list of criteria for comparison, perhaps a taxonomy of the features and approaches. This would make the review more structured and comprehensive.\n\nFrom a review, I would expect some structured summary and comparison of systems, approaches, and solutions in the from of a table, with clearly defined set of criteria. The discussion section is quite superficial in the current form.\n\nTo systematically and fairly compare all the WMS, a common reference architecture should be defined, in a more detailed way than presented in Fig. 1, taking into account not only the container layer, but also the cloud (IaaS/VM) layer.\n\nThe paper does not provide an appropriate introduction to Kubernetes, which seems to be a central orchestration in this review. An introduction including the architecture and definition of pods, helms, etc. should be provided to make the paper self-contained. Scoping the paper on Kubernetes-based approaches would make the paper more focused.\n\nThe description of Nextflow seems to be incomplete, as it does not describe how it is integrated with Kubernetes, only a diagram is provided in Fig. 3. Fig 3, however, is a copy of the documentation from here, which is provided without a reference.\n\nAn important issue is how the WMS handles the dynamic allocation of resources: can the additional VMs be added to the Kubernetes cluster, or additional pods added to the workflow during execution? This is mentioned briefly in the discussion, but in my opinion this is worth emphasizing as a separate criterion for evaluation.\n\nAn interesting discussion is provided regarding error management, but there is only one paragraph on that which touches the problem, so a more detailed discussion would be much appreciated.\n\nThe performance and scalability of the systems is an important criteria, which can be measured and compared, especially in the cloud environment and for large-scale applications. The paper mentions some performance issues with Luigi, but there is no similar discussion regarding other systems. Does it mean that there are no scalability issues, or not? To make a comparison fair, all the systems should be evaluated regarding the performance and scalability.\n\nIn conclusions, there should be some recommendations on which system is better for which purpose, in which application scenario or for which infrastructure.\n\nTo make a discussion more complete, it would be worth referring to other general-purpose scientific WMS, in which container technologies have been employed. These may include Pegasus, Condor, Makeflow or Airflow.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] }, { "id": "136766", "date": "06 May 2022", "name": "Roberto Vera Alvarez", "expertise": [ "Reviewer Expertise Computational biology", "NGS data analysis", "scientific workflow", "cloud computing" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript “Approaches for containerized scientific workflows in cloud environments with applications in life science” by Spjuth et. al. presents a review of containerized workflow managers currently available. The manuscript is well written in a clear and concise way. In my opinion, the manuscript’s sections are organized correctly giving to the readers the idea of how these complex systems are organized. However, I consider, as this manuscript intend to review scientific workflows in cloud environments, that some introduction should be added describing the most important public cloud providers and the tools they provide for cloud computing and workflow execution.\nIn page 5 paragraph 2, the terms \"Amazon Web Services (AWS) Batch service and the Google Genomics Pipelines service\" are used without previous definition or explanation of what they are. I would also highlight that “Google Genomics Pipelines service” no longer exist. The current solution in GCP is named “Cloud Life Sciences” (https://cloud.google.com/life-sciences/).\nAdditionally, the review should describe what is a workflow language, which is at the end, the core of each manager. It should also include main advantages and differences for each workflow language. Users choose the workflow manager depending on the capability of the workflow language to run their specific pipeline.\nThis review is not mentioning a set of established workflow managers based on the Common Workflow Language (CWL, https://www.commonwl.org) like Arvados (https://arvados.org/), Toil (https://github.com/BD2KGenomics/toil), CWL-Airflow (https://github.com/Barski-lab/cwl-airflow) and StreamFlow (https://streamflow.di.unito.it/). There are also other CWL based manager like REANA (https://docs.reana.io/), Calrissian (https://github.com/Duke-GCB/calrissian) and Mariner (https://github.com/uc-cdis/mariner) that use Kubernetes as well. They also should mention that Galaxy is able to work with CWL and describe the advantages of running Galaxy with CWL.\nIn my opinion, the manuscript is presenting an incomplete review of workflow managers for scientific computing using the cloud. The discussion is incomplete as important workflow managers like CWL are not mentioned. Also, there should be an introduction and description of workflow language.\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? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? No", "responses": [] } ]	1	https://f1000research.com/articles/10-513
https://f1000research.com/articles/9-1287/v1	30 Oct 20	{ "type": "Research Article", "title": "Impact evaluation of national nutrition policies to address obesity through implementation of sin taxes in Gulf Cooperation Council countries: Bahrain, Saudi Arabia, Oman, United Arab Emirates, Kuwait and Qatar", "authors": [ "Ayoub Al-Jawaldeh", "Rania Megally", "Ayoub Al-Jawaldeh" ], "abstract": "Background: Around 7% of under-five aged children in the Eastern Mediterranean are overweight, and there are higher rates in Gulf Cooperation Council (GCC) countries. This had led the GCC to impose policies that aim to decrease obesity, overweight, and diabetes rates. The objective of this research is to measure the impact of one such implemented policy to reduce obesity, i.e. sin taxes applied to sugar-sweetened beverages (SSB) in GCC. Methods: The impact of sin taxes on SSB has been measured using a panel data set that covers sales volumes of soft drinks in GCC countries from 2010 to 2020. Results: Growth rate of sales volumes decreased from 5.44% to 1.33% in Saudi Arabia, 7.37% to 5.93% in United Arab Emirates, and 5.25% to 5.09% in Bahrain from 2016 to 2017; sin taxes were implemented in these countries in 2017. In Qatar and Oman, sin taxes were implemented in 2019, and a reduction in sales volumes was observed from 2018 to 2019 (Qatar: 2.30% to 3.78%; Oman: 3.60% to 2.99%). Kuwait was the last GCC country to implement sin taxes in 2020. Growth rate of sales volumes decreased from 6.31% to 5.47% from 2019 to 2020. Conclusions: Awareness campaigns should promote the reduction of the consumption of SSB and substitute with more consumption of water, unsweetened milk for children, fresh fruits and vegetables. These recommendations align with the recommended priority actions by the World Health Organization for the strategy on nutrition for the Eastern Mediterranean Region 2020-2030.", "keywords": [ "Health and Economic Development", "Health Behavior", "Government Policy", "Regulation", "Public Health" ], "content": "Introduction\n\nThe prevalence rate of obesity and overweight is high in the Eastern Mediterranean Region (EMR). More than half of the adult women (50.1%) and more than 40% of men are obese or overweight in the EMR. Around 7% of under-five aged children in are overweight, which is greater than the global average rate of 6.2% (WHO, 2016a). Furthermore, the rate is even higher for children living in Gulf Cooperation Council (GCC) countries (Abdul-Rasoul, 2012). Obesity in children increases the risk of experiencing difficulties in breathing and mental health issues and is an early cardiovascular disease marker (Pizzi & Vroman, 2013; WHO, 2016a). Moreover, obese children can be highly affected concerning their educational attainments and quality of life. In addition, obesity in childhood is related to a high risk of obesity in adulthood, diabetes, and cardiovascular diseases (WHO, 2016c). There is a correlation between the high prevalence rates of overweight and obesity with the high prevalence rates of diabetes (World Health Statistics, 2016). Figure 1–Figure 3 show the increase of obesity, overweight, and deaths due to diabetes in the GCC from 2010 to 2016.\n\nSource: World Health Organization (http://apps.who.int/gho/data/view.main.CTRY2430A; http://apps.who.int/gho/data/view.main.CTRY2450A?lang=en; http://apps.who.int/gho/data/view.main.2464ESTSTANDARD; http://apps.who.int/gho/data/view.main.NCDRGLUCAv).\n\nSource: World Health Organization (http://apps.who.int/gho/data/view.main.CTRY2430A; http://apps.who.int/gho/data/view.main.CTRY2450A?lang=en; http://apps.who.int/gho/data/view.main.2464ESTSTANDARD; http://apps.who.int/gho/data/view.main.NCDRGLUCAv\n\nSource: World Health Organization (http://apps.who.int/gho/data/view.main.NCDDEATHCAUSESNUMBERv).\n\nAccordingly, GCC countries have imposed policies that aim to decrease obesity, overweight, and diabetes rates. Such policies are part of an operational policy for diabetes that has been implemented in all GCC countries, except Oman, and an operational policy to reduce unhealthy diet related to non-communicable diseases (NCDs), which were imposed in 2017 in all GCC countries (WHO, 2016b). One of the most important policies that have been implemented is sin taxes on sugar-sweetened beverages (SSB), which aims to reduce significant increases in the prevalence rates of obesity and overweight that have drawbacks on health and whole economy in the short and long-run. Saudi Arabia was the pioneer in implementing the sin tax policy, followed by United Arab Emirates (UAE) (these two countries implemented sin taxes on SSB in 2017), followed by Bahrain also in 2017. Oman and Qatar proceeded with the implementation at the beginning of 2019, and Kuwait in 2020 (Table 1) (Whitehead, 2019).\n\nIntroduction of sin tax policy does not negate the importance of the government’s role in running awareness campaigns. They are crucial to raise children’s awareness of good nutrients, for instance forbidding TV advertisements of the sweetened beverages in the times when children are most likely to be watching TV. Applying the latter will support the policy of applying sin taxes on sweetened beverages to discourage the sugar intake. These campaigns are important because children’s food preferences are mainly affected by their parent’s preferences, mass media, peer group, nutritional knowledge, and socio-economic factors (McCullough et al., 2004). Educating children about proper nutrients is essential via nutrition education programs in schools and pre-schools. Nutrition education programs should be run for long periods as it has been shown that short-term education had no impacts on changing dietary intake and behaviour in spite of the fact that it raises nutritional knowledge (Kim et al., 2018; Yeom & Cho, 2016).\n\nGiven the fact that food preferences have an impact on lifelong eating habits and health, proper nutrition and adequate selection of food is crucial in early childhood (Okubo et al., 2016; Ventura & Worobey, 2013). The World Health Organization (WHO) suggests a decrease of the intake of free sugar to less than 10% of the total consumed calories given the increasing rate of added sugar intake (WHO, 2015a). Children usually are exposed to sweetness when they are infants, which increases their intake of sweets when they grow up (Foterek, 2016; Okubo et al., 2016), so it is crucial to construct an environment that decreases their intake of added sugar.\n\nHence, a policy and an action plan for sugar reduction has been developed by the EMR of the World Health Organization based on WHO guidelines (Alwan et al., 2017) in order to reduce sugar intake by more than 50% for children and adults (WHO, 2020). Accordingly, one important initiative of the EMR Office is the implementation of fiscal measures that have been constructed to support the actions for obesity prevention 2019–2023. These measures include taxes on SSB, in addition to other taxes and subsidies that promote healthier diets (WHO, 2019).\n\nThis research has the following objectives:\n\n1. Provide an overview of the interventions to discourage sugar intake and reduce consumption level;\n\n2. Measure the impact of sin taxes on the consumption and sales level of SSB in GCC countries, who applied sin taxes on SSB (Table 1).\n\n\nMethods\n\nThis paper measured the impact of sin taxes on SSB using a panel data set that covers sales volumes of soft drinks in the GCC from 2010 to 2020. The data were secondary data collected by Global Company Intelligence (GCI) (Underlying data (Megally, 2020)), which is a company that specializes in collecting data from national governments and international industrial companies. GCI created a report for the authors with the following variables for the period 2010–2020 of the GCC countries Saudi Arabia, UAE, Bahrain, Oman, Qatar and Kuwait: consumption volumes of soft drinks in million liters per year, percentage growth from previous period to current (PP Growth %), percentage growth from previous period to current period in million litres, and value of soft drinks in million dollars and local currency of each country per year.\n\nThe results have been analysed using STATA 15.0 starting with descriptive statistics, then testing the normal distribution of the time series of both independent and dependent variables using the Shapiro-Wilk test. Finally, the impact of sin taxes on sales volumes has been tested via t-tests, average treatment effects, difference-in-difference estimation, and separate regression analysis.\n\nMeasuring the average treatment effect and the difference-in-difference necessitate a random selection of the treatment and control groups conditioning on some observed characteristics X. This enhances an unbiased estimation of the treatment effect. Ravallion (2008) has illustrated a model to simplify the idea by assuming Yi (1) as YiT and Yi (0) as YiC where the following equation can be applied to a subsample of treated and untreated as follows:\n\n\n\n\n\nOne single regression can be estimated by pooling the data for both treatment and control groups ending up with the following:\n\n\n\nwhere εi=Ti(μiT−μiC)+μiC. Hence, the treatment effect that can be derived from equation 5.13 can be represented in ATT = E(Yi\|Ti = 1,X) = E[αT - αC + Xi(βT - βC)]. ATT refers to the treatment effect on the treated only. Given equation 5.13, the treatment effect can be consistently estimated with OLS under the following assumption E(μiT\|X,T=t)=E(μiC\|X,T=t)=0,t={0,1} that predicts no selection bias because of randomization. Practically, the common impact-model is usually assuming βT = βC, resulting with the average treatment effect ATE as αT - αC.\n\nFor the difference-in-difference, let’s assume the binary regressor explained as follows\n\n\n\nAssuming that yit fixed effects model with\n\n\n\nWhere αi is an individual-specific is fixed effect, and δt is a time-specific fixed effect. This is equivalent to regressing yit on Dit as well as fixed effects of set of time dummies and individual-specific effects. If there are no other regressors for simplicity, the individual effects αi can be reduced by first differencing concluding\n\n\n\nIn this view, the impact of the treatment ϕ can be estimated by pooled OLS regression of Δyit on ΔDit as well as time dummies set.\n\nIf we considered two period of time only instead of set of time periods assuming that treatment takes place in period 2 only, so for all individuals in period 1, Di1=0, but in period 2, Di2 = 0 only for the untreated individuals and Di2 = 1 for the treated. Hence, t subscript can be dropped from (6) to end up with\n\n\n\nWhere Di is a binary variable indicates whether the individual in the treated or untreated group. In that view, OLS regression of Δy on the binary regressor D and an intercept can be used to estimate the treatment effect. If we defined the sample average of Δyi for the treated by Δy¯tr where Di =1 and the sample average of Δyi for the nontreated is defined by Δy¯nt where Di = 0. Accordingly, the estimator of the OLS will be reduced to\n\n\n\nThis estimator represents the differences-in-differences (DID) estimator. It is called difference in difference as one of the differences estimates the difference in time for both the treated and nontreated groups and then this difference is taken in the time differences. Definitely, this can be extended from panel data to separate cross sections data if they are available in the two periods. The averages for the treated and untreated groups in the first period will be denoted in y¯1tr and y¯1nt and similar averages for both groups in period 2 can be denoted in y¯2tr and y¯2nt. This will be applicable if it possible that the individual has been identified as treated or untreated in the first period. Hence, the estimator will be as follows:\n\n\n\nA consistent estimation of ϕ for preceding formulation of the DID estimator requires certain assumptions. First assumption states that the time effects δt are common between the untreated and treated groups. Second assumption assumes stability of composing treated and untreated groups before and after the assignment of the treatment as the fixed effects αi is eliminated with panel data differencing. For the repeated cross-section data, it is implied from model (5) that y¯ttr and y¯tnt are denoted as follows:\n\n\n\n\n\nConsidering the occurrence of the treatment in the second period only, the following will bring about:\n\n\n\nWhere v=(ε¯2tr−ε¯1tr)−(ε¯2nt−ε¯1nt), The consistency of ϕ^ as in Equation (9) will occur if the assignment of the treatment is random and if plim (α¯2tr−α¯1tr)=0 and (α¯2nt−α¯1nt)=0\n\n\nResults\n\nTable 2 shows the decline in percentage change of SSB consumption due to imposing sin taxes, from 2016 to 2020 in all countries assessed. This reflects the preliminary effect of excise taxes on consumption that will be explained further in the following sub-sections of the Results. Saudi Arabia and UAE had implemented excise taxes on SSB in 2017, followed by Bahrain. Oman and Qatar had implemented the policy by the beginning of 2019, then, Kuwait was the last country in 2020 (Table 1) (Whitehead, 2019).\n\nThe growth rate of sales volumes decreased from 5.44% to 1.33% in Saudi Arabia, 7.37% to 5.93% in UAE, and 5.25% to 5.09% in Bahrain from 2016 to 2017. The growth rate of sales volumes in Qatar and Oman showed a drop from 2018 to 2019 (Qatar: 2.30% to 3.78%; Oman: 3.60% to 2.99%). In Kuwait, the growth rate of sales volumes decreased from 6.31% to 5.47% from 2019 to 2020.\n\nSales volume of SSB decreased when sin taxes have been applied on energy and soft drinks as the figures showed a difference in the sales volumes in the GCC countries from 2010 to the 2020.\n\nThe trend of sales volume of SSB between 2010 and 2020 is shown in Figure 4 where it increases over this time period. However, the rate of change of sales volume starts to decrease sharply in 2017 in Saudi Arabia, Bahrain and UAE, and 2019 in Qatar and Oman, which is when sin taxes were applied on the prices of SSB (Figure 5).\n\nThe normal distribution of sales volume, the growth rate of sales volume, as well as the value in million dollars had been tested before estimating the model using the Shapiro Wilk test, where H0 assumes a normal distribution of the variables. The results in Table 3 show that the time series of the variables are normally distributed, which qualifies them to be used in the regression model except for the sales volume of Bahrain and the growth rate of sales volume of Oman.\n\nBefore measuring the impact of sin taxes on each country separately, the difference of average change in sales volumes between the GCC countries who applied sin taxes in 2017 as a treatment group with the remaining GCC countries who applied the policy later in 2019 and 2020 as a control group has been measured via t-tests. The results of the first t-test observed that there is a significant decrease in the change of consumption volumes in the countries that applied the policy versus the control group who had not applied the policy in 2017 with significance <1%. The average decrease of sales volumes of SSB is represented by 2.637% less in the treatment group versus the control group (Table 4). In addition, the difference of average change in sales volumes for all GCC countries before the implementation of the excise tax policy in 2017 in some GCC countries versus after implementation. The results show that there is a significant decrease by 2.577% in the change of consumption volumes after 2017 relative to the change of consumption before 2017 in all GCC countries.\n\n* The coefficient is significant at <1% significance level\n\nTable 5 shows the estimation of average treatment effect (ATE). The average treatment effect of applying sin taxes on the growth rate of the consumption level of SSB has been measured using one outcome represented in the change in sales volume. The model observed that the potential means of the growth rate of consumption level controlling for the price of the beverages in dollars. The potential mean of the growth rate of consumption in the control group is 6.10%; while, the mean is lower for the countries in the treatment group. Such results estimated a negative impact of applying sin taxes on the growth rate of consumption levels that is represented by 2.87% less. This implies that the difference between the potential means of the growth rate of consumption of the treatment group countries versus the control is 2.87% less. The ATE is significant, which gives enough evidence to reject the null-hypothesis that states there is no difference between the means of the growth rate of consumption of both the treatment and control groups. These results support the estimated observations of the t-test.\n\n* The coefficient is significant at <1% significance level\n\nThe impact of the treatment has been also measured by difference-in-difference estimator comparing the difference in the growth rate of consumption level between the treated and control groups. In addition, the difference in the growth rate of consumption level have been compared over time with the variable time taking value of 1 starting from 2017 and later and value of 0 when year is before 2017. The difference in both differences is measured by the coefficient of difference-in-difference. The estimator shows a significant negative impact of sin taxes on the growth rate of consumption level, which showed a decrease in the growth rate by 3.03% in the treatment group countries relative to the control group countries after imposing the excise taxes policy after 2017 (Table 6).\n\n* The coefficient is significant at <1% significance level\n\nThe impact of sin taxes for each country has been tested via the following model:\n\n\n\nWhere SalesVolt refers to the change in sales volume in million litres and Pricet refers to the price of soft drinks in million US dollars.\n\nThe results show that sin taxes have a significant negative impact on the change of sales volume over years with significance level <5% and high R2. This implies that the decrease in rate of sales volumes is explained by the change in price due to the implemented sin taxes by around 51%, 40%, 55%, 99%, 71% in Qatar, Oman, UAE, Kuwait, and Saudi Arabia, respectively. This reflects that the implemented sin taxes decreased the rate of sales volumes of SSB in Qatar, Oman, UAE, and Saudi Arabia by 0.8%, 1%, 0.1%, <0.1%, respectively. However, the estimated impact of sin taxes did not show significant impact of sin taxes on sales volumes in Bahrain, which is supported by zero R2, This implies that the model does not estimate the impact of sin taxes on sales volumes. For Kuwait, the estimated model showed that the higher the value of soft drinks, the higher the consumption level. However, the estimated model of Kuwait should be estimated later since the policy had just been implemented in 2020 during the lockdown of COVID-19, so the sin taxes did not show an impact on Kuwaiti consumption volumes (see Table 7).\n\n The coefficient is significant at <5% significance level; *** The coefficient is significant at <1% significance level\n\n\nDiscussion\n\nNCDs are responsible for more than half of deaths every year in Qatar. More than two-thirds of the population (70.1%) are overweight or obese, and women are more likely than men to be obese. More than quarter (27%) of school children suffer from one or more forms of malnutrition (overweight, obesity, underweight or stunted growth). Elsewhere in the EMR, Qatar suffers from a high burden of NCD-related risk factors, such as physical inactivity, tobacco use, and unhealthy diets high in salt, sugar and fat (Al-Kaabi & Atherton, 2015). Similarly, UAE children are facing increased risk of obesity and overweight, and the frequency of obesity among youth is two to three times more than international standards. The implications of childhood obesity on public health are profoundly increased for UAE children and adults since adult chronic diseases increase the obesity rates (Al-Haddad et al., 2005; Malik & Bakir, 2007).\n\nIn addition, it had been shown that 25.7% of 15-19 aged Omani girls were obese/overweight. Based on the high consumption of sugary drinks among adolescents, as well as other practices that are categorized as unhealthy dietary practices, obesity among children becomes an increasing serious concern (Alasfoor et al., 2007; WHO & Oman MOH, 2010; WHO & Oman MOH, 2017). Similar prevalence rates were observed in Bahrain where the prevalence of obese and overweight males ranged from 15.7% to 28.9% and from 21.1% to 30.7% among females. High consumption of fast food, sugary beverages, chocolates and sweets are expected to have the highest contribution to the high prevalence rates of obese children in Bahrain (Musaiger et al., 2011). It has been also observed that obesity is considered a problem among Bahrani school children that led them to call for interventions that eradicate obesity among schoolchildren (Musaiger et al., 2014).\n\nFurthermore, Kuwait also has an increasing trend of overweight and obesity. Prevalence of overweight adults increased from 71.7% to 73.4% from 2012 to 2016, and prevalence of obesity increased from 35.6% to 37.9% from 2012 to 2016 (WHO, 2016f). Similarly in Saudi Arabia, the obesity and overweight prevalence rate has increased among Saudi Arabian children alarmingly (Al-Hussaini et al., 2019).\n\nPrevalence rate of obesity, overweight and deaths due to diabetes have increased in the last decade in the EMR and the rate is higher for children living in GCC countries (Abdul-Rasoul, 2012). NCDs play a major role in the high rate of deaths in GCC countries annually. It does not only affect adults, but schoolchildren suffer from diabetes, obesity, overweight, underweight and stunting growth (WHO, 2016d).\n\nThese facts prompted GCC countries to introduce an operational policy for diabetes, and an operational policy to reduce unhealthy diet related to NCDs, which was imposed in 2017 (WHO, 2016e). Consequent, the current study aimed to measure the effect of implemented excise taxes on the reduction of SSB consumption. One of the main aims of the operational policies is increasing the consumption of healthy food in schools. Reaching such results requires cooperation between the Ministry of Health of GCC countries and the WHO. Considerable progress to implement the action plan has been achieved between 2011 and 2018 (as shown in our study, Figure 5), and one significant action was implementing a new tax on carbonated drinks (50%) and energy drinks (100%) (Whitehead, 2019).\n\nOur results showed that the rate of change in sales volume over the last decade in GCC countries started to decrease sharply in the year when sin taxes were applied to the prices of soft drinks. Sales volumes were increasing but at decreasing rates, and the sin tax had a significant negative impact on the change of sales volumes over the past 10 years. However, Kuwait has applied the sin taxes policy in 2020, and accordingly, the estimated results can not be relied on as the model needs to be estimated in a few years to be valid enough to estimate the impact of the sin taxes policy on the consumption of SSB. In addition, the policy had been applied in the time of the COVID-19 lockdown, which may play a role in making the policy ineffective. Also, the results showed that the model did not estimate the impact of sin taxes on the sales volumes of SSBs in Bahrain as the model may require more explanatory variables to explain the variation of consumption due to the variation in SSB price. Generally, the estimated difference between the potential means of the growth rate of consumption of the treatment group countries versus the control group is 2.87% less. The growth rate of sales volumes decreased (Alsukait, et al., 2020; Megally & Al-Jawaldeh, 2020) from 5.44% to 1.33% in Saudi Arabia, 5.25% to 5.09% in Bahrain, and 7.37% to 5.93% in UAE from 2016 to 2017. Also, the growth rate of sales volumes in Oman and Qatar showed a decrease from 2018 to 2019 as their excise taxes have been implemented in 2019 (Oman: 3.60% to 2.99%; Qatar: 2.30% to 3.78%). Kuwait was the last GCC country to implement the excise taxes in 2020 and the figures shows that the growth rate of sales volumes decreased slightly from 6.31% to 5.47% from 2019 to 2020. These results suggest the application of sin taxes for countries in the EMR.\n\nThese results align with the nutrition strategies of the United Nations of Action on Nutrition that was based on evidence and experimental studies. Such studies observed effective fiscal measures of taxes and subsidies and proved its effectiveness in shifting habits of promotion and purchase of dietary change (Thow et al., 2014; WHO, 2016g; WHO, 2015b). Also, the implemented excise taxes are expected to decrease the obesity levels among GCC’s children over the coming years. Such expected improvements in the health of the children will improve intelligence cognition and schooling performance of the children. Furthermore, these studies showed a decrease of the percentage change of sales volume, which supports the evidence of the countries that applied taxes and decreased the purchases of SSB in a range of 20–50% (Colchero et al., 2016; Ells et al., 2015; Mozaffarian et al., 2012; Powell et al., 2013; Thow et al., 2014; WHO, 2016g; WHO, 2015b).\n\n\nConclusions and recommendations\n\nReducing obesity among children in the EMR received great attention recently given its high prevalence rate. One health economic action is imposing excise taxes on SSB (Lobstein, 2014). The main aim of the current study was to measure the impact of applying such taxes on the consumption of SSBs. The estimated results showed a positive effect of applying sin taxes in terms of decreasing the consumption level of SSB in Qatar, Oman, UAE, and Saudi Arabia, but not in Bahrain and Kuwait.\n\nAccordingly, such policy is recommended to be implemented in other EMR countries. Furthermore, GCCs should proceed with promoting nutritional quality of foods as proposed by the roadmap of the WHO, as well as generating measures for education, marketing, and promotion for nutritious foods. Awareness campaigns should take place to promote reducing the consumption of SSB and substitute with more consumption of water, unsweetened milk for children, fresh fruits and vegetables. These recommendations align with the recommended priority actions by the WHO for the strategy on nutrition for the EMR 2020–2030 (WHO, 2019).\n\n\nData availability\n\nHarvard Dataverse: Soft Drinks Volumes - GCC, https://doi.org/10.7910/DVN/6OFWQE (Megally, 2020).\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\nAbdul-Rasoul MM: Obesity in Children and Adolescents in Gulf Countries: Facts and Solutions. Avences en Diabetologia. 2012; 28(3): 64–69. Publisher Full Text\n\nAlasfoor DH, Rajab H, Al-Rassasi B: Food Based Dietary Guidelines, Technical Background and Description. Oman Ministry of Health, Editor. Muscat: Ministry of Health. 2007. Reference Source\n\nAl-Haddad FH, Little BB, Abdul Ghafoor AG: Childhood obesity in United Arab Emirates schoolchildren: a national study. Ann Hum Biol. 2005; 32(1): 72–79. 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Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/6OFWQE\n\nMegally R, Al-Jawaldeh A: Impact of sin taxes on consumption volumes of sweetened beverages and soft drinks in Saudi Arabia [version 1; peer review: 2 approved]. F1000Res. 2020; 9: 1117. Publisher Full Text\n\nMozaffarian D, Afshin A, Benowitz NL, et al.: Population approaches to improve diet, physical activity, and smoking habits: a scientific statement from the American Heart Association. Circulation. 2012; 126(12): 1514–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMusaiger AO, Al-Mannai M, Al-Marzog Q: Overweight and Obesity among Children (10-13 years) in Bahrain:A comparison between Two International Standards. Pak J Med Sci. 2014; 30(3): 497–500. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMusaiger AO, Bader Z, Al-Roomi K, et al.: Dietary and Lifestyle Habits amongst Adolescents in Bahrain. Food Nutr Res. 2011; 55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOkubo H, Miyake Y, Sasaki S, et al.: Feeding practices in early life and later intake of fruit and vegetables among Japanese toddlers: the Osaka Maternal and Child Health Study. Public Health Nutr. 2016; 19(4): 650–657. PubMed Abstract \| Publisher Full Text\n\nPizzi MA, Vroman K: Childhood obesity: effects on children's participation, mental health, and psychosocial development. Occup Ther Health Care. 2013; 27(2): 99–112. PubMed Abstract \| Publisher Full Text\n\nPowell LM, Chriqui JF, Khan T, et al.: Assessing the Potential Effectiveness of Food and Beverage Taxes and Subsidies for Improving Public Health: A Systematic Review of Prices, Demand and Body Weight Outcomes. Obesity Reviews. 2013; 14(2): 110–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRavallion M: Evaluating Anti-Poverty Programs. Handbook of Development Economics, ed. T. Paul Schultz and John Strauss, Amsterdam: North-Holland. 2008; 4: 3787–3846. Publisher Full Text\n\nThow AM, Downs S, Jan S: A Systematic Review of The Effectiveness of Food Taxes and Subsidies to Improve Diets: Understanding the Recent Evidence. Nutr Rev. 2014; 72(9): 551–65. PubMed Abstract \| Publisher Full Text\n\nVentura AK, Worobey J: Early influences on the development of food preferences. Curr Biol. 2013; 23(9): R401–R408. PubMed Abstract \| Publisher Full Text\n\nWhitehead R: Things Have Just Got Tougher for Gulf Beverage Brands with Expansion of Sugar Tax. William Reed Business Media Ltd. Retrieved on September-2-2020. 2019. Reference Source\n\nWorld Health Organization: Policy Statement and Recommended Actions for Lowering Sugar Intake and Reducing Prevalence of Type 2 Diabetes and Obesity in The Eastern Mediterranean Region. Retrieved on May 29, 2020. 2020. Reference Source\n\nWorld Health Organization: Strategy on Nutrition for the Eastern Mediterranean Region 2020–2030. Cairo: WHO Regional Officer for the EasternMediterranean. License: CC BY-NC-SA 3.0 IGO. 2019. Reference Source\n\nWorld Health Organization: Consideration of The Evidence on Childhood Obesity for The Commission on Ending Childhood Obesity: Report of The Ad Hoc Working Group on Science and Evidence for Ending Childhood Obesity. World Health Organization: Geneva; 2016a. Reference Source\n\nWorld Health Organization: Fiscal Policies for Diet and The Prevention of Noncommunicable Diseases.Technical Meeting Report 5 – 6 May 2015, Geneva, Switzerland. Geneva: World Health Organization Regional Office for Europe; 2016b. Reference Source\n\nWorld Health Organization: Global Report on Diabetes. World Health Organization: Geneva; 2016c. Reference Source\n\nWorld Health Organization: Number of Deaths due to Cancer, Diabetes Mellitus, Cardiovascular Diseases and Chronic Pulmonary Diseases. Global Health Observatory Data Repository. 2016d. 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Publisher Full Text" }	[ { "id": "74057", "date": "18 Nov 2020", "name": "Nasrin Omidvar", "expertise": [ "Reviewer Expertise I am a nutritionist with experience in program planning and evaluation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is very interesting and is addressing an important public health issue. Overall, it is well organized and properly presented. However, there are a few minor points that is worth revising before indexing:\nIntroduction: In line 3, in the sentence that starts with “Around 7% of under-five aged children ..” there is a proposition “in” that probably needs to be omitted (highlighted in the attachment). In the same paragraph, the sentence that I underlined, needs a reference (Please see attached).\nResults: In the first line, is table two on SSB consumption or sales? The second paragraph of the result is repetition of table two. It is recommended to instead just highlight some of the significant points in the table.\nDiscussion: On page 9, 2nd column, what is the point of the last line of the first paragraph (highlighted). It is recommended to mention the study strengths and limitations at the end of discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\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": "79275", "date": "08 Mar 2021", "name": "Sally Sawaya", "expertise": [ "Reviewer Expertise Nutrition and dietetics." ], "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 general, the purpose of this study is important as it explores the impact of implementation of SSB taxes in GCC countries on sales volume and growth rate of soft drinks. It aims to show a positive impact of sin taxes on purchases of SSB as harmful foods for mitigating NCDs. However, there are important aspects that need to be clarified throughout the paper. E.g. Childhood obesity was dealt with as the main expected outcome tackled in the article, yet SSB taxes reflects on both childhood and adult overweight and obesity. It is important that the authors first introduce the link between SSB and childhood obesity, to conform with objective of the article The below paragraph from WHO website is suggested: \"The consumption of sugar-sweetened beverages has been suggested as a contributing factor to the rising levels of childhood obesity in many countries worldwide. Recent systematic reviews of the literature confirm the link between increased intake of free sugars, particularly in the form of sugar-sweetened beverages and unhealthy weight gain in both children and adults (1, 2) while reducing consumption of sugar-sweetened beverages has been shown to reduce weight gain in children, particularly in those who are already overweight (2–4)\".\n\nThe data provided in the study were restricted to industry data which did not attempt to rule out other confounding socioeconomic and demographic variables that might have impacted the sales. The study did not also attempt to show the positive effect of sin taxes on obesity or other NCDs in the population following implementing the taxes for few years.\n\nIt is recommended that the study methodology and different equations proposed be evaluated by an expert statistician to confirm if they provide the sought after answers.\n\nRegression analysis ruling out factors, other than Sin taxes, that might have impacted sales are also needed before this paper would provide the needed answers to the research question.\nThis paper cannot be accepted for indexing before ruling out the relevance of the statistical results to the purpose of the study.\nSpecific observations\nThe abstract It is reported in the abstract that: \"reduction in sales volumes was observed from 2018 to 2019 (Qatar: 2.30% to 3.78%; Oman: 3.60% to 2.99%)”, However the figures for Qatar show an increase in sales.\nIn the abstract, the conclusion does not refer to the objective of the paper which is documenting the influence of sin taxes on the purchases of SSB and reduction of NCDs and then possibly complement them with the idea of awareness campaigns for a more pronounced and beneficial effect on food habits and curbing obesity in the region, especially childhood obesity.\nIntroduction : Lines 1 and 2 “More than half of the adult women (50.1%) and more than 40% of men are obese or overweight in the EMR” need a reference. The “y” axis in figures 1,2 and 3 need to be labelled. Line 12: need to be re-phrased: “Moreover, obese children can be highly affected in their educational attainments and quality of life.” Line 15: “There is a correlation between the high prevalence rates of overweight and obesity with the high prevalence rates of diabetes”. To prevent repetition, it can be re-phrased simply as: Notably, there is a strong correlation between obesity and prevalence of diabetes. Line 18: Missing words: Figure 1–Figure 3 show the increase in prevalence of childhood obesity, overweight, and deaths due to diabetes in the GCC from 2010 to 2016. Line 28: “which aims to reduce significant increases in the prevalence rates of obesity and overweight that have drawbacks on health and whole economy in the short and long run”. Can be re-phrased and corrected as: which aims to reduce the prevalence rates of obesity and overweight, which have drawbacks on health and the whole economy on the short and long run. 3rd paragraph (line 37 onwards): The example of forbidding TV advertisement is not an example of raising awareness about importance of good nutrition in kids. It’s protecting them from marketing of these products. Raising awareness in children can be through school educational programs as highlighted at the end of the paragraph. Please can the authors align the ideas for better flow. “Nutrition education programs should be run for long periods as it has been shown that short-term education had no impacts on changing dietary intake and behaviour in spite of the fact that it raises nutritional knowledge” can be changed to: Essentially, nutrition education programs are more effective when they are implemented for long periods, as it has been shown that short-term education had no impact on changing dietary intake and behaviour in spite of the fact that it raises nutritional knowledge. Please can the author highlight this idea in the Discussion: did any of the countries implement these educational programs or ban SSB ads to kids in parallel to the taxes?\nWorld Health Organization recommendations and fiscal policies 1st paragraph: “The World Health Organization (WHO) suggests a decrease of the intake of free sugar to less than 10% of the total consumed calories given the increasing rate of added sugar intake” may be rephrased as: “With the rise in the intake of added sugar, the World Health Organization (WHO) recommends a decrease in free sugar intake to less than 10% of the total calories consumed”. 2nd paragraph: “Children usually are exposed to sweetness when they are infants, which increases their intake of sweets when they grow up (Foterek, 2016; Okubo et al., 2016), so it is crucial to construct an environment that decreases their intake of added sugar.” Consider changing to: \"Children are usually exposed to sweet taste when they are infants, which increases their intake of sugary foods when they grow up (Foterek, 2016; Okubo et al., 2016). Hence, it is crucial to construct an environment that discourages the intake of added sugar”. Paragraph 3 recommending long term nutrition education is out of context and does not provide the background needed for the paper. The introduction should cover success rate or impact of sin taxes in other countries and provide data on what interventions proved to be more impactful such as long term nutrition education. Paragraph 4 does not include the % sugar intake in GCC countries which provoked the intervention to decrease it, or provide the evidence for WHO recommendation on limiting energy from sugar to 10%. A background on evidence of a the link between SSB and obesity is needed.\nObjectives:\nThe first objective is not evident. Only the second objective is tackled in the paper.\nMethods: The authors wrote “equation 5.13”, is it equation (3)?\nResults: Throughout the paper the description of results on Qatar need to be modified. Qatar did not experience reductions in sales volumes from 2018 to 2019 when the taxes were implemented (first pointed out in Abstract and line #6 of Results (Qatar: 2.30% to 3.78%), so it actually increased); the reduction was seen before the sin taxes were implemented and only decreased between 2019 and 2020 (Table 2). The author need to address this change where applicable and highlight this delayed effect in the Discussion. The write-up needs improvement as specifically shown in the upcoming comments. Table 2: Is the change in percent growth from year to year significant? Can the author please indicate with symbols where significant differences lie? 1st paragraph: “This reflects the preliminary effect of excise taxes on consumption that will be explained further in the following sub-sections of the Result.” Suggested change: “This reflects the preliminary effect of excise taxes on consumption that will be further explained in the following sub-sections.” 2nd paragraph: “The growth rate of sales volumes in Qatar and Oman showed a drop from 2018 to 2019 (Qatar: 2.30% to 3.78%; Oman: 3.60% to 2.99%)”. It cannot be stated that Qatar showed a drop, the growth rate of sales volumes actually increased. Estimating the impact of sin taxes on the change in sales volume suggest changing over years to overtime\n1st paragraph: “Sales volume of SSB decreased when sin taxes have been applied on energy and soft drinks as the figures showed a difference in the sales volumes in the GCC countries from 2010 to the 2020.” I thought we were talking about soft drinks; does that including energy drinks? In the methods section, it was written that the data measured soft drinks. So, if it included energy drinks and sweetened juices, this needs to be highlighted. Figure 5: Again, it’s shown in this figure that Qatar shows an increase upon first implementation. The authors need to amend the text.\nTesting normal distribution of the time series variables suggested changes: 2nd paragraph: “The results of the first t-test observed that there is a significant decrease in the change of consumption volumes in the countries that applied the policy versus the control group who had not applied the policy in 2017 with significance <1%.” To be consistent, the author should be talking about sales volume rather than consumption volume throughout this paragraph. “The average decrease of sales volumes of SSB is represented by 2.637% less in the treatment group versus the control group (Table 4).” Instead of less, can be “reduction”. “In addition, the difference of average change in sales volumes for all GCC countries before the implementation of the excise tax policy in 2017 in some GCC countries versus after implementation. The results show that there is a significant decrease by 2.577% in the change of consumption volumes after 2017 relative to the change of consumption before 2017 in all GCC countries.” The authors need to combine these sentences as the first sentence is incomplete and need to be rephrased. 3rd paragraph: The statement “The model observed that the potential means of the growth rate of consumption level controlling for the price of the beverages in dollars.” is not clear. The authors need to explain what is meant by number of observations in Tables 4 and 5 (to be highlighted in the footnotes). Table 5 title: is repetitive and needs to align with text explanation, suggestion as follows: “Estimation of average treatment effect on the growth rate of SSB consumption”. “The potential mean of the growth rate of consumption in the control group is 6.10%; while, the mean is lower for the countries in the treatment group.” What is the mean in the treatment group? The author need to add this to Table 5. The statement “This implies that the difference between the potential means of the growth rate of consumption of the treatment group countries versus the control is 2.87% less.” May be rephrased to: This implies that the potential mean of growth rate of SSB consumption was reduced by 2.87%. 5th Paragraph: “The impact of the treatment has been also measured by difference-in-difference estimator comparing the difference in the growth rate of consumption level between the treated and control groups.” Better to use treatment instead of treated and the explanation of difference-in-difference should be in Methods section, not Results. Table 6 title needs to be more comprehensive, suggestion: “Difference-in-difference estimation in the growth rate of consumption.” Again, the explanation model of Sales Vol should be done in Methods section. 6th Paragraph: The statement “This implies that the decrease in rate of sales volumes is explained by the change in price due to the implemented sin taxes by around 51%, 40%, 55%, 99%, 71% in Qatar, Oman, UAE, Kuwait, and Saudi Arabia, respectively.” Would fit better in the Introduction. The results section should report the findings of Table 7, rather than explain them. Explanations should appear in the discussion section. Methods used in this section needs to be reviewed by an expert statistician to determine whether an increase in population has occurred and was taken into consideration. It is not clear what the equations were used for. There was no attempt to measure change in obesity prevalence in countries that implemented taxes for long periods which would provide evidence of impact.\nDiscussion: In the discussion, the authors expect a decrease in obesity due to decrease in SSB consumption without providing the evidence showing that lower consumption of SSB will impact obesity. The first 4 paragraphs in the discussion need to be in the Introduction as they are relevant to the topic at hand. In the discussion, the authors need to summarize the statistics of GCC countries childhood obesity and not go into lengthy discussion of adult obesity and its effects. It is important here to discuss the findings of the current study. Were there any previous studies in the GCC that measured consumption after certain sin taxes were implemented? Relate their findings to this study if applicable. Can the author discuss why Saudi Arabia had a more pronounced effect on sales and consumption? Also propose why there was a delayed effect in Qatar, whereas immediate effects in other countries. Could it be that national awareness campaigns were put into effect later on? The authors also need to discuss why a year after the implementation of sin taxes, the numbers started increasing again. 7th paragraph: This paragraph is what the Discussion should be about. It supports the study’s findings and states its expectations. “Furthermore, these studies showed a decrease of the percentage change of sales volume, which supports the evidence of the countries that applied taxes and decreased the purchases of SSB in a range of 20–50%.” (Colchero et al., 2016; Ells et al., 2015; Mozaffarian et al., 2012; Powell et al., 2013; Thow et al., 2014; WHO, 2016g; WHO, 2015b). It is important that the authors go into more details and discuss examples from these papers and relate them to their study findings. Additionally, it would be good to know if any of the countries implemented educational programs or banned SSB ads to kids in parallel to the taxes? If not, then it can be added as a recommendation in order to have a more pronounced effect on SSB consumption.\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": [ { "c_id": "6719", "date": "29 Jun 2021", "name": "Rania Megally", "role": "Author Response", "response": "First of all, I do like to express our appreciation for your helpful supporting comments that added a great value to improve the paper. Almost all your comments have been taken into considerations and the paper has been modified accordingly and re-submitted after modifications. We are looking for your feedback on the latest version of the paper" } ] } ]	1	https://f1000research.com/articles/9-1287
https://f1000research.com/articles/10-65/v1	03 Feb 21	{ "type": "Software Tool Article", "title": "iCAT: diagnostic assessment tool of immunological history using high-throughput T-cell receptor sequencing", "authors": [ "Ahmad Rajeh", "Kyle Wolf", "Courtney Schiebout", "Nabeel Sait", "Tim Kosfeld", "Richard J. DiPaolo", "Tae-Hyuk Ahn", "Ahmad Rajeh", "Kyle Wolf", "Courtney Schiebout", "Nabeel Sait", "Tim Kosfeld" ], "abstract": "The pathogen exposure history of an individual is recorded in their T-cell repertoire and can be accessed through the study of T-cell receptors (TCRs) if the tools to identify them were available. For each T-cell, the TCR loci undergoes genetic rearrangement that creates a unique DNA sequence. In theory these unique sequences can be used as biomarkers for tracking T-cell responses and cataloging immunological history. We developed the immune Cell Analysis Tool (iCAT), an R software package that analyzes TCR sequencing data from exposed (positive) and unexposed (negative) samples to identify TCR sequences statistically associated with positive samples. The presence and absence of associated sequences in samples trains a classifier to diagnose pathogen-specific exposure. We demonstrate the high accuracy of iCAT by testing on three TCR sequencing datasets. First, iCAT successfully diagnosed smallpox vaccinated versus naïve samples in an independent cohort of mice with 95% accuracy. Second, iCAT displayed 100% accuracy classifying naïve and monkeypox vaccinated mice. Finally, we demonstrate the use of iCAT on human samples before and after exposure to SARS-CoV-2, the virus behind the COVID-19 global pandemic. We were able to correctly classify the exposed samples with perfect accuracy. These experimental results show that iCAT capitalizes on the power of TCR sequencing to simplify infection diagnostics. iCAT provides the option of a graphical, user-friendly interface on top of usual R interface allowing it to reach a wider audience.", "keywords": [ "T-cell receptor sequencing", "diagnostic classification", "R-package", "biomarkers" ], "content": "Introduction\n\nT- and B-cell responses are responsible for long-lasting immune memory responses to infectious agents, such as bacteria and viruses. Expansion of pathogen specific T-cells provide us with a robust resource for understanding whether an individual has been infected with a pathogen. The T-cell receptor (TCR), located on the surface of T-cells, is responsible for recognizing pathogen-specific peptides, leading to immune response and development of protective immune memory 1 . During T-cell development, the loci that encode TCRs α and β-chains are rearranged by recombination of the variable (TCRV), diversity (TCRD), and joining (TCRJ) gene segments, encoding the complementary determining region 3 (CDR3) 2 . These genetic rearrangement events result in a high degree of diversity in the CDR3 regions of individual TCR loci 3 .\n\nDuring an infection or vaccination, T-cells that carry receptors recognizing pathogen associated peptides become activated and undergo rapid clonal expansion. The clonally expanded T-cells carry the same unique TCR rearrangement and a portion remain in circulation long after the pathogen has been cleared to provide long-lived immunological memory. The persistence of memory T-cells in circulation make the genetically rearranged TCR loci a stable biomarker documenting an individual’s immunological history. To utilize the diverse TCR repertoire as a potential biomarker for specific pathogen exposure, pathogen-specific TCR sequences common to different individuals exposed to the same pathogen need to be identified. This poses significant challenges given the diversity and magnitude of the TCR repertoire. On average, ∼ 107 unique TCRβ chains can be identified from the ∼ 1012 circulating T-cells present in a healthy human adult 4 . A healthy human adult can have 1018 mathematically possible TCR recombinations resulting from the genetic rearrangement 4 , 5 . The potential diversity of the repertoire coupled with the limited number of T-cells present in individuals makes identifying identical TCR sequences among multiple individuals exceptionally challenging. However, by analyzing the large and diverse TCR repertoire using high-throughput TCRβ sequencing, it is possible to identify pathogen specific TCRs shared among different individuals exposed to the same infectious agent 6 .\n\nRecently, high-throughput next-generation sequencing (NGS) techniques were employed to analyze the diverse immune cell repertoire 7‐9 . Additionally, recent publications described an analytical approach for computationally identifying common/public TCR sequences 5 , 9 , 10 . However, analyses of the TCR repertoire for diagnostic purposes have remained largely resource- and time-intensive efforts.\n\nUtilizing the diagnostic methodologies described in Wolf, et al. (2018) 5 , we have developed an R package with a user-friendly interface, the immune Cell Analysis Tool (iCAT), to identify TCR sequences statistically associated with pathogen exposure and to distinguish infected from non-infected samples. By providing an interactive interface through iCAT, sample exposure can be assessed and predicted conveniently without requiring command-line skills. iCAT has an ability to classify target-associated receptor sequences (TARSs) and diagnose exposure with a high accuracy.\n\n\nMethods\n\nWe developed iCAT, an R package utilizing high-throughput TCR sequencing data to analyze TCR sequences and to diagnose infection in a user-friendly format (Figure 1). iCAT provides both a graphical user interface (GUI) in the form of a web-application utilizing R-Shiny 11 and a command-line R interface for batch processing of large-scale data. The simplest method to install iCAT on a system is directly from GitHub using devtools and install_github:\n\nA) Flow chart depicting the purification of genomic DNA from blood samples and the production of TCR repertoires after TCR-specific amplification and sequencing. B) Visual representation of the iCAT methodology.\n\nIn addition to shiny, iCAT also uses shinyjs, data.table, ggplot2, DT, hash, and magrittr. Required packages will be installed through the install_github step. Alternatively, users can clone or download the repository from GitHub and run devtools::install(\"iCAT/\").\n\nA user can upload multiple TCR sequence repertoires from negative (control) and positive (experimental) cohorts. iCAT accepts tab-delimited files with the size limit of 10 gigabytes per file with multiple options to define TCR clonotypes within samples. The iCAT shiny app has three tabs, separating major functionalities: Training, Library, and Prediction. Under the “Training” tab, clicking ‘Train Model’ will start the pipeline to statistically identify a subset of TARSs that will act as feature selections for training the diagnostic classifier, diagnosing samples as either negative or positive. Upon training, iCAT’s main tab provides a table summary of the data, a figure shows the distribution of TARSs between the positive and negative samples, and a classification matrix predicting the exposure status of samples used in the training data (Figure 2). All figures and tables can be downloaded to the user’s machine. A progress bar will show on the bottom-right corner to update on the status of training.\n\nAfter samples are uploaded, clicking “Training” will start training to select features for the diagnostic classifier from the negative and positive samples.\n\nA separate tab, “Library”, is unlocked upon training and shows a table where each row describes a TARS and its presence in the positive and negative samples. All tables and figures are supplemented with a custom button for easy download (Figure 3).\n\nThe library tab shows a table of target-associated receptor sequences (TARS).\n\nThe third tab of iCAT, “Prediction”, also unlocks after training and allows the user to upload one or more independent TCR-sequencing samples for classification (Figure 4).\n\nThe prediction tab allows the user to upload one or more independent TCR-sequencing samples for classification.\n\nSamples, such as from blood or lymph tissue, are collected and genetic material is purified. TCR sequences present in the sample are selectively amplified and then sequenced (Figure 1A). The first step of iCAT is the “Training” step. A user should provide multiple negative training samples (naïve, unexposed, uninfected, etc.) using the Browse button. Then, repeat the step for positive training samples (exposed, infected, vaccinated, etc.). The user should select the type of training feature. iCAT provides three options: (1) CDR3 Amino Acid Sequence (TCRs will need the same CDR3 region to be called ”Identical”), (2) TCRV-CDR3-TCRJ (TCRs will need the same TCRBV segment, CDR3 region, and TCRJ segment to be called ”Identical”), (3) Nucleic Acid (DNA) (TCRs will need the exact same DNA rearrangements/sequence across TCRBV, CDR3, and TCRJ). Selecting TCRV-CDR3-TCRJ is recommended as a balance between sensitivity and specificity and this option has been used for all the use cases in this paper. In addition, users can customize the range acceptable of copies per clonotype, and the minimum threshold of public sequences, which determines the minimum samples a TCR sequence must be observed in to be considered for analysis.\n\nOne important option of this “Training” tab is Max p-value (default: 0.1), which determines the minimal degree of statistical significance that iCAT will accept as being potentially ”associated” with the positive group. The statistical methodology of iCAT is based on identifying a subset of TARSs that informs classification 5 . TCR sequences significantly associated with positive samples as opposed to negative samples are identified by performing a one-tailed Fisher’s exact test. iCAT determines the optimal p-value cutoff to generate the TARS library based on the idea of coverage ratio. To determine an optimal p-value threshold for identifying vaccine-associated TCRβ sequences, we applied a heuristic test that selected the optimal p-value threshold based on the ”coverage” provided by the library for both vaccinated (C v ) and naïve samples (C n ) 5 . “Coverage” is defined as the summation of the number of samples containing each VATS divided by the number of samples. In the equations below, x i denotes the number of vaccinated samples a single TCRβ is identified and n v represents the number of positive samples in the training data. y i also denotes for naïve samples and n n represents the number of naïve samples.\n\nFor the classification of vaccinated and naïve samples, iCAT calculates the percentage of TARS (% TARS) present in a sample. The % TARS for each sample in the training data is compared against the % TARS normal distribution for each group to predict if each sample is ”positive” or ”negative”, determined by which group a sample is more closely associated with 5 . Such a normal distribution has been adopted to calculate the distance of a sample to the mean. In detail, the normal distributions for the naïve and vaccinated populations in our training data were calculated based on a function of the difference between a single sample value (x) and the mean of a set of data (μ) over the standard deviation of that set of data (σ) utilizing a correction constant to remove negative selection bias of the naïve training sample data from the below equation.\n\nThe “Library” tab displays a table consisting of the TARS, determined to be statistically associated with exposure to the target/agent/pathogen (Figure 3). The table displays each sequence, number of positive and negative training samples the sequence is present in/absent from, and how statistically associated the sequence is to the positive training data (p-value). The table can be downloaded to the user’s computer for further analysis (Figure 3).\n\nTo allow the diagnostic classifier to test independent cohorts of samples, a third tab in iCAT, “Prediction” tab, also unlocks after training and allows the user to upload one or more independent TCR-sequencing samples for classification using the parameters generated by the training data (Figure 4). The “Prediction” tab allows the user to diagnose unknown samples (e.g. not included in the previous training data) for classification as “Positive” or “Negative” and determining the accuracy of the diagnostic assay. Use the Browse button to upload such independent samples for prediction. Multiple samples can be uploaded simultaneously. Click Predict Independent Sample will analyze the dataset. A table will appear after analysis is complete. The table displays sample names along with the prediction ”Positive” (red) or ”Negative” (blue), and displays the %TARS that is the percent of individual sequences from the sample that are included in the TARS library. The prediction results can be downloaded as a table.\n\niCAT requires R 3.4.0 or upper and can be run on any operating system with common specifications (1 GB disk space, 4 GB memory, and multicore CPU is recommended).\n\n\nUse cases\n\nTo evaluate the efficacy of iCAT, we used three TCR sequencing data sets that are publicly available. The first viral data set consists of 148 training and 20 independent mouse samples. The training set has 32 mouse pre-treatment naïve group and 116 vaccinated samples, each cohort inoculated intranasally with the ACAM2000 smallpox vaccine. The second viral data set consists of 133 (27 negative and 96 positive) training and 15 (5 negative and 20 positive) monkeypox virus. The two mouse datasets are publicly available from https://doi.org/10.17632/cf92gt44zf.1. The third data set is human TCR samples exposed to the novel SARS-CoV- 2 virus which is the cause of the ongoing COVID-19 global pandemic 12 . The sample size is small (two cohorts), but those two cohorts’ TCR repertoires were obtained for other projects one and two years prior to infection. Therefore, this negative and positive SARS-CoV-2 human TCR sequencing data set from same cohorts can be a great example to show the great potential of iCAT as a diagnostic assay. This data is available at https://doi.org/10.5281/zenodo.3835956.\n\n32 pre-exposure (naïve) samples were analyzed, which included 2,049,383 unique TCR sequences (clonotypes). We setup iCAT options to analyze by either the CDR3 amino acid sequence or the V-gene and J-gene names. 714,522 amino acid sequence-gene name combinations were found in the naïve samples. 58 samples taken 2- and 8-weeks post-vaccination for smallpox were analyzed, which included 1,581,619 clonotypes and 573,612 unique amino acid sequence-gene name combinations. After training, iCAT accurately generated the same virus-associated TCR library (314 TCR sequences) identified in Wolf, et al., 2018 5 . From the training data, iCAT correctly classified 32 of 32 naïve samples as ”unexposed” and 58 of 58 vaccinated samples as ”exposed” (100% accuracy). We utilized TCR-sequencing files from 10 mice pre- and post-smallpox vaccination that were not involved in the training of the diagnostic classifier to act as independent cohorts to test the diagnostic accuracy of the iCAT generated classifier. The classification results are displayed in the “Prediction” tab and can be downloaded as a .txt file. From a total of 20 samples, 90% of pre-vaccination samples (9 of 10) were correctly classified as “negative” and 100% of samples post-smallpox vaccination (10 of 10) were classified as ”positive”. Overall, this data displays that the iCAT platform computationally identifies target-associated public TCRs, utilized to train a diagnostic classifier capable of distinguishing between exposed and unexposed samples with a high degree of accuracy.\n\nWe tested iCAT using another TCR-sequencing mouse dataset which included 27 naïve samples and 48 samples 2- and 8-weeks post infection with monkeypox. We chose to analyze based on CDR3 amino acid sequence in addition to V-gene and J-gene names. The p-value cutoff was set to 0.1 and the minimum number of public sequences was set to 1. Those parameters produced the best separation experimentally. Naïve samples included 1,772,085 clonotypes and 630,381 unique amino acid sequence-gene name combinations. Exposed samples included 1,070,615 clonotypes and 382,906 unique amino acid sequence-gene name combinations. iCAT correctly classified this training data with 100% accuracy. When tested on an independent monkey pox data set – set up by excluding 5 samples from the naïve group and 10 samples from the exposed group pre-training – iCAT correctly classified the 5 naïve samples as negative and the 10 exposed samples as positive. Thus, we demonstrated a 100% classification accuracy using iCAT on this monkeypox dataset.\n\nWe further tested iCAT on TCR-sequencing data from two human individuals exposed to the novel SARS-CoV-2 virus 12 . Data included 4 naïve samples from 2018 and 2019, and 4 samples collected 15- and 30-days post-infection. iCAT was configured to analyze by CDR3 amino acid sequence only. The p-value cutoff was set to 0.1 and the minimum number of public sequences was set to 1. The naïve data included 2,935,893 clonotypes and 1,120,606 unique CDR3 amino acid sequences. The exposed data included 1,987,608 clonotypes and 541,111 unique amino acid sequences. iCAT achieved a perfect classification accuracy on the training data, correctly assigning the 4 naïve and 4 exposed samples. Further, iCAT correctly classified 4 independent exposed samples as positive. This demonstrates the wide utility of iCAT and the methodology it implements.\n\n\nConclusions\n\nIn this article, we have presented iCAT, a powerful software tool for determining pathogen exposure through TCR sequencing data. It has significant clinical applications in disease diagnosis, surveillance, as well as for determining potential vaccine efficacy. Once data interpretation is fully automated, the TCR sequencing analysis and other types of NGS will likely become a standard tool for diagnosis and management of disease. Our current datasets are from pre- and post- exposure to viruses, and serve as a proof of principle that TCR sequencing analysis can be utilized to identify individuals exposed to infectious agents or vaccines with great accuracy, speed, and accessibility. We demonstrated the use of iCAT for accurately detecting exposure to the SARS-CoV-2, the virus behind COVID-19. Although this use case was based on a few number of samples, it shows the immense potential of our software the utilization of TCRs as a biomarker. This type of analysis may be used to distinguish between two different but highly related infections, such as Zika virus and Dengue, which is one of the global concerns considering Zika virus’s association with fetal complications in infected pregnant women, and current laboratory testing cannot distinguish between the two. Parallel endeavors in our group show promising results in identifying virus-associated TCR sequences uniquely associated with a prior Zika versus Dengue virus infection in mice using iCAT. Further, the iCAT platform may prove useful for diagnosing individuals in the early stages of autoimmunity, by identifying auto-reactive TCRs before symptoms and significant tissue damage occurs. Earlier diagnosis may allow for preventative measures, better treatment, and better outcomes. Broadly, our approach can be used to diagnose autoimmune disease and possibly immune responses to cancer before or after immunotherapy.\n\n\nData availability\n\nMendeley Data: Identifying and Tracking Low Frequency Virus-Specific TCR Clonotypes Using High-Throughput Sequencing, https://doi.org/10.17632/cf92gt44zf.1 13 .\n\nThis project contains the raw sequencing data from HLA-A2 transgenic mice before and after infection with either the ACAM2000 smallpox virus or highly releated monkeypox virus.\n\nZenodo: Longitudinal high-throughput TCR repertoire profiling reveals the dynamics of T cell memory formation after mild COVID-19 infection, https://doi.org/10.5281/zenodo.3835956 14 .\n\nThis project contains the third human TCR samples exposed to the novel SARS-CoV-2 virus is available from zenodo in mixcr format.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSource code available from: https://github.com/BioHPC/iCAT.\n\nArchived sourced code as at time of publication: http://doi.org/10.5281/zenodo.4436485 15 .\n\nLicense: MIT", "appendix": "References\n\nRosati E, Dowds CM, Liaskou E, et al.: Overview of methodologies for t-cell receptor repertoire analysis. BMC Biotechnol 2017; 17(1): 61. ISSN 1472-6750. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVenturi V, Kedzierska K, Turner SJ, et al.: Methods for comparing the diversity of samples of the t cell receptor repertoire. J Immunol Methods 2007; 321. PubMed Abstract \| Publisher Full Text\n\nCabaniols JP, Fazilleau N, Casrouge A, et al.: Most alpha/beta t cell receptor diversity is due to terminal deoxynucleotidyl transferase. J Exp Med. 2001; 194(9): 1385–1390. ISSN 0022-1007 1540-9538. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobins HS, Campregher PV, Srivastava SK, et al.: Comprehensive assessment of t-cell receptor beta-chain diversity in alphabeta t cells. Blood 2009; 114(19): 4099–107. ISSN 1528-0020 (Electronic) 0006-4971 (Linking). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolf K, Hether T, Gilchuk P, et al.: Identifying and tracking low-frequency virus-specific tcr clonotypes using high-throughput sequencing. Cell Rep 2018; 25(9): 2369–2378, e4, ISSN 2211-1247. Reference SourcePubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEmerson RO, DeWitt WS, Vignali M, et al.: Immunosequencing identifies signatures of cytomegalovirus exposure history and hla-mediated effects on the t cell repertoire. Cell Rep 2017; 49(5): 659–665. ISSN 1546-1718. PubMed Abstract \| Publisher Full Text\n\nDeWitt WS, Emerson RO, Lindau P, et al.: Dynamics of the cytotoxic t cell response to a model of acute viral infection. J Virol 2015; 89(8): 4517–4526. Reference SourcePubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKirsch I, Vignali M, Robins H: T-cell receptor profiling in cancer. Mol Oncol 2015; 9(10): 2063–2070. ISSN 1574-7891. Reference SourcePubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGerritsen B, Pandit A, Andeweg AC, et al.: Rtcr: a pipeline for complete and accurate recovery of t cell repertoires from high throughput sequencing data. Bioinformatics 2016; 32(20): 3098–3106, ISSN 1367-4811 (Electronic) 1367-4803 (Linking). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNazarov VI, Pogorelyy MV, Komech EA, et al.: tcr: an r package for t cell receptor repertoire advanced data analysis. BMC Bioinformatics 2015; 16: 175, ISSN 1471-2105 (Electronic) 1471-2105 (Linking). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang W, Cheng J, Allaire J, et al.: shiny: Web Application Framework for R.2018. Reference Source\n\nMinervina AA, Komech EA, Titov A, et al.: Longitudinal high-throughput tcr repertoire profiling reveals the dynamics of t cell memory formation after mild covid-19 infection. bioRxiv 2020. Reference SourcePubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolf K: Identifying and Tracking Low Frequency Virus-Specific TCR Clonotypes Using High-Throughput Sequencing. Mendeley Data 2018; V1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinervina AA, Komech EA, Titov A, et al.: Longitudinal high-throughput TCR repertoire profiling reveals the dynamics of T cell memory formation after mild COVID-19 infection (Version 1.0) [Data set].Zenodo; 2020Reference SourcePubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajeh A, Ahn TH: BioHPC/iCAT: First release of iCAT (Version v1.0.0).Zenodo; 2021, January 13Reference Source" }	[ { "id": "82160", "date": "12 Apr 2021", "name": "Shuo-Wang Qiao", "expertise": [ "Reviewer Expertise Immunology", "T cells", "TCR", "single-cell receptor sequencing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDiagnostic methods based on the detection of disease-specific or disease-associated T-cell receptor (TCR) sequences are novel approaches whose development is still in its infancy. This paper introduces an intuitive, easy-to-use, software package for the analysis of TCR deep sequencing data with the purpose of grouping data into ‘naïve/healthy’ or ‘diseased/exposed’ groups. The statistical methods used is rather rudimentary, but serve the purpose. It demonstrates remarkably high accuracy in three use cases, including a small dataset of COVID-19. However, the choice of parameter settings and how data is split into training and test datasets, is not well explained. Thus, one is left with a feeling that some over-fitting and over-tuning may have occurred. Nevertheless the shortcomings, overall this software package is first of its kind and will be welcomed by the growing community of scientists working with immune receptors and disease prediction.\nMajor comments:\nIntroduction: Aside from the immensity of TCR diversity, both theoretical and within each individual, by far the largest challenge to identifying signals of T-cell memory to a particular antigen, is the rarity of specific T cells to any given antigen. In circulation during the homeostatic memory phase, this number can be as low as 1 in 100 000 for CD4+ T cells, and may be up to 1% in the CD8 compartment.\n\nOperation: Does iCAT support both TCRB and TCRA data, separately, or in the same dataset? Or does it only support TCRB data?\n\nSetting (2) during training: at which level of TRBV and TRBJ allelic similarity is the tool tuned? In other words, would human TRBV7-201 and TRBV7-202 be considered the same, or different? How about human TRBV4-2 and TRBV4-3 that are very similar, especially at the 3’-end where the last 40 amino acids are identical. Many HTS short reads would not be able to distinguish between these two gene segments.\n\nUse Case 1: The authors wrote that they analysed this dataset either by the CDR# amino acid sequence only, or with the V- and J-gene identity in addition. Did these two options behave differently?\n\nIn the use cases, it is not common to report the accuracy on training dataset. it is not clear how the data was split into the training and testing dataset. More common practice is to do cross validation, and report the averaged precision, sensitivity, maybe F-score, especially for unbalanced samples. For Use case 2 for example, how were the 5 naïve and 10 exposed samples in the test group chosen, randomly? Would one get the same 100% accuracy if another random set of 5 naïve and 10 exposed samples was chosen?\n\nIn the training tab, the default Max p-value is 0.1. Usually in disease associated studies, a more stringent cut-off p value is recommended. Considering the main purpose of iCAT is for diagnosis, the specificity is not as crucial, 0.1 might be a good choice. Is there any analysis supporting the choice, e.g. in the use cases or on other public available data which antigen specific sequences is known?\n\nIn the prediction tab, it would be better to provide a measurement of uncertainty in addition to %TARS\n\nSome studies such as, Schneider-Hohendorf et al. (2018)1, Britanova et al. (2014)2, and DeWitt et al. (2018)3 suggested that MHC, age and sex also effect the immune receptor repertoire. Incorporating such meta information should be useful, even if in cases where the sample size is too limited for these factors to be modelled in the training, they are better to be randomized in training and testing datasets.\n\nHow about computing time? How long did it take to do the training, and classification of for instance the use case 2 data?\n\nThere are substantial public resources including some antigen-specific database and repertoire database. It might be useful to allow loading some commonly used datasets as controls in case the user has limited sample size.\nMinor comments:\nFigure legend 1: TCR sequencing of blood samples can be done with, but not limited to, genomic DNA. Many groups also use cDNA.\n\nWhy does the tip of the iCAT’s tail has an antibody? If this were a TCR analysis tool, would it not be more appropriate with a cartoon drawing of TCR?\n\nWhat is VATS in last paragraph in Page 5, is it the same as TARS?\n\nIn the library tab, pattern for significant TARS would make more sense, e.g. are they all similar or there were numbers of clusters based on the similarity?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6776", "date": "09 Jul 2021", "name": "Tae-Hyuk Ahn", "role": "Author Response", "response": "Major comments: 1. Introduction: Aside from the immensity of TCR diversity, both theoretical and within each individual, by far the largest challenge to identifying signals of T-cell memory to a particular antigen, is the rarity of specific T cells to any given antigen. In circulation during the homeostatic memory phase, this number can be as low as 1 in 100 000 for CD4+ T cells, and may be up to 1% in the CD8 compartment. Response: We really appreciate all the great and thoughtful comments from the reviewer. As the reviewer commented, the rarity of specific T cells to a specific antigen is also a challenge in addition to the diversity and magnitude of the TCR repertoire. We updated the Introduction from the comment cited a reference for it. 2. Operation: Does iCAT support both TCRB and TCRA data, separately, or in the same dataset? Or does it only support TCRB data? Response: iCAT has been developed and tested to analyze TCR beta chain of the T-cell receptor in the alpha/beta T cells. The column call for the V and J are vGeneName and jGeneName and do not distinguish between alpha and beta. So, as long as the remaining formatting is the same, there is no reason we couldn’t do either TCRA or TCRB. However, it is not designed to do so in tandem. 3. Setting (2) during training: at which level of TRBV and TRBJ allelic similarity is the tool tuned? In other words, would human TRBV7-201 and TRBV7-202 be considered the same, or different? How about human TRBV4-2 and TRBV4-3 that are very similar, especially at the 3’-end where the last 40 amino acids are identical. Many HTS short reads would not be able to distinguish between these two gene segments. Response: We do not look that deeply into the different V and J chains. TRBV7-201 and TRBV7-202 are in the column vMaxResolved or jMaxResolved, which we do not use for the V and J calling. We use the vGeneName and jGeneName, which for this example, both would be called as TRBV7-2. But TRBV4-2 and TRBV4-3 will be called differently as those would be distinguished in the vGeneName column. You can see this in the data table of the generated library, the selected V and J regions do not include the 01 or 02 details. This is also true when you look in the TSV file. 4. Use Case 1: The authors wrote that they analysed this dataset either by the CDR# amino acid sequence only, or with the V- and J-gene identity in addition. Did these two options behave differently? Response: Using the V- and J-gene identity in addition to the CDR3 amino acid sequence results in significantly higher prediction accuracy in our testing, likely due to identifying more unique markers. Specifically, it results in fewer false positives. For example, in use case 1, using the amino acid sequence only resulted in correctly identifying only 1 / 10 of the pre-vaccination samples, while still correctly identifying 10/10 of the post-vaccination samples. 5. In the use cases, it is not common to report the accuracy on training dataset. it is not clear how the data was split into the training and testing dataset. More common practice is to do cross validation, and report the averaged precision, sensitivity, maybe F-score, especially for unbalanced samples. For Use case 2 for example, how were the 5 naïve and 10 exposed samples in the test group chosen, randomly? Would one get the same 100% accuracy if another random set of 5 naïve and 10 exposed samples was chosen? Response: We agree it is not common to report the accuracy on the training data and it is not as useful as the accuracy on the independent testing data in evaluating a classification tool. However, we chose to include it to demonstrate this feature of the iCAT interface. This feature may be useful as a baseline-check after training in the usual scenarios where the ground truth for the testing data is not available. The independent samples in use case 2 were chosen randomly before training and excluded from the training set. We also repeated the test three times after receiving this question using a script to randomly select 15 samples for testing. The accuracy was at a 100% for all the new random tests. 6. In the training tab, the default Max p-value is 0.1. Usually in disease associated studies, a more stringent cut-off p value is recommended. Considering the main purpose of iCAT is for diagnosis, the specificity is not as crucial, 0.1 might be a good choice. Is there any analysis supporting the choice, e.g. in the use cases or on other public available data which antigen specific sequences is known? Response: The max P-value is set at default to 0.1, it can be changed to any value. We found through our own testing groups that it was a reasonable cut-off (for a default value) as p-values above this tended to generate libraries that were not stringent enough. However, the user can manually alter the p-value to more stringent conditions. It is worth noting that while the max p-value of the assay may be defaulted at 0.1, iCAT includes a feature for determining the optimal p-value threshold for library generation, which is likely to be well below the max allowed p-value. 7. In the prediction tab, it would be better to provide a measurement of uncertainty in addition to %TARS Response: This is a great comment and we totally agree that a measurement of uncertainty in addition to %TARS might be useful for iCAT users. We considered an uncertainty metric based on the difference between the probability density in the pre-exposure distribution and the probability density in the post-exposure distribution. This new feature has been implemented and now available on GitHub iCAT and we updated the README screenshot of the prediction tab. 9. Some studies such as, Schneider-Hohendorf et al. (2018)1, Britanova et al. (2014)2, and DeWitt et al. (2018)3 suggested that MHC, age and sex also effect the immune receptor repertoire. Incorporating such meta information should be useful, even if in cases where the sample size is too limited for these factors to be modelled in the training, they are better to be randomized in training and testing datasets. Response: We agree this meta information can provide insightful context for different use cases. This is one of our goals for future versions of iCAT (iCAT 2.0). For example, we are especially excited to study the effect of clustering data based on HLA subtypes. In our opinion, this would be better explored in a separate paper. manuscript modification10. How about computing time? How long did it take to do the training, and classification of for instance the use case 2 data? Response: Following are the results from use case 2. Training time: 47.81 seconds Classification time: 6.83 seconds We updated the manuscript to reflect this information. 11. There are substantial public resources including some antigen-specific database and repertoire database. It might be useful to allow loading some commonly used datasets as controls in case the user has limited sample size. Response: Providing commonly used datasets as default controls would be a convenient feature for some use cases. We considered including such datasets with the iCAT software package, but decided against it in the current version due to size consideration. We aimed to create a versatile and easy to use software tool. Using the instructions in this paper and on GitHub, we believe loading public datasets as needed can be performed conveniently by the average iCAT user. We will consider this feature in iCAT 2.0. Minor comments: 1. Figure legend 1: TCR sequencing of blood samples can be done with, but not limited to, genomic DNA. Many groups also use cDNA. Response: Fixed in the new manuscript version. 2. Why does the tip of the iCAT’s tail has an antibody? If this were a TCR analysis tool, would it not be more appropriate with a cartoon drawing of TCR? Response: While it is true that a TCR tail would be more descriptive of our tool, it is our opinion that an antibody makes a more aesthetic cat tail than a TCR. Its shape is also more widely recognizable and can give a general idea to non-specialists about what iCAT deals with at first glance. 3. What is VATS in last paragraph in Page 5, is it the same as TARS? Response: Yes. Fixed in the new manuscript version. 4. In the library tab, pattern for significant TARS would make more sense, e.g. are they all similar or there were numbers of clusters based on the similarity? Response: The current version of iCAT does not perform clustering of the target-associated receptor sequences based on similarity, but this can be developed and incorporated in future versions. We agree that it would be a useful addition for exploratory analyses. iCAT currently focuses on using the sequences for prediction, but provides a starting point for further analyses by allowing the download of those significant sequences and some statistical information about their frequency in the training data." } ] }, { "id": "82161", "date": "13 Apr 2021", "name": "Se-Ran Jun", "expertise": [ "Reviewer Expertise Genomic epidemiology", "Multiomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a valuable resource for T-cell receptor (TCR) sequencing data-based infection diagnostics. The authors provided a R software package named iCAT which provides a step-by-step analysis of TCR sequencing data to predict if individual subjects are infected or not. First, iCAT takes positive and negative pathogen-specific TCR datasets to estimate parameters involved in in model. Second, iCAT provides the Library tab which summarizes the target associated receptors sequences with p-values. Third, iCAT provides prediction tab which takes test TCR datasets and make diagnostic decision. I enjoyed reading this article and github tutorial.\nMajor comments: It seems like that the performance of this tool does depend on the number of samples. Please provide discussion or guideline on the number of negative and positive samples relating to the performance.\nIt seems like that this is the first tool available for TCR data-based diagnostic tool. Please clarify if this is the first tool which uses TCR sequencing data for the purpose of diagnostic. If this is not the first tool, then comparison results with other tools should be included.\nMinor comments: First, I have a question for clarification purpose. Before TCR sequencing data is uploaded, does TCR sequencing data need any preprocessing step to make input compatible with iCAT?\nInstall.packages(“devtools”) does not work with R4.0.3 on Mac OS X. The error I got is as follows:\nDownloading GitHub repo BioHPC/iCAT@HEAD Error in utils::download.file(url, path, method = method, quiet = quiet, : download from 'https://api.github.com/repos/BioHPC/iCAT/tarball/HEAD' failed\nEach input file is limited to 10Gigabyte in size. Where does this limitation come from?\nI downloaded one of test cases which contains many files. It was not easy to figure out which files to be uploaded. It would be nice that this R package provides example datasets along with the package so that they can be played in R directly.\nThere is an option named ‘Other’ for ‘analyze clonotypes by (column names in parantheses)’. What is other?\nPlease include information of computational time for training and prediction steps for each case.\nAlthough feature from an option of ‘TCRV-CDR3-TCRJ’ is recommended, please include the performance for other features for each test case which could help readers understand and compare features.\nWhat does TCRBV stand for?\nWhat does VATS stand for?\nA correction constant is not shown in equation (2).\nWhole genome sequences can distinguish DENGU from ZIKE with 100% accuracy. For the sentence ‘current laboratory testing cannot distinguish between the two’, are you excluding whole genome sequencing data?\nIs it possible to make change range of acceptable into something like 1:99 or 1~99 instead of 1 99 to indicate range?\nDoes the max p-value change automatically according to the training data provided? Or should it be changed manually based on the selected one by Fisher’s t-test?\nPlease revise typo in Figure 1(B)\nPlease revise typo with ‘the minimum samples’\nPlease revise the following sentences: - The ratio of Cv to Cn is determined for each p-value. The p-value with the largest Cv:Cn ratio and offers significant coverage to distinguish (v)accinated from (n)aïve samples was chosen. - iCAT was configured to analyze by CDR3 amino acid sequence only\nIf the value is bigger, we can conclude that the sample is more associated to the the training group. Is this a correct sentence?\nFrom the training data, iCAT correctly classified 32 of 32 naïve samples as ”unexposed” and 58 of 58 vaccinated samples as ”exposed” (100% accuracy). Is this a correct sentence?\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6777", "date": "09 Jul 2021", "name": "Tae-Hyuk Ahn", "role": "Author Response", "response": "Major comments: 1. It seems like that the performance of this tool does depend on the number of samples. Please provide discussion or guideline on the number of negative and positive samples relating to the performance. Response: We really appreciate all the great and thoughtful comments from the reviewer. The sample size is of course very important in determining the power and accuracy of the diagnostic. We have generated positive and negative datasets of various sample sizes to determine the impact of sample numbers on accuracy. The conclusion was that the number of samples required to generate an accurate diagnosis is determined by the nature of the samples. For example, a dataset from Zika infected mice produced a small but conserved virus-specific TCR repertoire due to the small number of immunogenic epitopes (Hassert and Wolf et. al. 2020) ; this allowed for the generation of a powerful diagnostic assay using relatively small numbers of samples. Comparatively, using a dataset from mice before and after smallpox vaccination resulted in a large pool of virus-specific TCRs due to a large number of potential epitopes; the less focused response required a greater number of samples in each group to generate sufficient coverage and diagnostic accuracy (Wolf et. al. 2018). 2. It seems like that this is the first tool available for TCR data-based diagnostic tool. Please clarify if this is the first tool which uses TCR sequencing data for the purpose of diagnostic. If this is not the first tool, then comparison results with other tools should be included. Response: To our limited knowledge, this is the first publicly available tool for diagnostic classification using TCR sequencing data. Minor comments: 1. First, I have a question for clarification purpose. Before TCR sequencing data is uploaded, does TCR sequencing data need any preprocessing step to make input compatible with iCAT? Response: iCAT takes in TCR data in tab-delimited clonotype abundance tables. These tables are the result of mapping of reads from Variable, Diversity, and Joining segments then the assembly of clonotypes. iCAT does not perform these steps so they may be considered pre-processing. Clonotype tables are provided through the immunoSEQ assay from Adaptive Technologies, which is the most commonly used immunosequencing pipeline in recently published studies. While iCAT input-parsing was built around immunoSEQ’s format, it also has a flexible interface that allows handling of different formats with different user-selected column names. 2. Install.packages(“devtools”) does not work with R4.0.3 on Mac OS X. The error I got is as follows: This issue is resolved and updated on iCAT. 3. Each input file is limited to 10Gigabyte in size. Where does this limitation come from? Response: This is an arbitrary limitation that we coded into the tool’s interface. It is not a required limitation for any of the underlying functions in iCAT. The reason we put this size limit in place is because one of our future goals with iCAT is to host it on a website where users can upload their data for analysis, and thus data size can become a limiting factor. 4. I downloaded one of test cases which contains many files. It was not easy to figure out which files to be uploaded. It would be nice that this R package provides example datasets along with the package so that they can be played in R directly. Response: Example negative, positive, and independent datasets are included in the R package under the path “inst/extdata/”. 5. There is an option named ‘Other’ for ‘analyze clonotypes by (column names in parantheses)’. What is other? Response: This option exists to allow users to input a custom column name to analyze by. TCR data can vary in formatting (e.g. some files might describe the amino acid column as “aminoAcid” while others can describe it as “aa”) so this option can help users load different data formats into iCAT. 6. Please include information of computational time for training and prediction steps for each case. Response: Use case 1: Training time: 2.36 minutes Classification time: 30.6 seconds Use case 2: Training time: 47.81 seconds Classification time: 6.83 seconds Use case 3: Training time: 1.50 minutes Classification time: 33.12 seconds The manuscript was updated to reflect this information. 7. Although feature from an option of ‘TCRV-CDR3-TCRJ’ is recommended, please include the performance for other features for each test case which could help readers understand and compare features. Response: In general, we found that using the J-gene and V-gene info improves accuracy in our testing, but other features are available in case researchers to believe they might get better results with them. Using the amino acid sequence for the monkeypox data, 1/5 negative samples were identified correctly, while 6/10 positive samples were identified correctly. Using the DNA sequence, 5/5 negative samples were identified correctly and 3/10 positive samples were identified correctly. 8. What does TCRBV stand for? Response: It stands for T cell receptor beta chain variable region. 9. What does VATS stand for? Response: It stood for vaccine-associated target sequences. This is now changed to target-associated receptor sequences (TARSs) for consistency in the manuscript. 10. A correction constant is not shown in equation (2). Response: That is correct We updated the sentence for the equation in the manuscript. 11. Whole genome sequences can distinguish DENGU from ZIKE with 100% accuracy. For the sentence ‘current laboratory testing cannot distinguish between the two’, are you excluding whole genome sequencing data? Response: We are limiting that comment to typical laboratory diagnostics which typically does not include whole-genome sequencing. 12. Is it possible to make change range of acceptable into something like 1:99 or 1~99 instead of 1 99 to indicate range? Response: We reflected this comment and iCAT now allows inputting either a space or a colon to delimit range values. 13. Does the max p-value change automatically according to the training data provided? Or should it be changed manually based on the selected one by Fisher’s t-test? Response: The max p-value can be changed manually. The purpose is to allow the user to modify the stringency of the diagnostic. This could be done after generating the diagnostic library and the user determines they would prefer more (or less) stringent parameters. 14. Please revise typo in Figure 1(B) Response: Fixed typo in the new manuscript version. 15. Please revise typo with ‘the minimum samples’ 16. Please revise the following sentences: - The ratio of Cv to Cn is determined for each p-value. The p-value with the largest Cv:Cn ratio and offers significant coverage to distinguish (v)accinated from (n)aïve samples was chosen. - iCAT was configured to analyze by CDR3 amino acid sequence only Response: Sentences were revised in the new manuscript version to make them more clear. 17. If the value is bigger, we can conclude that the sample is more associated to the the training group. Is this a correct sentence? Response: The sentence was revised in the new manuscript version. 18. From the training data, iCAT correctly classified 32 of 32 naïve samples as ”unexposed” and 58 of 58 vaccinated samples as ”exposed” (100% accuracy). Is this a correct sentence? Response: The sentence was revised in the new manuscript version." } ] } ]	1	https://f1000research.com/articles/10-65
https://f1000research.com/articles/10-510/v1	28 Jun 21	{ "type": "Research Article", "title": "Publication analysis in Bay for Bengal Initiative for Multi-Sectoral Technical and Economic Cooperation Nations", "authors": [ "Manthan Janodia", "Aparna I. Narayan", "Santhosh Krishnan Venkata", "Bharti Chogtu", "Manthan Janodia", "Aparna I. Narayan", "Santhosh Krishnan Venkata" ], "abstract": "Background: Research output provides an insight into the development of the scientific capability of a country. Budget allocation for research and development (R&D) is directly proportional to the research output of a country. While developed countries spend a significant percentage of their GDP on R&D, developing countries do not have enough resources to invest in R&D. Countries in the Bay of Bengal Initiative for Multi-Sectoral Technical and Economic Cooperation (BIMSTEC) Nations has received significantly less attention from outside the region in studying R&D and research publication scenario of the region. The research output of BIMSTEC countries was analyzed using various metrics in this paper. Methods: Data on citation per paper, Field Weight Citation Impact (FWCI), paper per researcher, collaborative publications, and output in top 10 percent journals was extracted from one of the largest abstract and citation database of peer-reviewed literature, Scopus and its affiliate SciVal, for a period of 6 years between 2012-2017. Percentage of R&D spend, researchers per million population, and total scientific output were extracted from World Bank data. Results: India and Thailand have a higher quantum of publications compared to other countries. Subjects like clinical, technology, Computer Science have a larger publication number as compared to other subject areas like Social Science, Arts, Education, Law, and Physiology. The researcher population and research spend of a nation have an evident implication on the publication though no direct relation can be derived. Conclusion: Huge disparities in terms of percentage of research spent, research output, papers per researcher, and output with national and international authorship differ for countries. Higher research spent and publication count are not positively correlated with better FWCI.", "keywords": [ "BIMSTEC", "Collaboration", "Scopus", "Field Weighted Citation Impact" ], "content": "Introduction\n\nThe formation of the South Asian Association of Regional Conference (SAARC) was a landmark step in developing economic and regional integration in this area but the outcomes in terms of strengthening regional cooperation even after three decades were not satisfactory.1 This forced the regional members to look for an alternative option and the search led to the commencement of the Bay of Bengal Initiative for Multi-Sectoral Technical and Economic Cooperation (BIMSTEC). Initiated as BIST-EC in June 1997 with the inclusion of Bangladesh, India, Sri Lanka, and the Thailand Economic Corporation, saw Myanmar being included late in the same year. In 2004, Nepal and Bhutan also became members of this group and the group was renamed to its present name BIMSTEC.2 It fosters socio-economic linkages between South Asia and South-East Asia and includes 1.5 billion people constituting around 22% of the global population with a combined Gross Domestic Product (GDP) of 2.7 trillion economy. Elementally, it started with cooperation in six sectors: trade, technology, energy, transport, tourism, and fisheries. Subsequently, other sectors were incorporated and there was a commitment for active collaboration in training and research facilities in fields of common interest. The member states felt that knowledge sharing, research and development, and capacity building in various fields like climate change and public health among the BIMSTEC nations can provide solutions for problems of the future.3\n\nThe bilateral and sub-regional projects within BIMSTEC nations were a uniting force to bring this region under one economy. The 15th BIMSTEC ministerial meeting held in Nepal in the year 2017 agreed upon enhancing cooperation in different fields like economic integration connectivity and mutual support in disaster management. This meeting also decided upon implementing policies aligning with the United Nation’s Sustainable Development Goals (SDGs), which addresses global concerns like climate change, environment, and reducing poverty. Also, India-Bangladesh Joint research in oceanography was discussed as a bilateral agreement.4\n\nBIMSTEC nations face diverse challenges to excel in the field of research and development. One of the many hurdles faced by developing countries like Sri Lanka is inaccessibility to research papers, which in turn decreases the scientific output.5 The Sri Lankan Government has launched initiatives to promote Research and Development, which include a presidential award at the national level for outstanding researchers based on data from the Science Citation Index (SCI), research allowance, and promotion benefits.6 On comparing the research output amongst different streams in Sri Lanka, Science and Medicine tops the chart followed by Engineering, Agriculture, and Management7 as mentioned in the National Research Council of Sri Lanka. In ten years (2000-2010) certain bodies like the National Research Council focused on the funding of researchers.8\n\nIn 2008, the National Science, Technology and Innovation policy (STI) was established in Thailand. National STI Master Plan 2012-2021 aims at collaboration between private sectors, research institutes, and academics. Thailand research fund is a leading funding agency and does not come under the umbrella of government bureaucracy and thus supports research efficiently.9 Secretary General of STI has put forth that the investment in research and development has increased by 36% in 2018 as compared to the previous year. The proportion of researchers is increasing over the years at an annual rate of 4/10,000 population. The government is encouraging private investors to invest in research and development by providing funding for startups, expanding economic innovation zones, and favorable funding conditions. In Thailand, artificial intelligence related research is carried out after 2011 and two exemplary projects were on Speech Technologies and Health Car Knowledge Engineering, and Agricultural Cyber Brain. With Smart Thailand 2020, the country is trying to move ahead in the field of research.10\n\nLow-income countries like Nepal lack national commitment for research and a dearth of resources to conduct research. The poor state of higher education has an adverse effect on the volume and growth of research and the prevailing political and economic situations contribute to the same.11 Earlier, scientometric analysis suggested areas like medicine, agriculture, and biological sciences were highly productive.12 In the years 2015 and 2016 there was a sudden upsurge of publications after the devastating Gorkha Earthquake that set off the collaborative research in seismology, engineering, geology, civil engineering, and other fields.11 Since 2017, smaller countries like Nepal and Sri Lanka have increased their research output by engaging in international collaborations and by this they are gaining expertise in addition.13\n\nAfter 2011, the research environment in Myanmar has changed with facilities for international researchers to carry out research in Myanmar by relaxing travel restrictions, increased research funding from the Myanmar government and other international organizations like World Bank,14 bibliometric analysis of social sciences and humanities from 2005 to 2014 in India show that research articles increased from 62.1% to 82.9%, collaborative research was done routinely and it received more citations.15 From 2000 onwards there has been an exponential growth in publications in India. But this increase in volume does not commensurate with the citations of Indian research.16 A large part of research in India is published in regional or local languages which are not included by the indexing agencies.15\n\nAs per Web of Science (WoS) data, India published 4780 highly cited papers from 1989-2015. Papers from international collaborators received more citations as compared to others. In this period, the United States of America was the major collaborator accounting for 22.29% of highly cited papers and the average citation was 238.92 per paper. Collaborative papers with the US were followed by Germany and China.17 In papers published in the research field from 2009-14, India stands at fifth position in Chemistry, eighth position in Physics, and ninth position in Material Science. In the subject area of Computer Science, which is considered a thrust area throughout the globe, India has improved its position from twelfth to third. However, India has still a long way to go in Medical and Biosciences research.18\n\nSouth Asian countries like Bhutan, Nepal, India, and Sri Lanka have common health challenges like chronic conditions, infectious diseases, and injuries. Health systems also encounter a lack of social accountability making these countries benefit from collaborative health research.19 Myanmar, the land bridge between ASEAN and India has a vital role in shaping the economic, political, and security environment in the region.20 With the rising interest on the BIMSTEC nations, this paper aims to discuss the outcomes of the BIMSTEC nations as a group in terms of research output such as the number of publications, citations per paper, spend on research and development (R&D), Field Weighted Citation Impact (FWCI) across different subject areas and international collaborations. Analyzing the aforementioned outcomes of the BIMSTEC countries for the period 2012-17.\n\nResearch outcomes of a country are often associated with the quantity and quality of higher education institutions (HEIs). This paper attempts to analyze the research outcomes of the BIMSTEC countries for the period 2012-17. A comparative representation of the number of HEIs in each of the BIMSTEC countries is shown in Figure 1. Based on the data it can be seen that India has the highest number of HEIs with 799, followed by Thailand with 170, then Myanmar with 163, Bangladesh with 105, Sri Lanka with 30, Nepal with 10 and Bhutan with 2. These numbers would also have a direct relation to the population of each of the countries. To further understand the research parameters of BIMSTEC countries data with regard to percentage GDP spent on research and researcher population per million is plot in Figures 2 and 3, respectively. The data is extracted from the World Bank report on 4th April 2020.\n\nIt is to be noted that the average percentage of GDP spent on research is highest in India, which is around 0.62%, followed by Thailand which is 0.51%, then Nepal which is 0.3%, Bhutan and Myanmar spend around 0.16%, and the least spent is from Sri Lanka with 0.1%. Bangladesh has not reported any data with regard to research spent in the World Bank repository. Data corresponding to each research population is derived from World Bank repository as on 4th April 2020 (Figure 3). It can be seen that Thailand has the greatest number of research population per million with is around 514, followed by India with 183, Sri Lanka with 107, Nepal with 61, and Myanmar with 17. Data related to Bangladesh and Bhutan were not available in the World Bank data.\n\nFrom the data, it can be seen that the source for research in terms of researcher population and GDP spending is higher in countries like India and Thailand. Bhutan, Myanmar, Sri Lanka, Nepal, and Bangladesh have a lesser researcher population and expenditure in terms of GDP percent. In this paper, a detailed analysis of the research outcomes is presented which would give a fair idea of the correlation of the research sources of those countries. The paper focuses on the research outcomes in terms of research publications, which includes article, review papers, conference proceedings, book chapters, books, case reports, etc. published in Scopus indexed publications. Does publication performance/quantum of publication have any relation with research spend or researcher population of the country? An attempt is therefore made to understand the publication outcome of BIMSTEC countries during the period 2012-17.\n\n\nMethods\n\nFor the analysis of publication outcome of BIMSTEC countries, data was obtained from one of the largest abstract and citation database of peer-reviewed literature, Scopus on 3rd of March 2020 and its affiliate SciVal on 3rd March 2020. These databases provide options to extract various scientific parameters through different permutations and combinations for each country as well as for the region, in the present work the data corresponding to seven BIMSTEC countries namely Bangladesh, India, Myanmar, Sri Lanka, Nepal, Thailand and Bhutan is obtained for the period 2012 to 2017. With respect to this reported work publications, research articles, reviews, conference proceedings and book chapters were only considered. Case reports, letter to editors, technical reports and brief/short communications were not considered. The publication types for inclusion and exclusion were considered based on the uniformity of data across all the countries and relevance to the reported study. Moreover, publication of articles, reviews, conference proceedings and book chapters are produced in higher volumes and have considerable higher citation metrics as compared to the other types.\n\nWorld Bank Indicators for identifying research expenditure as a percentage of GDP, researchers per million population was extracted on 4th April 2020. Country-wide data corresponding to these two parameters are taken for the period 2013-2017 and the average value of these data for the period is chosen as the figure for all computations.\n\nBIMSTEC countries were selected for analysis as it is one of the leading associations for regional cooperation in South East Asia. The analysis of scientific disciplines was based on subject categories as Scopus distributes publications into various subject categories. Around 300 subject categories are divided among the five major subject areas namely Art & Humanities, Engineering & Technology, Life Science & Medicine, Physical Science and Social Science & Management. This paper presents eleven subject groupings that were common for ease of analysis based on subject categories provided by Times Higher Education (THE). These subjects are Arts & Humanities, Business & economics, Clinical, Computer Science, Education, Engineering & Technology, Law, Life science, Physical sciences, Psychology and Social sciences.\n\nPublication related data for the period 2021-2017 was exported from Scopus to SciVal analytics tool by Elsevier. SciVal is an online tool used to analyze research performance. Data is analyzed on 4th of April 2020. Publication data is analyzed for the period 2012-17 across the BIMSTEC countries for eleven subjects mentioned; outcome based on collaborative publication is also studied. Once analyzed data is it was exported to Excel, which was used to represent the data in the form of graph and plots.21\n\n\nResults\n\nTable 1 shows the scholarly output of each of the BIMSTEC countries in Scopus indexed publication for 2012-17. It can be seen that India has a maximum number of publications with around 0.8 million publication, followed by Thailand, which is around 80 thousand, Bangladesh around 24 thousand, Sri Lanka around 10 thousand, Nepal around 7 thousand, Myanmar with around 1000 publication, and Bhutan has the least number of five hundred. Figure 4 shows the magnitude-wise representation of the scholarly output which can be seen that publications from India constitute almost 80% of BIMSTEC publications, with Thailand being the nearest country with around 10% of its number. Publication outcome and quality of publication shows correlation with researcher population, percentage GDP on research, and the number of HEIs.\n\nIndia (IND), Thailand (TL), Bangladesh (BAN), Sri Lanka (SL), Nepal (NEP), Myanmar (MY), Bhutan (BHU).\n\nThe important measure of research along with the quantity of publication is also quality of the publication. To measure the quality of the publication, the most common parameter considered is the number of citations. But the absolute number of citations is biased across the different subject areas, as the global average of citation per publication is not uniform across the subjects. So, we have considered FWCI as the measure of the quality of research publication. FWCI is a normalized number of citations per paper across the globe where the average citation index for a given subject area across the globe is normalized to one.\n\nFigure 5 shows the relation of the average FWCI of the country with respect to its total scholarly output during the period 2012-17. It is seen that publications from Bhutan (2.5) and Myanmar (2.3) have larger FWCI as compared to other BIMSTEC countries, which is surprising looking at the source data of these countries in terms of the number of HEIs or researcher population or research spent. Publications of Thailand and India have the least FWCI of the BIMSTEC countries with around 0.9 and 0.75, respectively. Nepal, Bangladesh, and Sri Lanka have a medium ranged FWCI with around 1.4, 1.2, and 1.7, respectively. From Figure 5 it is evident that the quality vs quantity has an inverse relation in BIMSTEC countries.\n\nIndia (IND), Thailand (TL), Bangladesh (BAN), Sri Lanka (SL), Nepal (NEP), Myanmar (MY), Bhutan (BHU).\n\nTo understand the reason for the change in quality index with quantity, we put forth a few other correlated parameters other than the commonly considered parameters like the number of HEIs or research population, and research spent. One such important parameter is the international collaborative publication. The international collaborative publications are those publications which are coauthored by other country researchers.\n\nThe percentage of collaborative publication of BIMSTEC countries is shown in Figure 6. A total of 16.1% of publications from India have international coauthors; Thailand, 39%; Sri Lanka, 50.9%; Bangladesh, 51% of publication; Nepal, 62.9%; Bhutan, 75.7%; and Myanmar, 77.8%.\n\nIndia (IND), Thailand (TL), Bangladesh (BAN), Sri Lanka (SL), Nepal (NEP), Myanmar (MY), Bhutan (BHU).\n\nTo understand the effect of international collaboration on the quality of the publication, a comparison between the percentages of international collaboration to that of FWCI of the nation is indicated in Figure 7. It is seen that countries with a higher number of collaborative publications, like Myanmar (77.8%) and Bhutan (75.7%), have higher FWCI of 2.3 and 2.5, respectively. India with the least collaborative publication of 16.1% has the lowest FWCI of 0.75. The rest of the BIMSTEC countries show similar behavior in the relationship where higher international collaborative publication yield in higher FWCI: Nepal 62.9% international collaborative publications, FWCI of 1.4; Bangladesh 51% international collaborative publications, FWCI of 1.2; Sri Lanka 50.9%, FWCI of 1.7. Therefore, it can be suggested that the quality of publication depends on international collaborations.\n\nIndia (IND), Thailand (TL), Bangladesh (BAN), Sri Lanka (SL), Nepal (NEP), Myanmar (MY), Bhutan (BHU).\n\nThe term collaborative publication refers to the coauthored publications from authors affiliated with different countries. An attempt is made in this work to analyze the breakup of coauthored publications in terms of six regions; Africa, Asia Pacific, Europe, Middle East, North America, and South America, to understand the collaborating partners of different BIMSTEC countries. Representation of the distribution of coauthored publications of BIMSTEC countries in the six regions is shown in Figure 8, and the comparison in terms of absolute number is reported in Table 2. It can be seen that all countries, except India, have a large share of collaborative publications within Asia Pacific region whereas India has the highest percent of collaborative publications with Europe, followed by North America. It is also seen that the least percent of collaborative publication by all the BIMSTEC nations is with South America. It is also observed that around 70-80% of the collaborative publication of all the BIMSTEC nations are from the Asia Pacific, Europe, and North American region. The variation in nature of collaboration may be influenced by demographic or political or research interests.\n\nIndia (IND), Thailand (TL), Bangladesh (BAN), Sri Lanka (SL), Nepal (NEP), Myanmar (MY), Bhutan (BHU).\n\nAlong with international collaboration, one more parameter that would influence the quality of the publication is the subject area of the research. As per the All Subject Journal Code (ASJC), every journal is mapped to any of the 300+ subject areas, grouped among 11 main subject areas. These subject areas are Arts & Humanities (AH), Business and Economics (BE), Clinical, pre-clinical and health (CH), Computer Science (CS), Education (ED), Engineering and Technology (ET), Law, Life Science (LS), Physical Science (PS), Psychology (PY), and Social Science (SS). A detailed analysis is presented in this section where each of BIMSTEC country’s outcome in terms of the number of publications, percent of collaboration, and FWCI are analyzed across each of these eleven subject areas. Data related to international collaboration publication, FWCI, and the number of scholarly outputs across all the BIMSTEC countries are analyzed.\n\nBangladesh\n\nAcross the eleven subject areas, the maximum number of publications from Bangladesh are in the subject area of ET (7673) (Figure 9a) with 42.2% of its publication with international collaboration (Figure 9b) and has a FWCI of 0.95 (Figure 9c). Around 6781 publications are in the subject area of PS with 56.6% of collaborative publication and FWCI of 0.91. In the subject area of CH there were 6316 publications with 55.8% of international coauthored publications having the highest FWCI of 1.99, which is as highest as compared to all other subject areas. In the subject area of LS, 5708 papers were published with 68.8% of it with international coauthors having a FWCI of 0.93. CS publication has a FWCI of 0.84 from 4994 publications with 31.7% of it with international collaboration. Publication share from the remaining six subject areas is minimal with SS at 1626 (FWCI, 0.87; international collaboration (IC), 51.1%), followed by BE with 992 (FWCI, 0.78; IC, 55.2%), AH with 389 (FWCI, 0.73; IC, 34.2%), ED with 155 (FWCI, 0.83; IC, 52.3%), PY with 134 (FWCI, 0.87; IC, 56.7%), and Law with 94 publications (FWCI, 0.85; 36.2%).\n\nArts & Humanities (AH), Business and Economics (BE), Clinical, pre-clinical and health (CH), Computer Science (CS), Education (ED), Engineering and Technology (ET), Life Science (LS), Physical Science (PS), Psychology (PY), Social Science (SS).\n\nBhutan\n\nAcross the eleven subject areas, the maximum number of publications were in the subject area LS with 209 publications (Figure 10a), with 81.8% international collaboration (Figure 10b), having a FWCI of 1.15 (Figure 10c). CH has the highest FWCI in comparison to all subject areas with 6.64 having 86.4% of its publication with international coauthored from 177 publications. Around 117 publications are in the area of PS with 75.2% of them with international collaboration having a FWCI of 0.81. Publication in SS is 75 with 61.3% of it with international collaboration has a FWCI of 0.92. Publication in the area of ET and CS has an equal number of publication (36) with 55.6% (FWCI, 0.45) and 52.8% (FWCI, 0.28) of its publication with international collaboration respectively. Around 39 publications are published in the area of BE with 59% of it has international coauthored publication and FWCI of 0.45. In total, 28 papers are published in the area of ED, with 50% of its publication with international coauthors and has a FWCI of 0.48. AH with a FWCI of 1.31 has around 28.6% of its 14 publications from international authors. The subject area of PY and Law have negligible outcome in terms of publication numbers.\n\nArts & Humanities (AH), Business and Economics (BE), Clinical, pre-clinical and health (CH), Computer Science (CS), Education (ED), Engineering and Technology (ET), Life Science (LS), Physical Science (PS), Psychology (PY), Social Science (SS).\n\nIndia\n\nIndia produces most of its publications in the subject area of PS (287965) (Figure 11a), with 21.1% of its publication with international collaboration (Figure 11b) having a FWCI of 0.89 (Figure 11c). A total of 279205 papers are published in the area of ET with 15.4% of them having international co-authorship with a FWCI of 0.91. Around 206718 papers are published in the area of CH with 14.3% of them with international authors (FWCI, 0.71). In LS, there are around 164266 publications with 16.9% international collaborative publications with a FWCI of 0.7. CS has a FWCI of 0.8 with 11.5% of the publications being co-authored by international collaborators (total, 135024 publications). SS published 26515 papers with 15% of them with international collaboration (FWCI, 0.69). In BE, there are around 24189 papers with 14.5% of them with international co-authorship (FWCI, 0.76). AH have 7473 publications 14.7% of which are internationally co-authored publications (FWCI of 0.79). In ED, there are 4309 publications with 11.9% being international co-authored with FWCI of 0.59. In total 25.3% of publications in PY out of 3909 publications are international co-authored with a FWCI of 0.76. The least number of publications are in Law with 2092 publications, 11.1% of which with international collaboration (FWCI, 0.66).\n\nArts & Humanities (AH), Business and Economics (BE), Clinical, pre-clinical and health (CH), Computer Science (CS), Education (ED), Engineering and Technology (ET), Life Science (LS), Physical Science (PS), Psychology (PY), Social Science (SS).\n\nMyanmar\n\nThe maximum number of publications are in the subject area of LS (448) (Figure 12a) with 95.3% of its publication having international coauthors (Figure 12b), with a FWCI of 0.92 (Figure 12c). CH (429 publications) had 87.4% of its publications with international coauthors, with FWCI of 6.16. In total, 280 papers are published in PS with 81.8% international co-authored, with FWCI of 0.98. ET have 166 publications with 52.4% international co-authored, and FWCI of 0.57. Around 166 papers are published in CS with 87.4% international coauthored, and 0.61 FWCI. Myanmar produced 110 SS papers (51.8% international collaborations; FWCI of 1.0). A total of 51 papers are published in AH, 50% of which are international coauthored (FWCI of 0.77). In ED, there are nine publications, 77.8% of which are international collaborative publications having a FWCI of 0.57. All the nine papers published in PY are with international coauthors (FWCI of 0.52). There a zero publications in the subject area of Law from Myanmar.\n\nArts & Humanities (AH), Business and Economics (BE), Clinical, pre-clinical and health (CH), Computer Science (CS), Education (ED), Engineering and Technology (ET), Life Science (LS), Physical Science (PS), Psychology (PY), Social Science (SS).\n\nNepal\n\nIt is seen that the highest number of publications are in the CH (3707 publications) (Figure 13a), 51.3% of which are international coauthored (Figure 13b), with FWCI of 1.62 (Figure 13c). In LS, 1630 papers are published, 79% of which are international collaborative, with a FWCI of 0.92. Around 1594 papers are published in PS, 81.7% are with international collaboration (FWCI of 1.36). In SS, there are 678 publications, 71.4% international co-authored, with FWCI of 1.28. In ET, there are 604 publications (76.5% international collaboration; FWCI of 1.06). In total, 155 papers are published in CS, out of which 45.2% are international coauthored publications, having a FWCI of 0.76. In ED, 42 papers are published 57.1% of which are international collaborative, with a FWCI of 1.42. A total of 41 papers are published in PY, of which 82.9% are coauthored with international authors, having a FWCI of 1.64. The least number of papers are from Law with 18 papers, of which 61.1% are international collaborative publications having a FWCI of 0.42.\n\nArts & Humanities (AH), Business and Economics (BE), Clinical, pre-clinical and health (CH), Computer Science (CS), Education (ED), Engineering and Technology (ET), Life Science (LS), Physical Science (PS), Psychology (PY), Social Science (SS).\n\nSri Lanka\n\nThe highest number of publications for Sri Lanka is in CH (2884 publications) (Figure 14a), 46% of its publications are with international coauthors (Figure 14b), with a FWCI of 3.03 (Figure 14c). In the PS, there were 2537 publications, 68% of which are with international collaboration having FWCI of 1.37. A total of 2148 papers are published in LS, with 64% international co-authored, with 1.02 FWCI. In ET, 1735 papers were published, with 52.6% with international collaboration, and a FWCI of 1.14. In CS, 1525 papers are published with 29.1% being international collaborations, with a FWCI of 0.83. A total of 671 publications in the field of SS had a FWCI of 1.25 with 49.3% of its publications with international collaborators. BE had a FWCI of 0.89 from 535 papers, with 45.6% of them being international coauthored. In ED, 170 papers are published with a FWCI of 0.91, and 36.5% of them had international collaboration. In total 150 publications are in the area of AH with 48.7% of them with international collaboration, with a FWCI of 0.95. In PY, 58 papers are published with 50% with international collaboration (FWCI of 1.06). In Law, 51 papers were published with 39.2% internationally coauthored with FWCI of 1.02.\n\nArts & Humanities (AH), Business and Economics (BE), Clinical, pre-clinical and health (CH), Computer Science (CS), Education (ED), Engineering and Technology (ET), Life Science (LS), Physical Science (PS), Psychology (PY), Social Science (SS).\n\nThailand\n\nET have the highest number of publications for Thailand at 5138 (Figure 15a). In total, 28.9% of these were international collaborations (Figure 15b), with FWCI of 0.85 (Figure 15c). In CH, there were 24821 publications with 46.2% having international co-authorship, and FWCI of 1.14. FWCI of papers published in PS is 0.97 with 22920 papers, of which 41.2% have international collaboration. In total, 50.3% of 22171 papers in LS are with international collaboration (FWCI, 0.93). In CS, 10424 papers were published with 23.2% of them international collaborations, with FWCI of 0.63. A total of 3782 papers were published in SS (35.3% international collaboration; FWCI of 0.84). In ED, 21% of papers had international coauthors, with FWCI of 0.62. In AH, 1214 papers were published with 25.4% of them international co-authored (FWCI, 0.93), while in PY, 484 papers were published with 70.5% of them international co-authored (FWCI, 1.15). The least numbers of papers were published in Law with 33.8% of 136 papers with international collaboration (FWCI of 0.86).\n\nArts & Humanities (AH), Business and Economics (BE), Clinical, pre-clinical and health (CH), Computer Science (CS), Education (ED), Engineering and Technology (ET), Life Science (LS), Physical Science (PS), Psychology (PY), Social Science (SS).\n\n\nDiscussion\n\nAs can be seen from Table 4, Bangladesh has the highest number of papers published in the LS followed by CH. While the highest percentage of international coauthored papers are published in the field of LS, the highest FWCI is in the field of CH with ET being a close second. For Bhutan, the absolute number of papers is highest in LS, while the percentage of international coauthored publications and FWCI being highest in CH. Since only one paper in the field of PY is published with international coauthors, it is an outlier and hence not considered for analysis. The FWCI of Bhutan in CH is not only highest among all subject categories but is also highest among all BIMSTEC countries. It can be inferred that for the international community, collaborating with Bhutan is more productive in CH, with LS being a close second.\n\nAmong the BIMSTEC countries, India is demographically largest and leads in quantitative output in terms of number of publications. However, qualitatively the performance by India is poor compared with other BIMSTEC countries. The highest number of papers are published in PS, and ET, yet international coauthored papers are the highest for PY. ET has the highest FWCI, but this is lower than the world average of 1. With the highest number of HEIs established among BIMSTEC countries, a lot of researchers in India collaborate within the country. This could probably be one of the reasons for the lower percentage of international co-authored publications and lower FWCI. For Myanmar, PS is the leading subject category for publication while CH is second. A direct relationship can be established for Myanmar in terms of the number of publications and the percentage of international coauthored papers while a qualitatively higher FWCI is observed for CH. Myanmar and Bhutan show similar patterns in terms of quantitative and qualitative output. Again, papers in PY are not considered for analysis due to lower volume with all international coauthored papers.\n\nNepal has the highest publication output for CH and corresponding highest FWCI among all subject categories. While PY has a higher FWCI than CH, the subject category is excluded for analysis due to the relatively lower publication count skewing the data. PS has the highest number of international coauthored papers. Sri Lanka’s largest publication output is CH, with a corresponding highest FWCI whereas PS exhibits the highest international coauthored publications. In the case of Thailand, the highest papers are published in ET followed by CH. While ET has the highest papers, corresponding international coauthored papers and FWCI are relatively lower. The percentage of international coauthored papers and FWCI are highest in the subject category PY. FWCI for CH is almost equal to PY yet the difference in quantitative output is significantly larger.\n\nFrom the detailed analysis of BIMSTEC countries in terms of eleven subject areas, it is observed that there is diversity in terms of publications. A few of the points perceived are:\n\n• The least numbers of papers are published in the subject area of Law in all BIMSTEC countries.\n\n• For all the BIMSTEC countries, CH, LS, PS, and ET constitutes a major chunk of their research output.\n\n• Smaller countries have higher output in CH and have higher international collaboration due to various funding schemes aimed at supporting capacity building in these small countries with comparatively lower HEIs.1,2\n\n\nConclusion\n\nThe Bay of Bengal Initiative for Multi-Sectoral Technical and Economic Cooperation (BIMSTEC) is an international organization of seven nations of South Asia and Southeast Asia. BIMSTEC was an initiative to accelerate economic growth and social progress. BIMSTEC also promotes partners to have active collaboration and excel in technical and scientific fields. The progress in a scientific field is usually measured in terms of publication, grant, and innovation. The objective of this paper was to analyze the publication output of these seven nations using various bibliometric parameters.\n\nDemographically and geographically larger countries like India and Thailand among BIMSTEC have better quantitative research outcomes. However, smaller countries like Bhutan, Myanmar, Sri Lanka, and Nepal have better qualitative outcomes such as percentage of international coauthored papers and FWCI While subject categories of Clinical, preclinical and Health, Physical Science, and Engineering and Technology are the focus areas of research, Arts and Humanities, Psychology, Education and Law are less preferred.\n\n\nData availability\n\nOpen Science Framework: BIMSTEC, https://doi.org/10.17605/OSF.IO/4D32C.22\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBhattacharjee J: SAARC vs. BIMSTEC: The Search for the Ideal Platform for Regional Cooperation. Observer Issue Brief. 2018; 226: 1–12.\n\nHossain SM: Impacts of BIMSTEC Free Trade Area: A CGE Analysis. J Econ Sustainable Develop. 2013; 4(13): 16–28.\n\nKamruzzaman MD, Action C: Climate Change and Healthcare in the BIMSTEC Countries: Potential for Cooperation.2019.\n\nMallempati S: The 15th BIMSTEC (Bay of Bengal Initiative for Multi Sectoral Technical and Economic Cooperation) Ministerial Meeting: Outcome.2017.\n\nMashroofa MM, Seneviratne W: Open access initiatives and institutional repositories: Sri Lankan scenario.2016.\n\nPratheepan T, Weerasooriya WA: The publication output and impact of various faculties in Sri Lankan Universities: A Scientometric Assessment and Policy Implications. J Univ Librarians Assoc Sri Lanka. 2016; 19(1). Publisher Full Text\n\nRanasinghe P, Jayawardena R, Katulanda P: Sri Lanka in global medical research: a scientific analysis of the Sri Lankan research output during 2000-2009. BMC Res Notes. 2012; 5(1): 1–8.\n\nMalalasekera A: Sri Lankan research output. Sri Lanka J Surgery. 2015; 32(4).\n\nCRDS J: Current status on science and technology in ASEAN countries. CRDS-FY2014-OR-02-EN. 2015.\n\nKawtrakul A, Praneetpolgrang P: A history of AI research and development in Thailand: Three periods, three directions. AI Magazine. 2014; 35(2): 83–92. Publisher Full Text\n\nGautam P: An overview of the Web of Science record of scientific publications (2004–2013) from Nepal: focus on disciplinary diversity and international collaboration. Scientometrics. 2017; 113(3): 1245–1267. Publisher Full Text\n\nGupta BM, Bala A: S&T publications output of Nepal: a quantitative analysis, 2001–10. Scientometrics. 2012; 93(3): 1029–1046. Publisher Full Text\n\nMoed H, Halevi G: Tracking scientific development and collaborations–The case of 25 Asian countries. Res Trends. 2014; 38.\n\nThawnghmung AM: Do consultancies compromise academic research and ethics? A case study of Burma/Myanmar. Asian J Political Sci. 2017; 25(2): 176–193. Publisher Full Text\n\nTripathi M, Kumar S, Babbar P: Bibliometrics of social science and humanities research in India. Curr Sci. 2018; 114(11): 2240–2247. Publisher Full Text\n\nShilpa AK: Scientific Research in India: Drawing Insights from Bibliometric Indicators.Publisher Full Text\n\nKolle SR, Shankarappa TH: Scientometric analysis of scientific papers from India (1989-2015) based on WoS data. SRELS J Information Management. 2016; 53(6): 2016. Publisher Full Text\n\nPaul G, Deoghuria S: Where we stand? A scientometric mapping of Indian Science & Technology research in some major research areas.\n\nSadana R, D’Souza C, Hyder AA, et al.: Importance of health research in South Asia. Bmj. 2004; 328(7443): 826–830. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHtun KW, Lwin NN, Naing TH, et al.: ASEAN-India connectivity: A Myanmar perspective. ASEAN-India Connectivity: The Comprehensive Asia Development Plan, Phase II; 2011.\n\nSanthosh k, Janodia v, M N, et al.: BIMSTEC.2021, April 18. Retrieved from osf.io/xs3m7\n\nJanodia ksv, N M, A I, et al.: BIMSTEC.2021, April 18. Publisher Full Text" }	[ { "id": "91025", "date": "06 Aug 2021", "name": "Shankar Reddy Kolle", "expertise": [ "Reviewer Expertise Bibliometric analysis", "scientometric analysis", "research evaluation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work entitled \"Publication analysis in Bay for Bengal Initiative for Multi-Sectoral Technical and Economic Cooperation Nations\" was carried out using the data from the Scopus database and analyzed using the SciVal tool in terms of scholarly output, field-based citations impact, collaboration with other countries, subject-wise contribution, along with international collaboration. The study's significant findings are that more significant countries like India and Thailand have contributed immensely than smaller countries. However, a more detailed analysis of the number of papers in the top ten high-impact factor journals would be helpful to see the research progress in the BIMSTEC countries. This work would be helpful to know for researchers for the research contribution of BIMSTEC countries.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "97163", "date": "25 Nov 2021", "name": "Samile Andréa de Souza Vanz", "expertise": [ "Reviewer Expertise Bibliometrics", "scientific collaboration." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper presents an interesting historical background of seven BIMSTEC countries which is very useful to understand the context of each country. Publication output from Bangladesh, India, Myanmar, Sri Lanka, Nepal, Thailand, and Bhutan is obtained for the period 2012 to 2017 from Scopus, a great database to identify papers published around the world. As an international database, Scopus is not specific and embracing for each country, but permits a comparison between nations. The World Bank Indicators are also adequate to compare different nations.\nData from SciVal coincide with the period 2021-2017 and the Scopus dataset period is 2012-2017. It is important to explain why a different period was chosen, why and when Scopus and SciVal data were used.\n\nThe descriptive analysis presented, figures and tables are very illustrative.\n\nThe results and discussion could improve with a review of previous bibliometric studies, especially those related to the 7 countries.\n\nAnother important ponderation must be the volume of journals indexed by Scopus. Everyone knows that it is a very selective database. I suggest the authors mention how many journals from each country are indexed compared to how many are published by each country. The output indexed by Scopus is just a part of the total produced by each country.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-510
https://f1000research.com/articles/10-509/v1	28 Jun 21	{ "type": "Research Article", "title": "Comparison of two multiplexed technologies for profiling >1,000 serum proteins that may associate with tumor burden", "authors": [ "Annie Ren", "Ioannis Prassas", "Vijithan Sugumar", "Antoninus Soosaipillai", "Marcus Bernardini", "Eleftherios P Diamandis", "Vathany Kulasingam", "Annie Ren", "Ioannis Prassas", "Vijithan Sugumar", "Antoninus Soosaipillai", "Marcus Bernardini", "Eleftherios P Diamandis" ], "abstract": "Background: In this pilot study, we perform a preliminary comparison of two targeted multiplex proteomics technologies for discerning serum protein concentration changes that may correlate to tumor burden in ovarian cancer (OC) patients. Methods: Using the proximity extension assay (PEA) and Quantibody® Kiloplex Array (QKA), we measured >1,000 proteins in the pre-surgical and post-surgical serum from nine OC patients (N=18 samples). We expect that proteins that have decreased significantly in the post-surgical serum concentration may correlate to tumor burden in each patient. Duplicate sera from two healthy individuals were used as controls (N=4 samples). We employed in-house ELISAs to measure five proteins with large serum concentration changes in pre- and post-surgical sera, from four of the original nine patients and the two original controls. Results: Both platforms showed a weak correlation with clinical cancer antigen 125 (CA125) data. The two multiplexed platforms showed a significant correlation with each other for >400 overlapping proteins. PEA uncovered 15 proteins, while QKA revealed 11 proteins, with more than a two-fold post-surgical decrease in at least six of the nine patients. Validation using single enzyme-linked immunosorbent assays (ELISAs) showed at least a two-fold post-surgical decrease in serum concentration of the same patients, as indicated by the two multiplex assays. Conclusion: Both methods identified proteins that had significantly decreased in post-surgical serum concentration, as well as recognizing proteins that had been implicated in OC patients. Our findings from a limited sample size suggest that novel targeted proteomics platforms are promising tools for identifying candidate serological tumor-related proteins. However further studies are essential for the improvement of accuracy and avoidance of false results.", "keywords": [ "Proteomics", "cancer biomarkers", "proximity extension assay", "antibody array", "multiplex", "immunoassay", "ovarian cancer" ], "content": "List of abbreviations\n\nACVR1, activin receptor type 1\n\nBCAM, basal cell adhesion molecule\n\nBMP9, bone morphogenetic protein 9\n\nBSA, bovine serum albumin\n\nCA125, cancer antigen 125\n\nCOMP, cartilage oligomeric matrix protein\n\nCV, coefficients of variation\n\nCYFRA21-1, keratin type I cytoskeletal 19\n\nPARP-1, poly [ADP-ribose] polymerase 1\n\nELISA, enzyme-linked immunosorbent assay\n\nEPCAM, epithelial cell adhesion molecule\n\nFC, fold change\n\nFOLR1, folate receptor 1\n\nGYN, gynecologic\n\nHE4, human epididymis protein\n\nIFNL1, interferon lambda 1\n\nIgG, immunoglobulin G\n\nKLK, kallikrein\n\nKRT19, keratin type I cytoskeletal 19\n\nLLD, lower limit of detection\n\nLOA, limits of agreement\n\nMS, mass spectrometry\n\nMSLN, mesothelin\n\nNAMPT, nicotinamide phosphoribosyltransferase\n\nNPX, normalized protein expression\n\nOC, ovarian cancer\n\nPAEP, glycodelin\n\nPCR, polymerase chain reaction\n\nPEA, proximity extension assay\n\nQKA, Quantibody® KiloplexArray\n\nSLAM, signaling lymphocytic activation molecule\n\nSTIP1, stress-induced phosphoprotein 1\n\nTLR, toll-like receptor\n\nTROP2, trophoblast antigen 2\n\nUHN, University Health Network\n\n\nIntroduction\n\nAdvancements in omics technologies have been instrumental in illuminating the heterogenous traits of cancer cells that harbor distinct genetic, epigenetic and/or phenotypic fingerprints.1 The further understanding of this intra- and inter-tumoral heterogeneity has propelled a new era of precision medicine, which seeks to personalize clinical practices, such as disease prognosis, treatment selection and monitoring treatment efficacy, according to the unique molecular makeup of each tumor.2,3 Among the multitudes of omics technologies, examining the proteome is the ultimate lens into the functional units of tumor cells. The blood proteome is a particularly rich mine for unearthing information on the pathophysiological traits of an individual. Tumors secrete a medley of tumor-derived proteins into the blood, which could be quantified to serve as proxies of tumor status. The concentration of tumor-related proteins is further propagated with highly increased expression during cancer progression. The detection and measurement of proteins in blood plasma/serum is thus very advantageous for mapping tumor activity in each patient. This would enable the discovery and validation of new biomarkers for the prediction of disease progression, treatment response, and effective personalized treatment for patients.\n\nThe proteomics technique, mass spectrometry (MS), conventionally centers around a system-wide, unbiased approach to protein detection. However, pre-analytical and analytical variation, detection of low abundance proteins, and the depth of proteome coverage remain major hurdles for the MS-based methods, in relation to analyzing complex biological fluids.4 Complementing a mounting interest in precision medicine is a timely advent of new, targeted proteomics platforms, aimed at aiding biomarker discovery with enhanced sensitivity, specificity, multiplexing, and throughput. These immunoassay-based multiplex technologies could potentially screen thousands of proteins, while detecting miniscule amounts of clinically relevant, tumor-associated proteins in the plasma or serum of individual patients.5 We previously described such tumor-associated proteins identified in cancer patients as personalized tumor markers, where each marker is highly specific and sensitive for cancer detection in 5-30% of patients.6,7 Cancer screening individual patients against a repertoire of personalized tumor markers, by using a targeted proteomics method could identify a distinct protein signature that best characterizes the mosaicism of each unique tumor. This would help guide treatment and surveillance.\n\nIn this study, we sought to conduct preliminary analyses examining the technical feasibility of two targeted multiplex proteomics technologies for detecting changes in serum protein concentrations that could correspond to tumor burden in individual patients, using advanced ovarian cancer (OC) as a model. OC is often diagnosed at late stages, where the survival rate is only 30%.8 Clinical practices for diagnosing, monitoring, and guiding treatment are reliant on imaging modalities and the serum-based biomarker, CA125.8 However, genomic studies have unveiled a diverse tumor mutational landscape, with advanced OC deriving from numerous genetic mutations and intra-individual genetic heterogeneity heavily accounting for relapse and acquired chemoresistance.9 The indisputable evidence on OC genetic heterogeneity espouses the need to build a library of personalized tumor markers that may better represent the diverse tumors found in patients.\n\nHerein, we performed an initial assessment of the technical reliability of two commercially available multiplex proteomics technologies, the proximity extension assay (PEA) and the Quantibody® KiloplexArray (QKA), for delineating changes in serum protein concentrations that may correlate to OC tumor burden. The platforms used in this study reflect two different ways of improving the sensitivity and throughput of traditional sandwich-type ELISAs. The PEA technology is based on immuno-polymerase chain reaction (PCR) and uses dual-recognition antibodies that are labelled with complementary oligonucleotides to generate quantitative PCR signals corresponding to protein concentration. The QKA works by precisely printing captured antibodies on glass slides and leveraging nanofluidics to perform 1000 micro-ELISAs in a single experiment. Over 1000 proteins analyzed by each platform span a wide biological and pathological relevance, such as their involvement in pro-inflammatory responses, immune response, cell regulation, and tumorigenesis. To evaluate these technologies, we employed each method to measure ≥1000 proteins in paired serum samples collected pre- and post-surgically from OC patients. In addition to performing technical comparisons for data derived from the ≥1000 proteins and >400 overlapping targets, we selected proteins that had the most decrease in serum after surgery, according to each proteomics method. We speculate significant decrease in serum proteins following the tumor debulking surgery, may be associated with changes in tumor burden in the patient. We further conducted orthogonal validation for five of the selected proteins using verified, in-house, single target ELISAs, to evaluate the reliability and accuracy of the platforms. Our study takes a comparative approach to serum protein profiling in cancer by using two different targeted proteomics methods. These methods offer a preliminary glimpse into the strengths and limitations of each technology for the identification of serum proteins that may correlate to tumor burden, and individualized serum protein signatures that could provide information on tumor heterogeneity.\n\n\nMethods\n\nSerum samples from nine advanced high-grade serous carcinoma OC patients were collected one week before optimal debulking surgery, and at four weeks after surgery (n = 18). These samples were retrospectively obtained from the University Health Network (UHN) Gynecologic (GYN) Blood Biobank. The patient inclusion criteria included patients who showed pre-surgical clinical CA125 values of at least 1000 U/mL, in order to select for patients with high tumor load prior to surgery, as well as those who received primary debulking surgery followed by six cycles of platinum/taxane-based chemotherapy as their first-line treatment. Samples were collected and stored at -80 °C, prior to analysis with a standardized protocol to avoid systemic biases. One serum sample from a healthy male and female were split into two identical aliquots (n = 4) for use as controls.\n\nSera from OC patients and controls were analyzed for intra-individual relative protein concentration changes using the multiplex PEA technology (Olink Proteomics, Uppsala, Sweden). A total of 1073 unique proteins were assayed across 13 panels (92-plex) , which span a broad dynamic range of eight to 10-log with the sensitivity of sub fg/mL to pg/mL. We have previously described the workflow of the PEA technology.10 In brief, 1μL of serum sample was incubated with 92 pairs of oligonucleotide-labeled antibodies. Upon binding of both antibodies to the target, the single-stranded DNA ends hybridize and act as a template for DNA polymerase-dependent extension. To ensure the analytical quality of the samples that ran in singlicates, three types of internal controls (for sample quality, immunoglobulin G (IgG) binding, oligonucleotide annealing, and PCR amplification) are spiked into each sample prior to conducting the assay. Control sera in duplicate, negative control samples in triplicate, and inter-plate sera in triplicate are also included in each assay plate to control for signal specificity, inter-well variation and inter-plate variation. The DNA amplicon which results from the extension step, is detected with qPCR on the BioMark HD Real-Time PCR System (Fluidigm, CA, USA). The signal from each unique amplicon is proportional to protein concentration [expressed as a relatively quantitative, arbitrary unit on a log2 scale termed, normalized protein expression (NPX)]. NPX values are determined by normalizing each signal to the median signal per plate. To minimize bias, the technical personnel were blinded to the sample status and only intra-plate NPX value comparisons were made.\n\nThe workflow of some panels uses serum at a pre-specified dilution (Cardiometabolic: 1:2025; Cardiovascular III: 1:100; Development: 1:100; Metabolism: 1:10) to detect high abundance proteins in expected physiological and pathological ranges. A very low post-surgical fold change (FC) in CA125 (in Oncology panel) was observed, when we ran undiluted sera as recommended by the manufacturer (Underlying data,11 therefore only the pre- and post-surgical sera with 1:10 dilution for the Oncology panel were performed. We identified three proteins from this panel [CA125, human epididymis protein (HE4), and folate receptor 1 (FOLR1)] that exhibited signs of the Hook effect in the undiluted analysis, as the pre-surgical serum concentrations were higher in the 1:10 dilution versus undiluted analysis. Therefore, we used the pre- and post-surgical serum levels of CA125, HE4, and FORL1 that were obtained from the 1:10 dilution, for the continuation of our analysis.\n\nWe employed the QKA (RayBiotech, GA, USA) to concurrently measure 1000 proteins in sera from OC patients and controls. The biological relevance of the 1000 target molecules is mainly inflammation-related. The platform, which combines 25 nonoverlapping 40-plex arrays to perform 1000 sandwich ELISAs simultaneously, possesses a dynamic range of around 3- to 4-log concentration and sensitivity ranging from 1 pg/mL to 100 ng/mL. The workflow of the array was previously described.12 In brief, each array included protein standards to generate a standard curve for absolute protein quantitation. The samples were analyzed in technical quadruplicates, and the technical personnel were blinded to the sample status. We diluted 750μL of serum four-fold to a final volume of 3 mL for use in all 25 arrays. Capture antibodies were bound to the glass surface in the multiplex. After blocking and sample incubation, nonspecific binding was washed away, and a biotin-labeled detection antibody was subsequently added. Finally, Streptavidin-conjugated Cy3 equivalent dye was added to yield fluorescent signals, which were read using a microarray laser scanner (GenePix 4200A, Molecular Dynamics, Sunnyvale, CA, USA). The Q-Analyzer software (Raybiotech) was used to compute protein concentration, after accounting for intra- and inter-slide normalization.\n\nWe analyzed the data from each multiplex proteomics method by first calculating the FC in serum concentration from the pre-surgical to post-surgical sample for each protein and patient. For all proteins, concentrations that were below the lower limit of detection (LLD) of the method were normalized to equal the value of the LLD. As a reference to the technical variation, we also calculated the FC between the technical duplicate measurements of the control samples for each technology. In the control samples, proteins with more than two-FC between the duplicate measurements were removed. In at least six of the nine patients, proteins with pre-specified two-fold or higher decrease in their post-surgical serum concentrations were selected as biomarker candidates.\n\nIn-house single target ELISA assays previously developed were used to validate the post-surgical serum concentration changes in the five proteins [kallikrein-5 (KLK5), KLK6, KLK7, KLK10, and KLK11] that were observed in the same four of the nine patients (Patient 3, 6, 7, 8 for the PEA analysis and patient 1, 3, 6, 7 for the QKA), and the two controls. The type and sources of all antibodies used in the immunoassays and the assay workflow are previously described.13 In brief, sandwich format immunoassays for KLK5, 6, 7, and 10 employed monoclonal-monoclonal antibody configurations. We coated each well in microtiter plates with 500ng of monoclonal antibody in 100μL coating buffer (50 mmol/L Tris-HCl, pH 7.8). We incubated the plates overnight before washing (10 mmol/L Tris-HCl, pH 7.4, containing 150 mmol/L NaCl and 0.5 mL/L Tween 20). Next, we added 50 μL recombinant protein standards or samples diluted in 6% bovine serum albumin (BSA), along with 50 μL assay buffer (6% BSA containing 25 mL/L normal mouse serum, 100 mL/L normal goat serum, and 10 g/L bovine IgG). After incubation and wash, we added 50ng of our in-house biotinylated monoclonal detection antibody diluted in 100 μL assay buffer, as previously described.13 Following incubation and wash, we added 5 ng alkaline phosphatase–conjugated streptavidin diluted in 100 μL 6% BSA. Final round of incubation and wash was followed by, incubation of each well with 100 μL diflunisal phosphate solution (0.1 mol/L Tris-HCl, pH9.1, containing 1 mmol/L diflunisal phosphate, 0.1 mol/LNaCl, and 1 mmol/L MgCl2). Finally, we added 100 μL developing solution (1 mmol/L Tris, 0.4 mol/L NaOH, 2 mmol/L TbCl3, and 3 mmol/L EDTA), before measuring the fluorescence with a time-resolved fluorometer, The EnVision® 2105 multimode plate reader (PerkinElmer Life Science, MA, USA). Standard curve and protein concentration calculations were performed automatically, as previously described.14 We used a monoclonal-polyclonal ELISA configuration for measuring KLK11. The assay followed the same procedures, except we added 50 ng of our in-house biotinylated polyclonal antibody.13\n\nStatistical analyses were performed using the GraphPad Prism software (version 4.02). Pearson correlation coefficients were computed to evaluate the correlation between serum protein concentrations obtained from QKA, PEA and ELISA. P values of less than 0.05 were considered statistically significant. Bland-Altman plots were used to assess the agreement of measurements between two methods. For the latter, we calculated the mean difference, or bias, between measurements by the two methods, and the 95% limits of agreement (LOA) using the formula, bias ± 1.96* Standard Deviation (SD). Agreement is determined by how close the bias is to zero.\n\n\nEthical considerations\n\nSerum samples from OC patients with high-grade serous carcinoma were retrospectively obtained from the UHNGYN Blood Biobank with approval by the Research Ethics Board (REB) of UHN, Toronto, Canada (REB #10-0591). All eligible patients attending the GYN Oncology Clinics at the Princess Margaret Cancer Centre (Toronto, ON, Canada) are approached at their initial appointment and offered the opportunity to participate in the UHN GYN Blood Biobank. The purpose of the biobank, procedures, and schedule of collections are reviewed and explained thoroughly, aiding in written informed consent (as per UHN Research Ethics Board guidelines and UHN REB-approved consent document). Upon consent, additional blood samples are collected for future research at the same time as clinical bloodwork. These samples are linked to high quality de-identified clinical data.\n\n\nResults\n\nWe compared the post-surgical FC in CA125 reported by the clinic and the two proteomics platforms in the nine OC patients (Figure 1). Scatterplot analysis of FC assessed by PEA and the clinical assay showed a weak correlation [Figure 1A, Pearson correlation coefficient (r) = 0.546, P = 0.066]. Bland-Altman plot for the two methods showed a bias of −9.64 in FC. Signifying PEA showed a lower post-surgical FC in CA125 overall. Measurement in one patient (P3) resided outside of the LOA (−41.55 to 22.27 difference in FC). Similarly, there is a weak correlation between post-surgical CA125 FC measured by QKA and clinical assay (Figure 1B, r = 0.470, P = 0.202). Bland-Altman analysis of QKA versus clinical data showed a bias of −5.43. The LOA ranged from −42.31 to 31.46, with all measurements within limit.\n\nA) Proximity extension assay (PEA): (Left) Scatterplot analysis of the post-surgical CA125 fold change assessed by PEA technology and clinical assay showed a weak correlation (Pearson correlation coefficient (r) = 0.546, P = 0.066). Solid red line in scatterplots shows the line of best fit. (Right) Bland-Altman plot of the post-surgical fold change in CA125 measured by PEA and the clinical assay. Solid red line shows the mean difference or bias in fold change measurements (−9.64). Dashed blue lines show the upper and lower limit of agreement (LOA; Mean ± 1.96 SD; ranges from -41.55 to 22.27 difference in fold change). B) Quantibody array: (Left) Similarly, there is a weak correlation between the post-surgical CA125 fold change measured by Quantibody array and clinical assay (r = 0.470, P = 0.202). (Right) Bland-Altman plot of the post-surgical CA125 fold change assessed by Quantibody array and clinical assay. Solid red line exhibits bias (−5.43). Dashed blue lines show the upper and lower LOA (−42.31 to 31.46).\n\nWe mined the target analytes of the PEA and QKA platforms and identified 444 overlapping proteins (Extended data).15 Scatterplot analysis of the post-surgical FC in the 444 proteins from nine OC patients, showed a weak but significant correlation between the methods (r = 0.275, P < 0.0001) (Figure 2A). Bland-Altman plot showed a minimal bias of −0.68 in the FC (-13.95% difference) between the methods (Figure 2B). The LOA ranged from −7.84 to 6.47 difference in the FC between the methods (−115.0% to 87.11% difference), where 94.3% of protein measurements were within this range. Differences in measurements are shown as percentages due to the large data range.\n\nA) Scatterplot showed a significant correlation between the post-surgical protein fold changes assessed by PEA technology and Quantibody array (Pearson correlation coefficient (r) = 0.275, **P < 0.0001). Solid red line shows the line of best fit. B) Bland-Altman plot of the post-surgical protein fold changes measured by PEA and Quantibody array. Solid red line shows the mean % difference or bias in fold change measurements (−13.95%). Dashed blue lines show the upper and lower limit of agreement (Mean ± 1.96 SD; −115.0% to 87.11% difference in fold change). The difference in measurements are shown as percentages to better visualize the large range of data.\n\nThe PEA analysis identified 15 proteins (including CA125) that had a two-fold decrease between the pre-surgical to post-surgical samples, in six out of the total of nine patients (66.7-100%) (Table 1). Thirteen proteins had at least five-fold decrease in two of the seven patients (22.2-77.6%) (Table 1). Nine proteins decreased by at least ten-fold in one of the five patients (11.1-55.6%) (Table 1). CA125, FOLR1, and keratin type I cytoskeletal 19 (KRT19) showed the highest median post-surgical fold decrease (over 10-fold) (Table 1).\n\nHeatmap of post-surgical fold decrease in protein serum concentration in 9 ovarian cancer patients.\n\n Fold change between technical duplicate measurements of healthy controls is shown.\n\n** FD, Fold decrease in protein serum concentration post-surgery.\n\nThe QKA data yielded 11 proteins that decreased by at least two-fold in six of the nine patients (66.7-100%) (Table 2). Ten of the proteins (Table 2) decreased by at least five-fold, in one of the nine patients (11.1-100%). Five proteins (Table 2) decreased by at least ten-fold, in one of the five patients (11.1-66.7%). CA125, KLK11, FOLR1, and KLK5 showed the highest median post-surgical fold decrease (Table 2).\n\nHeatmap of post-surgical fold decrease in protein serum concentration in 9 ovarian cancer patients.\n\n* Fold change between technical duplicate measurements of healthy controls is shown.\n\n** FD, Fold decrease in protein serum concentration post-surgery.\n\nAmong the proteins with the largest post-surgical decrease in serum concentration, eight proteins [CA125, FOLR1, KLK11, mesothelin (MSLN), basal cell adhesion molecule (BCAM), trophoblast antigen 2 (TROP2), HE4, and cartilage oligomeric matrix protein (COMP)], were targeted by both PEA and QKA. There were significant correlation between the platforms in the post-surgical FC observed for CA125 (r = 0.785, P = 0.0121), FOLR1 (r = 0.861, P = 0.0028), and BCAM (r = 0.845, P = 0.0041) (Figure 3). KLK11, MSLN, TROP2, HE4, and COMP did not show a significant correlation for the post-surgical FC. Bias in CA125 FC was at −4.21, where PEA data showed lower FC overall, compared to QKA (Figure 3A). Measurement of one patient (P4) was outside of the LOA (−20.50 to 12.08). Bias in FOLR1 FC was at 2.88, with all data within the LOA range of −11.36 to 17.12 (Figure 3B). BCAM FC showed a bias of −3.33, LOA, where all measurements were within the LOA range of −16.26 to 9.60 (Figure 3C).\n\nA) (Top) CA125 showed a significant correlation in the post-surgical fold change obtained from proximity extension assay (PEA) and Quantibody array in 9 ovarian cancer patients (Pearson correlation coefficient (r) = 0.785, P = 0.0121). Solid red line in scatterplots shows the line of best fit. (Bottom) Bland-Altman plot of the post-surgical CA125 fold change measured by PEA and Quantibody array. The solid red line shows the mean difference or bias in fold change assessments (−4.21). Dashed blue lines show the upper and lower limit of agreement (LOA, Mean ± 1.96 SD; −20.50 to 12.08). B) (Top) Folate receptor 1 (FOLR1) showed a highly significant correlation in the post-surgical fold change assessed by PEA and Quantibody array (r = 0.861, P = 0.0028). (Bottom) Bland-Altman analysis of the post-surgical FOLR1 fold change observed by PEA and Quantibody array. Solid red line shows bias (2.88). Dashed blue lines show the upper and lower LOA (−11.36 to 17.12). C) (Top) Basal cell adhesion molecule (BCAM) also showed a highly significant correlation in the post-surgical fold change assessed by PEA and Quantibody array (r = 0.845, P = 0.0041). (Bottom) Bland-Altman plot of the post-surgical fold change in BCAM observed by PEA and Quantibody array. Solid red line shows bias (−3.33). Dashed blue lines show upper and lower LOA (−16.26 to 9.60).\n\nBoth the PEA and ELISA data showed that KLK6, 10, and 11 decreased by at least two-fold in the post-surgical samples of four patients (Figure 4A and B). However, the correlation between the FC in KLKs shown by the two methods was not significant (Figure 4C), with KLK10 relatively showing the strongest correlation (r = 0.855, P = 0.065). The Bland-Altman plot demonstrated minimal difference between the average of the FC obtained by PEA and QKA data for all three proteins. All data points for FC as measured by the two methods were also within the upper and lower limits of agreement (LOA), which is the 95% confidence interval that shows the maximum allowed difference between two methods (Figure 4D).\n\nA-B) Bar graphs depict the pre- and post-surgical serum level of three proteins (Kallikrein (KLK) 6, 10, 11) in four ovarian cancer patients and two controls (C1 and C2), as assessed by PEA technology (A) and ELISA (B). C) Scatterplot analysis of the post-surgical fold change of KLK6, 10, and 11 observed by PEA and ELISA showed a weak correlation. Solid red line in scatterplots shows the line of best fit. D) Bland-Altman plot of the post-surgical fold change in KLK6, 10, and 11 assessed by PEA and ELISA. Solid red line shows the mean difference or bias in fold change assessments. Dashed blue lines show the upper and lower limit of agreement (Mean + 1.96 SD). Post-surgical decrease of at least two-fold. Post-surgical decrease of at least five-fold. Post-surgical decrease of at least ten-fold.\n\nThe QKA and ELISA results for KLK5, 7, and 11 showed at least a two-fold decrease in post- surgical samples of all four patients (except KLK7 in P3 in the QKA data and P6 in the ELISA data) (Figure 5A and B). As both methods yield absolute protein quantitation, we correlated their serum concentration (in pg/mL) in the pre- and post-surgical samples in four patients (N = 8 samples, Figure 5C). KLK5 and KLK7 did not show a significant correlation, while KLK11 showed a significant correlation in serum concentration measured by the two methods (r = 0.940, P = 0.001). Bland-Altman analysis showed bias in FC for all three proteins ranging from -5.48 to 0.62 between the QKA and ELISA results, with all data within the LOA range (Figure 5D).\n\nA-B) Bar graphs depict the serum level of three proteins (Kallikrein (KLK) 5, 7, 11), pre- and post-surgery, in four ovarian cancer patients and two controls (C1 and C2), as assessed by Quantibody array (A) and ELISA (B). C) Scatterplot analysis of the post-surgical fold change of KLK5 and 7 observed by PEA and ELISA showed a weak correlation, while KLK11 showed a significant correlation (Pearson correlation coefficient (r) = 0.940, P = 0.001). Solid red line in scatterplots depicts the line of best fit. D) Bland-Altman plot of the post-surgical fold change in KLK5, 7, and 11 measured by Quantibody array and ELISA. Solid red line demonstrates the mean difference or bias in fold change assessments. Dashed blue lines show the upper and lower limit of agreement (Mean ± 1.96 SD).\n\n\nDiscussion\n\nThe dynamic progression of cancer gives rise to a non-uniform dispersal of molecularly distinct cell subpopulations across and within tumors.1 This innate cancer heterogeneity is manifested through alterations in protein expression within the tumor and it is reflected in the composition of tumor-related proteins that are released into the bloodstream. Investigation of the blood proteome of individual cancer patients is hence a non-invasive modality to uncover the tumor-derived proteins and their distinctive tumor makeup. Latest innovations in multiplex proteomics technologies have become attractive tools for mapping the circulating cancer proteome, with the hopes of giving novel insight into tumor progression and discovery of potential biomarkers to improve clinical management.16 As the concept of personalized tumor markers were kept in mind,6,7 we aimed to compare the feasibility of two fundamentally different targeted multiplex proteomics technologies, PEA and QKA, to measure serological proteins that may correspond to tumor burden in specific patients. To accomplish this aim, we employed the PEA and QKA technologies to simultaneously measure 1073 and 1000 proteins, respectively, in before and after surgical sera from nine OC patients (18 samples total), with two control duplicates (4 samples in total). We compared the reliability of the two methods for detecting changes in serum protein concentration in relation to an abrupt change in tumor burden, which was achieved by tumor debulking surgery.\n\nWe previously published the intra-assay reproducibility of the PEA and QKA technology, finding that PEA showed high precision with a strong correlation between the technical duplicate measurements for the two controls (r = 0.95 to 0.97).10 Our previous evaluation of QKA demonstrated lower precision and a weaker correlation between the technical duplicates (r = 0.853 to 0.862).12 A closer look at intra-assay variation observed that 97.6% of the 1073 proteins analyzed by PEA showed a favorable coefficient of variation (CV) below 20%, 89.3% had a CV below 10%, and 0.3% showed a CV over 100% (data not shown). In contrast, 67.9% of the 1000 proteins assayed by QKA showed a CV below 20%, 47.6% had a CV below 10%, while 20.5% exhibited high variation with a CV over 100% (data not shown). We also assessed the concordance of the multiplex measurements and clinical values for CA125. The Post-surgical FC in CA125, derived from both multiplex assays, did not have a significant correlation with the clinical CA125 trends. PEA and QKA showed much lower FC in CA125 post-surgery compared to the clinical data, with the mean differences of -9.64 and -5.43–fold. Because the PEA and QKA assays measure in 92-plex and 40-plex respectively with ultrasensitivity (down to fg/mL – pg/mL), there may be a bias for low abundance proteins, while highly abundant proteins, such as large quantities of CA125 secreted by OC tumors, may be beyond the upper limit of the dynamic range. The Hook effect can occur in these situations, where very high concentrations of an analyte can cause excessive binding to both the capture and detection antibodies, thereby obstructing the antibodies from forming a sandwich immunocomplex.17 This causes assay signals to decrease at a very high analyte concentrations, leading to a misconceived lower serum level.17 Although we tried to correct for the Hook effect seen in three proteins, CA125, HE4 and FOLR1, in the PEA analysis after re-running sera at 1:10 dilution, some pre-surgical samples may have benefited from further dilution such as at 1:50 or 1:100, to elucidate the true post-surgical FC which would correspond better to clinical patterns. Although a previous study reported a strong correlation between CA125 concentrations as measured by PEA and clinical CA125 concentrations,18 we did not see this result. This is likely because we selected for advanced OC patients whose pre-surgical clinical CA125 values exceeded 1000 U/mL, which we found showed Hook effect. Future considerations for avoiding the possible Hook effect when using multiplex proteomics technologies, such as PEA and QKA, is to analyze pre-surgical samples that may contain high concentrations of tumor markers at varying dilutions. However, this will increase the cost of analysis.\n\nWe next assessed the concordance of measurements between the two platforms and observed a significant correlation between the post-surgical FC of the 444 proteins measured by both methods. PEA and QKA showed a comparable FC in the overlapping proteins, with mean differences of −0.68–fold. We noted 36.9% (164/444) of the overlapping proteins are covered by the PEA panels that require pre-dilution of plasma/serum (from 1:10 up to 1:2025) to enrich high abundance proteins. The other overlapping proteins are in PEA panels that use undiluted plasma/serum and encompass mainly low abundance proteins. In our analysis, the PEA and QKA results showed a correlation for a range of low and high abundance proteins.\n\nUpon the selection of proteins that may correlate to tumor burden, we considered the intra-individual FC in protein concentration observed from the pre- to post-surgical samples in each patient. We propose that protein levels which decrease significantly after tumor debulking may be informative of tumor burden in the patient. The PEA data revealed 15 proteins (including CA125 and the FDA-approved marker HE4) that had at least two-fold decrease in their serum concentrations in at least six of the nine patients.CA125, FOLR1, and KRT19 had the highest median FC (Table 1). Each patient exhibited a unique protein tumor signature consisting of 7 to 14 proteins that decreased by at least two-fold post-surgery, which may be informative of their heterogenous tumor (Table 1). We previously employed an integrated approach that had combined proteomics, transcriptomics, and bioinformatics to identify FOLR1, as a new OC biomarker.19,20 Since then, a FOLR1-targeting antibody-drug conjugate has shown encouraging efficacy.21,22 Interestingly, tumors with overexpression in FORL1 and KRT19 have indicated increased sensitivity to cisplatin treatment, the main form of chemotherapy in OC,23,24 which could serve as predictive biomarkers of drug response. A recent study integrating PCR arrays, genome-wide microarray, and proteome analyses reaffirmed KRT19 and MSLN as OC-related proteins.25 Another OC study confirmed the overexpression of KRT19 and epithelial cell adhesion molecule (EPCAM) mRNA in circulating tumor cell-fraction samples, compared to peripheral blood samples.26 KRT19 is also known as CYFRA21-1 and has been extensively studied as a prognostic biomarker for OC that is complementary to CA125.27,28 Our PEA analysis yielded four KLKs (KLK6, 8, 10, 11) that had a considerable decrease in 67-78% of OC patients after surgery. Over the past decades, we have discovered these KLKs as new OC biomarkers, demonstrated their link to shorter progression-free-survival, and highlighted their potential as targets for novel therapeutics.29–31 Mainly, KLK6 is recently considered as a promising target for inhibition in OC.32 Among the other proteins in Table 1, poly [ADP-ribose] polymerase 1 (PARP-1) is extensively studied for promoting angiogenesis and invasion in OC, and it is the target for novel PARP-1 inhibitors used as maintenance therapy for OC.33–35 A multi-platform analysis using PEA to measure 368 plasma proteins identified that CA125, KLK11, MSLN, KLK6, BCAM, KLK8, and HE4 among 176 proteins were elevated in OC compared to non-malignant gynecologic disorders.36 The PEA results were independently validated using ELISA and an aptamer-based multiplex proteomics assay, SOMAscan, demonstrating high concordance.36 Finally, glycodelin (PAEP) and TROP2 have been shown to promote proliferation and invasion of OC cells and are prognostic markers for poor OC outcome.37–40 A novel antibody-drug conjugate targeting TROP2 is currently under validation for inhibiting OC tumor growth.41,42 Although interferon lambda 1 (IFNL1) has not been studied in relation to OC thus far, this new class of interferons showed anti-tumor properties, and has been scrutinized as a new immunotherapy agent for certain types of malignancies.43,44 Overall, the PEA analysis of pre- and post-surgical samples in individual OC patients was able to identify proteins with clinical importance. As new immunotherapies and targeted therapies are revealed,45,46 our findings suggest value in further investigations into a panel of personalized tumor markers, which may include proteins with therapeutic relevance such as FOLR1, KRT19, KLK6, PARP-1, TROP2, and IFNL1. This could be an instrumental tool for identifying the heterogenous makeup of individual tumors to select the best treatment options for individual patients.\n\nThe QKA data indicated 11 proteins (including CA125) with at least two-fold serum concentration decrease, in at least six of the nine patients (Table 2). Each patient showed a distinct protein tumor signature consisting of 4 to 10 proteins that showed at least two-fold post-surgical decrease, which may be informative of their tumor burden (Table 2). From the candidate proteins that were selected by separate PEA and QKA analysis, there were eight candidate proteins in common. However, only CA125, FOLR1 and BCAM from the eight common candidate proteins showed significant correlation in the trend of post-surgical FC (Figure 3), although the lack of statistical significance in other proteins could be due to the limited sample size, since KLK11 was selected as a top protein by both analyses (Tables 1 and 2). In fact, the three proteins that showed the highest median FC based on QKA analysis, (CA125, KLK11, and FOLR1) were also selected in the PEA analysis. QKA data also identified two other KLKs (KLK5 and 7), which we previously discovered to be indicators of poor prognosis in OC.47–49 Recent clinical and tumor-biological studies also recognized KLK5 and 7 as favorable drug targets for OC.32 Among the other proteins in Table 2, renin has been studied in relation to an increased activity in the hormonal renin-angiotensin system in OC.50 However, extrarenal renin-secreting tumors represent a subpopulation of OC tumors.51 DNA microarray analysis has revealed upregulation of activin receptor type 1 (ACVR1) in OC tissue compared to normal ovarian tissue.52 Furthermore, the binding of stress-induced phosphoprotein 1 (STIP1) to ACVR1 and bone morphogenetic protein 9 (BMP9) signaling of ACVR1 have both shown to promote OC proliferation.53,54 Notably, microarray analysis of OC tumor tissue revealed just 29% of tumors expressed BMP9, which induces ACVR1 pathway hyperactivation.54 In recent years, unravelling the over-expression of nicotinamide phosphoribosyltransferase (NAMPT), a critical enzyme in the NAD salvage pathway, had prompted the development of new therapeutic strategies for NAMPT inhibition in subsets of OC patients.55,56 The immunosuppressive receptor, signaling lymphocytic activation molecule (SLAM), has also shown heterogeneous expression, where it was overexpressed in a subset of CD3+ T lymphocytes isolated from the blood of OC patients.57 One study thus far has described an upregulation of COMP in OC through cancer stem cells analysis.58 Nevertheless, COMP has been widely studied for inducing cancer stem cell formation and proliferation,59,60 as well its correlation with poor survival and recurrence in breast cancer, colon cancer, and prostate cancer.59,61,62 Finally, one study linked toll-like receptor 1 (TLR1), a pattern recognition receptor for innate immunity, to inflammation in the OC microenvironment.63 Remarkably, several studies have uncovered that genetic polymorphisms in the toll-like receptor gene cluster (TLR6, TLR1, and TLR10) promote chronic inflammation and enhance prostate cancer risk, in a subpopulation of individuals,64,65 warranting further investigation in other cancer subpopulations. Collectively, the QKA analysis was able to identify proteins associated with OC and tumorigenesis, where many proteins exhibited varied upregulations in subsets of OC patients. Our results support the idea that a panel of tumor-related proteins, including heterogeneously expressed proteins such as renin, ACVR1, TLR1, and KLKs,5,7,11 may encompass tumor heterogeneity in ovarian cancer patients.\n\nIn order to assess the concordance of the multiplexed data with our validated, in-house ELISAs, we performed single-target ELISA assays to quantitatively measure the change in serum expression levels of five proteins (KLK5, 6, 7, 10, and 11), in the same four patients and two controls. The two multiplex assays showed at least two-fold post-surgical decrease in serum concentrations of all KLKs (Figures 4 and 5). However, only KLK11 measurements by QKA showed statistical significance for correlation with ELISA (Figures 4 and 5), which could be due to limitations in our sample size. Visually assessing the agreement in multiplexed measurements with ELISA assays revealed that PEA, compared to QKA, showed a lower mean difference in post-surgical KLKs FC in the ELISA results (bias ranged from -1.49 to 0.18-fold for PEA versus -5.48 to 0.62-fold for QKA) (Figures 4 and 5). Two studies have reported a favorable correlation of PEA data and ELISA for validating candidate cancer-related proteins in OC and glioma.36,66 One recent study had employed Quantibody arrays, akin to QKA, for the identification of differentially expressed plasma proteins in OC, which resulted in validating nine candidates successfully by using ELISA.67 However, we previously reported discrepancies between QKA and ELISA results for validating nine candidate proteins that may correlate to tumor burden in pancreatic cancer patients.12 The analytical precision of multiplexed platforms may be highly analyte-dependent, and multiplexed data may benefit from independent validation to verify the protein quantitation.\n\n\nConclusions\n\nOur preliminary investigation demonstrated the initial potential of two targeted multiplex proteomics platforms, PEA and QKA, for identifying changes in serum protein concentrations that may associate with tumor burden in OC patients. While multiplex technologies possess high sensitivity, which is key to discovering clinically relevant proteins that are present at extremely low concentrations in the plasma/serum, there may be a concern for missing highly abundant tumor-derived proteins, such as CA125 for OC, which increase by several orders of magnitude in cancer plasma/serum. This bias can generally be avoided by analyzing serum at varying dilutions to enhance accurate detection of high abundance analytes. Although our study represented a limited sample size and cannot conclusively determine the technical reliability of the PEA and QKA assays, the multiplexed data in general, were in concordance with each other and, single target ELISAs for identifying general patterns of large changes in serum protein concentration. Our results showed the multiplatform approach was complementary for detecting proteins that were associated with pathobiological and clinical significance for OC, including encompassing a heterogenous array of known OC biomarkers, new therapeutic targets, and emerging markers of polymorphic gene/protein expression. Each patient may bear a unique tumor protein signature, a concept that warrants future investigation in larger cohorts. In the future, personalized tumor protein profiles could be monitored for changes in expression that may trigger clinically actionable events, such as selecting the best treatment, calculating prognostic risk, and detecting cancer recurrence in the patient. Our conclusions from a small sample size suggest that novel targeted proteomics methods can serve as valuable complementary tools to each other for comprehensively identifying diverse tumor protein signatures in the cancer proteome. However, it is beneficial to corroborate the data with independent, orthogonal methods such as MS and ELISA for accurate and validated protein quantitation, in order to minimize false results and promote further advancements in the biomarker discovery pipeline.\n\n\nData availability\n\nHarvard Dataverse: Comparison of two multiplexed technologies for profiling >1,000 serum proteins that may associate with tumor burden – a pilot study\n\nhttps://doi.org/10.7910/DVN/KRROIC.11\n\nThis project contains the following underlying data:\n\n1. Clinical CA125 vs Olink and Raybiotech (CA125 measurements from clinical assay, PEA technology and QKA)\n\n2. ELISA raw data results for KLK5, 7, 11 (KLK5, 7, 11 ELISA raw data)\n\n3. ELISA raw data results for KLK6 and KLK10 (KLK6 and KLK10 ELISA raw data)\n\n4. Raw data from Olink_1 in 10 dilution of Oncology panel (PEA technology data for 92 proteins with sample 1:10 dilution)\n\n5. Raw data from Olink_1076 unique protein measurements (PEA technology data for 1076 unique proteins)\n\n6. Raw data from Raybiotech_1000 unique protein measurements (QKA data for 1000 proteins)\n\nData are available under the terms of the CC BY-NC-SA (CC0 1.0 Universal).\n\nHarvard Dataverse: Extended data for Comparison of two multiplexed technologies for profiling >1,000 serum proteins that may associate with tumor burden\n\nhttps://doi.org/10.7910/DVN/7C3SV0.15\n\nThis project contains the following extended data:\n\n• Extended table 1. (List of overlapping protein targets for proximity extension assay and Quantibody® Kiloplex Array).", "appendix": "Acknowledgements\n\nNot applicable.\n\n\nReferences\n\nDagogo-Jack I, Shaw AT: Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol. 2018; 15(2): 81–94. PubMed Abstract \| Publisher Full Text\n\nDoll S, Gnad F, Mann M: The Case for Proteomics and Phospho-Proteomics in Personalized Cancer Medicine. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "89025", "date": "28 Jul 2021", "name": "Nivedita Chowdhury", "expertise": [ "Reviewer Expertise Single-cell technologies", "tumor biology", "biomarkers", "tumor immunology", "histology", "translational 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\nRen et al. have demonstrated a comparative study of identifying serum proteins that may associate with tumor burden in ovarian cancers, using two multiplexed proteomic technologies. This is an interesting study and the authors have elucidated the two methods distinctly in a patient cohort. Overall, the paper is well written, researched and structured. However, there are a few shortcomings, especially the justification for choosing the two methods and the clinical relevance. I also find that the authors have not stressed on the novelty of this study enough. Below, I have provided numerous comments on the text and suggestions for more in-depth analysis of the data. Given these, the manuscript requires major revisions.\nAbstract:\nWell summarized in terms of the methodology including validation using ELISAs. However, please use assertive language. Use of \"pilot\" and \"preliminary\" is not only redundant but also indicates low conviction/doubts on their own study. Also, change “expect that proteins have decreased…”. For eg. “speculate/hypothesize that proteins with reduced concentrations in the post-surgery sera, correlate to tumor burden in each patient”.\n\nIn the Results section, the authors need to emphasize on the novel findings of serum proteins previously not associated with tumor burden and/or OC. Also remove “novel proteomic platforms” from the conclusion - PEA is fairly well known and a few groups, including the authors' have published QKA results in the past.\n\nIntroduction:\n\nThe authors have included most of the relevant background with respect to tumor-associated proteins, the two technologies, drawbacks of the conventional ELISA/MS methods and OC. They also link their previous work to show a natural progression to their current work. The research aim has been clearly outlined. I have two comments:\nAdd more about other methods currently used to profile serum concentrations, including multiplex technologies. Eg. SOMAscan. Include their limitations and justify your choice of testing PEA and QKA only - if they are more sensitive, specific, personalized, comprehensive, cost-effective, etc. If they are highly personalized, how do they compare to proteomics including single-cell methods such as CyTOF?\n\nPlease add more relevant references for other techniques such as Gool et al. (20201) and Makridakis et al. (20202). Please also include more information on the already established circulating tumor markers in OC such as EPCAM, MUC1, etc. Also, include a reference for Paragraph 1, Line 7, \"Tumors secrete a medley…\".\n\nMethods:\nThe study is well designed to address the research objectives. The methods are described in detail and the process of subject selection is well-outlined. I am interested in knowing about the criteria for selection fo the 5 proteins to validate using ELISA. Since there was an overlap of many proteins between the two technologies, on what basis were the 5 chosen for further validation?\n\nResults and discussion:\nThe results are stated clearly and the figure legends are detailed and self explanatory. I have the following questions:\nConsidering that the pearson coefficient, r > 0.7 is considered strong positive correlation, how do your results add to what was previously known? Other groups (that you have kindly referenced) have shown high correlation in their findings with clinical values. Moreover, your previous work (Ren et al., 20203) also showed no-to-low correlation in a different tumor - please elaborate on the possible reasons behind this discrepancy, besides small patient cohort.\n\nThe conclusions the authors draw about the decrease in proteins after surgery being related to tumor burden may be overestimated. In the methods, it is stated that the patients were also given adjuvant chemotherapy. We know that chemotherapy can result in changes in extra-cellular matrix proteins and in those related to inflammation. How are the authors certain that the changes observed are as a result of surgery alone, and thus, relate to tumor burden?\n\nIs there a way to demonstrate clinical relevance of this study? Since this is a retrospective study of patients with advanced disease, the survival data may be available. I suggest the authors do a survival analysis of selected proteins to check for their correlation with survival or progression. These results can then be further validated in a larger, publicly available dataset of serum proteins. If a correlation between them and clinical outcome is observed, it lends more credibility to the authors' claim.\n\nLastly, I have the following comments on the paper as a whole:\nThe limitations addressed in the conclusion are great opportunities to inform further research. They also shed light on possible bias based on abundance of proteins. The authors' efforts to control for this are commendable!\n\nPlease stress on the novelty of this study more! For eg. in the discussion, the authors elude to the various functions of the proteins previously identified in OC and other tumors. Explain how this is a novel finding using these platforms and how it can help change the field to move towards similar technologies. Also, the authors identify FOLR1, which could be a novel finding in relation to OC prognosis.\n\nThe title can be better phrased. For eg. \"Evaluation and comparison of two multiplexed platforms profiling serum proteins and its possible correlation with tumor burden (or clinical outcome) in human ovarian 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": [] }, { "id": "88486", "date": "11 Nov 2021", "name": "Anna E Lokshin", "expertise": [ "Reviewer Expertise Cancer biomarkers" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the first to compare two targeted multiplex proteomics technologies, proximity extension assay (PEA) and Quantibody® Kiloplex Array (QKA), for analysis of serum protein concentration changes.\n\nThe results show that the data obtained by platforms highly correlate. Authors have identified proteins that decrease following debulking surgery for ovarian cancer.\n\nData were validated by ELISA. This study is of great importance for future biomarker research using highly novel targeted proteomics platforms.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "88484", "date": "22 Nov 2021", "name": "Stefan Holdenrieder", "expertise": [ "Reviewer Expertise Biomarkers in cancer diagnostics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 pilot study, the authors compared two multiplexed technologies (proximity extension assay, PEA and quantibody kiloplex array, QKA), for profiling a large number of serum proteins. Tested samples were collected prior and after surgery of ovarian cancer patients. Thus, the authors hypothesized that markers with significant decreases in serum concentrations were associated with tumor burden.\nHowever, they clearly state that they performed this study only as preliminary investigation justifying the low number of patients and controls. While this limits the findings substantially, great changes in marker concentrations may indicate candidate markers that should be further validated.\nWithin the large number of markers (>1000) measured by both proteomics technologies, more than 400 were overlapping. The most relevant one, the ovarian cancer marker CA 125, showed only weak correlation between both methods, and also the postsurgical changes in the 400 common markers were only weakly correlated. Among all markers, 15 showed an at least two-fold postsurgical decrease in two thirds of the patients by the use of PEA and 11 by the use of QKA; 8 of them were detected by both methods of which only 3 showed correlations in postsurgical changes by PEA and QKA. Subsequently, 5 kallikrein markers were validated by ELISA. The postsurgical changes correlated with PEA for 3 markers, absolute values only for one.\nThe authors conclude that both novel targeted proteomics platforms are promising tools for identifying candidate serological tumor-related proteins. However, they also admit several limitations of their study and some further ones have to be added:\nThe small number of patients and controls that could lead to missing or overestimating single markers.\n\nThe lack of sufficient reproducibility at least for one platform.\n\nThe only weak correlation between both platforms.\n\nThe need for different dilutions and the challenge to identify and avoid hook effects.\n\nThe difference in absolute value levels for markers if different platforms were used.\n\nThe weak correlation results in the validation experiment with single markers ELISAs.\nObviously, the authors are aware of the limitations and the long way to identify and validate new diagnostic markers for ovarian cancer. They are encouraged to do these validation steps on larger cohorts with proper assays for the new markers identified by their proteomics approach in a subsequent 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/10-509
https://f1000research.com/articles/9-1499/v1	23 Dec 20	{ "type": "Brief Report", "title": "Expression of TNF, IL1B, and NOS2 in the neural cell after induced by Porphyromonas gingivalis with and without coating antibody anti-Porphyromonas gingivalis", "authors": [ "Endang Winiati Bachtiar", "Citra F. Putri", "Retno D. Soejoedono", "Boy M. Bachtiar", "Citra F. Putri", "Retno D. Soejoedono", "Boy M. Bachtiar" ], "abstract": "Porphyromonas gingivalis has virulence factors such as gingipain and lipopolysaccharide, causing bacteremia to reach the brain and activate neuroinflammatory release cytokines. This study analyzed the effect of the co-culture of neuron cells with P. gingivalis coated with anti-P. gingivalis antibodies against cytokines produced by neuron cells. The gene expressions of the TNF, IL1B, NOS2 in neurons was evaluated using RT-qPCR. The results showed that P. gingivalis coated with anti-P. gingivalis antibody before co-culture with neuron cells could decrease the gene expression of TNF, IL1B, and NOS2 of neuron cells.", "keywords": [ "Porphyromonas gingivalis", "Blocking Antibody", "Neuroinflammation", "TNF-α", "IL-1β", "iNOS" ], "content": "Introduction\n\nPeriodontitis is an infectious disease that causes inflammation of the tooth-supporting tissue, loss of bone adhesions, initiated by the main pathogens, Porphyromonas gingivalis. These bacteria are Gram-negative and have virulence factors such as fimbriae, gingipain, and lipopolysaccharide (LPS), which play a critical role in inducing periodontitis. With this virulence factor, P. gingivalis and its products not only damage the periodontal tissue but can also enter the blood circulation or bacteremia and cause systemic spread1,2. P. gingivalis can move to other organs such as the heart and brain. Sophie's research found the presence of LPS P. gingivalis in the brains of Alzheimer's patients3. The mechanism for invading P. gingivalis bacteria into brain tissue is by penetrating the blood-brain barrier and damaging neuron cells4. When entering the central nervous system, these bacteria will first activate defense cells in the brain, namely the microglia, and astrocytes. Activation of both then releases neuroinflammatory mediators such as TNF-α and IL-1β. Several studies have stated that neuron cells themselves can also release the neuroinflammatory mediators TNF-α and IL-1β triggered by foreign bodies such as bacteria. This excessive release of neuroinflammation is toxic to neuron cells and can cause their damage and death5,6. Besides, the excessive release of inducible nitric oxide synthase (iNOS) molecule due to antigen by neuron, microglia, and astrocyte cells, may induce human brain neurodegeneration7.\n\nAs a form of defense against bacterial attack, the body will naturally produce antibodies to eliminate bacteria. The antibodies produced by the host can specifically recognize certain bacterial species. Either monoclonal or polyclonal antibodies can recognize the lipid A region of the LPS of Gram-negative bacteria, such as P. gingivalis8. Animal studies by Barekzi et al. stated that pooled human polyclonal antibodies that are injected locally in the area of injury in mice have broad-spectrum antimicrobial effects against Gram-negative bacteria9. This study aims to evaluate the effect of anti-P. gingivalis antibodies on TNF, IL1B, and NOS2 gene expression when bacteria interact with neuron cells. We hypothesized that there are differences in the gene expression of TNF, IL1B and NOS2 in SHSY-5Y cells that have been exposed to P. gingivalis with and without antibody coating.\n\n\nMethods\n\nThis research is an experimental laboratory study with post test only control group design. This study used the neuron cell line SHSY-5Y (Elabscience, USA), originating from a four-year-old human’s bone marrow neuroblastoma. The cell culture medium was DMEM High Glucose with L-glutamine (Caisson Labs, USA), 15% FBS (Gibco, South America), and 1% Antibiotic-Antimycotic (Gibco, USA). The cultured condition was 5% CO2 at 37°C incubator until 90% confluency was achieved (Figure 1)10.\n\n(A) Cell image of 3 days culture (40x enlargement). (B) Cell image after 7 days, showing elongation of neuron cell bodies and cells growing in groups (40x magnification). (C) There is an increase in cell proliferation and group cell growth (20x magnification). (D) The cells have reached 80% confluence and are ready to be harvested (20x magnification), (E and F) SHSY-5Y cells have undergone differentiation, seen the presence of axons from cells and the cell proliferation process begins to decline (40x magnification). Underlying data shows raw, unprocessed images used to generate this figure12.\n\nPorphyromonas gingivalis ATCC 33277 was cultured in Brain Heart Infusion (BHI) agar as a growth medium and incubated under anaerobic conditions with a temperature of 37°C for 24 hours. Then cultured into BHI broth and incubated again under anaerobic conditions with a temperature of 37°C for 24 hours. Then stored at 4°C until ready to use.\n\nThis study also used P. gingivalis ATCC 33277 bacterial culture. The multiplicity of infection (MOI) used was 1:100, the number of bacteria was 3.6 × 107 CFU/mL, and the number of neuron cells was 8 × 105 cells/well. In addition, this research used serum anti-P. gingivalis antibodies obtained from rabbits after immunization of killed P. gingivalis. P. gingivalis antisera were obtained from one-month-old rabbits that have been immunized with 1 mL of 1.7 × 108 CFU/mL of P. gingivalis culture. The bacteria were inactivated at 60°C for 30 min before being injected intravenously to the rabbit for 8 weeks with two boosters in intervals of 2 weeks. The animals were euthanized by anesthetic ether inhalation and injection by overdose of anesthetic drug (ketamine 50 mg/kg IM and xylazine 10 mg/kg IM), which caused the animal to fall asleep then slowed and eventually stopped the heart. The blood serum was determined by agar gel precipitation test (AGPT) and the antibody was purified using the Qiagen (QIAGEN, Inc., Valencia, Calif.) protein purification kit, following the manufacturer's protocol. Ethical clearance was given by the Ethical Research Committee of Medical Faculty Universitas Indonesia (2020, number 19-11-1402).\n\nThe antisera coated P. gingivalis (3.6 × 107 CFU/mL) was prepared by 1:300 diluted rabbit antibody serum in 150 µL growth medium (DMEM High Glucose with L-glutamine (Caisson Labs, USA), 15% FBS (Gibco, South America), and 1% Antibiotic-Antimycotic (Gibco, USA)) for the treatment group; the control was P. gingivalis (3.6 × 107 CFU/mL) in 150 µL growth medium and the growth medium only without addition of bacteria. The tubes were then incubate for 1 hour in an incubator with a temperature of 37°C11.\n\nThe experiment design as follows: group A was the neurons plus bacteria with antibody coating, and group B for the neuron group plus bacteria without antibody coating and medium only, with 6 replications of each group.\n\nNeuron cell cultures that had reached 80% confluence were harvested using 0.25% trypsin-EDTA (Gibco, Canada). The number of cells harvested was counted using a hemocytometer (number of cells 8×105 cells/well). The cells were then transferred to a 15 mL tube and resuspended in 2 mL growth medium and then divided into well plates that have been designed with each well containing 100 µL (4×103 cells/well) of SHSY5Y cells. The neuron cell line SHSY-5Y (Elabscience, USA) is a cell that has epithelial-like cell and neuronal-like cell morphology and has a cell density of more than 1×106 cells/cm2. In this study, cell culture was carried out with two subcultures in January 2020 and February 2020 until the number of cells reached 8×105 cells/well. Observation with a microscope was carried out every 2–3 days to identify neuron cells and determine the stage of neuron cell differentiation (Figure 1)\n\nEach well of 96 well culture plate filled with SHSY-5Y cells and antibody-coated P. gingivalis bacteria and incubated for one hour were added. Group A was filled with 30 µL (1×105 CFU/mL) P. gingivalis coated with antibody, while group B was filled with 30 µL of bacterial P. gingivalis without antibodies. After that, cells were incubated for 24 hours at 37°C. All cells present in the well plate were evaluated for the morphological features, then harvested for RNA extraction.\n\nRNA extraction and RT-qPCR\n\nThe neural cell culture was harvested, and RNA extracted for cDNA synthesis using a Reverse Transcription Kit (ReverTra Ace®, Toyobo, Japan) in line with the manufacturer’s instructions. The cDNA sample is ready for use in the Real-Time PCR tool, with the selected primers as Table 1. RT-PCR was performed using the SYBR Premix Ex Taq TM kit. Relative expression of the target gene normalized to GAPDH, gene expression was analyzed using the 2-ΔΔCt method and compared to control. The gene expression of TNF, IL1B and NOS2 were evaluated by RT-qPCR as previously reported15\n\n\nResults\n\nFigure 2 shows the microscopic morphology of the SHSY-5Y cells that were not exposed to P. gingivalis, and those exposed to P. gingivalis and coated with anti-P. gingivalis antibodies. From these figures, it is known that cells not exposed to P. gingivalis grew more than cells exposed to P. gingivalis, both with and without antibodies.\n\n(A) SHSY-5Y cell morphology before exposure to P. gingivalis, (B) After exposure to P. gingivalis without antibodies, (C) After exposure to P. gingivalis with antibodies. (Light microscope, 20x magnification).\n\nFrom qPCR analysis, it was observed that there are differences in the gene expression of TNF-α, IL-1β and iNOS in SHSY-5Y cells that have been exposed to P. gingivalis with and without antibody coating. Based on this analysis, it can be concluded that the research hypothesis is accepted. This is shown in Figure 3, where the expression of TNF-α and IL-1β genes in the antibody-coated group was lower than in the antibody-coated group. Ct values are available as Underlying data16.\n\n\nDiscussion\n\nThe SHSY-5Y neuron cell line (Elabscience, USA) is a cell derived from human neuroblastoma and taken from bone marrow tissue. These cells have epithelial-like cell and neuronal-like cell morphology. During culture, SHSY-5Y cells can grow into two types of cells, namely adherent cells and floating cells, both of which are viable. However, in this study, adherent cells were used because they were clearer in morphology and proliferation development, and were easy to evaluate after a routine medium change17,18.\n\nMicroscopy images of SHSY-5Y cells (Figure 2) showed significant growth changes over time. According to Kovalevich and Langford, one of the considerations for the success of SHSY-5Y cell culture is the growth medium used18. In this study, DMEM growth medium containing L-glutamine was used. Glutamine can help increase neuron cell viability and increase neuron cell density, so that it can be seen on microscopy images that neuron cell cultures grow well. However, the number of cells collected until the end of cell culture is 8×105, where this number is limited for the study sample. This may occur because cells have started to enter the differentiation stage, so that the cell proliferation process tends to decrease17.\n\nTNF-α and IL-1β are inflammatory mediators released by immune cells when a stimulus triggers the cells. In the nervous system, TNF-α and IL-1β are usually released by astrocytes and microglia cells. However, a number of studies suggest that these inflammatory mediators are also released in large numbers by neuron cells when there are intrinsic or extrinsic triggers19. Extrinsic triggers such as LPS presence from P. gingivalis bacteria can trigger the expression of TNF-α and IL-1β by neuron cells so that it can damage neuron cells20–22. In the incidence of Alzheimer's disease, the release of this inflammatory mediator can cause neuronal cell death, according to a study by Janelsins et al., which stated that the inflammatory mediators TNF-α and IL-1β appears to be directly proportional to Alzheimer's disease severity22–24.\n\nThis study is in line with the research of Janelsins et al., who found that neuron cells can express TNF-α in brain injury in experimental animals. This is evidenced by the detection of the molecules NeuN and TNF-α in the brain of six-month-old mice. In this study, SHSY-5Y neuron cells can also express TNF-α. In addition, Janelsins et al. also found that TNF-α contributed to neuron cell death in the brain with Alzheimer's condition. The signaling mechanism is still unknown, but Janelsins et al. stated that there was an increase in the expression of TNFRII and Jun transcript as pro-apoptotic signals mediated by TNF-α24,25.\n\nThe expression of iNOS has been characterized in various cell types as an inflammatory mediator during infection, disease, or tissue damage. INOS is expressed by astrocytes, microglia, and a small portion of endothelial cells in the brain. However, under conditions of increased inflammatory activation in neuron cells, neurons can also express these cytotoxic agents and other reactive oxidative species. The main component that regulates the signaling pathway of iNOS in neurons is the transcription factor NF-κb. The results of this study indicate that anti-P. gingivalis antibodies can suppress iNOS expression in neuron cell cultures exposed to P. gingivalis. Blocking carried out by antibodies to P. gingivalis LPS was thought to suppress bacterial pathogenicity so that iNOS expression in neurons was lower than that of the control group. We assume the antibody use reduced neuronal damage. This is in line with Heneka and Feinstein's research, which states that increased expression of iNOS in neurons can affect neurodegeneration and inflammation in the brain7,26.\n\nP. gingivalis have secreted and non-secreted virulence factors. Secreted virulence factors, for example, gingipain, are virulence factors secreted by bacteria to carry out their activities. Meanwhile, non-secreted virulence factors are virulence factors that are not secreted by bacteria, usually attached to the bacterial structure, such as LPS. In this study, the antibodies used were from injections of killed P. gingivalis in rabbits. This will result in the formation of polyclonal antibodies against non-secreted virulence factors, namely LPS, because when it is turned off, the bacteria are unable to secrete other virulence factors such as gingipain. The anti-P. gingivalis polyclonal antibodies can recognize P. gingivalis bacterial cells and these bacteria's LPS structure8,9,25. Therefore, coating this antibody with P. gingivalis bacteria for 1 hour before exposure to neuronal cells is thought to block LPS P. gingivalis bacteria not to infect neuron cells.\n\nIn contrast to the control group that did not use antibodies, P. gingivalis was exposed to neuron cells, infecting neuron cells with secreted and non-secreted virulence factors. This occurs because there are no antibodies that block the two types of P. gingivalis virulence factors. Therefore, in qPCR analysis results, neuron cell culture with anti P. gingivalis antibody showed lower TNF-α and IL-1β expression than the control group. The study (Figure 3) show that the use of antibodies can suppress the expression of TNF-α and IL-1β. The low expression of TNF-α and IL-1β with the use of antibodies is thought to prevent neuronal damage and is expected to prevent the occurrence of Alzheimer's disease or other cognitive disorders. However, different research results may occur because of the MOI value used. In this study, the MOI used was 1:100.\n\nThis study's findings indicate that there is good potential for the development of the anti-P. gingivalis vaccine. The anti-P. gingivalis antibody used in this study was able to block the development of bacteria in vitro so that the neuroinflammatory response can also be minimized. Further research at the in vivo level and clinical trials can be developed to see the positive effects of administering antibodies locally or systemically. In the case of local infection of P. gingivalis in the oral cavity, the local administration of antibodies may have more potential to suppress bacterial development.\n\nIn addition, long-term research involving the role of neuron cells and damage to the central nervous system also needs to be done. With this research, it is hoped that it can become a reference to increase the level of research so that in the future, the prevention of P. gingivalis infection can be done so that it can prevent neurodegeneration in the incidence of Alzheimer's disease.\n\n\nConclusion\n\nThe cultured SHSY-5Y neuron cells exposed to P. gingivalis bacteria after anti-P. gingivalis antibody coating exhibited a reduction in the expression of the TNF, IL1B, and NOS2. Further research to see the effectiveness of anti-P. gingivalis antibodies still needs to be developed, especially in vivo. The success of anti-P. gingivalis antibodies in suppressing factors that can damage neuronal cells can be used as a guideline for developing a P. gingivalis vaccine, since it is one of the oral bacteria that triggers Alzheimer's disease.\n\n\nData availability\n\nOpen Science Framework: Expression of TNF-α, IL-1β, and iNOS in the neural cell after induced by Porphyromonas gingivalis with and without coating antibody anti-Porphyromonas gingivalis. https://doi.org/10.17605/OSF.IO/Q5CVW16.\n\nThis project contains the following underlying data:\n\nBeta actin GAPDH 2506202_data (1).xls. (qPCR data for housekeeping gene GAPDH.)\n\nIL1b TNFa_data(1).xls. (qPCR data for IL1B and TNF.)\n\niNOS 23062020_data.xls. (qPCR data for NOS2.)\n\nOpen Science Framework: Expression of TNF, IL1B, and NOS2 in the neural cell after induced by Porphyromonas gingivalis. https://doi.org/10.17605/OSF.IO/JFG3T12.\n\nThis project contains the raw images used to produce Figure 1.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe thank Vivi, Asti, and Anissa for their help in the laboratory work.\n\n\nReferences\n\nDing Y, Ren J, Yu H, et al.: Porphyromonas gingivalis, a periodontitis causing bacterium, induces memory impairment and age-dependent neuroinflammation in mice. Immun Ageing. 2018; 15: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamer AR, Craig RG, Dasanayake AP, et al.: Inflammation and Alzheimer's disease: possible role of periodontal diseases. Alzheimers Dement. 2008; 4(4): 242–50. 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J Oral Microbiol. 2019; 11(1): 1563405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJanelsins MC, Mastrangelo MA, Park KM, et al.: Chronic neuron-specific tumor necrosis factor-alpha expression enhances the local inflammatory environment ultimately leading to neuronal death in 3xTg-AD mice. Am J Pathol. 2008; 173(6): 1768–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang J, Yu C, Zhang X, et al.: Porphyromonas gingivalis lipopolysaccharide induces cognitive dysfunction, mediated by neuronal inflammation via activation of the TLR4 signaling pathway in C57BL/6 mice. J Neuroinflammation. 2018; 15(1): 37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSonar SA, Lal G: The iNOS activity during an immune response controls the CNS pathology in experimental autoimmune encephalomyelitis. Front Immunol. 2019; 10: 710. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "76422", "date": "25 Jan 2021", "name": "Hadhimulya Asmara", "expertise": [ "Reviewer Expertise Neuroscience (any works with neurons involvement)", "Molecular Biology (any works related to DNA and protein)." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Bachtiar et al. presents some potentially interesting findings on preventing the neurons from damage and death by reducing excessive release of neuroinflammatory mediators such as TNF-α and IL-1β and other molecules such as iNOS. This paper highlights the potential success of using antibody specially anti- P. gingivalis antibodies for suppressing factors that can damage neuronal cells can be used as a guideline for developing a P. gingivalis vaccine since it is one of the oral bacteria that triggers Alzheimer's disease. Overall, the work presents an interesting idea regarding possible antibody use to decrease the release of neuroinflammatory agents and other molecules that are toxins and cause harm to the neurons. If this finding is rigorously proven, it could be a great contribution to the prevention and treatment of bacterial infection in Neurodegenerative disease. At this stage, however, there are several comments that need to be clarified in the manuscript that substantively influences the significance of the findings.\nSpecific comments:\nIn the paper, the authors did not mention the efficacy of other treatments such as antibiotic treatment for P. gingivalis infection and its effect on the release of neuroinflammatory agents. It is important to highlight the antibiotic’s effect since if the antibiotic is effective enough to kill the bacteria and prevent the infection on neurons then this paper must add more reasons why this paper or the antibody approach is better than the antibiotic treatment.\n\nIn the figure 2. The authors used the word “morphology” of the neurons, but the authors did not explain the morphology aspect of the neurons (such as elongation axons or shape of the cell body, etc). It only described the change of the growth or number of the viable cells that was decreased by the exposure of P. gingivalis with or without antibody coating. It is better to describe how the exact morphological changes of the neurons as clearly shown in figure 1 before the exposure P. gingivalis. Even better if the authors can add the qualitative and quantitative analysis of those differences between the groups.\n\nThe correlation between figure 2 and figure 3. The reducing number of viable cells in figure 2 and the reducing the expression of TNF-α, IL-1β, and iNOS in figure 3 seems does not fit with the hypothesis in the paper. If the expression of TNF-α, IL-1β, and iNOS were decreased on the neurons that were exposed by coating antibody P. gingivalis compared to the ones without coating (figure 3) then the number of viable neurons in the figure 2c (expose with antibody) must be higher than figure 2b (without antibody) since the antibody will decrease TNF-α, IL-1β, and iNOS and it means to prevent the neuronal damage and death on figure 2b. What is the clarification or the explanation of this confusion?\n\nIt is necessary to state the P values or statistical significance of the differences between the three groups in figure 3 (with coating antibody, without coating anti, and neuron only). It is important to conclude that one group is higher or lower than the others groups based on statistical significance or P values.\n\nAs a minor comment, I think there is a typo on the first line of the second paragraph in the discussion section. In my opinion, I think the authors want to show figure 1 instead of figure 2 (as stated in that line) for describing the growth changes of neurons over time.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6412", "date": "17 Mar 2021", "name": "Boy Muchlis Bachtiar", "role": "Author Response", "response": "Dear Reviewer, Thank you for the valuables suggestion in improving of the quality our manuscript. Here we would like to respond your feedback. Specific comments: In the paper, the authors did not mention the efficacy of other treatments such as antibiotic treatment for P. gingivalis infection and its effect on the release of neuroinflammatory agents. It is important to highlight the antibiotic’s effect since if the antibiotic is effective enough to kill the bacteria and prevent the infection on neurons then this paper must add more reasons why this paper or the antibody approach is better than the antibiotic treatment. Author’s responses: We thank for this valuable comments. We have inserted the argumentation in the manuscript of this suggestion as follow: P gingivalis reside in a structured community of biofilm attached to surfaces embedded in the extracellular matrix which they produce themselves and they are difficult to eradicate due to their resistance to antimicrobials and the body's defense mechanisms 10. The passive immunization approach using polyclonal antibodies to inhibit P gingivalis adhesion to the periodontium tissue is a strategy to prevent biofilm formation and periodontium tissue damage which can lead to deeper tissue invasion so that P gingivalis can enter the systemic circulation. In the figure 2. The authors used the word “morphology” of the neurons, but the authors did not explain the morphology aspect of the neurons (such as elongation axons or shape of the cell body, etc). It only described the change of the growth or number of the viable cells that was decreased by the exposure of P. gingivalis with or without antibody coating. It is better to describe how the exact morphological changes of the neurons as clearly shown in figure 1 before the exposure P. gingivalis. Even better if the authors can add the qualitative and quantitative analysis of those differences between the groups. Author’s responses: We have deleted ‘morphology’ In Figure 2, it can be seen that there is a decrease in the number of neuron cells after being exposed to P. gingivalis bacteria. The correlation between figure 2 and figure 3. The reducing number of viable cells in figure 2 and the reducing the expression of TNF-α, IL-1β, and iNOS in figure 3 seems does not fit with the hypothesis in the paper. If the expression of TNF-α, IL-1β, and iNOS were decreased on the neurons that were exposed by coating antibody P. gingivalis compared to the ones without coating (figure 3) then the number of viable neurons in the figure 2c (expose with antibody) must be higher than figure 2b (without antibody) since the antibody will decrease TNF-α, IL-1β, and iNOS and it means to prevent the neuronal damage and death on figure 2b. What is the clarification or the explanation of this confusion? Author’s responses: Qualitatively, from the microscope image (Figure 2), there was no difference between the antibody group and the non-antibody group. In our opinion, the number of cells seen on a microscope does not necessarily indicate the expression of neuroinflammation. Based on the results of the real time PCR analysis, it showed that the expression of neuroinflammation was more in the group without antibodies (Figure 3). It is necessary to state the P values or statistical significance of the differences between the three groups in figure 3 (with coating antibody, without coating anti, and neuron only). It is important to conclude that one group is higher or lower than the others groups based on statistical significance or P values. Author’s responses: We used pooled samples (we have added this information in the methods section) as there was insufficient amount of RNA from each individual replication of the experiments. But we think the value of gene expression presented here is equal to de average of 6 replications of experiment. Hence, we couldn’t get a statistical significance. Some studies also use this kind of data interpretation (Shu-Dong Zhang, Timothy W. Gant, Effect of pooling samples on the efficiency of comparative studies using microarrays, Bioinformatics, Volume 21, Issue 24, 15 December 2005, Pages 4378–4383) As a minor comment, I think there is a typo on the first line of the second paragraph in the discussion section. In my opinion, I think the authors want to show figure 1 instead of figure 2 (as stated in that line) for describing the growth changes of neurons over time. Author’s responses: Thank you, we have fixed this typo. Again thanks With warm regards Bachtiar EW" } ] }, { "id": "76420", "date": "16 Apr 2021", "name": "Widya Lestari", "expertise": [ "Reviewer Expertise Molecular Biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report provides new insight into dentistry. P. gingivalis is not only related to Periodontitis but also express in neuron cells, which may give additional knowledge to clinicians. Pathways of expression of TNF alpha and IL-1B area clearly explained. Methodology and results are well established and, we believe these findings will benefit clinicians and also researchers as well.\nThe methodology:\n\nThis is an in vitro study using a nerve cell culture. The experimental design has used a treatment group and a control group which, in my opinion, is good enough to observe the effect of administering anti-P. gingivalis antibodies on the expression of neuroinflammatory cytokines.\nThe results:\nThe results have been presented clearly. I suggest analyzing them descriptively without using statistics. This is a preliminary study, in my opinion, it has described how the expression of neuroinflammatory cytokines in nerve cell cultures after exposure to P. gingivalis which has been treated with anti-P. gingivalis antibody.\nOverall, a minor revision is needed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1499
https://f1000research.com/articles/10-414/v1	24 May 21	{ "type": "Research Article", "title": "Association of subgingival Epstein–Barr virus and periodontitis", "authors": [ "Chaerita Maulani", "Sri Lelyati C. Masulili", "Widayat Djoko Santoso", "Nurtami Soedarsono", "Lindawati Kusdhany", "Elza Ibrahim Auerkari", "Chaerita Maulani", "Sri Lelyati C. Masulili", "Widayat Djoko Santoso", "Nurtami Soedarsono", "Lindawati Kusdhany" ], "abstract": "Background: The Epstein–Barr virus (EBV) is gaining interest as a possible agent in the etiology of periodontitis. Previous studies have shown controversy on whether EBV DNA in the subgingival periodontal pockets is associated with periodontitis. The aim of the present study was to seek the potential relationship between EBV and periodontitis. Methods: Data on socio-demographics, oral health, and periodontal health were recorded, and samples were collected from gingival crevicular fluid, using sterile paper point. This case–control study of 118 participants included 59 subjects with severe periodontitis and 59 control subjects with mild periodontitis. The EBV load was determined by quantitative real-time PCR. Results: EBV DNA was detected in 37.3% of the case samples and in 18.6% of the control samples. There was no significant difference in the load of EBV DNA between severe and mild periodontitis (p>0.05). The observed load of EBV DNA was up to 4.55x105 copies/mL. The detected EBV DNA was significantly associated with the plaque index and the oral hygiene index (all p<0.05). Conclusions: A significant association was not found, but EBV might contribute to periodontitis. Gingival crevicular fluid is useful for monitoring the EBV load by the real-time PCR technique.", "keywords": [ "Epstein-Barr Virus", "Periodontitis", "Gingival Crevicular Fluid" ], "content": "Introduction\n\nPeriodontal diseases consist of chronic inflammatory conditions that affect the supporting tissue of the teeth. Localized inflammation within the gingiva caused by microorganisms in the dental plaque leads to gingivitis. Untreated gingivitis can progress to chronic periodontitis with the advanced loss of gingiva, bone and periodontal ligament.1 The periodontitis process is a multifactorial interplay between microbial, host and environmental factors. In the development of periodontitis, microbial agents are the important key. However, microbial communities in periodontitis compared with healthy individuals showed that the presence of bleeding was not associated with a specific microbiome although total bacterial load tends to be higher in bleeding sites.2 Periodontal breakdown is site specific, and can be explained not just by bacterial specificity or immunopathology, but also by a combined herpesvirus–bacterial infection.3 With this contribution, herpesvirus has emerged as a significant periodontal pathogen.4 Epstein–Barr virus (EBV) is one of eight herpesviruses that can cause disease in humans. EBV is found in more than 90% of the world populations with oral transmission as the primary route of the EBV infection.5\n\nHowever, variable findings have been shown on the presence of EBV in subgingival pockets. Some studies have reported higher prevalence of EBV DNA with increasing severity of periodontitis6–9 while others have shown weak or no relationship between them.10–12 For these purposes, presence of EBV DNA was determined by techniques such PCR,13 nested PCR6,7 or quantitative real-time PCR.9,11\n\nThe aim of this study was to examine using quantitative real-time PCR whether EBV DNA in subgingival pockets has higher prevalence in systemically healthy patients with severe periodontitis than in those with mild periodontitis.\n\n\nMethods\n\nThis case–control study included 118 systematically healthy, 18 to 66 years old individuals with at least 14 erupted teeth12 and with either healthy periodontal condition, gingivitis or periodontitis. Subjects were excluded if they had systemic disease, periodontal treatment in the last six months, pregnancy, lactation, used antibiotic therapy or immunosuppressants in the last 3 months, had acute illness, detectable inflammation within oral mucosa, or refused to take part in the research. All participants were given a thorough verbal and written explanation of the study and were required to sign an informed consent form. The study was approved by the Institutional Review Board, protocol number 070390418. The study was conducted within three different district offices in Central Jakarta between June 2018 and March 2019. The case and control subjects were recruited from the same districts. The subjects not meeting the inclusion criteria were excluded and the final study consisted of 59 case patients with severe periodontitis and 59 controls with mild periodontitis. The sample size was considered acceptable in comparison to previous studies.9,13 The sample size also give 80% power with the effect size 0.3 (medium) with a 0.05 two tails significance level.\n\nClinical periodontal examinations were performed by two investigators (CM, PW) for each subject with measurement of plaque index (PI),14 bleeding on probing (BOP), pocket depth (PD), clinical attachment level (CAL), and oral hygiene index–simplified (OHIS).15,16 The plaque index was measured at four sites per tooth according to Loe.14 The UNC-15 probe (Hu-Friedy, USA) was used to measure PD at six sites per tooth (disto-buccal, mid-buccal, mesio-buccal, disto-lingual, mid-lingual and mesio-lingual). The distance between the cementoenamel junction and the base of the periodontal pocket was used to measure clinical attachment loss. BOP was measured at one site per tooth using papillary bleeding index by Mühlemann.15 OHIS was recorded by addition debris index and calculus index, measured at two sites per tooth.16\n\nPatient grouping was according to classification of periodontal and peri-implantitis condition.17 Stage 3 and stage 4 periodontitis cases with CAL ≥5 mm and PD ≥6 mm were included in the severe periodontitis case category. The control group included gingivitis, stage 1 and stage 2 periodontitis cases of CAL <5 mm and PD ≤5 mm.15,18\n\nThe examiner reliability was verified with interclass correlation coefficient (ICC) of 0.741 (95% CI 0.507–0.846) and 0.837 (95% CI 0.784–0.875), indicating good and excellent agreement for PD and CAL respectively. The kappa value for PI and BOP ranged from 0.737 to 0.805, indicating substantial agreement.\n\nGingival crevicular fluid (GCF) was collected from four sites of the deepest periodontal pocket and randomly at one site of each quadrant in healthy sulcus. Saliva and supragingival plaque were cleaned gently with sterile cotton pellets, separated with cotton rolls, and air dried before sampling. A sterile paper point ISO 30 (Roeko, Germany) was inserted gently into the periodontal pocket until it reached the base of pocket depth with mild resistance and held for about 30 seconds. Bleeding sites were carefully avoided. The collected samples were pooled then put in a plastic tube with 1 mL of sterile phosphate buffered saline. The vial was vortexed for about 1 min and then centrifuged for approximately 20 min at 3000 rpm. The supernatants were collected and samples stored at −20°C until further processing.\n\nAfter centrifugation, DNA was extracted from GCF using a commercial kit (QIAamp DNA Mini Kit, Qiagen, Germany) according to the instructions. The presence and quantity of extracted DNA was tested by NanoQuant Infinite® M200Pro. The real-time PCR method was used to determine the amount of EBV DNA in the samples. The analysed amount of EBV DNA in each sample was in a volume of 10 μL. The single copy gene encoding the EBV nuclear antigen 1 (EBNA1) was the target sequence (EBNA1). Real-time PCR LightCycler® 480 II (Roche Diagnostics GmbH, Mannheim, Germany) was used with a commercial kit (EBV/ISIN/100, GeneProof, Brno, CZ). The kit has four standards containing EBV calibrator from 101 to 104 copies of EBNA1 per μL. From a linear regression to standard curves, quantification was conducted by extrapolation. The PCR conditions were set as directed by the manufacturer.\n\nThe findings were analyzed using IBM SPSS v.23 (RRID:SCR_019096), for which an open-access alternative would be JASP (https://jasp-stas.org; RRID:SCR_015823) To determine the association between EBV and periodontitis, the Chi-squared test was used. Statistical significance was assumed at a p-value of 0.05 or less.\n\n\nResults\n\nIn total, 118 subjects with mild-to-severe periodontitis, ranging from 18 to 66 years old, mean 38.15 ± 14.4 years, were evaluated for periodontal status including EBV DNA in gingival crevicular fluid. Table 1 shows the demographic and clinical periodontal parameters.\n\na Mann–Whitney test.\n\nb Continuity correction.\n\nc Independent t-test\n\nThe EBV DNA was detected in 28.0% of all subjects, in 37.3% of cases of severe periodontitis, and in 18.6% of the control cases of mild periodontitis. The highest EBV DNA level was 4.55 × 105 copies/mL in severe periodontitis. No statistically significant difference in the detected EBV DNA was found between the case and control groups (Table 2).\n\na Mann–Whitney test.\n\nIn Table 3, clinical parameters of periodontitis were compared with EBV DNA detection. It showed that the EBV DNA load was significantly associated with the plaque index and OHIS, but not with age, gender and other clinical parameters of periodontitis. Although also not statistically significant, more EBV DNA was detected from subjects with severe periodontitis than from those with mild periodontitis.\n\na Mann–Whitney test.\n\nb Independent t-test\n\n\nDiscussion\n\nThe etiology of periodontal disease was previously thought to be bacteria driven, but later studies have suggested that herpesviruses, especially EBV, are also involved.19,20 This hypothesis emerged because some periodontal disease cannot be explained by bacterial activity alone.4 The results of our study do not indicate a role for EBV to play in periodontitis, similarly as in findings made in some previous studies.10,12 In cases of severe periodontitis, the prevalence of EBV detected at periodontal pockets was about 37.3%. This was only slightly lower than in a previous study in India with 44.2% in aggressive periodontitis9 but clearly lower than observed values for chronic periodontitis: 72.5% in Iran;6 74.49% in Indonesia;21 and 66–68% in Japan.7,8 The differences in terms of detected EBV DNA between the cases or severe periodontitis and the controls of mild periodontitis were not statistically significant, but showed significant association with the clinical indicators of plaque index and OHIS.\n\nAge, gender and smoking status were associated significantly with periodontitis, so were the clinical parameters of periodontitis (Table 1). The results are in line with a previous study that smoking has been determined in periodontal classification as an important risk factor in periodontitis.18\n\nThe detected EBV DNA was not significantly associated with periodontitis, age, gender and clinical parameters (pocket depth, clinical attachment level, bleeding on probing) of periodontitis (p > 0.05). However, the increasing plaque index and OHIS were significantly associated with detected EBV DNA (Table 3). This is in agreement with the findings of a previous study.13\n\nLow rates of EBV detection could have various reasons including study methodology. In the present study, the periodontitis group had a mean of clinical attachment level of 2.74 ± 0.92, while in other studies, a mean clinical attachment level of 6.7 ± 1.6 mm did not indicate association between prevalence of EBV and periodontitis.12 Similarly, the probing depth was 1.9 ± 0.52 mm, well below of the 5.8 ± 1.2 mm in the comparison study.\n\nWe analysed the presence of EBV DNA by sterile paper points in 118 patients from gingival crevicular fluid (GCF) as done previously using paper strips.13 In other studies, EBV DNA has been detected from subgingival plaque with sterile paper point7,8,12,21 or curettage.6,10,22 GCF can only be obtained in small amounts in healthy sulcus, but during inflammation, GCF flow increases 5.5-fold as shown in experimental gingivitis.23 The resting void volume in GCF is highly dependent upon the pocket depth; as a pocket increases from 3 to 6 mm, the resting void volume increases by 50%.24 When compared with baseline, the GCF level in periodontitis decreased after 6 weeks posttreatment of phase-I periodontal therapy.13 Although GCF samples could be one reason for relatively low detection rates, subgingival plaque with curette technique can also infrequently detect EBV DNA that was not correlated with increased probing depth in another study.11 A proposed mechanism for the EBV role suggests that periodontal pockets harbour EBV and contain excessive reactive oxygen species, which induce excessive development of receptor activator of nuclear factor κB ligand (RANKL) that activates osteoclasts.25\n\nThe lowest positively detected EBV DNA was indicated at 3.4 copies/mL. The range for the controls or mild periodontitis patients was up to 7.98 × 104 copies/mL and for the severe periodontitis cases up to 4.55 × 105 copies/mL. In a previous study in Japan on chronic periodontitis, the observed upper range of detected EBV DNA was about 103–109 copies/mL and an order of magnitude higher in deep pockets (PD ≥ 5 mm) than in shallower pockets (PD ≤3 mm).8\n\nAnother reason for variability is the grouping of periodontitis that does not present a good contrast between cases and controls when healthy subjects are uncommon, so that subjects with gingivitis and mild periodontitis provide the control group. As the recent classification of periodontitis does not differentiate between aggressive and chronic periodontitis, the severity of periodontitis is based on staging.18\n\nFurthermore, sensitive methods like nested PCR6,7 to identify EBV DNA have the risk of overestimating the result, while other methods like automated real-time PCR assays in previous studies found EBV DNA less frequently,10–12 including in our investigation.\n\nA previous study about EBV in another Indonesian population of Bandung, West Java, found all subjects seropositive for EBV with 75% detection rate in subgingival EBV DNA using the same technique of qRT-PCR.21 Some differences in environmental, ethnic and immunogenetic factors may contribute, but are not expected to be large. Limitations of the study include the relatively small sample size and the unclear difference between the detection rates of EBV DNA from GCF samples and indicated high seroprevalence for EBV in the previous study.21\n\n\nConclusions\n\nOur findings showed no significant association between EBV and periodontitis, although in cases of severe periodontitis EBV DNA was detected more frequently and at higher maximum level than in cases of mild periodontitis. Gingival crevicular fluid is useful to check the EBV load using a real-time PCR technique.\n\n\nData availability\n\nHarvard Dataverse: Underlying data for ‘Association of subgingival Epstein–Barr virus and periodontitis’. https://doi.org/10.7910/DVN/AR3Y00\n\nThis project contains the following underlying data: F1000_EBV_RawData_EIA_2021.csv\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 patient details was obtained from the patients.\n\n\nAuthor contributions\n\nCM: Data Curation, Formal Analysis, Investigation, Visualization, Writing-Original Draft Preparation; SLM: Conceptualization, Project Administration, Supervision; WDS: Methodology, Supervision, Validation; NS: Validation, Visualization; LK: Formal Analysis, Validation; EIA: Conceptualization, Funding Acquisition, Methodology, Supervision, Writing-Review & Editing.", "appendix": "Acknowledgments\n\nThe authors wish to gratefully acknowledge the expert technical assistance of Pitu Wulandari in clinical examination, and Universitas Indonesia for funding the work.\n\n\nReferences\n\nKinane DF, Stathopoulou PG, Papapanou PN: Periodontal diseases. Nat Rev Dis Primers. 2017; 3: 17038. PubMed Abstract \| Publisher Full Text\n\nAbusleme L, Dupuy AK, Dutzan N, et al.: The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation. ISME J. 2013; 7(5): 1016–1025. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSlots J: Periodontitis: facts, fallacies and the future. Periodontol 2000 . 2017; 75: 7–23. PubMed Abstract \| Publisher Full Text\n\nSlots J, Slots H: Periodontal herpesvirus morbidity and treatment. Periodontol 2000. 2019; 79(1): 210–220. PubMed Abstract \| Publisher Full Text\n\nSmatti MK, Al-Sadeq DW, Ali NH, et al.: Epstein-Barr Virus Epidemiology, Serology, and Genetic Variability of LMP-1 Oncogene Among Healthy Population: An Update. Front Oncol. 2018; 8: 211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChalabi MMR, Mogharehabed A, Rezael MM, et al.: Periodontopathic bacteria and herpesviruses in chronic periodontitis. 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PubMed Abstract \| Publisher Full Text\n\nGao Z, Lv J, Wang M: Epstein-Barr virus is associated with periodontal diseases: A meta-analysis based on 21 case-control studies. Medicine (Baltimore). 2017; 96(6): e5980. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu C, Li F, Wong M, et al.: Association between Herpesviruses and Chronic Periodontitis: A Meta-Analysis Based on Case-Control Studies. PLoS One. 2015; 10(12): e0144319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmaliya A, Laine ML, Delanghe JR, et al.: Java project on periodontal diseases: periodontal bone loss in relation to environmental and systemic conditions. J Clin Periodontol. 2015; 42(4): 325–332. PubMed Abstract \| Publisher Full Text\n\nSharma S, Tapashetti RP, Patil SR, et al.: Revelation of Viral – Bacterial Interrelationship in Aggressive Periodontitis via Polymerase Chain Reaction: A Microbiological Study. J Int Oral Health. 2015; 7(9): 101–107. PubMed Abstract \| Free Full Text\n\nKowashi Y, Jaccard F, Cimasoni G: Sulcular polymorphonuclear leucocytes and gingival exudate during experimental gingivitis in man. J Peridontol Res. 1980; 15: 151–158. PubMed Abstract \| Publisher Full Text\n\nBarros SP, Williams R, Offenbacher S, et al.: Gingival crevicular fluid as a source of biomarkers for periodontitis. Periodontol 2000. 2016; 70(1): 53–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJakovljevic A, Andric M, Miletic M, et al.: Epstein-Barr virus infection induces bone resorption in apical periodontitis via increased production of reactive oxygen species. Med Hypotheses. 2016; 94: 40–42. PubMed Abstract \| Publisher Full Text" }	[ { "id": "86020", "date": "16 Jun 2021", "name": "Muhammad Sohail Zafar", "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\nI have reviewed the manuscript “Association of subgingival Epstein–Barr virus and periodontitis” submitted to F1000Research for publication. In this study, authors have investigated the potential relationship between Epstein–Barr virus and periodontitis. This is a well conducted study and the manuscript fits well within the scope of the journal; it needs some improvements; there are a few suggestions that authors may consider to improve it further:\nThe use of English language is reasonable, however, there are a number of punctuation and grammatical errors; that should be corrected and rephrased using academic English for a better flow of text for reader.\nAuthors should clarify that whether the STROBE checklist was used or not.\nAuthors should mention more information regarding the non-respondents (i.e., how many individuals were willing to participant and how many were not?).\nAuthors have used GCF in this study to measure EBV level. Is there any study that showed the use of saliva to measure the EBV?\nThe introduction section is very brief and barely cover the rationale of the study and background of GCF in context. Authors should expand the introduction section a bit more adding GCF details. The following articles on GCF may be included (which I am a co-author):\nHuman gingival crevicular fluids (GCF) proteomics: an overview.\" Dentistry Journal 5.1 (2017): 121.\n\nBacteriological Evaluation of Gingival Crevicular Fluid in Teeth Restored Using Fixed Dental Prostheses: An In Vivo Study.\" International Journal of Molecular Sciences 22.11 (2021): 54632.\nPlease add; What was the main purpose of using GCF in this study?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6827", "date": "28 Jun 2021", "name": "Elza Ibrahim Auerkari", "role": "Author Response", "response": "\"I have reviewed the manuscript “Association of subgingival Epstein–Barr virus and periodontitis” submitted to F1000Research for publication. In this study, authors have investigated the potential relationship between Epstein–Barr virus and periodontitis. This is a well conducted study and the manuscript fits well within the scope of the journal; it needs some improvements; there are a few suggestions that authors may consider to improve it further: The use of English language is reasonable, however, there are a number of punctuation and grammatical errors; that should be corrected and rephrased using academic English for a better flow of text for reader.\" Answer: We check again the punctuation and grammatical errors. \"Authors should clarify that whether the STROBE checklist was used or not.\" Answer: STROBE checklist was not used in our study but our manuscript was checked against the STROBE checklist by the academic editor journal, and we completed the manuscript according to that checklist. \"Authors should mention more information regarding the non-respondents (i.e., how many individuals were willing to participant and how many were not?).\" Answer: We added in the non-respondents in the methods. \"Authors have used GCF in this study to measure EBV level. Is there any study that showed the use of saliva to measure the EBV?\" Answer: Yes, some study did measure EBV level in GCF or saliva or subgingival plaque. We added in the introduction. \"The introduction section is very brief and barely cover the rationale of the study and background of GCF in context. Authors should expand the introduction section a bit more adding GCF details. The following articles on GCF may be included (which I am a co-author): Human gingival crevicular fluids (GCF) proteomics: an overview.\" Dentistry Journal 5.1 (2017): 121. Bacteriological Evaluation of Gingival Crevicular Fluid in Teeth Restored Using Fixed Dental Prostheses: An In Vivo Study.\" International Journal of Molecular Sciences 22.11 (2021): 54632.\" Answer: Thank you for the suggestion, we added the references in the introduction section. \"Please add; What was the main purpose of using GCF in this study?\" Answer: We added in the purpose of using GCF in the discussion." } ] } ]	1	https://f1000research.com/articles/10-414

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